U.S. patent application number 10/277232 was filed with the patent office on 2003-11-13 for opioid receptors: compositions and methods.
This patent application is currently assigned to Arch Development Corporation. Invention is credited to Bell, Graeme I., Reisine, Terry, Yasuda, Kazuki.
Application Number | 20030211537 10/277232 |
Document ID | / |
Family ID | 38178728 |
Filed Date | 2003-11-13 |
United States Patent
Application |
20030211537 |
Kind Code |
A1 |
Bell, Graeme I. ; et
al. |
November 13, 2003 |
Opioid receptors: compositions and methods
Abstract
The invention relates generally to compositions of and methods
for obtaining opioid receptor polypeptides. The invention relates
as well to polynucleotides encoding opioid receptor polypeptides,
the recombinant vectors carrying those sequences, the recombinant
host cells including either the sequences or vectors, and
recombinant opioid receptor polypeptides. By way of example, the
invention discloses the cloning and functional expression of at
least three different opioid receptor polypeptides. The invention
includes as well, methods for using the isolated, recombinant
receptor polypeptides in assays designed to select and improve
substances capable of interacting with opioid receptor polypeptides
for use in diagnostic, drug design and therapeutic
applications.
Inventors: |
Bell, Graeme I.; (Chicago,
IL) ; Reisine, Terry; (Philadelphia, PA) ;
Yasuda, Kazuki; (Tokyo, JP) |
Correspondence
Address: |
Gina N. Shishima
FULBRIGHT & JAWORSKI L.L.P.
Suite 2400
600 Congress Avenue
Austin
TX
78701
US
|
Assignee: |
Arch Development
Corporation
|
Family ID: |
38178728 |
Appl. No.: |
10/277232 |
Filed: |
October 21, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10277232 |
Oct 21, 2002 |
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08455683 |
May 31, 1995 |
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08455683 |
May 31, 1995 |
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08292694 |
Aug 19, 1994 |
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6319686 |
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08292694 |
Aug 19, 1994 |
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PCT/US94/05747 |
May 20, 1994 |
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PCT/US94/05747 |
May 20, 1994 |
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08147592 |
Nov 5, 1993 |
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6096513 |
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08147592 |
Nov 5, 1993 |
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08100694 |
Jul 30, 1993 |
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08100694 |
Jul 30, 1993 |
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08066296 |
May 20, 1993 |
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Current U.S.
Class: |
435/7.1 ;
530/350 |
Current CPC
Class: |
C07K 14/705 20130101;
G01N 33/9486 20130101; G01N 2500/00 20130101; A61K 38/00 20130101;
G01N 33/566 20130101; C07K 2319/00 20130101 |
Class at
Publication: |
435/7.1 ;
530/350 |
International
Class: |
G01N 033/53; C07K
014/705 |
Goverment Interests
[0002] The research for the information disclosed herein was
supported by the Howard Hughes Medical Institute, American Diabetes
Association and United States Public Health Service Grants
DK-20595, DK-42086, MH-45533 and MH-48518. These organizations and
the United States government may own certain rights to the
invention disclosed herein.
Claims
What is claimed is:
1. An isolated and purified polynucleotide that encodes an opioid
receptor polypeptide.
2. The isolated and purified polynucleotide of claim 1, comprising
an isolated and purified polynucleotide that encodes a truncated
opioid receptor polypeptide.
3. The polynucleotide of claim 2, wherein said truncated opioid
receptor polypeptide is a truncated kappa or delta opioid receptor
polypeptide.
4. The polynucleotide of claim 3, wherein said truncated opioid
receptor polypeptide comprises amino acid residues 79 to 380 of a
kappa opioid receptor polypeptide.
5. The polynucleotide of claim 4, wherein said truncated opioid
receptor polypeptide comprises amino acid residues 167 to 228 of a
kappa opioid receptor polypeptide.
6. The polynucleotide of claim 4, wherein said truncated opioid
receptor polypeptide comprises amino acid residues 271 to 318 of a
kappa opioid receptor polypeptide.
7. The polynucleotide of claim 3, wherein said truncated opioid
receptor polypeptide comprises amino acid residues 70 to 372 of a
delta opioid receptor polypeptide.
8. The isolated and purified polynucleotide of claim 1, further
defined as encoding a chimeric opioid receptor polypeptide.
9. The polynucleotide of claim 8, wherein the polypeptide comprises
the second extracellular loop of delta opioid receptor.
10. The polynucleotide of claim 8, wherein the polypeptide
comprises the third extracellular loop of delta opioid
receptor.
11. The polynucleotide of claim 8, wherein the polypeptide portions
of both kappa and delta opioid receptors.
12. The polynucleotide of claim 1 1, wherein said chimeric
polypeptide is designated as .kappa..sub.1-78/.delta..sub.70-372 or
.delta..sub.1-69.kappa..sub.79-380.
13. The isolated and purified polynucleotide sequence of claim 1,
further defined as a mutant opioid receptor polypeptide.
14. The isolated and purified polynucleotide sequence of claim 13,
wherein in said mutant opioid receptor polypeptide is a mORD1
polypeptide having an asparagine at residue 95 instead of an
aspartate.
15. The isolated and purified polynucleotide sequence of claim 13,
wherein in said mutant opioid receptor polypeptide is a mutant
opioid receptor polypeptide having the amino acid residue sequence
of MORD1 of FIG. 1 except that residue number 128 is an asparagine
residue.
16. The isolated and purified polynucleotide sequence of claim 13,
wherein in said mutant opioid receptor polypeptide is a mutant
opioid receptor polypeptide having the amino acid residue sequence
of MORD1 of FIG. 1 except that residue number 278 is an asparagine
residue.
17. An isolated and purified opioid receptor polypeptide.
18. The opioid receptor polypeptide of claim 17, wherein the
polypeptide is a recombinant polypeptide.
19. The opioid receptor polypeptide of claim 17, wherein the
polypeptide is a delta, a kappa, or mu opioid receptor
polypeptide.
20. The opioid receptor polypeptide of claim 19, wherein said
polypeptide is a delta opioid receptor.
21. The opioid receptor polypeptide of claim 20, wherein said delta
opioid receptor comprises the amino acid residue sequence of SEQ ID
NO:4.
22. The opioid receptor polypeptide of claim 19, wherein said
polypeptide is a kappa opioid receptor.
23. The opioid receptor polypeptide of claim 22, wherein the kappa
opioid receptor comprises the amino acid sequence of SEQ ID
NO:2.
24. The opioid receptor polypeptide of claim 22, wherein the kappa
opioid receptor comprises the amino acid sequence of SEQ ID NO:
12.
25. The opioid receptor polypeptide of claim 17, comprising a
truncated opioid receptor polypeptide.
26. The opioid receptor polypeptide of claim 25, wherein said
truncated opioid receptor polypeptide is a truncated kappa or a
delta opioid receptor polypeptide.
27. The opioid receptor polypeptide of claim 25, wherein said
truncated opioid receptor polypeptide comprises amino acid residues
79 to 380 of a kappa opioid receptor polypeptide.
28. The opioid receptor polypeptide of claim 25, wherein said
truncated opioid receptor polypeptide comprises amino acid residues
167 to 228 of a kappa opioid receptor polypeptide.
29. The opioid receptor polypeptide of claim 25, wherein said
truncated opioid receptor polypeptide comprises amino acid residues
271 to 318 of a kappa opioid receptor polypeptide.
30. The opioid receptor polypeptide of claim 25, wherein said
truncated opioid receptor polypeptide comprises amino acid residues
70 to 372 of a delta opioid receptor polypeptide.
31. The opioid receptor polypeptide of claim 1, comprising a
chimeric opioid receptor polypeptide.
32. The polypeptide of claim 31, wherein the polypeptide comprises
the second extracellular loop of kappa opioid receptor.
33. The polypeptide of claim 31, wherein the polypeptide comprises
the third extracellular loop of delta opioid receptor.
34. The polypeptide of claim 31, wherein the polypeptide portions
of both kappa and delta opioid receptors.
35. The opioid receptor polypeptide of claim 31, wherein said
chimeric polypeptide is designated as
.kappa..sub.1-78/.delta..sub.70-372 or
.delta..sub.1-69/.kappa..sub.79-380.
36. The opioid receptor polypeptide sequence of claim 1, further
defined as a mutant opioid receptor polypeptide.
37. The opioid receptor polypeptide sequence of claim 36, wherein
in said mutant opioid receptor polypeptide is a mORD1 polypeptide
having an asparagine at residue 95 instead of an aspartate.
38. The opioid receptor polypeptide sequence of claim 36, wherein
in said mutant opioid receptor polypeptide is a mutant opioid
receptor polypeptide having the amino acid residue sequence of
MORD1 of FIG. 1 except that residue number 128 is an asparagine
residue.
39. The opioid receptor polypeptide sequence of claim 36, wherein
in said mutant opioid receptor polypeptide is a mutant opioid
receptor polypeptide having the amino acid residue sequence of
MORD1 of FIG. 1 except that residue number 278 is an asparagine
residue.
40. The isolated and purified polynucleotide sequence of claim 1,
wherein the encoded opioid receptor polypeptide has
pharmacologically altered properties relative to the
pharmacological properties of previously defined opioid
receptors.
41. The opioid receptor polypeptide of claim 40, comprising the
amino acid residue sequence of SEQ ID NO: 6.
42. An antibody immunoreactive with an opioid receptor
polypeptide.
43. A process of detecting an opioid receptor polypeptide, wherein
the process comprises: (a) immunoreacting the polypeptide with the
antibody of claim 42 to form an antibody-polypeptide conjugate; and
(b) detecting the conjugate.
44. A process of detecting a messenger RNA transcript that encodes
an opioid receptor polypeptide, wherein the process comprises: (a)
hybridizing the messenger RNA transcript with a polynucleotide
sequence that encodes the opioid receptor polypeptide to form a
duplex; and (b) detecting the duplex.
45. A diagnostic assay kit for detecting the presence of an opioid
receptor polypeptide in a biological sample, said kit comprising a
first container containing a first antibody capable of
immunoreacting with said opioid receptor polypeptide, wherein said
first antibody is present in an amount sufficient to perform at
least one assay.
46. A diagnostic assay kit for detecting the presence, in a
biological sample, of an antibody immunoreactive with an opioid
receptor polypeptide, said kit comprising a first container
containing an opioid receptor polypeptide that immunoreacts with
said antibody, and wherein said polypeptide is present in an amount
sufficient to perform at least one assay.
47. A process of screening a substance for its ability to interact
with an opioid receptor, said process comprising the steps of: a)
providing an opioid receptor polypeptide; b) testing the ability of
said substance to interact with said opioid receptor.
48. The process according to claim 47, wherein said opioid receptor
polypeptide is a chimeric opioid receptor polypeptide.
49. The process of claim 48, wherein the polypeptide comprises the
second extracellular loop of delta opioid receptor.
50. The process of claim 48, wherein the polypeptide comprises the
third extracellular loop of delta opioid receptor.
51. The process of claim 48, wherein the polypeptide portions of
both kappa and delta opioid receptors.
52. The process according to claim 48, wherein said chimeric opioid
receptor polypeptide is designated as
.kappa..sub.1-78/.delta..sub.70-372 or
.delta..sub.1-69/.kappa..sub.79-380.
53. The process according to claim 47, wherein said opioid receptor
polypeptide is a truncated opioid receptor polypeptide.
54. The process of claim 53, wherein said truncated opioid receptor
polypeptide is a truncated kappa or a delta opioid receptor
polypeptide.
55. The process of claim 53, wherein said truncated opioid receptor
polypeptide comprises amino acid residues 79 to 380 of a kappa
opioid receptor polypeptide.
56. The process according to claim 47, wherein said opioid receptor
polypeptide is a mutant opioid receptor polypeptide.
57. The process according to claim 56, wherein said mutant opioid
receptor polypeptide is a mORD1 polypeptide having an asparagine at
residue 95 instead of an aspartate.
58. The process according to claim 47, wherein providing said
opioid receptor polypeptide is transfecting a host cell with a
polynucleotide that encodes an opioid receptor polypeptide to form
a transformed cell and maintaining said transformed cell under
biological conditions sufficient for expression of said opioid
receptor polypeptide.
59. A process of making a product with an ability to act as a
specific agonist of a kappa opioid receptor, said process
comprising the steps of: a) providing an opioid receptor
polypeptide; and b) obtaining a candidate specific kappa opioid
receptor agonist; c) testing the ability of said substance to
interact with said opioid receptor; and d) providing a product that
has the ability to interact with the opioid receptor.
60. The process of claim 59, wherein the opioid receptor
polypeptide comprises a portion of a kappa opioid receptor
polypeptide.
61. The process of claim 60, wherein the opioid receptor
polypeptide comprises a portion of a second extracellular loop of
the kappa opioid receptor polypeptide.
62. The process of claim 61, wherein the opioid receptor
polypeptide comprises a negatively charged region of the second
extracellular loop of the kappa opioid receptor.
63. The process of claim 59, wherein the opioid receptor
polypeptide comprises a chimeric opioid receptor polypeptide.
64. The process of claim 63, wherein the polypeptide comprises the
second extracellular loop of kappa opioid receptor.
65. The process of claim 63, wherein the polypeptide comprises the
third extracellular loop of delta opioid receptor.
66. The process of claim 63, wherein the polypeptide comprises
portions of both kappa and delta opioid receptors.
67. The process of claim 63, wherein said chimeric polypeptide is
designated as .kappa..sub.1-78/.delta..sub.70-372 or
.delta..sub.1-69/.kappa..sub.79-380.
68. The process of claim 59, wherein the opioid receptor
polypeptide comprises a truncated opioid receptor polypeptide.
69. The process of claim 68, wherein said truncated opioid receptor
polypeptide is a truncated kappa opioid receptor polypeptide.
70. The process of claim 69, wherein the truncated opioid receptor
polypeptide comprises amino acid residues 79 to 380 of a kappa
opioid receptor polypeptide.
71. The process of claim 69, wherein the truncated opioid receptor
polypeptide comprises amino acid residues 167 to 228 of a kappa
opioid receptor polypeptide.
72. The process of claim 59, wherein the candidate specific kappa
opioid receptor agonist is pre-screened determining whether the
candidate has a positive charge.
73. The process according to claim 59, wherein providing said
opioid receptor polypeptide is transfecting a host cell with a
polynucleotide that encodes an opioid receptor polypeptide to form
a transformed cell and maintaining said transformed cell under
biological conditions sufficient for expression of said opioid
receptor polypeptide.
74. A specific kappa opioid receptor agonist isolatable by the
process of claim 59.
Description
[0001] This application is a continuation of PCT/US94/05747, filed
May 20, 1994, which was a continuation-in-part of U.S. patent
application Ser. No. 08/147,592, filed Nov. 5, 1993, which
application was itself a continuation-in-part of U.S. patent
application Ser. No. 08/100,694, filed Jul. 30, 1993, which
application was a continuation-in-part of U.S. patent application
Ser. No. 08/066,296, filed May 20, 1993. The disclosures of all of
the above applications are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] This invention relates generally to compositions of and
methods for obtaining opioid receptors. The invention relates as
well to the DNA sequences encoding opioid receptors, the
recombinant vectors carrying those sequences, the recombinant host
cells including either the sequences or vectors, and recombinant
opioid receptor polypeptides. The invention includes as well
methods for using the isolated, recombinant receptor polypeptides
in assays designed to select and improve among candidate substances
such as agonists and antagonists of opioid receptors and
polypeptides for use in diagnostic, drug design and therapeutic
applications.
[0005] 2. Description of Related Art
[0006] Opioid drugs have various effects on perception of pain,
consciousness, motor control, mood, and autonomic function and can
also induce physical dependence (Koob, et al 1992). The endogenous
opioid system plays an important role in modulating endocrine,
cardiovascular, respiratory, gastrointestinal and immune functions
(Olson, et al 1989). Opioids exert their actions by binding to
specific membrane-associated receptors located throughout the
central and peripheral nervous system (Pert, et al. 1973). The
endogenous ligands of these opioid receptors have been identified
as a family of more than 20 opioid peptides that derive from the
three precursor proteins proopiomelanocortin, proenkephalin, and
prodynorphin (Hughes, et al. (1975); Akil, et al. (1984)). Although
the opioid peptides belong to a class of molecules distinct from
the opioid alkaloids, they share common structural features
including a positive charge juxtaposed with an aromatic ring that
is required for interaction with the receptor (Bradbury, et al.
(1976)).
[0007] Pharmacological studies have suggested that there are at
least four major classes of opioid receptors, designated .delta.,
.kappa., .mu. and .sigma. (Simon 1991; Lutz, et al. 1992). The
classes differ in their affinity for various opioid ligands and in
their cellular distribution. The different classes of opioid
receptors are believed to serve different physiological functions
(Olson, et al., 1989; Simon 1991; Lutz & Pfister 1992).
However, there is substantial overlap of function as well as of
distribution. Biochemical characterization of opioid receptors from
many groups reports a molecular mass of .apprxeq.60,000 Da for all
three subtypes, suggesting that they could be related molecules
(Loh, et al. (1990)). Moreover, the similarity between the three
receptor subtypes is supported by the isolation of (i)
antiidiotypic monoclonal antibodies competing with both .mu. and
.delta. ligands but not competing with .kappa. ligands (Gramsch, et
al. (1988); Coscia, et al. (1991)) and (ii) a monoclonal antibody
raised against the purified .mu. receptor that interacts with both
.mu. and .kappa. receptors (Bero, et al. (1988)).
[0008] Opioids are used clinically in the management of pain, but
their use is limited by a constellation of undesirable side
effects, including respiratory depression, miosis, decreased
gastrointestinal motility, sedation, nausea and vomiting (Jaffe et
al., (1990)). A concern of the use of opioids in the treatment of
chronic pain is their potential for dependence and abuse. Studies
suggest the clinical effects of opioids are mediated via a variety
of receptors and that the therapeutic effects and the undesirable
side effects of opioids are mediated by different receptor
(sub)types (Jaffe et al, (1990); Pasternack, (1993)). Therefore,
the therapeutic and side effects of opioids can be separated with
the use of more selective agents for receptor subtypes. The present
invention discloses the pharmacological properties of the cloned
.kappa., .delta., and .mu. opioid receptors and receptor
selectivity of widely employed opioid ligands.
[0009] The .delta. receptors bind with the greatest affinity to
enkephalins and have a more discrete distribution in the brain than
either .mu. or .kappa. receptors, with high concentrations in the
basal ganglia and limbic regions. Although morphine interacts
principally with .mu. receptors, peripheral administration of this
opioid induces release of enkephalins (Bertolucci, et al. (1992)).
Thus, enkephalins may mediate part of the physiological response to
morphine, presumably by interacting with .delta. receptors. Despite
pharmacological and physiological heterogeneity, at least some
types of opioid receptors inhibit adenylate cyclase, increase
K.sup.+ conductance, and inactivate Ca.sup.2+ channels through a
pertussis toxin-sensitive mechanism (Puttfarcken, et al. 1988;
Attali, et al. 1989; Hsia, et al., 1984). These results and others
suggest that opioid receptors belong to the large family of cell
surface receptors that signal through G proteins (Di Chiara, et al.
(1992); Loh, et al. (1990)).
[0010] Several attempts to clone cDNAs encoding opioid receptors
have been reported. A cDNA encoding an opioid-binding protein
(OBCAM) with .mu. selectivity was isolated (Schofield, et al.
(1989)), but the predicted protein lacked transmembrane domains,
presumed necessary for signal transduction. More recently, the
isolation of another cDNA was reported, which was obtained by
expression cloning (Xie, et al. (1992)). The deduced protein
sequence displays seven putative transmembrane domains and is very
similar to the human neuromedin K receptor. However, the affinity
of opioid ligands for this receptor expressed in COS cells is two
orders of magnitude below the expected value, and no subtype
selectivity can be shown.
[0011] Many cell surface receptor/transmembrane systems consist of
at least three membrane-bound polypeptide components: (a) a
cell-surface receptor; (b) an effector, such as an ion channel or
the enzyme adenylate cyclase; and (c) a guanine nucleotide-binding
regulatory polypeptide or G protein, that is coupled to both the
receptor and its effector.
[0012] G protein-coupled receptors mediate the actions of
extracellular signals as diverse as light, odorants, peptide
hormones and neurotransmitters. Such receptors have been identified
in organisms as evolutionarily divergent as yeast and man. Nearly
all G protein-coupled receptors bear sequence similarities with one
another, and it is thought that all share a similar topological
motif consisting of seven hydrophobic (and potentially
.alpha.-helical) segments that span the lipid bilayer (Dohlman et
al. 1987; Dohlman et al. 1991).
[0013] G proteins consist of three tightly associated subunits,
.alpha., .beta. and .gamma. (1:1:1) in order of decreasing mass.
Following agonist binding to the receptor, a conformational change
is transmitted to the G protein, which causes the G.alpha.-subunit
to exchange a bound GDP for GTP and to dissociate from the
.beta..gamma.-subunits. The GTP-bound form of the .alpha.-subunit
is typically the effector-modulating moiety. Signal amplification
results from the ability of a single receptor to activate many G
protein molecules, and from the stimulation by G.alpha.-GTP of many
catalytic cycles of the effector.
[0014] The family of regulatory G proteins comprises a multiplicity
of different .alpha.-subunits (greater than twenty in man), which
associate with a smaller pool of .beta.- and .gamma.-subunits
(greater than four each) (Strothman and Simon 1991). Thus, it is
anticipated that differences in the .alpha.-subunits probably
distinguish the various G protein oligomers, although the targeting
or function of the various .alpha.-subunits might also depend on
the .beta. and .gamma. subunits with which they associate
(Strothman and Simon 1991).
[0015] Improvements in cell culture and in pharmacological methods,
and more recently, use of molecular cloning and gene expression
techniques, have led to the identification and characterization of
many seven-transmembrane segment receptors, including new sub-types
and sub-sub-types of previously identified receptors. The
.alpha..sub.1 and .alpha..sub.2-adrenergic receptors, once thought
to each consist of single receptor species, are now known to each
be encoded by at least three distinct genes (Kobilka et al. 1987;
Regan et al. 1988; Cotecchia et al. 1988; Lomasney 1990). In
addition to rhodopsin in rod cells, which mediates vision in dim
light, three highly similar cone pigments mediating color vision
have been cloned (Nathans et al. 1986A; and Nathans et al. 1986B).
All of the family of G protein-coupled receptors appear to be
similar to other members of the family of G protein-coupled
receptors (e.g., dopaminergic, muscarinic, serotonergic,
tachykinin, etc.), and each appears to share the characteristic
seven-transmembrane segment topography.
[0016] When comparing the seven-transmembrane segment receptors
with one another, a discernible pattern of amino acid sequence
conservation is observed. Transmembrane domains are often the most
similar, whereas the amino and carboxyl terminal regions and the
cytoplasmic loop connecting transmembrane segments V and VI can be
quite divergent (Dohlman et al. 1987).
[0017] Interaction with cytoplasmic polypeptides, such as kinases
and G proteins, was predicted to involve the hydrophobic loops
connecting the transmembrane domains of the receptor. The
challenge, however, has been to determine which features are
preserved among the seven-transmembrane segment receptors because
of conservation of function, and which divergent features represent
structural adaptations to new functions. A number of strategies
have been used to test these ideas, including the use of
recombinant DNA and gene expression techniques for the construction
of substitution and deletion mutants, as well as of hybrid or
chimeric receptors (Dohlman et al. 1991).
[0018] With the growing number of receptor sub-types, G-protein
subunits, and effectors, characterization of ligand binding and G
protein recognition properties of these receptors is an important
area for investigation. It has long been known that multiple
receptors can couple to a single G protein and, as in the case of
epinephrine binding to .beta..sub.2- and .alpha..sub.2-adrenergic
receptors, a single ligand can bind to multiple,
functionally-distinct, receptor sub-types. Moreover, G proteins
with similar receptor and effector coupling specificities have also
been identified. For example, three species of human G.sub.1 have
been cloned (Itoh et al. 1988), and alternate mRNA splicing has
been shown to result in multiple variants of G.sub.s (Kozasa et al.
1988). Cloning and over production of the muscarinic and
.alpha..sub.2-adrenergi- c receptors led to the demonstration that
a single receptor sub-type, when expressed at high levels in the
cell, will couple to more than one type of G protein.
[0019] Opioid receptors are known to be sensitive to reducing
agents, and the occurrence of a disulfide bridge has been
postulated as essential for ligand binding (Gioannini, et al.
1989). For rhodopsin, muscarinic, and .beta.-adrenergic receptors,
two conserved cysteine residues in each of the two first
extracellular loops have been shown to be critical for stabilizing
the functional protein structure and are presumed to do so by
forming a disulfide bridge. Structure/function studies of opioid
ligands have shown the importance of a protonated amine group for
binding to the receptor with high affinity. The binding site of the
receptor might, therefore, possess a critical negatively charged
counterpart. Catecholamine receptors display in their sequence a
conserved aspartate residue that has been shown necessary for
binding the positively charged amine group of their ligands.
[0020] Given the complexity and apparent degeneracy of function of
various opioid receptors, a question of fundamental importance is
how, and under what circumstances, do specific sub-type and
sub-sub-type receptors exert their physiological effect in the
presence of the appropriate stimulatory ligand. A traditional
approach to answering this question has been to reconstitute the
purified receptor and G protein components in vitro. Unfortunately,
purification schemes have been successful for only a very limited
number of receptor sub-types and their cognate G-proteins.
Alternatively, heterologous expression systems can be of more
general usefulness in the characterization of cloned receptors and
in elucidating receptor G protein coupling specificity (Marullo et
al. 1988; Payette et al. 1990; King et al. 1990).
[0021] One such system was recently developed in yeast cells, in
which the genes for a mammalian .beta..sub.2-adrenergic receptor
and G.sub.s .alpha.-subunit were co-expressed (King et al. 1990).
Expression of the .beta..sub.2-adrenergic receptor to levels
several hundred-fold higher than in any human tissue was attained,
and ligand binding was shown to be of the appropriate affinity,
specificity, and stereoselectivity. Moreover, a
.beta..sub.2-adrenergic receptor-mediated activation of the
pheromone signal transduction pathway was demonstrated by several
criteria, including imposition of growth arrest, morphological
changes, and induction of a pheromone-responsive promoter (FUS1)
fused to the Escherichia coli lacz gene (encoding
.beta.-galactosidase) (King et al. 1990).
[0022] Finally, expression of a single receptor in the absence of
other related sub-types is often impossible to achieve, even in
isolated, non-recombinant mammalian cells. Thus, there has been
considerable difficulty in applying the standard approaches of
classical genetics or even the powerful techniques of molecular
biology to the study of opioid receptors. In particular, means are
needed for the identification of the DNA sequences encoding
individual opioid receptors. Given such isolated, recombinant
sequences, it is possible to address the heretofore intractable
problems associated with design and testing of isoform-specific
opioid receptor agonists and antagonists. The availability of cDNAs
encoding the opioid receptors will permit detailed studies of
signal-transduction mechanisms and reveal the anatomical
distribution of the mRNAs of these receptors, providing information
on their expression pattern in the nervous system. This information
should ultimately allow better understanding of the opioid system
in analgesia, and also the design of more specific therapeutic
drugs.
[0023] Availability of polynucleotide sequences encoding opioid
receptors, and the polypeptide sequences of the encoded receptors,
will significantly increase the capability to design pharmaceutical
compositions, such as analgesics, with enhanced specificity of
function. In general, the availability of these polypeptide
sequences will enable efficient screening of candidate
compositions. The principle in operation through the screening
process is straightforward: natural agonists and antagonists bind
to cell-surface receptors and channels to produce physiological
effects; certain other molecules bind to receptors and channels;
therefore, certain other molecules may produce physiological
effects and act as therapeutic pharmaceutical agents. Thus, the
ability of candidate drugs to bind to opioid receptors can function
as an extremely effective screening criterion for the selection of
pharmaceutical compositions with a desired functional efficacy.
[0024] Prior methods for screening candidate drug compositions
based on their ability to preferentially bind to cell-surface
receptors has been limited to tissue-based techniques. In these
techniques, animal tissues rich in the receptor type of interest
are extracted and prepared; candidate drugs are then allowed to
interact with the prepared tissue and those found to bind to the
receptors are selected for further study. However, these
tissue-based screening techniques suffer from several significant
disadvantages. First, they are expensive because the source of
receptor cell tissue--laboratory animals--is expensive. Second,
extensive technical input is required to operate the screens. And,
third, the screens may confuse the results because there are no
tissues where only one receptor subtype is expressed exclusively.
With traditional prior art screens you are basically looking at the
wrong interactions or, at best, the proper interactions mixed in
with a whole variety of unwanted interactions. An additional
fundamental deficiency of animal tissue screens is that they
contain animal receptors--ideal for the development of drugs for
animals but of limited value in human therapeutic agents.
[0025] A solution to this problem is provided by the present
invention. A polynucleotide of the present invention, transfected
into suitable host cells, can express polypeptide sequences
corresponding to opioid receptors, both in large quantities and
through relatively simple laboratory procedures. The result is the
availability of extremely specific receptor-drug interactions free
from the competitive and unwanted interactions encountered in
tissue-based screens. Further expression in a microorganism where
no such endogenous receptors exist (e.g. yeast cells or mutant
mammalian cell lines) can be useful for screening and evaluating
sub-type-selective drugs (Marullo et al. 1988; Payette et al. 1990;
and King et al. 1990).
SUMMARY OF THE INVENTION
[0026] Generally, the present invention concerns opioid receptors.
The inventors have isolated and cloned genes that code various
opioid receptors, including the delta and the kappa receptors.
These genes have been expressed into opioid receptor polypeptides.
These polypeptides have a variety of utility, with one of the most
important being the ability to serve as the basis for screening
assays that allow for the determination of substances that can be
used as opioid receptor agonists and antagonists. Such agonists and
antagonists have pharmaceutical utility. There is a great need for
new opioid receptor agonists and antagonists, since those currently
used have rather severe side-effects.
[0027] The present invention provides an isolated and purified
polynucleotide that encodes an opioid receptor polypeptide. In a
preferred embodiment, a polynucleotide of the present invention is
a DNA molecule. More preferably, a polynucleotide of the present
invention encodes a polypeptide that is a delta, kappa, mu or sigma
opioid receptor. Even more preferred, a polynucleotide of the
present invention encodes a polypeptide comprising the amino acid
residue sequence of kappa opioid receptor, e.g. mORK1 (SEQ ID NO:
2) or human kappa opioid receptor (SEQ ID NO: 12) or delta opioid
receptor, e.g. mORD1 (SEQ ID NO: 4). Most preferably, an isolated
and purified polynucleotide of the invention comprises the
nucleotide base sequence of kappa opioid receptor, e.g. mORK1 (SEQ
ID NO: 1) or human kappa opioid receptor (SEQ ID NO: 11), or delta
opioid receptor (SEQ ID NO: 3).
[0028] The present invention provides isolated and purified
polynucleotides that encode opioid receptor polypeptides with
pharmacologically altered properties relative to the
pharmacological properties of previously defined opioid receptor
polypeptides, for example, MOP2 (SEQ ID NO: 6). One such opioid
receptor polypeptide encoding polynucleotide of the invention
comprises the nucleotide base sequence of SEQ ID NO: 5.
[0029] The present invention also contemplates and allows for the
production of mutant opioid receptors. These mutant receptors have
altered binding activities and pharmacological activities relative
to the naturally occurring opioid receptors from which they are
mutated. For example, it has been shown that inserting a histidine
at certain amino acid residues of the mORD1 sequence can prevent
the binding of opioid agonists to that receptor. Examples of mutant
opioid receptor polypeptides include mORD1 polypeptide having an
asparagine at residue 128 instead of an aspartate and mORD1
polypeptide having an asparagine at residue 278 instead of a
histidine. These mutated receptors have utility in screening
assays. Of course, the invention also contemplates nucleotide
sequences encoding these mutant opioid receptors.
[0030] The present invention contemplates an isolated and purified
polynucleotide comprising a base sequence that is identical or
complementary to a segment of at least 15, 20, 25, 30, 35, 40, 45,
50 or more contiguous bases of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID
NO: 5, or SEQ ID NO: 11, wherein the polynucleotide hybridizes to a
polynucleotide that encodes an opioid receptor polypeptide.
Preferably, the isolated and purified polynucleotide comprises a
base sequence that is identical or complementary to a segment of at
least 15 to 100 contiguous bases of SEQ ID NO: 1, SEQ ID NO: 3, SEQ
ID NO: 5, or SEQ ID NO: 11. Of course, the isolated and purified
polynucleotide comprises a shorter base sequence that is identical
or complementary to a segment of at least 25 to 75 contiguous bases
of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 11.
Further, the isolated and purified polynucleotide comprises a
shorter base sequence that is identical or complementary to a
segment of at least 35 to 60 contiguous bases of SEQ ID NO: 1, SEQ
ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 11. Also, the polynucleotide
of the invention can comprise a segment of bases identical or
complementary to 40 or 55 contiguous bases of the disclosed
nucleotide sequences.
[0031] The present invention enables one to obtain an isolated and
purified polynucleotide comprising a base sequence that is
identical or complementary to a segment of at least 10 contiguous
bases of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO:
11. The polynucleotide of the invention hybridizes to SEQ ID NO: 1,
SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 11, or a complement of
SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 11.
Preferably, the isolated and purified polynucleotide comprises a
base sequence that is identical or complementary to a segment of at
least 25 to 70 contiguous bases of SEQ ID NO: 1, SEQ ID NO: 3, SEQ
ID NO: 5, or SEQ ID NO: 11. For example, the polynucleotide of the
invention can comprise a segment of bases identical or
complementary to 40 or 55 contiguous bases of SEQ ID NO: 1 (the
coding portion of which encodes SEQ ID NO:2).
[0032] The present invention further contemplates an isolated and
purified opioid receptor polypeptide. Preferably, an opioid
receptor polypeptide of the invention is a recombinant polypeptide.
More preferably, an opioid receptor polypeptide of the present
invention is delta, kappa, mu or sigma opioid receptor polypeptide.
Even more preferably, an opioid receptor polypeptide of the present
invention comprises the amino acid residue sequence of SEQ ID NO:
2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 12.
[0033] The present invention contemplates an expression vector
comprising a polynucleotide that encodes an opioid receptor
polypeptide. Preferably, an expression vector of the present
invention comprises a polynucleotide that encodes a polypeptide
comprising the amino acid residue sequence of SEQ ID NO: 2, SEQ ID
NO: 4, SEQ ID NO: 6, or SEQ ID NO: 12. More preferably, an
expression vector of the present invention comprises a
polynucleotide comprising the nucleotide base sequence of SEQ ID
NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 11. Even more
preferably, an expression vector of the invention comprises a
polynucleotide operatively linked to an enhancer-promoter. More
preferably still, an expression vector of the invention comprises a
polynucleotide operatively linked to a prokaryotic promoter.
Alternatively, an expression vector of the present invention
comprises a polynucleotide operatively linked to an
enhancer-promoter that is a eukaryotic promoter, and the expression
vector further comprises a polyadenylation signal that is
positioned 3' of the carboxy-terminal amino acid and within a
transcriptional unit of the encoded polypeptide.
[0034] The present invention includes recombinant host cells
transfected with a polynucleotide that encodes an opioid receptor
polypeptide. Preferably, a recombinant host cell of the present
invention is transfected with the polynucleotide of SEQ ID NO: 1,
SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 11. In one aspect, a host
cell of the invention is a eukaryotic host cell. It is contemplated
that virtually any eukaryotic cell that is know to those of skill
as being a suitable recombinant host will be useful in this regard.
For example, a recombinant host cell of the present invention can
be a yeast cell, a COS-1 cell, a PC12 cell, or a CHO-D644 cell, or
the like.
[0035] Recombinant host cells of the present invention can be
prokaryotic host cells. Those of skill will understand that
prokaryotic cells are very useful in the practice of cloning and
performing genetic manipulations. Therefore, there may be clear
advantages to using prokaryotes containing the genetic sequences of
the invention. There are also methods whereby active polypeptides
can be obtained from prokaryotes, i.e. some systems allow for
proper polypeptide folding and many allow for the translation of a
polypeptide that can then be denatured to remove improper folding
and renatured to an active form. For use in peptide expression, a
recombinant host cell typically comprises a polynucleotide under
the transcriptional control of regulatory signals functional in the
recombinant host cell, wherein the regulatory signals appropriately
control expression of an opioid receptor polypeptide in a manner to
enable all necessary transcriptional and post-transcriptional
modification. Prokaryotic methods for producing active polypeptides
are included within the ambit of this invention. An, exemplary
recombinant host cell of the invention is a bacterial cell of the
DH5.alpha. strain of Escherichia coli.
[0036] The present invention also contemplates a process of
preparing an opioid receptor polypeptide comprising transfecting a
cell with polynucleotide that encodes an opioid receptor
polypeptide to produce a transformed host cell; and maintaining the
transformed host cell under biological conditions sufficient for
expression of the polypeptide. Preferably, the transformed host
cell is a eukaryotic cell, for example, a COS-1 cell.
Alternatively, the host cell is a prokaryotic cell, for example
DH5.alpha. strain of Escherichia coli cell. In a preferred
embodiment, a polynucleotide transfected into the transformed cell
comprises the nucleotide base sequence of SEQ ID NO: 1, SEQ ID NO:
3, SEQ ID NO: 5, or SEQ ID NO: 11.
[0037] The present invention provides for antibodies that are
immunoreactive with an opioid receptor polypeptide. Preferably, an
antibody of the invention is a monoclonal antibody. More
preferably, an opioid receptor polypeptide comprises the amino acid
residue sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or
SEQ ID NO: 12.
[0038] The invention contemplates a process of producing an
antibody immunoreactive with an opioid receptor polypeptide
comprising the steps of (a) transfecting a recombinant host cell
with a polynucleotide that encodes an opioid receptor polypeptide;
(b) culturing the host cell under conditions sufficient for
expression of the polypeptide; (c) recovering the polypeptide; and
(d) preparing the antibody to the polypeptide. Preferably, the host
cell is transfected with the polynucleotide of SEQ ID NO: 1, SEQ ID
NO: 3, SEQ ID NO: 5, or SEQ ID NO: 11. Even more preferably, the
present invention provides an antibody prepared according to the
process described above.
[0039] The present invention provides a process of detecting an
opioid receptor polypeptide, wherein the process comprises
immunoreacting the polypeptide with an antibody prepared according
to the process described above, to form an antibody-polypeptide
conjugate, and detecting the conjugate.
[0040] The present invention contemplates a process of detecting a
messenger RNA transcript that encodes an opioid receptor
polypeptide, wherein the process comprises (a) hybridizing the
messenger RNA transcript with a polynucleotide sequence that
encodes the opioid receptor polypeptide to form a duplex; and (b)
detecting the duplex. Alternatively, the present invention provides
a process of detecting a DNA molecule that encodes an opioid
receptor polypeptide, wherein the process comprises (a) hybridizing
DNA molecules with a polynucleotide that encodes an opioid receptor
polypeptide to form a duplex; and (b) detecting the duplex.
[0041] The present invention provides a diagnostic assay kit for
detecting the presence of an opioid receptor polypeptide in a
biological sample, where the kit comprises a first container
containing a first antibody capable of immunoreacting with an
opioid receptor polypeptide, with the first antibody present in an
amount sufficient to perform at least one assay. Preferably, an
assay kit of the invention further comprises a second container
containing a second antibody that immunoreacts with the first
antibody. More preferably, the antibodies used in an assay kit of
the present invention are monoclonal antibodies. Even more
preferably, the first antibody is affixed to a solid support. More
preferably still, the first and second antibodies comprise an
indicator, and, preferably, the indicator is a radioactive label or
an enzyme.
[0042] The present invention provides a diagnostic assay kit for
detecting the presence, in biological samples, of a polynucleotide
that encodes an opioid receptor polypeptide, the kits comprising a
first container that contains a second polynucleotide identical or
complementary to a segment of at least 10 contiguous nucleotide
bases of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO:
11.
[0043] In a further embodiment, the present invention contemplates
a diagnostic assay kit for detecting the presence, in a biological
sample, of an antibody immunoreactive with an opioid receptor
polypeptide, the kit comprising a first container containing an
opioid receptor polypeptide that immunoreacts with the antibody,
with the polypeptide present in an amount sufficient to perform at
least one assay.
[0044] The present invention contemplates a process of screening
substances for their ability to interact with an opioid receptor
polypeptide comprising the steps of providing an opioid receptor
polypeptide, and testing the ability of selected substances to
interact with the opioid receptor polypeptide. In a preferred
embodiment, the opioid receptor polypeptide is a chimeric opioid
receptor polypeptide. The invention contemplates a virtually
endless array of possible chimeric receptors, and certain of these
receptors will have advantages depending upon whether one wishes to
screen for specific agonists, non-specific agonists, or antagonists
to a particular opioid receptor. The inventors have discovered that
there are specific binding regions for each type of opioid receptor
ligand. By employing a chimera that has one particular ligand
binding site, for example, a kappa-specific agonist binding site,
and lacks the non-specific binding site, one can screen for
kappa-specific ligands without worrying about false signals from
non-specific ligands.
[0045] In one embodiment, the invention contemplates transfecting a
host cell with a polynucleotide that encodes an opioid receptor
polypeptide to form a transformed cell and maintaining the
transformed cell under biological conditions sufficient for
expression of the opioid receptor polypeptide.
[0046] In another aspect, the present invention provides an
isolated and purified polynucleotide that encodes a truncated
opioid receptor polypeptide. A "truncated" opioid receptor
polypeptide comprises a portion of the amino acid sequence of a
full length opioid receptor. Truncated receptors typically are
created by standard genetic manipulations of genetic material that
encodes a longer amino acid sequence. Truncated receptors will have
great utility in screening assays. Since it is possible to truncate
a receptor so that one or more of the ligand binding sites is
missing. For example, the opioid receptor can be a kappa or a delta
opioid receptor polypeptide. In specific examples, the opioid
receptor comprises amino acid residues 79 to 380 of a kappa opioid
receptor polypeptide or amino acid residues 70 to 372 of a delta
opioid receptor polypeptide.
[0047] The invention provides for isolated and purified
polynucleotides that encode chimeric opioid receptor polypeptides.
A "chimeric" opioid receptor polypeptide is a polypeptide that
comprises amino acid sequences from two or more sources, wherein at
least one of the sources is an opioid receptor polypeptide. For
example, chimeras consisting of portions of the amino acid
sequences of one or more of delta opioid receptor, mu, opioid
receptor, kappa opioid receptor, sigma opioid receptor, MOP2, or
one of the somatostatin receptors are possible. Exemplary chimeras
can be kappa-delta (carboxy-amino terminus), delta-kappa,
kappa-delta-kappa, etc. The inventors have made numerous chimeras
during the course of their studies, and certain of these have
advantages in screening assays. Examples of such chimeras that have
been or could be constructed are
.kappa..sub.1-78/.delta..sub.70-372,
.delta..sub.1-69/.kappa..sub.79-380,
.kappa..sub.1-74/.delta..sub.65-372 or
.delta..sub.1-64/.kappa..sub.75-38- 0, and the like.
[0048] Chimeras of the present invention are very useful in
screening assays designed to allow for the detection and
elucidation of opioid ligands that perform a desired purpose. As a
class, the opioid receptors comprise extracellular loops,
transmembrane regions, intracellular loops, and an extracellular
amino terminus. The inventors have shown that the extracellular
portions of the receptors, the extracellular loops and the amino
terminus, serve as the binding sites for opioid receptor ligands.
For example, with regards to the kappa receptor, it has been shown
that kappa-specific agonists bind to the second extracellular loop
while antagonists bind to the amino terminus. The inventors
strongly suspect that non-specific agonists bind to the third
extracellular loop of the kappa receptor, and studies are in
progress that should prove this. With this knowledge, it is
possible to design chimeras that are very useful as specific
screening tools. For example, if one wishes to screen for
kappa-specific agonists, a chimera having the second extracellular
loop of the kappa receptor should be used. Further, a chimera
having the second extracellular loop of the kappa receptor but
lacking the third extracellular loop could have the advantage of
detecting kappa specific agonists without any fear of detecting
non-specific agonists. Of course, a chimera having all of the
regions of the kappa receptor except the second extracellular loop
can be used as a negative control in assays designed to screen for
kappa-specific agonists. The inventors have constructed many such
chimeras, and are in the process of constructing more. It is
possible to create an almost endless array of chimeras using
standard genetic manipulations and the knowledge that the inventors
have derived concerning the ligand binding sites of the opioid
receptors. All such chimeras, the polynucleotides encoding them,
and methods of using them in assays are contemplated within the
scope of the invention.
[0049] The present invention further provides a process of
screening a substance for its ability to interact with an opioid
receptor, the process comprising the steps of:
[0050] a) providing a chimeric opioid receptor polypeptide; and
[0051] b) testing the ability of the substance to interact with the
chimeric opioid receptor polypeptide. A preferred chimeric is the
same as set forth above.
[0052] The present invention still further provides a process of
screening a substance for its ability to interact with an opioid
receptor, the process comprising the steps of:
[0053] a) providing a truncated opioid receptor polypeptide;
and
[0054] b) testing the ability of the substance to interact with the
truncated opioid receptor polypeptide. A preferred truncated
receptor polypeptide is the same as set forth above.
[0055] Other aspects of the invention include assays that are
useful to screen suitable candidates for the ability to act as a
specific agonist of a kappa opioid receptor. These assays have been
made possible by the inventors' discovery that specific agonists of
the kappa opioid receptor bind to a different region of the
receptor than do non-specific agonists. Such screening assays
involve the steps of providing an opioid receptor polypeptide,
obtaining a candidate specific kappa opioid receptor agonist, an
assaying the ability of the candidate substance to interact with
the opioid receptor. Those of skill in the art will recognize that
the ability of the candidate substance to interact with the kappa
receptor may be assayed in any number of ways, including, but not
limited to, those describe in detail in the Detailed Description of
the Invention section of the application. These screening processes
allow for the elucidation of specific kappa receptor agonists that
do not have the negative side-effects of present less-specific
kappa agonists.
[0056] In a preferred embodiment, the opioid receptor polypeptide
used in the screening assay comprises a portion of a kappa opioid
receptor polypeptide. More preferably, the opioid receptor
polypeptide comprises a portion of a second extracellular loop of
the kappa opioid receptor polypeptide, which has been shown to have
a binding site for kappa receptor-specific agonists. It is expected
that opioid receptor polypeptides comprising a negatively charged
region of the second extracellular loop of the kappa opioid
receptor will be particularly preferred for use in these screening
procedures, since kappa receptor specific agonist-kappa receptor
binding appears to be based, at least in part, on charge
interactions between the negatively-charged portions of the second
extracellular loop and positively charged portions of the
agonists.
[0057] Chimeric opioid receptor polypeptides will be usable in the
above-described assays. In fact, the studies that led to the
elucidation of these assays were carried out with chimeric
receptors. In preferred embodiments, the chimeric receptor
comprises the second extracellular loop of the kappa opioid
receptor. The kappa second extracellular loop is located between
amino acid residues 167-228 of the kappa opioid receptor
polypeptide. Other preferred chimeras will have the second
extracellular loop of the kappa receptor, but lack the third
extracellular loop. Since the third extracellular loop contains the
putative non-specific agonist binding region, a chimera lacking
this region will be expected to not be able to detect non-specific
agonist activity. Therefore, any agonism seen for such a chimera
will have to be the result of a kappa-specific agonist binding to
the second extracellular loop. Chimeras lacking the second
extracellular loop will be useful as negative controls. When
provided with the teachings of this specification, those of skill
will be able to formulate chimeras and controlled screening
strategies that allow for the screening of all forms of opioid
receptor agonists and antagonists. All such assays are within the
scope of the invention. Specific examples of chimeric opioid
receptors that are useful in such screening assays are:
.kappa..sub.1-78/.delta..sub.70-372,
.delta..sub.1-69/.kappa..sub.79-380,
.kappa..sub.1-74/.delta..sub.65-372 or
.delta..sub.1-64/.kappa..sub.75-38- 0, and the like.
[0058] Truncated opioid receptor polypeptides will be useful in the
above-described candidate screening assays. It is to be anticipated
that shorter polypeptides that exhibit kappa receptor-specific
agonist binding will have certain advantages over longer
polypeptide. Preferably, the truncated opioid receptor polypeptide
is a truncated kappa opioid receptor polypeptide. For example, a
truncated opioid receptor polypeptide comprising amino acid
residues 79 to 380 of a kappa opioid receptor polypeptide is
expected to be useful in this regard. Truncated kappa opioid
receptors comprising the second extracellular loop of the receptor
will be useful in these assays. For example, a truncated kappa
receptor comprising amino acid residues 167 to 228 will be useful
in the invention. Of course, it is possible to use specifically
those amino acid residues which correspond to the kappa
receptor-specific agonist binding region of the kappa receptor
second extracellular loop.
[0059] Potential kappa receptor-specific agonists can be
pre-screened prior to being tested with the described assays by
determining whether the candidate has a positive charge. Charge
relationships influence the kappa receptor-specific agonist binding
mechanism, with the negatively charged binding region binding
positively charged agonists. Of course, it is possible for an
effective agonist not to be positively charged, however, the
assessment of charge will provide one mechanism for narrowing of
the range of agonists to be tested.
[0060] One embodiment of the assay methods described above entails
transfecting a host cell with a polynucleotide that encodes an
opioid receptor polypeptide to form a transformed cell and
maintaining the transformed cell under biological conditions
sufficient for expression of the opioid receptor polypeptide. The
polypeptides thus obtained may be used in the screening assay.
[0061] Other aspects of the invention include specific kappa opioid
receptor agonists that are isolatable and/or isolated by the by the
process described above.
BRIEF DESCRIPTION OF THE DRAWINGS
[0062] In the drawings, which form a portion of the
specification:
[0063] FIG. 1 shows a comparison of amino acid sequences of mouse
kappa opioid receptor (mORK1) and mouse delta opioid receptor
(mORD1). Asterisks denote identical amino acids, and bars indicate
similar residues. Gaps introduced to generate this alignment are
represented by colons. The seven predicted transmembrane domains
(TM1-TM7) are noted. The potential sites for N-linked glycosylation
in the NH.sub.2-terminal extracellular domain are underlined. There
are potential phosphorylation sites for cAMP dependent protein
kinase in mORK1 and mORD1 at residues 274 and 260, respectively.
Potential protein kinase C phosphorylation sites are present in
mORK1 at residues 242, 255, 344 and 352, and in mORD1 at residues
255, 357 and 369.
[0064] FIG. 2A and FIG. 2B show that mouse kappa (a) and delta (b)
opioid receptors mediate opioid inhibition of cAMP formation. COS-1
cells transiently expressing mouse kappa and delta opioid receptors
were treated with forskolin (10 .mu.M).+-.1 .mu.M opioid agonist or
1 .mu.M agonist and 10 .mu.M naloxone. For these studies, equal
numbers of cells (5.times.10.sup.5) were plated. In cells
expressing kappa and delta opioid receptors, the basal cAMP levels
were 40.+-.3 p/mol/well and forskolin stimulated cAMP formation
5-fold (203.+-.10 pmol/well). The values are expressed as percent
of forskolin-stimulated cAMP formation and are the mean.+-.SEM of
three different experiments. The asterisks indicate significant
(p<0.05) difference in cAMP levels between forskolin and opioid
agonist/antagonist-treated cells. Nal, naloxone; EKC,
ethylketocyclazocine.
[0065] FIG. 3 shows a partial genomic sequence for a human kappa
opioid receptor. Intron 1 begins at residue 1 and ends at residue
101. Intron 2 begins at residue 454. The length of intron 2 is
undetermined presently. The 13 colons after residue 455 does not
represent 13 unknown nucleotides. The colons signify that intron 2
contains more nucleotide residues than is set out in FIG. 4A and
FIG. 4B starts at residue 503 and ends at residue 435. The stop
codon begins at residue 436. In exon 2, there are several
undetermined nucleotide residues. These residues are at 656, 657,
691, 692, 945, and 955.
[0066] FIG. 4A and FIG. 4B show a comparison of the amino acid
sequences of human kappa and mouse kappa (mORK1) opioid receptors.
Gaps introduced to generate this alignment are represented by
colons. Amino acid residues 255, 267, 351 and 355 are unidentified
because the corresponding nucleotide sequences are as yet
unidentified. The mouse sequence begins with amino acid residue 1,
and the human sequence begins with amino acid residue 87.
[0067] FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E and FIG. 5F show
saturable binding of [.sup.3H]U-69,593, [.sup.311]naltrindole, or
[.sup.3H]DAMGO to the cloned .kappa., .delta. and .mu. opioid
receptors. Membranes from PC12 cells stably expressing the cloned
.kappa. receptor (FIG. 5A and FIG. 5B), CHO-DG44 cells stably
expressing the cloned 6 receptor (FIG. 5C and FIG. 5D), or COS-7
cells transiently expressing the cloned .mu. receptor (FIG. 5E and
FIG. 5F) were incubated for 40 min at 25.degree. C. with increasing
concentrations of [.sup.3H]U-69,593, [.sup.3H]naltrindole, or
[.sup.3H]DAMGO, respectively, in the presence (.DELTA.) or absence
(.quadrature.) of 10 uM naloxone in order to determine specific
binding (.circle-solid.). Upper, saturation isotherms of
representative experiments; lower, linearization of the saturation
isotherm data. Analysis of the saturable binding to the .kappa.
receptor revealed that [.sup.3H]U-69,593 bound to a single site
with a K.sub.D of 2.8 nM and a B.sub.max of 3346 fmol/mg protein.
Analysis of the saturable binding to the 6 receptor revealed that
[.sup.3H]DAMGO bound to a single site with a K.sub.D of 0.18 nM and
a B.sub.max of 633 fmol/mg protein. Analysis of the saturable
binding to the .mu. receptor revealed that [.sup.3H]DAMGO bound to
a single site with a K.sub.D of 0.57 nM and a B.sub.max of 444
fmol/mg protein. Experiments were conducted in triplicate and the
results of two to three independent experiments were similar.
[0068] FIG. 6A and FIG. 6B show the correlation of the potencies of
opioid ligands to inhibit radioligand binding to the cloned
.kappa., .delta., and .mu. opioid receptors with opioid receptors
characterized in heterogenous tissues. Correlation analyses were
performed by plotting the logarithm of the affinities of opioid
ligands for the cloned .kappa. (a) and cloned .mu. (b) receptors
vs. the logarithm of the potencies of these compounds to inhibit
subtype-selective radioligand binding to these opioid receptor
types in heterogenous tissues. The affinities of ligands for the
.kappa. and .mu. receptors were highly correlated with literature
values, with r values of 0.954 and 0.879, respectively. The
correlation of potencies at the .delta. receptor was much poorer
(r=0.185) (not plotted).
[0069] FIG. 7A shows a schematic of wild-type .delta. receptor.
[0070] FIG. 7B shows a schematic of wild-type .kappa. receptor.
[0071] FIG. 7C shows a schematic of
.kappa..sub.1-78/.delta..sub.70-372 chimeric receptor.
[0072] FIG. 7D shows a schematic of
.delta..sub.1-69/.kappa..sub.79-380 chimeric receptor.
[0073] FIG. 8 shows the binding properties of the chimeric
.kappa..sub.1-78/.delta..sub.70-372 receptor. Binding if 6- and
K-selective agonists and antagonists to the chimeric
.kappa..sub.1-78/.delta..sub.70-372 receptor. COS-7 cells were
transfected by the calcium phosphate precipitation method with the
wild-type .delta. or .kappa. or .kappa..sub.1-78/.delta..sub.70-372
receptor cDNAs. .delta.- and .kappa.-selective agonists
([.sup.3H]DPDPE and [.sup.3H]U-69,593, respectively) and
antagonists ([.sup.3H]naltrindole and [.sup.3H]naloxone,
respectively) were tested for their abilities to bind to the
.kappa..sub.1-78/.delta..sub.70-372 receptor. Values are express as
percent [.sup.3H]DPDPE and [.sup.3H]naltrindole binding to
wild-type .delta. and [.sup.3H]U-69,593 and [.sup.3H]naloxone
binding to wild-type .kappa. receptors. The average binding of
[.sup.3H]DPDPE and [.sup.3H]naltrindole to wild-type 6 receptor was
1987 fmol/mg protein and 2404 fmol/mg protein, respectively; the
average binding of [.sup.3H]U-69,593 and [.sup.3H]naloxone to
wild-type .kappa. receptor was 998 fmol/mg protein and 2085 fmol/mg
protein, respectively. These are the average results of 3-4
different experiments.
[0074] FIG. 9A and FIG. 9B show the inhibition of [.sup.3H]DPDPE
(a) and [.sup.3H]naloxone (b) binding to the
.kappa..sub.1-78/.delta..sub.70-372 chimera by .kappa.- and
.delta.-selective agents. The .delta.-selective agonists DSLET
(.box-solid.) and DPDPE (.circle-solid.) and the .delta.-selective
antagonist naltrindole () were tested for their abilities to
inhibit [.sup.3H]DPDPE binding to this chimera (top). IC.sub.50
values for inhibition of [.sup.3H]DPDPE binding were 5.8, 2.0 and
0.25 nM for DPDPE, DSLET and naltrindole, respectively. The
IC.sub.50 value for inhibition of [.sup.3H]naloxone binding was 14
nM for naloxone (.diamond-solid.), but the .kappa.-selective
agonist U-50,488 () did not inhibit [.sup.3H]naloxone binding to
the .kappa..sub.1-78/.delta..sub.70-- 372 chimera (bottom).
[0075] FIG. 10A and FIG. 10B show the inhibition of
forskolin-stimulated cAMP accumulation. COS-7 cells were
transfected by the calcium phosphate precipitation method with
wild-type (solid bars), chimeric (open bars) or truncated (hatched
bars) receptor cDNA. .kappa.- and .delta.-selective agonists (1
.mu.M U-50,488 and DSLET, respectively) were tested for their
abilities to inhibit 10 .mu.M forskolin-stimulated cAMP
accumulation. The abilities of .kappa.- and .delta.-selective
antagonists (1 .mu.M naloxone and naltrindole, respectively) to
block the effects of agonists were also examined. Results were
calculated as a percent of forskolin-stimulated cAMP accumulation
(173 pmol/well for wild-type .delta. receptor, 244 pmol/well for
wild-type .kappa. receptor, 172 pmol/well for
.delta..sub.1-69/.kappa..sub.79-380 receptor, 205 pmol/well for
.kappa..sub.1-78/.delta..sub.70-372 receptor, 100 pmol/well for
.delta..sub.70-372 receptor, and 51 pmol/well for
.kappa..sub.79-380 receptor). Basal cAMP levels, which were <5%
of forskolin-stimulated cAMP levels, were subtracted for all
values. The results are the means.+-.S.E.M. of 3 different
experiments.
[0076] FIG. 11A, FIG. 11B, and FIG. 11C show the saturable binding
of .sup.3H-naltrindole to the wild-type and D128N and H278N mutant
delta receptors. Saturable binding of 3H-naltrindole to the
wild-type (A, open squares) D128N mutant (B, filled circles) and
H278N mutant (C, open circles) was determined to assess the
affinity (K.sub.d) and density (B.sub.max) of each receptor
expressed in COS-7 cells.
[0077] FIG. 12A, FIG. 12B, and FIG. 12C show inhibition of
.sup.3H-naltrindole binding to the wild-type and mutant delta
receptors by an antagonist (NTB), a delta-selective agonist (DPDPE)
and a non-selective agonist (levorphanol). .sup.3H-Naltrindole
binding to membranes from COS-7 cells expressing the wild-type
(open squares), D128N mutant (filled circles) and H278N mutant
(open circles) was inhibited by delta-selective antagonist NTB (A),
the delta-selective agonist DPDPE (B) and the non-selective opioid
agonist levorphanol (C). These are representative examples of 3
different determinations.
[0078] FIG. 13 shows the inhibition of Forskolin-stimulated cAMP
accumulation by the delta agonist DSLET in COS-7 cells expressing
the wild-type and mutant delta opioid receptors. cAMP accumulation
was measured in COS-7 cells expressing the wild-type (open bars),
D128N mutant (dark bars) and H278N mutant (hatched bars) as
described in the Methods. Basal levels and levels stimulated by 10
uM forskolin in the absence (FORSKOLIN) or presence of 1 uM DSLET
(DSLET) or 1 uM DSLET together with 1 uM naltrindole
(DSLET+NALTRINDOLE) were assessed. Results are the mean.+-.SEM of
three different determinations.
[0079] FIG. 14A and FIG. 14B show saturable binding of [3H]DAMGO to
the cloned human .mu. opioid receptor. Membranes from COS-7 cells
transiently expressing the cloned human .mu. receptor were
incubated for 40 min at 25.degree. C. with increasing
concentrations of [3H]DAMGO in the presence or absence of 1 mM
naloxone in order to determine specific binding. Shown are
saturation isotherms of a representative experiment (A) and
linearization of the saturation isotherm data (B). Analysis of the
saturable binding to the human .mu. receptor revealed that
[3H]DAMGO bound to a single site with a KD of 1.0 nM and a
B.sub.max of 232 fmol/mg protein. Experiments were conducted in
triplicate and the results of three independent experiments were
similar.
[0080] FIG. 15 shows regulation of agonist binding to the cloned
human and rat .mu. receptors by the stable GTP analogue, GTPgS, and
by pretreatment of cells with pertussis toxin. Mu receptors in
membranes from COS-7 cells transiently expressing human (hatched)
or rat (open) .mu. receptors were labelled with [3H]DAMGO in the
presence and absence of 100 mM GTPgS (GTPgS). Separate flasks of
cells were treated for 18 hr with 100 ng/ml pertussis toxin (PTX).
These are the means'SEM for three separate experiments.
[0081] FIG. 16 shows the effect of opioids on forskolin-stimulated
cAMP accumulation in cells expressing the human .mu. receptor.
Forskolin-stimulated (10 mM) cAMP accumulation in COS-7 cells
transiently expressing human .mu. receptor was tested in the
presence and absence of levorphanol, dextrorphan, or leu-enkephalin
(1 mM) with or without naloxone (10 mM). Leu-enkephalin and
levorphanol inhibited forskolin-stimulated cAMP accumulation to a
similar maximal extent, by 41% and 31%, respectively. These are the
results of three independent experiments.
[0082] FIG. 17 shows a lack of regulation of agonist binding to the
cloned human and rat .mu. receptor by morphine. COS-7 cells
transiently expressing human (hatched) or rat (open) .mu. receptor
were treated with or without 1 mM morphine for 4 hrs. The cells
were washed twice, harvested, and membranes prepared for the
[3H]naloxone and [3H]DAMGO binding assay as described in the
Methods section. Residual radioligand binding after pre-exposure to
agonist is plotted as a percent of control. These are the means+SEM
for three separate experiments.
[0083] FIG. 18 shows a Northern blot analysis of the distribution
of human mu receptor in human brain. The human brain RNA blot was
obtained from CLONTECH laboratories. Each lane contained 2 mg of
poly A-selected mRNA which was hybridized with 32P-labelled human
.mu. opioid receptor cDNA probe. The lanes are 1-amygdala,
2-caudate nucleus, 3-corpus callosum, 4-hippocampus,
5-hypothalamus, 6-substantia nigra, 7-subthalamic nucleus, and
8-thalamus. The blots were exposed to film at -80.degree. C. for
5-7 days.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0084] Brief Description of Sequences
[0085] The following list briefly identifies the sequences
discussed in the specification and claims:
1 SEQ ID NO:1 Mouse kappa opioid receptor cDNA SEQ ID NO:2 Mouse
kappa opioid receptor amino acid sequence (mORK1) SEQ ID NO:3 Mouse
delta opioid receptor cDNA SEQ ID NO:4 Mouse delta opioid receptor
amino acid sequence (mORD1) SEQ ID NO:5 cDNA of opioid receptor
like receptor from mouse SEQ ID NO:6 Amino acid sequence of opioid
receptor like receptor from mouse (MOP2) SEQ ID NO:7
Oligonucleotide used to generate Spe I restriction site in mouse
kappa receptor SEQ ID NO:8 Oligonucleotide used to generate Spe I
restriction site in mouse delta receptor SEQ ID NO:9
Oligonucleotide used to screen a mouse library SEQ ID NO:10
Oligonucleotide used to screen a mouse library SEQ ID NO:11 Partial
genomic sequence of a human opioid receptor SEQ ID NO:12 Partial
amino acid sequence of a human opioid receptor SEQ ID NO:13 Amino
acid sequence of the third intracellular loop of somatostatin
receptor subtype SSTR1. SEQ ID NO:14 Amino acid sequence of the
third intracellular loop of the mouse kappa receptor. SEQ ID NO:15
Amino acid sequence of the third intracellular loop of the mouse
delta receptor. SEQ ID NO:16 Amino acid sequence of the second
intracellular loop of somatostatin receptor subtype SSTR1. SEQ ID
NO:17 Amino acid sequence of the second intracellular loop of the
mouse kappa and delta receptor. SEQ ID NO:18 Forward PCR primer
from the amino terminal of somatostatin receptor subtype SSTR1. SEQ
ID NO:19 Reverse PCR primer from the amino terminal of somatostatin
receptor subtype SSTR1. SEQ ID NO:20 Forward PCR primer from the
amino terminal of the mouse delta receptor. SEQ ID NO:21 Reverse
PCR primer from the amino terminal of the mouse delta receptor. SEQ
ID NO:22 Forward PCR primer from the amino terminal of the mouse
kappa receptor. SEQ ID NO:23 Reverse PCR primer from the amino
terminal of the mouse kappa receptor. SEQ ID NO:24 Forward PCR
primer from the third intracellular loop of somatostatin receptor
subtype SSTR1. SEQ ID NO:25 Reverse PCR primer from the third
intracellular loop of somatostatin receptor subtype SSTR1. SEQ ID
NO:26 Forward PCR primer from the third intracellular loop of the
mouse delta receptor. SEQ ID NO:27 Reverse PCR primer from the
third intracellular loop of the mouse delta receptor. SEQ ID NO:28
Forward PCR primer from the third intracellular loop of the mouse
kappa receptor. SEQ ID NO:29 Reverse PCR primer from the third
intracellular loop of the mouse kappa receptor. SEQ ID NO:30
Forward PCR primer from the carboxy terminal of somatostatin
receptor subtype SSTR1. SEQ ID NO:31 Reverse PCR primer from the
carboxy terminal of somatostatin receptor subtype SSTR1. SEQ ID
NO:32 Forward PCR primer from the carboxy terminal of the mouse
delta receptor. SEQ ID NO:33 Reverse PCR primer from the carboxy
terminal of the mouse delta receptor. SEQ ID NO:34 Forward PCR
primer from the carboxy terminal of the mouse kappa receptor. SEQ
ID NO:35 Reverse PCR primer from the carboxy terminal of the mouse
kappa receptor. SEQ ID NO:36 Amino acid sequence of antigen used to
generate antisera against Go.alpha. subunit of G-proteins. SEQ ID
NO:37 Amino acid sequence of antigen used to generate antisera
against Go.alpha. subunit of G-proteins. SEQ ID NO:38 Amino acid
sequence of antigen used to generate antisera against the carboxy
terminus of the mouse kappa receptor. SEQ ID NO:39 Amino acid
sequence of antigen used to generate antisera against the amino
terminus of the mouse kappa receptor. SEQ ID NO:40 Amino acid
sequence of antigen used to generate antisera against the amino
terminus of the mouse delta receptor. SEQ ID NO:41 Amino acid
sequence of antigen used to generate antisera against the carboxy
terminus of the mouse delta receptor. SEQ ID NO:42 Oligonucleotide
sequence from mouse delta receptor used to generate chimeric opioid
receptors. SEQ ID NO:43 Oligonucleotide sequence from mouse kappa
receptor used to generate chimeric opioid receptors. SEQ ID NO:44
Oligonucleotide sequence from mouse delta receptor used to
mutagenize aspartic acid 128 to asparagine. SEQ ID NO:45
Oligonucleotide sequence from mouse delta receptor used to
mutagenize histidine 128 to asparagine. SEQ ID NO:46 Peptide
employed to obtain polysonal antiserum against C- terminus of delta
opioid receptor.
[0086] Detailed Description of the Invention
[0087] I. The Invention
[0088] The present invention provides DNA segments, purified
polypeptides, methods for obtaining antibodies, methods of cloning
and using recombinant host cells necessary to obtain and use
recombinant opioid receptors. Thus, the difficulties encountered
with applying the standard approaches of classical genetics or
techniques in molecular biology evident in the prior art to opioid
receptors, have been overcome. Accordingly, the present invention
concerns generally compositions and methods for the preparation and
use of opioid receptors.
[0089] II. Polynucleotide
[0090] A. Isolated and Purified Polynucleotide that Encode Opioid
Receptor Polypeptides.
[0091] In one aspect, the present invention provides an isolated
and purified polynucleotide that encodes an opioid receptor
polypeptide. In a preferred embodiment, the polynucleotide of the
present invention is a DNA molecule. More preferably, the
polynucleotide of the present invention encodes polypeptides that
are delta, kappa, mu or sigma opioid receptors. Even more
preferred, a polynucleotide of the present invention encodes a
polypeptide comprising the amino acid residue sequence of a human,
mouse, or mouse-like opioid receptor for example, the sequence of
SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO 12. Most
preferably, an isolated and purified polynucleotide of the
invention comprises the nucleotide base sequence of SEQ ID NO:
1,SEQ ID NO: 3,SEQ ID NO: 5,AND SEQ ID NO: 11.
[0092] As used herein, the term "polynucleotide" means a sequence
of nucleotides connected by phosphodiester linkages.
Polynucleotides are presented herein in the direction from the 5'
to the 3' direction. A polynucleotide of the present invention can
comprise from about 680 to about several hundred thousand base
pairs. Preferably, a polynucleotide comprises from about 680 to
about 150,000 base pairs. Preferred lengths of particular
polynucleotide are set forth hereinafter.
[0093] A polynucleotide of the present invention can be a
deoxyribonucleic acid (DNA) molecule or ribonucleic acid (RNA)
molecule. Where a polynucleotide is a DNA molecule, that molecule
can be a gene or a cDNA molecule. Nucleotide bases are indicated
herein by a single letter code: adenine (A), guanine (G), thymine
(T), cytosine (C), inosine (I) and uracil (U).
[0094] A polynucleotide of the present invention can be prepared
using standard techniques well known to one of skill in the art.
The preparation of a cDNA molecule encoding an opioid receptor
polypeptide of the present invention is described hereinafter in
Examples 1 and 2. A polynucleotide can also be prepared from
genomic DNA libraries using lambda phage technologies.
[0095] In another aspect, the present invention provides an
isolated and purified polynucleotide that encodes an opioid
receptor polypeptide, where the polynucleotide is preparable by a
process comprising the steps of constructing a library of cDNA
clones from a cell that expresses the polypeptide; screening the
library with a labelled cDNA probe prepared from RNA that encodes
the polypeptide; and selecting a clone that hybridizes to the
probe. Preferably, the polynucleotide of the invention is prepared
by the above process. More preferably, the polynucleotide of the
invention encodes a polypeptide that has the amino acid residue
sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO
12. More preferably still, the polynucleotide comprises the
nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or
SEQ ID NO: 11.
[0096] B. Probes and Primers.
[0097] In another aspect, DNA sequence information provided by the
present invention allows for the preparation of relatively short
DNA (or RNA) sequences having the ability to specifically hybridize
to gene sequences of the selected polynucleotide disclosed herein.
In these aspects, nucleic acid probes of an appropriate length are
prepared based on a consideration of a selected nucleotide
sequence, e.g., a sequence such as that shown in SEQ ID NO: 1, SEQ
ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 11. The ability of such
nucleic acid probes to specifically hybridize to a polynucleotide
encoding an opioid receptor lends them particular utility in a
variety of embodiments. Most importantly, the probes can be used in
a variety of assays for detecting the presence of complementary
sequences in a given sample.
[0098] In certain embodiments, it is advantageous to use
oligonucleotide primers. The sequence of such primers is designed
using a polynucleotide of the present invention for use in
detecting, amplifying or mutating a defined segment of a gene or
polynucleotide that encodes an opioid receptor polypeptide from
mammalian cells using polymerage chain reactive (PCR)
technology.
[0099] To provide certain of the advantages in accordance with the
present invention, a preferred nucleic acid sequence employed for
hybridization studies or assays includes probe molecules that are
complementary to at least a 10 to 70 or so long nucleotide stretch
of a polynucleotide that encodes an opioid receptor polypeptide,
such as that shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or
SEQ ID NO 12. A size of at least 10 nucleotides in length helps to
ensure that the fragment will be of sufficient length to form a
duplex molecule that is both stable and selective. Molecules having
complementary sequences over stretches greater than 10 bases in
length are generally preferred, though, in order to increase
stability and selectivity of the hybrid, and thereby improve the
quality and degree of specific hybrid molecules obtained. One will
generally prefer to design nucleic acid molecules having
gene-complementary stretches of 25 to 40 nucleotides, 55 to 70
nucleotides, or even longer where desired. Such fragments can be
readily prepared by, for example, directly synthesizing the
fragment by chemical means, by application of nucleic acid
reproduction technology, such as the PCR technology of U.S. Pat.
No. 4,603,102, herein incorporated by reference, or by excising
selected DNA fragments from recombinant plasmids containing
appropriate inserts and suitable restriction enzyme sites.
[0100] In another aspect, the present invention contemplates an
isolated and purified polynucleotide comprising a base sequence
that is identical or complementary to a segment of at least 10
contiguous bases of SEQ ID NO: 5, wherein the polynucleotide
hybridizes to a polynucleotide that encodes an opioid receptor
polypeptide. Preferably, the isolated and purified polynucleotide
comprises a base sequence that is identical or complementary to a
segment of at least 25 to 70 contiguous bases of SEQ ID NO: 1, SEQ
ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 11. For example, the
polynucleotide of the invention can comprise a segment of bases
identical or complementary to 40 or 55 contiguous bases of the
disclosed nucleotide sequences.
[0101] Accordingly, a polynucleotide probe molecule of the
invention can be used for its ability to selectively form duplex
molecules with complementary stretches of the gene. Depending on
the application envisioned, one will desire to employ varying
conditions of hybridization to achieve varying degree of
selectivity of the probe toward the target sequence. For
applications requiring a high degree of selectivity, one will
typically desire to employ relatively stringent conditions to form
the hybrids. For example, one will select relatively low salt
and/or high temperature conditions, such as provided by 0.02 M-0.15
M NaCl at temperatures of 50.degree. C. to 70.degree. C. Those
conditions are particularly selective, and tolerate little, if any,
mismatch between the probe and the template or target strand.
[0102] Of course, for some applications, for example, where one
desires to prepare mutants employing a mutant primer strand
hybridized to an underlying template or where one seeks to isolate
an opioid receptor polypeptide coding sequence from other cells,
functional equivalents, or the like, less stringent hybridization
conditions are typically needed to allow formation of the
heteroduplex. In these circumstances, one can desire to employ
conditions such as 0.15 M-0.9 M salt, at temperatures ranging from
20.degree. C. to 55.degree. C. Cross-hybridizing species can
thereby be readily identified as positively hybridizing signals
with respect to control hybridizations. In any case, it is
generally appreciated that conditions can be rendered more
stringent by the addition of increasing amounts of formamide, which
serves to destabilize the hybrid duplex in the same manner as
increased temperature. Thus, hybridization conditions can be
readily manipulated, and thus will generally be a method of choice
depending on the desired results.
[0103] In certain embodiments, it is advantageous to employ a
polynucleotide of the present invention in combination with an
appropriate label for detecting hybrid formation. A wide variety of
appropriate labels are known in the art, including radioactive,
enzymatic or other ligands, such as avidin/biotin, which are
capable of giving a detectable signal.
[0104] In general, it is envisioned that a hybridization probe
described herein is useful both as a reagent in solution
hybridization as well as in embodiments employing a solid phase. In
embodiments involving a solid phase, the test DNA (or RNA) is
adsorbed or otherwise affixed to a selected matrix or surface. This
fixed nucleic acid is then subjected to specific hybridization with
selected probes under desired conditions. The selected conditions
depend as is well known in the art on the particular circumstances
and criteria required (e.g., on the G+C contents, type of target
nucleic acid, source of nucleic acid, size of hybridization probe).
Following washing of the matrix to remove nonspecifically bound
probe molecules, specific hybridization is detected, or even
quantified, by means of the label.
[0105] III. Opioid Receptor Polypeptide
[0106] In one embodiment, the present invention contemplates an
isolated and purified opioid receptor polypeptide. Preferably, an
opioid receptor polypeptide of the invention is a recombinant
polypeptide. More preferably, an opioid receptor polypeptide of the
present invention is a delta, kappa, mu or sigma opioid receptor
polypeptide. Even more preferably, an opioid receptor polypeptides
of the present invention comprises the amino acid residue sequence
of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO 12. An
opioid receptor polypeptide preferably comprises less than about
500 amino acid residues and, more preferably less than about 400
amino acid residues.
[0107] Polypeptides are disclosed herein as amino acid residue
sequences. Those sequences are written left to right in the
direction from the amino to the carboxy terminus. In accordance
with standard nomenclature, amino acid residue sequences are
denominated by either a single letter or a three letter code as
indicated below.
2 Amino Acid Residue 3-Letter Code 1-Letter Code Alanine Ala A
Arginine Arg R Asparagine Asn N Aspartic Acid Asp D Cysteine Cys C
Glutamine Gln Q Glutamic Acid Glu E Glycine Gly G Histidine His H
Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M
Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T
Tryptophan Trp W Tyrosine Tyr Y Valine Val V
[0108] Modifications and changes can be made in the structure of a
polypeptide of the present invention and still obtain a molecule
having like opioid receptor characteristics. For example, certain
amino acids can be substituted for other amino acids in a sequence
without appreciable loss of receptor activity. Because it is the
interactive capacity and nature of a polypeptide that defines that
polypeptide's biological functional activity, certain amino acid
sequence substitutions can be made in a polypeptide sequence (or,
of course, its underlying DNA coding sequence) and nevertheless
obtain a polypeptide with like properties.
[0109] In making such changes, the hydropathic index of amino acids
can be considered. The importance of the hydropathic amino acid
index in conferring interactive biologic function on a polypeptide
is generally understood in the art (Kyte & Doolittle, J. Mol.
Biol., 157:105-132, 1982). It is known that certain amino acids can
be substituted for other amino acids having a similar hydropathic
index or score and still result in a polypeptide with similar
biological activity. Each amino acid has been assigned a
hydropathic index on the basis of its hydrophobicity and charge
characteristics. Those indices are: isoleucine (+4.5); valine
(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine
(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4);
threonine -0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3);
proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine
(-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and
arginine (-4.5).
[0110] It is believed that the relative hydropathic character of
the amino acid determines the secondary structure of the resultant
polypeptide, which in turn defines the interaction of the
polypeptide with other molecules, such as enzymes, substrates,
receptors, antibodies, antigens, and the like. It is known in the
art that an amino acid can be substituted by another amino acid
having a similar hydropathic index and still obtain a functionally
equivalent polypeptide. In such changes, the substitution of amino
acids whose hydropathic indices are within .+-.2 is preferred,
those which are within .+-.1 are particularly preferred, and those
within .+-.0.5 are even more particularly preferred.
[0111] Substitution of like amino acids can also be made on the
basis of hydrophilicity, particularly where the biological
functional equivalent polypeptide or peptide thereby created is
intended for use in immunological embodiments. U.S. Pat. No.
4,554,101, incorporated herein by reference, states that the
greatest local average hydrophilicity of a polypeptide, as governed
by the hydrophilicity of its adjacent amino acids, correlates with
its immunogenicity and antigenicity, i.e. with a biological
property of the polypeptide.
[0112] As detailed in U.S. Pat. No. 4,554,101, the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate
(+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); proline (-0.5.+-.1); threonine (-0.4); alanine (-0.5);
histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine
(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4). It is understood that an
amino acid can be substituted for another having a similar
hydrophilicity value and still obtain a biologically equivalent,
and in particular, an immunologically equivalent polypeptide. In
such changes, the substitution of amino acids whose hydrophilicity
values are within .+-.2 is preferred, those which are within .+-.1
are particularly preferred, and those within .+-.0.5 are even more
particularly preferred.
[0113] As outlined above, amino acid substitutions are generally
therefore based on the relative similarity of the amino acid
side-chain substituents, for example, their hydrophobicity,
hydrophilicity, charge, size, and the like. Exemplary substitutions
which take various of the foregoing characteristics into
consideration are well known to those of skill in the art and
include: arginine and lysine; glutamate and aspartate; serine and
threonine; glutamine and asparagine; and valine, leucine and
isoleucine (See Table 1, below). The present invention thus
contemplates functional or biological equivalents of an opioid
receptor polypeptide as set forth above.
3 TABLE 1 Original Residue Exemplary Substitutions Ala Gly; Ser Arg
Lys Asn Gln; His Asp Glu Cys Ser Gln Asn Glu Asp Gly Ala His Asn;
Gln Ile Leu; Val Leu Ile; Val Lys Arg Met Met; Leu; Tyr Ser Tyr Thr
Ser Trp Tyr Tyr Trp;Phe Val Ile; Leu
[0114] Biological or functional equivalents of a polypeptide can
also be prepared using site-specific mutagenesis. Site-specific
mutagenesis is a technique useful in the preparation of second
generation polypeptides, or biologically functional equivalent
polypeptides or peptides, derived from the sequences thereof,
through specific mutagenesis of the underlying DNA. As noted above,
such changes can be desirable where amino acid substitutions are
desirable. The technique further provides a ready ability to
prepare and test sequence variants, for example, incorporating one
or more of the foregoing considerations, by introducing one or more
nucleotide sequence changes into the DNA. Site-specific mutagenesis
allows the production of mutants through the use of specific
oligonucleotide sequences which encode the DNA sequence of the
desired mutation, as well as a sufficient number of adjacent
nucleotides, to provide a primer sequence of sufficient size and
sequence complexity to form a stable duplex on both sides of the
deletion junction being traversed. Typically, a primer of about 17
to 25 nucleotides in length is preferred, with about 5 to 10
residues on both sides of the junction of the sequence being
altered.
[0115] In general, the technique of site-specific mutagenesis is
well known in the art, as exemplified by Adelman, et al. (1983). As
will be appreciated, the technique typically employs a phage vector
which can exist in both a single stranded and double stranded form.
Typical vectors useful in site-directed mutagenesis include vectors
such as the M13 phage (Messing, et al. 1981). These phage are
commercially available and their use is generally known to those of
skill in the art.
[0116] In general, site-directed mutagenesis in accordance herewith
is performed by first obtaining a single-stranded vector which
includes within its sequence a DNA sequence which encodes all or a
portion of the opioid receptor polypeptide sequence selected. An
oligonucleotide primer bearing the desired mutated sequence is
prepared, generally synthetically, for example, by the method of
Crea, et al. (1978). This primer is then annealed to the
singled-stranded vector, and extended by the use of enzymes such as
E. coli polymerase I Klenow fragment, in order to complete the
synthesis of the mutation-bearing strand. Thus, a heteroduplex is
formed wherein one strand encodes the original non-mutated sequence
and the second strand bears the desired mutation. This heteroduplex
vector is then used to transform appropriate cells such as E. coli
cells and clones are selected which include recombinant vectors
bearing the mutation. Commercially available kits come with all the
reagents necessary, except the oligonucleotide primers.
[0117] An opioid receptor polypeptide of the present invention is
understood to be any opioid receptor polypeptide capable of binding
opioid in any of its forms or analogs of opioid. In addition, an
opioid receptor polypeptide of the invention is not limited to a
particular source. As disclosed herein, the techniques and
compositions of the present invention provide, for example, the
identification and isolation of msls 1-3 from mouse sources. Thus,
the invention provides for the general detection and isolation of
the genus of opioid receptor polypeptides from a variety of sources
while identifying specifically three species of that genus. It is
believed that a number of species of the family of opioid receptor
polypeptides are amenable to detection and isolation using the
compositions and methods of the present inventions. For example,
the present invention also discloses
[0118] A polypeptide of the present invention is prepared by
standard techniques well known to those skilled in the art. Such
techniques include, but are not limited to, isolation and
purification from tissues known to contain that polypeptide, and
expression from cloned DNA that encodes such a polypeptide using
transformed cells (See Examples 1 and 2, hereinafter).
[0119] In another embodiment, the present invention contemplates an
opioid-like receptor polypeptide. Such a polypeptide comprises the
amino acid residue sequence of, for example, SEQ ID NO: 6. A
polynucleotide encoding opioid-like receptor polypeptide comprises
the nucleotide base sequence of SEQ ID NO: 5, for example.
[0120] Opioid receptor polypeptides are found in virtually all
mammals including human. The sequence of a mouse delta opioid
receptor has been previously described (Kieffer, et al., 1992 and
Evans, et al., 1992). As is the case with other receptors, there is
likely little variation between the structure and function of
opioid receptors in different species. Where there is a difference
between species, identification of those differences is well within
the skill of an artisan. Thus, the present invention contemplates
an opioid receptor polypeptide from any mammal. A preferred mammal
is a rodent or a human.
[0121] IV. Expression Vectors
[0122] In an alternate embodiment, the present invention provides
expression vectors comprising polynucleotide that encode opioid
receptor polypeptides. Preferably, the expression vectors of the
present invention comprise polynucleotide that encode polypeptides
comprising the amino acid residue sequence of SEQ ID NO: 2, SEQ ID
NO: 4, SEQ ID NO: 6, or SEQ ID NO 12. More preferably, the
expression vectors of the present invention comprise polynucleotide
comprising the nucleotide base sequence of SEQ ID NO: 1, SEQ ID NO:
3, SEQ ID NO: 5, or SEQ ID NO: 11. Even more preferably, the
expression vectors of the invention comprise polynucleotide
operatively linked to an enhancer-promoter. More preferably still,
the expression vectors of the invention comprise polynucleotide
operatively linked to a prokaryotic promoter. Alternatively, the
expression vectors of the present invention comprise polynucleotide
operatively linked to an enhancer-promoter that is a eukaryotic
promoter, and the expression vectors further comprise a
polyadenylation signal that is positioned 3' of the
carboxy-terminal amino acid and within a transcriptional unit of
the encoded polypeptide.
[0123] A promoter is a region of a DNA molecule typically within
about 100 nucleotide pairs in front of (upstream of) the point at
which transcription begins (i.e., a transcription start site). That
region typically contains several types of DNA sequence elements
that are located in similar relative positions in different genes.
As used herein, the term "promoter" includes what is referred to in
the art as an upstream promoter region, a promoter region or a
promoter of a generalized eukaryotic RNA Polymerase II
transcription unit.
[0124] Another type of discrete transcription regulatory sequence
element is an enhancer. An enhancer provides specificity of time,
location and expression level for a particular encoding region
(e.g., gene). A major function of an enhancer is to increase the
level of transcription of a coding sequence in a cell that contains
one or more transcription factors that bind to that enhancer.
Unlike a promoter, an enhancer can function when located at
variable distances from transcription start sites so long as a
promoter is present.
[0125] As used herein, the phrase "enhancer-promoter" means a
composite unit that contains both enhancer and promoter elements.
An enhancer-promoter is operatively linked to a coding sequence
that encodes at least one gene product. As used herein, the phrase
"operatively linked" means that an enhancer-promoter is connected
to a coding sequence in such a way that the transcription of that
coding sequence is controlled and regulated by that
enhancer-promoter. Means for operatively linking an enhancer-
promoter to a coding sequence are well known in the art. As is also
well known in the art, the precise orientation and location
relative to a coding sequence whose transcription is controlled, is
dependent inter alia upon the specific nature of the
enhancer-promoter. Thus, a TATA box minimal promoter is typically
located from about 25 to about 30 base pairs upstream of a
transcription initiation site and an upstream promoter element is
typically located from about 100 to about 200 base pairs upstream
of a transcription initiation site. In contrast, an enhancer can be
located downstream from the initiation site and can be at a
considerable distance from that site.
[0126] An enhancer-promoter used in a vector construct of the
present invention can be any enhancer-promoter that drives
expression in a cell to be transfected. By employing an
enhancer-promoter with well-known properties, the level and pattern
of gene product expression can be optimized.
[0127] A coding sequence of an expression vector is operatively
linked to a transcription terminating region. RNA polymerase
transcribes an encoding DNA sequence through a site where
polyadenylation occurs. Typically, DNA sequences located a few
hundred base pairs downstream of the polyadenylation site serve to
terminate transcription. Those DNA sequences are referred to herein
as transcription-termination regions. Those regions are required
for efficient polyadenylation of transcribed messenger RNA (mRNA).
Transcription-terminating regions are well known in the art. A
preferred transcription-terminating region used in an adenovirus
vector construct of the present invention comprises a
polyadenylation signal of SV40 or the protamine gene.
[0128] An expression vector comprises a polynucleotide that encodes
an opioid receptor polypeptide. Such a polypeptide is meant to
include a sequence of nucleotide bases encoding an opioid receptor
polypeptide sufficient in length to distinguish said segment from a
polynucleotide segment encoding a non-opioid receptor polypeptide.
A polypeptide of the invention can also encode biologically
functional polypeptides or peptides which have variant amino acid
sequences, such as with changes selected based on considerations
such as the relative hydropathic score of the amino acids being
exchanged. These variant sequences are those isolated from natural
sources or induced in the sequences disclosed herein using a
mutagenic procedure such as site-directed mutagenesis.
[0129] Preferably, the expression vectors of the present invention
comprise polynucleotide that encode polypeptides comprising the
amino acid residue sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID
NO: 6, or SEQ ID NO 12. An expression vector can include an opioid
receptor polypeptide coding region itself of any of the opioid
receptor polypeptides noted above or it can contain coding regions
bearing selected alterations or modifications in the basic coding
region of such an opioid receptor polypeptide. Alternatively, such
vectors or fragments can code larger polypeptides or polypeptides
which nevertheless include the basic coding region. In any event,
it should be appreciated that due to codon redundancy as well as
biological functional equivalence, this aspect of the invention is
not limited to the particular DNA molecules corresponding to the
polypeptide sequences noted above.
[0130] Exemplary vectors include the mammalian expression vectors
of the pCMV family including pCMV6b and pCMV6c (Chiron Corp.,
Emeryville Calif.). In certain cases, and specifically in the case
of these individual mammalian expression vectors, the resulting
constructs can require co-transfection with a vector containing a
selectable marker such as pSV2neo. Via co-transfection into a
dihydrofolate reductase-deficient Chinese hamster ovary cell line,
such as DG44, clones expressing opioid polypeptides by virtue of
DNA incorporated into such expression vectors can be detected.
[0131] A DNA molecule of the present invention can be incorporated
into a vector by a number of techniques which are well known in the
art. For instance, the vector pUC18 has been demonstrated to be of
particular value. Likewise, the related vectors M13mp18 and M13mp19
can be used in certain embodiments of the invention, in particular,
in performing dideoxy sequencing.
[0132] An expression vector of the present invention is useful both
as a means for preparing quantities of the opioid receptor
polypeptide-encoding DNA itself, and as a means for preparing the
encoded polypeptide and peptides. It is contemplated that where
opioid receptor polypeptides of the invention are made by
recombinant means, one can employ either prokaryotic or eukaryotic
expression vectors as shuttle systems. However, in that prokaryotic
systems are usually incapable of correctly processing precursor
polypeptides and, in particular, such systems are incapable of
correctly processing membrane associated eukaryotic polypeptides,
and since eukaryotic opioid receptor polypeptides are anticipated
using the teaching of the disclosed invention, one likely expresses
such sequences in eukaryotic hosts. However, even where the DNA
segment encodes a eukaryotic opioid receptor polypeptide, it is
contemplated that prokaryotic expression can have some additional
applicability. Therefore, the invention can be used in combination
with vectors which can shuttle between the eukaryotic and
prokaryotic cells. Such a system is described herein which allows
the use of bacterial host cells as well as eukaryotic host
cells.
[0133] Where expression of recombinant opioid receptor polypeptides
is desired and a eukaryotic host is contemplated, it is most
desirable to employ a vector such as a plasmid, that incorporates a
eukaryotic origin of replication. Additionally, for the purposes of
expression in eukaryotic systems, one desires to position the
opioid receptor encoding sequence adjacent to and under the control
of an effective eukaryotic promoter such as promoters used in
combination with Chinese hamster ovary cells. To bring a coding
sequence under control of a promoter, whether it is eukaryotic or
prokaryotic, what is generally needed is to position the 5' end of
the translation initiation side of the proper translational reading
frame of the polypeptide between about 1 and about 50 nucleotides
3' of or downstream with respect to the promoter chosen.
Furthermore, where eukaryotic expression is anticipated, one would
typically desire to incorporate into the transcriptional unit which
includes the opioid receptor polypeptide, an appropriate
polyadenylation site.
[0134] The pCMV plasmids are a series of mammalian expression
vectors of particular utility in the present invention. The vectors
are designed for use in essentially all cultured cells and work
extremely well in SV40-transformed simian COS cell lines. The
pCMV1, 2, 3, and 5 vectors differ from each other in certain unique
restriction sites in the polylinker region of each plasmid. The
pCMV4 vector differs from these 4 plasmids in containing a
translation enhancer in the sequence prior to the polylinker. While
they are not directly derived from the pCMV1-5 series of vectors,
the functionally similar pCMV6b and c vectors are available from
the Chiron Corp. of Emeryville, Calif. and are identical except for
the orientation of the polylinker region which is reversed in one
relative to the other.
[0135] The universal components of the pCMV plasmids are as
follows. The vector backbone is pTZ18R (Pharmacia), and contains a
bacteriophage f1 origin of replication for production of single
stranded DNA and an ampicillin-resistance gene. The CMV region
consists of nucleotides -760 to +3 of the powerful
promoter-regulatory region of the human cytomegalovirus (Towne
stain) major immediate early gene (Thomsen et al., 1984; Boshart et
al., 1985). The human growth hormone fragment (hGH) contains
transcription termination and poly-adenylation signals representing
sequences 1533 to 2157 of this gene (Seeburg, 1982). There is an
Alu middle repetitive DNA sequence in this fragment. Finally, the
SV40 origin of replication and early region promoter-enhancer
derived from the pcD-X plasmid (HindII to PstI fragment) described
in (Okayama et al., 1983). The promoter in this fragment is
oriented such that transcription proceeds away from the CMV/hGH
expression cassette.
[0136] The pCMV plasmids are distinguishable from each other by
differences in the polylinker region and by the presence or absence
of the translation enhancer. The starting pCMV1 plasmid has been
progressively modified to render an increasing number of unique
restriction sites in the polylinker region. To create pCMV2, one of
two EcoRI sites in pCMV1 were destroyed. To create pCMV3, pCMV1 was
modified by deleting a short segment from the SV40 region (StuI to
EcoRI), and in so doing made unique the PstI, SalI, and BamHI sites
in the polylinker. To create pCMV4, a synthetic fragment of DNA
corresponding to the 5'-untranslated region of a mRNA transcribed
from the CMV promoter was added C. The sequence acts as a
translational enhancer by decreasing the requirements for
initiation factors in polypeptide synthesis (Jobling et al., 1987);
Browning et al., 1988). To create pCMV5, a segment of DNA (HpaI to
EcoRI) was deleted from the SV40 origin region of pCMV1 to render
unique all sites in the starting polylinker.
[0137] The pCMV vectors have been successfully expressed in simian
COS cells, mouse L cells, CHO cells, and HeLa cells. In several
side by side comparisons they have yielded 5- to 10-fold higher
expression levels in COS cells than SV40-based vectors. The pCMV
vectors have been used to express the LDL receptor, nuclear factor
1, G.sub.s alpha polypeptide, polypeptide phosphatase,
synaptophysin, synapsin, insulin receptor, influenza
hemmagglutinin, androgen receptor, sterol 26-hydroxylase, steroid
17- and 21 -hydroxylase, cytochrome P-450 oxidoreductase,
beta-adrenergic receptor, folate receptor, cholesterol side chain
cleavage enzyme, and a host of other cDNAs. It should be noted that
the SV40 promoter in these plasmids can be used to express other
genes such as dominant selectable markers. Finally, there is an ATG
sequence in the polylinker between the HindIII and PstI sites in
pCMU that can cause spurious translation initiation. This codon
should be avoided if possible in expression plasmids. A paper
describing the construction and use of the parenteral pCMV1 and
pCMV4 vectors has been published (Anderson et al., 1989b).
[0138] V. Transfected Cells.
[0139] In yet another embodiment, the present invention provides
recombinant host cells transformed or transfected with
polynucleotide that encode opioid receptor polypeptides, as well as
transgenic cells derived from those transformed or transfected
cells. Preferably, the recombinant host cells of the present
invention are transfected with polynucleotide of SEQ ID NO: 1, SEQ
ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 11. Means of transforming or
transfecting cells with exogenous polynucleotide such as DNA
molecules are well known in the art and include techniques such as
calcium-phosphate- or DEAE-dextran-mediated transfection,
protoplast fusion, electroporation, liposome mediated transfection,
direct microinjection and adenovirus infection (Sambrook, Fritsch
and Maniatis, 1989).
[0140] The most widely used method is transfection mediated by
either calcium phosphate or DEAE-dextran. Although the mechanism
remains obscure, it is believed that the transfected DNA enters the
cytoplasm of the cell by endocytosis and is transported to the
nucleus. Depending on the cell type, up to 90% of a population of
cultured cells can be transfected at any one time. Because of its
high efficiency, transfection mediated by calcium phosphate or
DEAE-dextran is the method of choice for experiments that require
transient expression of the foreign DNA in large numbers of cells.
Calcium phosphate-mediated transfection is also used to establish
cell lines that integrate copies of the foreign DNA, which are
usually arranged in head-to-tail tandem arrays into the host cell
genome.
[0141] In the protoplast fusion method, protoplasts derived from
bacteria carrying high numbers of copies of a plasmid of interest
are mixed directly with cultured mammalian cells. After fusion of
the cell membranes (usually with polyethylene glycol), the contents
of the bacteria are delivered into the cytoplasm of the mammalian
cells and the plasmid DNA is transported to the nucleus. Protoplast
fusion is not as efficient as transfection for many of the cell
lines that are commonly used for transient expression assays, but
it is useful for cell lines in which endocytosis of DNA occurs
inefficiently. Protoplast fusion frequently yields multiple copies
of the plasmid DNA tandemly integrated into the host
chromosome.
[0142] The application of brief, high-voltage electric pulses to a
variety of mammalian and plant cells leads to the formation of
nanometer-sized pores in the plasma membrane. DNA is taken directly
into the cell cytoplasm either through these pores or as a
consequence of the redistribution of membrane components that
accompanies closure of the pores. Electroporation can be extremely
efficient and can be used both for transient expression of cloned
genes and for establishment of cell lines that carry integrated
copies of the gene of interest. Electroporation, in contrast to
calcium phosphate-mediated transfection and protoplast fusion,
frequently gives rise to cell lines that carry one, or at most a
few, integrated copies of the foreign DNA.
[0143] Liposome transfection involves encapsulation of DNA and RNA
within liposomes, followed by fusion of the liposomes with the cell
membrane. The mechanism of how DNA is delivered into the cell is
unclear but transfection efficiencies can be as high as 90%.
[0144] Direct microinjection of a DNA molecule into nuclei has the
advantage of not exposing DNA to cellular compartments such as
low-pH endosomes. Microinjection is therefore used primarily as a
method to establish lines of cells that carry integrated copies of
the DNA of interest.
[0145] The use of adenovirus as a vector for cell transfection is
well known in the art. Adenovirus vector-mediated cell transfection
has been reported for various cells (Stratford-Perricaudet, et al.
1992).
[0146] A transfected cell can be prokaryotic or eukaryotic.
Preferably, the host cells of the invention are eukaryotic host
cells. More preferably, the recombinant host cells of the invention
are COS-1 cells. Where it is of interest to produce a human opioid
receptor polypeptides, cultured mammalian or human cells are of
particular interest.
[0147] In another aspect, the recombinant host cells of the present
invention are prokaryotic host cells. Preferably, the recombinant
host cells of the invention are bacterial cells of the DH5.alpha.
strain of Escherichia coli. In general, prokaryotes are preferred
for the initial cloning of DNA sequences and constructing the
vectors useful in the invention. For example, E. coli K12 strains
can be particularly useful. Other microbial strains which can be
used include E. coli B, and E. coli X1776 (ATCC No. 31537). These
examples are, of course, intended to be illustrative rather than
limiting.
[0148] Prokaryotes can also be used for expression. The
aforementioned strains, as well as E. coli W3110 (F-, lambda-,
prototrophic, ATCC No. 273325), bacilli such as Bacillus subtilus,
or other enterobacteriaceae such as Salmonella typhimurium or
Serratus marcesans, and various Pseudomonas species can be
used.
[0149] In general, plasmid vectors containing replicon and control
sequences which are derived from species compatible with the host
cell are used in connection with these hosts. The vector ordinarily
carries a replication site, as well as marking sequences which are
capable of providing phenotypic selection in transformed cells. For
example, E. coli can be transformed using pBR322, a plasmid derived
from an E. coli species (Bolivar, et al. 1977). pBR322 contains
genes for ampicillin and tetracycline resistance and thus provides
easy means for identifying transformed cells. The pBR plasmid, or
other microbial plasmid or phage must also contain, or be modified
to contain, promoters which can be used by the microbial organism
for expression of its own polypeptides.
[0150] Those promoters most commonly used in recombinant DNA
construction include the .beta.-lactamase (penicillinase) and
lactose promoter systems (Chang, et al. 1978; Itakura, et al 1977;
Goeddel, et al. 1979; Goeddel, et al. 1980) and a tryptophan (TRP)
promoter system (EPO Appl. Publ. No. 0036776; Siebwenlist et al.,
1980). While these are the most commonly used, other microbial
promoters have been discovered and utilized, and details concerning
their nucleotide sequences have been published, enabling a skilled
worker to introduce functional promoters into plasmid vectors
(Siebwenlist, et al. 1980).
[0151] In addition to prokaryotes, eukaryotic microbes such as
yeast can also be used. Saccharomyces cerevisiase or common baker's
yeast is the most commonly used among eukaryotic microorganisms,
although a number of other strains are commonly available. For
expression in Saccharomyces, the plasmid YRp7, for example, is
commonly used (Stinchcomb, et al. 1979; Kingsman, et al. 1979;
Tschemper, et al. 1980). This plasmid already contains the trpl
gene which provides a selection marker for a mutant strain of yeast
lacking the ability to grow in tryptophan, for example ATCC No.
44076 or PEP4-1 (Jones, 1977). The presence of the trp1 lesion as a
characteristic of the yeast host cell genome then provides an
effective environment for detecting transformation by growth in the
absence of tryptophan.
[0152] Suitable promoter sequences in yeast vectors include the
promoters for 3-phosphoglycerate kinase (Hitzeman, et al. 1980) or
other glycolytic enzymes (Hess, et al. 1968; Holland, et al. 1978)
such as enolase, glyceraldehyde-3-phosphate dehydrogenase,
hexokinase, pyruvate decarboxylase, phosphofructokinase,
glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate
kinase, triosephosphate isomerase, phosphoglucose isomerase, and
glucokinase. In constructing suitable expression plasmids, the
termination sequences associated with these genes are also
introduced into the expression vector downstream from the sequences
to be expressed to provide polyadenylation of the mRNA and
termination. Other promoters, which have the additional advantage
of transcription controlled by growth conditions are the promoter
region for alcohol dehydrogenase 2, isocytochrome C, acid
phosphatase, degradative enzymes associated with nitrogen
metabolism, and the aforementioned glyceraldehyde-3-phosphate
dehydrogenase, and enzymes responsible for maltose and galactose
utilization. Any plasmid vector containing a yeast-compatible
promoter, origin or replication and termination sequences is
suitable.
[0153] In addition to microorganisms, cultures of cells derived
from multicellular organisms can also be used as hosts. In
principle, any such cell culture is workable, whether from
vertebrate or invertebrate culture. However, interest has been
greatest in vertebrate cells, and propagation of vertebrate cells
in culture (tissue culture) has become a routine procedure in
recent years (Kruse and Peterson, 1973). Examples of such useful
host cell lines are AtT-20, VERO and HeLa cells, Chinese hamster
ovary (CHO) cell lines, and W138, BHK, COSM6, COS-7, 293 and MDCK
cell lines. Expression vectors for such cells ordinarily include
(if necessary) an origin of replication, a promoter located
upstream of the gene to be expressed, along with any necessary
ribosome binding sites, RNA splice sites, polyadenylation site, and
transcriptional terminator sequences.
[0154] For use in mammalian cells, the control functions on the
expression vectors are often derived from viral material. For
example, commonly used promoters are derived from polyoma,
Adenovirus 2, Cytomegalovirus and most frequently Simian Virus 40
(SV40). The early and late promoters of SV40 virus are particularly
useful because both are obtained easily from the virus as a
fragment which also contains the SV40 viral origin of replication
(Fiers, et al. 1978). Smaller or larger SV40 fragments can also be
used, provided there is included the approximately 250 bp sequence
extending from the HindIII site toward the BglI site located in the
viral origin of replication. Further, it is also possible, and
often desirable, to utilize promoter or control sequences normally
associated with the desired gene sequence, provided such control
sequences are compatible with the host cell systems.
[0155] An origin of replication can be provided with by
construction of the vector to include an exogenous origin, such as
can be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV,
BPV, CMV) source, or can be provided by the host cell chromosomal
replication mechanism. If the vector is integrated into the host
cell chromosome, the latter is often sufficient.
[0156] VI. Preparing Recombinant Opioid Receptor Polypeptides.
[0157] In yet another embodiment, the present invention
contemplates a process of preparing opioid receptor polypeptides
comprising transfecting cells with polynucleotide that encode
opioid receptor polypeptides to produce transformed host cells; and
maintaining the transformed host cells under biological conditions
sufficient for expression of the polypeptide. Preferably, the
transformed host cells are eukaryotic cells. More preferably still,
the eukaryotic cells are COS-1 cells. Alternatively, the host cells
are prokaryotic cells. More preferably, the prokaryotic cells are
bacterial cells of the DH5.alpha. strain of Escherichia coli. Even
more preferably, the polynucleotide transfected into the
transformed cells comprise the nucleotide base sequence of SEQ ID
NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 11. Most
preferably transfection is accomplished using a hereinbefore
disclosed expression vector.
[0158] A host cell used in the process is capable of expressing a
functional, recombinant opioid receptor polypeptide. A preferred
host cell is a Chinese hamster ovary cell. However, a variety of
cells are amenable to a process of the invention, for instance,
yeasts cells, human cell lines, and other eukaryotic cell lines
known well to those of skill in the art.
[0159] Following transfection, the cell is maintained under culture
conditions for a period of time sufficient for expression of an
opioid receptor polypeptide. Culture conditions are well known in
the art and include ionic composition and concentration,
temperature, pH and the like. Typically, transfected cells are
maintained under culture conditions in a culture medium. Suitable
medium for various cell types are well known in the art. In a
preferred embodiment, temperature is from about 20.degree. C. to
about 50.degree. C., more preferably from about 30.degree. C. to
about 40.degree. C. and, even more preferably about 37.degree. C.
pH is preferably from about a value of 6.0 to a value of about 8.0,
more preferably from about a value of about 6.8 to a value of about
7.8 and, most preferably about 7.4. Osmolality is preferably from
about 200 milliosmols per liter (mosm/L) to about 400 mosm/l and,
more preferably from about 290 mosm/L to about 310 mosm/L. Other
biological conditions needed for transfection and expression of an
encoded protein are well known in the art.
[0160] Transfected cells are maintained for a period of time
sufficient for expression of an opioid receptor polypeptide. A
suitable time depends inter alia upon the cell type used and is
readily determinable by a skilled artisan. Typically, maintenance
time is from about 2 to about 14 days.
[0161] Recombinant opioid receptor polypeptide is recovered or
collected either from the transfected cells or the medium in which
those cells are cultured. Recovery comprises isolating and
purifying the opioid receptor polypeptide. Isolation and
purification techniques for polypeptides are well known in the art
and include such procedures as precipitation, filtration,
chromatography, electrophoresis and the like.
[0162] VII. Antibodies.
[0163] In still another embodiment, the present invention provides
antibodies immunoreactive with opioid receptor polypeptides.
Preferably, the antibodies of the invention are monoclonal
antibodies. More preferably, the opioid receptor polypeptides
comprise the amino acid residue sequence of SEQ ID NO: 2, SEQ ID
NO: 4, SEQ ID NO: 6, or SEQ ID NO 12. Means for preparing and
characterizing antibodies are well known in the art (See, e.g.,
Antibodies "A Laboratory Manual, E. Howell and D. Lane, Cold Spring
Harbor Laboratory, 1988).
[0164] Briefly, a polyclonal antibody is prepared by immunizing an
animal with an immunogen comprising a polypeptide or polynucleotide
of the present invention, and collecting antisera from that
immunized animal. A wide range of animal species can be used for
the production of antisera. Typically an animal used for production
of anti-antisera is a rabbit, a mouse, a rat, a hamster or a guinea
pig. Because of the relatively large blood volume of rabbits, a
rabbit is a preferred choice for production of polyclonal
antibodies.
[0165] As is well known in the art, a given polypeptide or
polynucleotide may vary in its immunogenicity. It is often
necessary therefore to couple the immunogen (e.g., a polypeptide or
polynucleotide) of the present invention with a carrier. Exemplary
and preferred carriers are keyhole limpet hemocyanin (KLH) and
bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse
serum albumin or rabbit serum albumin can also be used as
carriers.
[0166] Means for conjugating a polypeptide or a polynucleotide to a
carrier protein are well known in the art and include
glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester,
carbodiimide and bis-biazotized benzidine.
[0167] As is also well known in the art, immunogenicity to a
particular immunogen can be enhanced by the use of non-specific
stimulators of the immune response known as adjuvants. Exemplary
and preferred adjuvants include complete Freund's adjuvant,
incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
[0168] The amount of immunogen used of the production of polyclonal
antibodies varies inter alia, upon the nature of the immunogen as
well as the animal used for immunization. A variety of routes can
be used to administer the immunogen (subcutaneous, intramuscular,
intradermal, intravenous and intraperitoneal. The production of
polyclonal antibodies is monitored by sampling blood of the
immunized animal at various points following immunization. When a
desired level of immunogenicity is obtained, the immunized animal
can be bled and the serum isolated and stored.
[0169] In another aspect, the present invention contemplates a
process of producing an antibody immunoreactive with an opioid
receptor polypeptide comprising the steps of (a) transfecting
recombinant host cells with polynucleotide that encode opioid
receptor polypeptides; (b) culturing the host cells under
conditions sufficient for expression of the polypeptides; (c)
recovering the polypeptides; and (d) preparing the antibodies to
the polypeptides. Preferably, the host cell is transfected with the
polynucleotide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ
ID NO: 11. Even more preferably, the present invention provides
antibodies prepared according to the process described above.
[0170] A monoclonal antibody of the present invention can be
readily prepared through use of well-known techniques such as those
exemplified in U.S. Pat. No 4,196,265, herein incorporated by
reference. Typically, a technique involves first immunizing a
suitable animal with a selected antigen (e.g., a polypeptide or
polynucleotide of the present invention) in a manner sufficient to
provide an immune response. Rodents such as mice and rats are
preferred animals. Spleen cells from the immunized animal are then
fused with cells of an immortal myeloma cell. Where the immunized
animal is a mouse, a preferred myeloma cell is a murine NS-1
myeloma cell.
[0171] The fused spleen/myeloma cells are cultured in a selective
medium to select fused spleen/myeloma cells from the parental
cells. Fused cells are separated from the mixture of non-fused
parental cells, for example, by the addition of agents that block
the de novo synthesis of nucleotides in the tissue culture media.
Exemplary and preferred agents are aminopterin, methotrexate, and
azaserine. Aminopterin and methotrexate block de novo synthesis of
both purines and pyrimidines, whereas azaserine blocks only purine
synthesis. Where aminopterin or methotrexate is used, the media is
supplemented with hypoxanthine and thymidine as a source of
nucleotides. Where azaserine is used, the media is supplemented
with hypoxanthine.
[0172] This culturing provides a population of hybridomas from
which specific hybridomas are selected. Typically, selection of
hybridomas is performed by culturing the cells by single-clone
dilution in microtiter plates, followed by testing the individual
clonal supernatants for reactivity with an antigen-polypeptide. The
selected clones can then be propagated indefinitely to provide the
monoclonal antibody.
[0173] By way of specific example, to produce an antibody of the
present invention, mice are injected intraperitoneally with between
about 1-200 .mu.g of an antigen comprising a polypeptide of the
present invention. B lymphocyte cells are stimulated to grow by
injecting the antigen in association with an adjuvant such as
complete Freund's adjuvant (a non-specific stimulator of the immune
response containing killed Mycobacterium tuberculosis). At some
time (e.g., at least two weeks) after the first injection, mice are
boosted by injection with a second dose of the antigen mixed with
incomplete Freund's adjuvant.
[0174] A few weeks after the second injection, mice are tail bled
and the sera titered by immunoprecipitation against radiolabeled
antigen. Preferably, the process of boosting and titering is
repeated until a suitable titer is achieved. The spleen of the
mouse with the highest titer is removed and the spleen lymphocytes
are obtained by homogenizing the spleen with a syringe. Typically,
a spleen from an immunized mouse contains approximately
5.times.10.sup.7 to 2.times.10.sup.8 lymphocytes.
[0175] Mutant lymphocyte cells known as myeloma cells are obtained
from laboratory animals in which such cells have been induced to
grow by a variety of well-known methods. Myeloma cells lack the
salvage pathway of nucleotide biosynthesis. Because myeloma cells
are tumor cells, they can be propagated indefinitely in tissue
culture, and are thus denominated immortal. Numerous cultured cell
lines of myeloma cells from mice and rats, such as murine NS-1
myeloma cells, have been established.
[0176] Myeloma cells are combined under conditions appropriate to
foster fusion with the normal antibody-producing cells from the
spleen of the mouse or rat injected with the antigen/polypeptide of
the present invention. Fusion conditions include, for example, the
presence of polyethylene glycol. The resulting fused cells are
hybridoma cells. Like mycloma cells, hybridoma cells grow
indefinitely in culture.
[0177] Hybridoma cells are separated from unfused myeloma cells by
culturing in a selection medium such as HAT media (hypoxanthine,
aminopterin, thymidine). Unfused myeloma cells lack the enzymes
necessary to synthesize nucleotides from the salvage pathway
because they are killed in the presence of aminopterin,
methotrexate, or azaserine. Unfused lymphocytes also do not
continue to grow in tissue culture. Thus, only cells that have
successfully fused (hybridoma cells) can grow in the selection
media.
[0178] Each of the surviving hybridoma cells produces a single
antibody. These cells are then screened for the production of the
specific antibody immunoreactive with an antigen/polypeptide of the
present invention. Single cell hybridomas are isolated by limiting
dilutions of the hybridomas. The hybridomas are serially diluted
many times and, after the dilutions are allowed to grow, the
supernatant is tested for the presence of the monoclonal antibody.
The clones producing that antibody are then cultured in large
amounts to produce an antibody of the present invention in
convenient quantity.
[0179] By use of a monoclonal antibody of the present invention,
specific polypeptides and polynucleotide of the invention can be
recognized as antigens, and thus identified. Once identified, those
polypeptides and polynucleotide can be isolated and purified by
techniques such as antibody-affinity chromatography. In
antibody-affinity chromatography, a monoclonal antibody is bound to
a solid substrate and exposed to a solution containing the desired
antigen. The antigen is removed from the solution through an
immunospecific reaction with the bound antibody. The polypeptide or
polynucleotide is then easily removed from the substrate and
purified.
[0180] VIII. Pharmaceutical Compositions.
[0181] In a preferred embodiment, the present invention provides
pharmaceutical compositions comprising opioid receptor polypeptides
and physiologically acceptable carriers. More preferably, the
pharmaceutical compositions comprise opioid receptor polypeptides
comprising the amino acid residue sequence of SEQ ID NO: 2, SEQ ID
NO: 4, SEQ ID NO: 6, or SEQ ID NO 12. Even more preferably, the
pharmaceutical compositions of the invention comprise
polynucleotide that encode opioid receptor polypeptides, and
physiologically acceptable carriers. Still more preferably, the
pharmaceutical compositions of the present invention comprise
opioid receptor polypeptides comprising the amino acid residue
sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO
12. Alternatively, the pharmaceutical compositions comprise
polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1,
SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 11.
[0182] A composition of the present invention is typically
administered parenterally in dosage unit formulations containing
standard, well-known nontoxic physiologically acceptable carriers,
adjuvants, and vehicles as desired. The term parenteral as used
herein includes intravenous, intramuscular, intraarterial
injection, or infusion techniques.
[0183] Injectable preparations, for example sterile injectable
aqueous or oleaginous suspensions, are formulated according to the
known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation can also be a
sterile injectable solution or suspension in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol.
[0184] Among the acceptable vehicles and solvents that may be
employed are water, Ringer's solution, and isotonic sodium chloride
solution. In addition, sterile, fixed oils are conventionally
employed as a solvent or suspending medium. For this purpose any
bland fixed oil can be employed including synthetic mono- or
di-glycerides. In addition, fatty acids such as oleic acid find use
in the preparation of injectables.
[0185] Preferred carriers include neutral saline solutions buffered
with phosphate, lactate, Tris, and the like. Of course, one
purifies the vector sufficiently to render it essentially free of
undesirable contaminants, such as defective interfering adenovirus
particles or endotoxins and other pyrogens such that it does not
cause any untoward reactions in the individual receiving the vector
construct. A preferred means of purifying the vector involves the
use of buoyant density gradients, such as cesium chloride gradient
centrifugation.
[0186] A carrier can also be a liposome. Means for using liposomes
as delivery vehicles are well known in the art [See, e.g. Gabizon
et al., 1990; Ferruti et al., 1986; and Ranade, V. V., 1989].
[0187] A transfected cell can also serve as a carrier. By way of
example, a liver cell can be removed from an organism, transfected
with a polynucleotide of the present invention using methods set
forth above and then the transfected cell returned to the organism
(e.g. injected intravascularly).
[0188] IX. Detecting Polynucleotide and the Polypeptides
Encoded.
[0189] Alternatively, the present invention provides a process of
detecting opioid receptor polypeptides, wherein the process
comprises immunoreacting the polypeptides with antibodies prepared
according to the process described above to form
antibody-polypeptide conjugates, and detecting the conjugates.
[0190] In yet another embodiment, the present invention
contemplates a process of detecting messenger RNA transcripts that
encode opioid receptor polypeptides, wherein the process comprises
(a) hybridizing the messenger RNA transcripts with polynucleotide
sequences that encode the opioid receptor polypeptides to form
duplexes; and (b) detecting the duplex. Alternatively, the present
invention provides a process of detecting DNA molecules that encode
opioid receptor polypeptides, wherein the process comprises (a)
hybridizing DNA molecules with polynucleotide that encode opioid
receptor polypeptides to form duplexes; and (b) detecting the
duplexes.
[0191] X. Screening Assays
[0192] In yet another aspect, the present invention contemplates a
process of screening substances for their ability to interact with
opioid receptor polypeptides comprising the steps of providing
opioid receptor polypeptides, and testing the ability of selected
substances to interact with the opioid receptor polypeptides.
[0193] Utilizing the methods and compositions of the present
invention, screening assays for the testing of candidate substances
such as agonists and antagonists of opioid receptors can be
derived. A candidate substance is a substance which potentially can
interact with or modulate, by binding or other intramolecular
interaction, an opioid receptor polypeptide. In some instances,
such a candidate substance will be an agonist of the receptor and
in other instances can exhibit antagonistic attributes when
interacting with the receptor polypeptide. In other instances, such
substances can have mixed agonistic and antagonistic properties or
can modulate the opioid receptor in other ways.
[0194] Recombinant receptor expression systems of the present
invention possess definite advantages over tissue-based systems.
The methods of the present invention make it possible to produce
large quantities of opioid receptors for use in screening assays.
More important, however, is the relative purity of the receptor
polypeptides provided by the present invention. A relatively pure
polypeptide preparation for assaying a protein-protein interaction
makes it possible to use elutive methods without invoking
competing, and unwanted, side-reactions.
[0195] Cloned expression systems such as those of the present
invention are also useful where there is difficulty in obtaining
tissue that satisfactorily expresses a particular receptor. Cost is
another very real advantage, at least with regard to the microbial
expression systems of the present invention. For antagonists in a
primary screen, microorganism expression systems of the present
invention are inexpensive in comparison to prior art
tissue-screening methods.
[0196] Traditionally, screening assays employed the use of crude
receptor preparations. Typically, animal tissue slices thought to
be rich in the receptor of interest was the source of the receptor.
Alternatively, investigators homogenized the tissue and used the
crude homogenate as a receptor source. A major difficulty with this
approach is that there are no tissue types where only one receptor
type is expressed. The data obtained therefore could not be
definitively correlated with a particular receptor. With the recent
cloning of receptor sub-types and sub-sub-types, this difficulty is
highlighted. A second fundamental difficulty with the traditional
approach is the unavailability of human tissue for screening
potential drugs. The traditional approach almost invariably
utilized animal receptors. With the cloning of human receptors,
there is a need for screening assays which utilize human
receptors.
[0197] With the availability of cloned receptors, recombinant
receptor screening systems have several advantages over tissue
based systems. A major advantage is that the investigator can now
control the type of receptor that is utilized in a screening assay.
Specific receptor sub-types and sub-sub-types can be preferentially
expressed and its interaction with a ligand can be identified.
Other advantages include the availability of large amounts of
receptor, the availability of rare receptors previously unavailable
in tissue samples, and the lack of expenses associated with the
maintenance of live animals.
[0198] Screening assays of the present invention generally involve
determining the ability of a candidate substance to bind to the
receptor and to affect the activity of the receptor, such as the
screening of candidate substances to identify those that inhibit or
otherwise modify the receptor's function. Typically, this method
includes preparing recombinant receptor polypeptide, followed by
testing the recombinant polypeptide or cells expressing the
polypeptide with a candidate substance to determine the ability of
the substance to affect its physiological function. In preferred
embodiments, the invention relates to the screening of candidate
substances to identify those that affect the enzymatic activity of
the human receptor, and thus can be suitable for use in humans.
[0199] As is well known in the art, a screening assay provides a
receptor under conditions suitable for the binding of an agent to
the receptor. These conditions include but are not limited to pH,
temperature, tonicity, the presence of relevant co-factors, and
relevant modifications to the polypeptide such as glycosylation or
prenylation. It is contemplated that the receptor can be expressed
and utilized in a prokaryotic or eukaryotic cell. The host cell
expressing the receptor can be used whole or the receptor can be
isolated from the host cell. The receptor can be membrane bound in
the membrane of the host cell or it can be free in the cytosol of
the host cell. The host cell can also be fractionated into
sub-cellular fractions where the receptor can be found. For
example, cells expressing the receptor can be fractionated into the
nuclei, the endoplasmic reticulum, vesicles, or the membrane
surfaces of the cell. pH is preferably from about a value of 6.0 to
a value of about 8.0, more preferably from about a value of about
6.8 to a value of about 7.8 and, most preferably about 7.4. In a
preferred embodiment, temperature is from about 20.degree. C. to
about 50.degree. C., more preferably from about 30.degree. C. to
about 40.degree. C. and, even more preferably about 37.degree. C.
Osmolality is preferably from about 5 milliosmols per liter
(mosm/L) to about 400 mosm/l and, more preferably from about 200
milliosmols per liter to about 400 mosm/l and, even more preferably
from about 290 mosm/L to about 310 mosm/L. The presence of
co-factors can be required for the proper functioning of the
receptor. Typical co-factors include sodium, potassium, calcium,
magnesium, and chloride. In addition, small, non-peptide molecules,
known as prosthetic groups can be required. Other biological
conditions needed for receptor function are well known in the
art.
[0200] It is well known in the art that proteins can be
reconstituted in artificial membranes, vesicles or liposomes.
(Danboldt, et al. 1990). The present invention contemplates that
the receptor can be incorporated into artificial membranes,
vesicles or liposomes. The reconstituted receptor can be utilized
in screening assays.
[0201] It is further contemplated that the receptor of the present
invention can be coupled to a solid support. The solid support can
be agarose beads, polyacrylamide beads, polyacrylic beads or other
solid matrices capable of being coupled to proteins. Well known
coupling agents include cyanogen bromide, carbonyldiimidazole,
tosyl chloride, and glutaraldehyde.
[0202] It is further contemplated that secondary polypeptides which
can function in conjunction with the receptor of the present
invention can be provided. For example, the receptor of the present
invention exerts its physiological effects in conjunction with a
G-protein and an effector polypeptide.
[0203] In a typical screening assay for identifying candidate
substances, one employs the same recombinant expression host as the
starting source for obtaining the receptor polypeptide, generally
prepared in the form of a crude homogenate. Recombinant cells
expressing the receptor are washed and homogenized to prepare a
crude polypeptide homogenate in a desirable buffer such as
disclosed herein. In a typical assay, an amount of polypeptide from
the cell homogenate, is placed into a small volume of an
appropriate assay buffer at an appropriate pH. Candidate
substances, such as agonists and antagonists, are added to the
admixture in convenient concentrations and the interaction between
the candidate substance and the receptor polypeptide is
monitored.
[0204] Where one uses an appropriate known substrate for the
receptor, one can, in the foregoing manner, obtain a baseline
activity for the recombinantly produced receptor. Then, to test for
inhibitors or modifiers of the receptor function, one can
incorporate into the admixture a candidate substance whose effect
on the receptor is unknown. By comparing reactions which are
carried out in the presence or absence of the candidate substance,
one can then obtain information regarding the effect of the
candidate substance on the normal function of the receptor.
[0205] Accordingly, it is proposed that this aspect of the present
invention provides those of skill in the art with methodology that
allows for the identification of candidate substances having the
ability to modify the action of opioid receptor polypeptides in one
or more manners.
[0206] In one embodiment, such an assay is designed to be capable
of discriminating those candidate substances with the desirable
properties of opioids but which lack the undesirable properties of
opioids. In another embodiment, screening assays for testing
candidate substances such as agonists and antagonists of opioid
receptors are used to identify such candidate substances having
selective ability to interact with one or more of the opioid
receptor polypeptides but which polypeptides are without a
substantially overlapping activity with another of the opioid
receptor polypeptides identified herein.
[0207] Additionally, screening assays for the testing of candidate
substances are designed to allow the investigation of structure
activity relationships of opioid with the receptors, e.g., study of
binding of naturally occurring hormones or other substances capable
of interacting or otherwise modulating with the receptor versus
studies of the activity caused by the binding of such molecules to
the receptor. In certain embodiments, the polypeptides of the
invention are crystallized in order to carry out x-ray
crystallographic studies as a means of evaluating interactions with
candidate substances or other molecules with the opioid receptor
polypeptide. For instance, the purified recombinant polypeptides of
the invention, when crystallized in a suitable form, are amenable
to detection of intra-molecular interactions by x-ray
crystallography.
[0208] An important aspect of the invention is the use of
recombinantly produced opioid receptor polypeptide in screening
assays for the identification of substances which can inhibit or
otherwise modify or alter the function of the receptor. The use of
recombinantly produced receptor is of particular benefit because
the naturally occurring receptor is present in only small
quantities and has proven difficult to purify. Moreover, this
provides a ready source of receptor, which has heretofore been
unavailable.
[0209] As described above, receptors in the presence of agonists
exert its physiological effects through a secondary molecule. A
screening assay of the invention, in preferred embodiments,
conveniently employs an opioid receptor polypeptide directly from
the recombinant host in which it is produced. This is achieved most
preferably by simply expressing the selected polypeptide within the
recombinant host, typically a eukaryotic host, followed by
preparing a crude homogenate which includes the enzyme. A portion
of the crude homogenate is then admixed with an appropriate
effector of the receptor along with the candidate substance to be
tested. By comparing the binding of the selected effector to the
receptor in the presence or absence of the candidate substance, one
can obtain information regarding the physiological properties of
the candidate substance.
[0210] The receptor can be expressed in a prokaryotic or a
eukaryotic cell. Receptors have been expressed in E. coli (Bertin,
et al. 1992), in yeast (King, et al. (1990) and in mammalian cells
(Bouvier, et. al 1988).
[0211] A cell expressing a receptor can be used whole to screen
agents. For example, cells expressing the receptor of the present
invention can be exposed to radiolabelled agent and the amount of
binding of the radiolabelled agent to the cell can be
determined.
[0212] The cell expressing the receptor can be fractionated into
sub-cellular components which contain the receptor of the present
invention. Methods for purifying sub-cellular fractions are well
known in the art. Sub-cellular fractions include but are not
limited to the cytoplasm, cellular membrane, other membranous
fractions such as the endoplasmic reticulum, golgi bodies, vesicles
and the nucleus. Receptors isolated as sub-cellular fractions can
be associated with cellular membranes. For example, if cellular
membrane vesicles are isolated from the cell expressing the
receptor, the receptor molecule can be membrane bound. It is
further contemplated that the receptor of the present invention can
be purified from a cell that expresses the receptor. Methods of
purification are well known in the art. The purified receptor can
be used in screening assays.
[0213] In that most such screening assays in accordance with the
invention are designed to identify agents useful in mimicking the
desirable aspects of opioids while eliminating the undesirable
aspects of the hormone, preferred assays employ opioids as the
normal agonist.
[0214] There are believed to be a wide variety of embodiments which
can be employed to determine the effect of the candidate substance
on the receptor polypeptides of the invention, and the invention is
not intended to be limited to any one such method. However, it is
generally desirable to employ a system wherein one can measure the
ability of the receptor polypeptide to bind to and or be modified
by the effector employed in the presence of a particular
substance.
[0215] The detection of an interaction between an agent and a
receptor can be accomplished through techniques well known in the
art. These techniques include but are not limited to
centrifugation, chromatography, electrophoresis and spectroscopy.
The use of isotopically labelled reagents in conjunction with these
techniques or alone is also contemplated. Commonly used radioactive
isotopes include .sup.3H, .sup.14C, .sup.22Na, .sup.32P, .sup.35S,
.sup.45Ca, .sup.60Co, .sup.125I, and .sup.131I. Commonly used
stable isotopes include .sup.2H, .sup.13C, .sup.15N, .sup.18O.
[0216] For example, if an agent can bind to the receptor of the
present invention, the binding can be detected by using
radiolabelled agent or radiolabelled receptor. Briefly, if
radiolabelled agent or radiolabelled receptor is utilized, the
agent-receptor complex can be detected by liquid scintillation or
by exposure to X-Ray film.
[0217] When an agent modifies the receptor, the modified receptor
can be detected by differences in mobility between the modified
receptor and the unmodified receptor through the use of
chromatography, electrophoresis or centrifugation. When the
technique utilized is centrifugation, the differences in mobility
is known as the sedimentation coefficient. The modification can
also be detected by differences between the spectroscopic
properties of the modified and unmodified receptor. As a specific
example, if an agent covalently modifies a receptor, the difference
in retention times between modified and unmodified receptor on a
high pressure liquid chromatography (HPLC) column can easily be
detected.
[0218] As a specific example, if an agent covalently modifies a
receptor, the spectroscopic differences between modified and
unmodified receptor in the nuclear magnetic resonance (NMR) spectra
can be detected. Alternatively, one can focus on the agent and
detect the differences in the spectroscopic properties or the
difference in mobility between the free agent and the agent after
modification of the receptor.
[0219] When a secondary polypeptide is provided, the
agent-receptor-secondary polypeptide complex or the
receptor-secondary polypeptide complex can be detected. Differences
in mobility or differences in spectroscopic properties as described
above can be detected.
[0220] It is further contemplated that when a secondary polypeptide
is provided the enzymatic activity of the effector polypeptide can
be detected. For example, many receptors exert physiological
effects through the stimulation or inhibition of adenylyl cyclase.
The enzymatic activity of adenylyl cyclase in the presence of an
agent can be detected.
[0221] The interaction of an agent and a receptor can be detected
by providing a reporter gene. Well known reporter genes include
.beta.-galactosidase (.beta.-Gal), chloramphenicol transferase
(CAT) and luciferase. The reporter gene is expressed by the host
and the enzymatic reaction of the reporter gene product can be
detected.
[0222] In preferred assays, an admixture containing the
polypeptide, effector and candidate substance is allowed to
incubate for a selected amount of time, and the resultant incubated
mixture subjected to a separation means to separate the unbound
effector remaining in the admixture from any effector/receptor
complex so produced. Then, one simply measures the amount of each
(e.g., versus a control to which no candidate substance has been
added). This measurement can be made at various time points where
velocity data is desired. From this, one can determine the ability
of the candidate substance to alter or modify the function of the
receptor.
[0223] Numerous techniques are known for separating the effector
from effector/receptor complex, and all such methods are intended
to fall within the scope of the invention. Use of thin layer
chromatographic methods (TLC), HPLC, spectrophotometric, gas
chromatographic/mass spectrophotometric or NMR analyses. It is
contemplated that any such technique can be employed so long as it
is capable of differentiating between the effector and complex, and
can be used to determine enzymatic function such as by identifying
or quantifying the substrate and product.
[0224] The effector/receptor complex itself can also be the subject
of techniques such as x-ray crystallography. Where a candidate
substance replaces the opioid molecule as the drug's mode of
action, studies designed to monitor the replacement and its effect
on the receptor will be of particular benefit.
[0225] A. Screening Assays for Opioid Receptor Polypeptides.
[0226] The present invention provides a process of screening a
biological sample for the presence of an opioid receptor
polypeptide. A biological sample to be screened can be a biological
fluid such as extracellular or intracellular fluid or a cell or
tissue extract or homogenate. A biological sample can also be an
isolated cell (e.g., in culture) or a collection of cells such as
in a tissue sample or histology sample. A tissue sample can be
suspended in a liquid medium or fixed onto a solid support such as
a microscope slide.
[0227] In accordance with a screening assay process, a biological
sample is exposed to an antibody immunoreactive with the opioid
receptor polypeptide whose presence is being assayed. Typically,
exposure is accomplished by forming an admixture in a liquid medium
that contains both the antibody and the candidate opioid receptor
polypeptide. Either the antibody or the sample with the opioid
receptor polypeptide can be affixed to a solid support (e.g., a
column or a microtiter plate).
[0228] The biological sample is exposed to the antibody under
biological reaction conditions and for a period of time sufficient
for antibody-polypeptide conjugate formation. Biological reaction
conditions include ionic composition and concentration,
temperature, pH and the like.
[0229] Ionic composition and concentration can range from that of
distilled water to a 2 molal solution of NaCl. Preferably,
osmolality is from about 100 mosmols/l to about 400 mosmols/l and,
more preferably from about 200 mosmols/l to about 300 mosmols/l.
Temperature preferably is from about 4.degree. C. to about
100.degree. C., more preferably from about 15.degree. C. to about
50.degree. C. and, even more preferably from about 25.degree. C. to
about 40.degree. C. pH is preferably from about a value of 4.0 to a
value of about 9.0, more preferably from about a value of 6.5 to a
value of about 8.5 and, even more preferably from about a value of
7.0 to a value of about 7.5. The only limit on biological reaction
conditions is that the conditions selected allow for
antibody-polypeptide conjugate formation and that the conditions do
not adversely affect either the antibody or the opioid receptor
polypeptide.
[0230] Exposure time will vary inter alia with the biological
conditions used, the concentration of antibody and polypeptide and
the nature of the sample (e.g., fluid or tissue sample). Means for
determining exposure time are well known to one of ordinary skill
in the art. Typically, where the sample is fluid and the
concentration of polypeptide in that sample is about 10.sup.-10 M,
exposure time is from about 10 minutes to about 200 minutes.
[0231] The presence of opioid receptor polypeptide in the sample is
detected by detecting the formation and presence of antibody-opioid
receptor polypeptide conjugates. Means for detecting such
antibody-antigen (e.g., receptor polypeptide) conjugates or
complexes are well known in the art and include such procedures as
centrifugation, affinity chromatography and the like, binding of a
secondary antibody to the antibody-candidate receptor complex.
[0232] In one embodiment, detection is accomplished by detecting an
indicator affixed to the antibody. Exemplary and well known such
indicators include radioactive labels (e.g., .sup.32 P, .sup.125I,
.sup.14C), a second antibody or an enzyme such as horse radish
peroxidase. Means for affixing indicators to antibodies are well
known in the art. Commercial kits are available.
[0233] B. Screening Assay for Anti-opioid Receptor Antibody.
[0234] In another aspect, the present invention provides a process
of screening a biological sample for the presence of antibodies
immunoreactive with an opioid receptor polypeptide (i.e., an
anti-opioid receptor antibody). In accordance with such a process,
a biological sample is exposed to an opioid receptor polypeptide
under biological conditions and for a period of time sufficient for
antibody-polypeptide conjugate formation and the formed conjugates
are detected.
[0235] C. Screening Assay for Polynucleotide that Encodes an Opioid
Receptor Polypeptide.
[0236] A DNA molecule and, particularly a probe molecule, can be
used for hybridizing as oligonucleotide probes to a DNA source
suspected of possessing an opioid receptor polypeptide encoding
polynucleotide or gene. The probing is usually accomplished by
hybridizing the oligonucleotide to a DNA source suspected of
possessing such a receptor gene. In some cases, the probes
constitute only a single probe, and in others, the probes
constitute a collection of probes based on a certain amino acid
sequence or sequences of the opioid receptor polypeptide and
account in their diversity for the redundancy inherent in the
genetic code.
[0237] A suitable source of DNA for probing in this manner is
capable of expressing opioid receptor polypeptides and can be a
genomic library of a cell line of interest. Alternatively, a source
of DNA can include total DNA from the cell line of interest. Once
the hybridization process of the invention has identified a
candidate DNA segment, one confirms that a positive clone has been
obtained by further hybridization, restriction enzyme mapping,
sequencing and/or expression and testing.
[0238] Alternatively, such DNA molecules can be used in a number of
techniques including their use as: (1) diagnostic tools to detect
normal and abnormal DNA sequences in DNA derived from patient's
cells; (2) means for detecting and isolating other members of the
opioid receptor family and related polypeptides from a DNA library
potentially containing such sequences; (3) primers for hybridizing
to related sequences for the purpose of amplifying those sequences;
(4) primers for altering the native opioid receptor DNA sequences;
as well as other techniques which rely on the similarity of the DNA
sequences to those of the opioid receptor DNA segments herein
disclosed.
[0239] As set forth above, in certain aspects, DNA sequence
information provided by the invention allows for the preparation of
relatively short DNA (or RNA) sequences (e.g., probes) that
specifically hybridize to encoding sequences of the selected opioid
receptor gene. In these aspects, nucleic acid probes of an
appropriate length are prepared based on a consideration of the
selected opioid receptor sequence (e.g., a sequence such as that
shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO:
11). The ability of such nucleic acid probes to specifically
hybridize to opioid receptor encoding sequences lend them
particular utility in a variety of embodiments. Most importantly,
the probes can be used in a variety of assays for detecting the
presence of complementary sequences in a given sample. However,
uses are envisioned, including the use of the sequence information
for the preparation of mutant species primers, or primers for use
in preparing other genetic constructions.
[0240] To provide certain of the advantages in accordance with the
invention, a preferred nucleic acid sequence employed for
hybridization studies or assays includes probe sequences that are
complementary to at least a 14 to 40 or so long nucleotide stretch
of the opioid receptor encoding sequence, such as that shown in SEQ
ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 11. A size of
at least 14 nucleotides in length helps to ensure that the fragment
is of sufficient length to form a duplex molecule that is both
stable and selective. Molecules having complementary sequences over
stretches greater than 14 bases in length are generally preferred,
though, to increase stability and selectivity of the hybrid, and
thereby improve the quality and degree of specific hybrid molecules
obtained. One will generally prefer to design nucleic acid
molecules having gene-complementary stretches of 14 to 20
nucleotides, or even longer where desired. Such fragments can be
readily prepared by, for example, directly synthesizing the
fragment by chemical means, by application of nucleic acid
reproduction technology, such as the PCR technology of U.S. Pat.
No. 4,603,102, herein incorporated by reference, or by introducing
selected sequences into recombinant vectors for recombinant
production.
[0241] Accordingly, a nucleotide sequence of the present invention
can be used for its ability to selectively form duplex molecules
with complementary stretches of the gene. Depending on the
application envisioned, one employs varying conditions of
hybridization to achieve varying degrees of selectivity of the
probe toward the target sequence. For applications requiring a high
degree of selectivity, one typically employs relatively stringent
conditions to form the hybrids. For example, one selects relatively
low salt and/or high temperature conditions, such as provided by
0.02M-0.15M NaCl at temperatures of 50.degree. C. to 70.degree. C.
Such conditions are particularly selective, and tolerate little, if
any, mismatch between the probe and the template or target
strand.
[0242] Of course, for some applications, for example, where one
desires to prepare mutants employing a mutant primer strand
hybridized to an underlying template or where one seeks to isolate
opioid receptor coding sequences from related species, functional
equivalents, or the like, less stringent hybridization conditions
are typically needed to allow formation of the heteroduplex. Under
such circumstances, one employs conditions such as 0.15M-0.9M salt,
at temperatures ranging from 20.degree. C. to 55.degree. C.
Cross-hybridizing species can thereby be readily identified as
positively hybridizing signals with respect to control
hybridizations. In any case, it is generally appreciated that
conditions can be rendered more stringent by the addition of
increasing amounts of formamide, which serves to destabilize the
hybrid duplex in the same manner as increased temperature. Thus,
hybridization conditions can be readily manipulated, and thus will
generally be a method of choice depending on the desired
results.
[0243] In certain embodiments, it is advantageous to employ a
nucleic acid sequence of the present invention in combination with
an appropriate means, such as a label, for determining
hybridization. A wide variety of appropriate indicator means are
known in the art, including radioactive, enzymatic or other
ligands, such as avidinibiotin, which are capable of giving a
detectable signal. In preferred embodiments, one likely employs an
enzyme tag such a urease, alkaline phosphatase or peroxidase,
instead of radioactive or other environmentally undesirable
reagents. In the case of enzyme tags, calorimetric indicator
substrates are known which can be employed to provide a means
visible to the human eye or spectrophotometrically, to identify
specific hybridization with complementary nucleic acid-containing
samples.
[0244] In general, it is envisioned that the hybridization probes
described herein are useful both as reagents in solution
hybridization as well as in embodiments employing a solid phase. In
embodiments involving a solid phase, the sample containing test DNA
(or RNA) is adsorbed or otherwise affixed to a selected matrix or
surface. This fixed, single-stranded nucleic acid is then subjected
to specific hybridization with selected probes under desired
conditions. The selected conditions depend inter alia on the
particular circumstances based on the particular criteria required
(depending, for example, on the G+C contents, type of target
nucleic acid, source of nucleic acid, size of hybridization probe,
etc.). Following washing of the hybridized surface so as to remove
nonspecifically bound probe molecules, specific hybridization is
detected, or even quantified, by means of the label.
[0245] D. Screening For Agonists and Antagonists
[0246] Delta receptors are one of the three major subtypes of
opioid receptors. The endogenous peptides that interact with this
receptor are methionine- and leucine-enkephalin. These receptors
are coupled to multiple cellular effector systems, including
adenylyl cyclase, Ca++ and K+ channels via pertussin-toxin
sensitive G proteins. Delta opioid receptors mediate analgesic
effects of opioids. While delta opioid receptor agonists can induce
analgesia, they have limited abuse potential. Therefore, highly
selective delta opioid receptor agonists can be clinically useful
in the treatment of chronic pain without the harmful side-effects
of addiction.
[0247] Development of highly selective, clinically useful delta
opioid receptor agonists would be facilitated by understanding the
specific sites within the delta receptor necessary for agonist
binding. The cloning of the mouse delta opioid receptor cDNA has
opened up the possibility to investigate the structural domains of
this receptor subtype that are responsible for its functioning. As
indicated below, a single amino acid in the second transmembrane
spanning region of the delta receptor is critical for the binding
of delta-selective opioid agonists.
[0248] To investigate structural components of the mouse delta
opioid receptor involved in ligand binding, an aspartate at residue
95 was converted to an asparagine by site-directed mutagenesis.
This aspartate is conserved among G protein-linked receptors and
has been proposed to mediate Na+ regulation of agonist binding. To
test the ligand binding characteristics of the delta receptor, the
mutant and wild-type receptors were expressed in COS-7 cells. Both
receptors could be labeled with the delta-selective agonist
[.sup.3H]-DPDPE and the antagonist [.sup.3H]-naltrindole. Na+ (90
mM) reduced [.sup.3H]-DPDPE binding to the wild-type delta receptor
but not to the mutant receptor. Na+ did not affect
[.sup.3H]-naltrindole binding but reduced the potency of agonists
to inhibit radiolabeled antagonist binding to the wild-type
receptor but not to the mutant receptor, indicating that Na+
selectively reduces the affinity of the wild-type receptor for
agonists.
[0249] The binding of [.sup.3H]-DPDPE to the mutant receptor was
reduced compared to the wild-type. The reduced binding could be due
to uncoupling of the receptor from G proteins, low expression of
the mutant receptor or an alteration in the ligand binding
properties of the receptor. The mutant receptor remained coupled to
G proteins since GTP.gamma.S could reduce [.sup.3H]-DPDPE binding
to the receptor. Furthermore, the mutant receptor could mediate
agonist inhibition of cAMP formation, a response requiring G
protein coupling. The mutant receptor was expressed at higher
levels than the wild-type receptor. Therefore, the mutant receptor
had a selective reduction in affinity for agonists.
[0250] This was further indicated by the diminished potencies of
the delta selective agonists DPDPE, DSLET, deltorphin and
met-enkephalin to inhibit [.sup.3H]-naltrindole binding to the
mutant receptor compared to the wild-type receptor. The affinity of
the mutant receptor was over 100-fold less for these peptides. In
contrast, the affinity of the mutant and wild-type receptors for
the delta selective antagonists naltrindole, NTB and BNTX were
similar, indicating that the mutant receptor had a specific
reduction in affinity for agonists.
[0251] The potency of a non-selective opioid agonist such as
bremazocine at binding to the mutant and wild-type delta receptors
was similar. This compound is an agonist at all opioid receptor
subtypes. The alkaloid buprenorphine is a compound being used to
treat opioid addiction, that has been reported to be a partial mu
opioid agonist and is a full agonist at the delta receptor since it
inhibits forskolin stimulated cAMP formation in COS cells
expressing either the wild-type or mutant delta receptor. This
non-peptide agonist potently binds to both the mutant and wild-type
delta opioid receptor with similar affinities. Since the mutant
receptor exhibits similar affinity as the wild-type receptor for
non-selective, non-peptide opioid agonists but had diminished
affinity for the delta-selective peptide agonists, differences in
ligand binding properties of the mutant and wild-type receptors
were examined relative to the peptide nature of the agonists or
their delta receptor-selective characteristics.
[0252] BW373U86 and SIOM are non-peptide, potent delta opioid
receptor selective agonists. Both compounds stimulate the wild-type
and mutant delta receptors to inhibit cAMP formation. BW373U86 and
SIOM potently inhibit [.sup.3H]-naltrindole binding to the
wild-type delta receptor. In contrast, BW373U86 is over 100-fold,
and SIOM is over 50-fold less potent at binding to the mutant
receptor. These findings indicate that the mutant delta receptor
has reduced affinity for delta opioid receptor-selective agonists
of different structures.
[0253] The data show that the aspartate at residue 95 of the mouse
delta opioid receptor is necessary for the high affinity binding of
the delta receptor selective agonists. This residue is not
necessary for antagonist binding nor for the binding of
non-selective opioid agonists. The ability of the non-selective
agonists to bind to and stimulate the mutant and wild-type delta
receptors equally well suggests that the single residue mutation
did not induce large conformational changes in the receptor that
would non-selectively alter the ligand binding domain or inhibit
the interaction of the receptor with G proteins, which is essential
for the receptor to bind agonists with high affinity. Because the
binding of non-selective agonists and the delta selective
antagonists to the delta receptor was not affected by the point
mutation, such agonists and antagonists may interact with similar
regions of the ligand binding domain of this opioid receptor that
are distinct from the site involved in delta opioid receptor
selective agonist binding. The aspartate 95 may facilitate the
binding of agonists selective for the delta receptor by providing a
negative charge for stabilization of ligand interaction with the
receptor that is not necessary for the binding of non-selective
agonists or delta opioid antagonists. Similarly an aspartate in the
beta-adrenergic receptor has been proposed to provide a charge for
stabilization of the binding of beta-adrenergic selective agonists.
Recent studies have also shown that a single amino acid in the
tachykinin and cholecytokinin receptors are responsible for subtype
selective antagonist binding, further indicating that a single
residue can be critical for specific ligand-receptor
interactions.
[0254] The data show that selective agonists and antagonists bind
differently to the delta opioid receptor. This suggests that they
may interact with distinct regions of this receptor. A domain of
the second transmembrane spanning region of the delta opioid
receptor containing the aspartate 95 is involved the selective
binding of agonists.
[0255] E. Chimeric Opioid Receptor Polypeptides
[0256] Kappa and delta opioid receptors exhibit distinct
pharmacological specificities. The high degree of amino acid
sequence similarly between the kappa and delta opioid receptors in
their transmembrane spanning regions suggests that extracellular
domains are likely involved in selective ligand binding to each
receptor. The amino-terminal extracellular regions of the two
receptors are divergent in amino acid sequence.
[0257] Standard mutagenesis techniques well known in the art are
used to create chimeric opioid receptor polypeptides comprising
portions from different receptor subtypes. In a preferred
embodiment, the amino-terminal region of a particular receptor
subtype (e.g.,kappa, delta) is linked to a portion
(non-amino-terminal) of a different receptor subtype. In this way,
amino-terminals of particular receptor subtypes can be exchanged. A
mutant polynucleotide (e.g., cDNA) that encodes such a chimeric
receptor polypeptide is then transfected into a host cell where the
chimeric receptor is expressed. A preferred host cell is a COS
cell. The recombinant chimeric receptor receptor polypeptide is
then tested for its ability to bind subtype-selective agonists and
antagonists.
[0258] An Spe I restriction site in the first transmembrane
spanning region was engineered into mouse kappa receptor and delta
receptor cDNAs using oligonucleotide-directed mutagenesis. The site
was engineered into identical locations in both cDNAs, thereby
avoiding frameshifts and/or deletions or additions in sequence.
There are no naturally occurring Spe I restriction sites in the
coding regions of either the kappa or delta opioid receptor cDNAs.
Fragments corresponding to the amino-termini of each receptor are
isolated by cutting at this newly engineered Spe I site in both
cDNAs. Those fragments are then ligated to purified cDNA
corresponding to the carboxy-terminus of the opposite receptor to
generate chimeric kappa .sub.1-74/delta.sub.65-372 and delta
.sub.1-64/kappa.sub.75-380 receptors.
[0259] The mutant DNA fragments are subcloned into a suitable
expression vector (e.g., the mammalian expression vector
pCMV6.sub.0) and either transiently transfected into or stably
expressed in a suitable host cell such as COS-7 cells or CHO cells.
The chimeric is then used in agonist, antagonist studies. By way of
example, a kappa .sub.1-74/delta.sub.65-372 chimeric is tested for
its ability to be labeled with the delta receptor selective agonist
[.sup.3H]-DPDPE and the antagonist [.sup.3H]-naltrindole, which
bind potently to the wild-type delta receptor and the kappa
selective agonist [.sup.3H]-U69,593 and the antagonist
[.sup.3H]-naloxone, which bind to the cloned kappa but not the
cloned delta opioid receptor. If the delta opioid receptor
radioligands do not bind to this chimeric receptor, but
[.sup.3H]-U69,593 and [.sup.3H]-naloxone do bind with high potency,
the ligand binding regions of both receptors is likely included in
the amino-terminus.
[0260] Mouse kappa and delta opioid receptor cDNA were mutated
using the Altered Site.TM. in vitro Mutagenesis System (Promega
Corp. Madison Wis.). To engineer in the Spe I restriction site at
residues 78-80 in the first transmembrane spanning regions of the
kappa receptor cDNA, the mouse kappa receptor cDNA was subcloned
into the phagemid pALTER.TM. and with the helper phage R408,
single-stranded template was produced. A 24-mer oligonucleotide
(GTGGGCAATTCACTAGTCATGTTT; SEQ ID NO:7) encoding the desired
mutation (TCTGGT to ACTAGT) was annealed to the single-stranded
template and elongated with T4 DNA polymerase. The heteroduplex DNA
was then used to transform the repair-minus E. coli strain BMH
71-18 mut S.
[0261] Transformants were selected by growth in LB plates
containing 125 .mu.g/ml ampicillin. Double-stranded plasmid DNA was
sequenced by the Sanger dideoxy chain termination method and
digested with Spe I to confirm the presence of the mutation. For
the delta receptor cDNA, a 24-mer oligonucleotide
(CTGGGCAACGTACTAGTCATGTTT; SEQ ID NO:8) encoding the desired
mutation (GCTCGT to ACTAGT) was used and similar procedures as
described above for the kappa receptor cDNA were employed. Each
mutated cDNA was excised from pALTER with EcoRI and Sal I in the
case of the delta receptor cDNA and Sal I and BamHI for the kappa
receptor cDNA and subcloned into the corresponding sites in the
mammalian expression vector pCMV6c. The 6' regions of each cDNA
corresponding to the N-terminal regions of each receptor (residues
1-75 in the kappa receptor and 1-65 in the delta receptor) were
excised with Eco RI/Spe I (delta receptor) and Sal I/Spe I (kappa
receptor) and gel purified. The N-terminal fragment of the kappa
receptor was ligated to the C-terminal fragment of the delta
receptor. The inserts were excised from the vector and their size
determined by agarose gel electrophoresis to establish whether
appropriate ligation occurred. The chimeric kappa-delta receptor
cDNA was then transiently transfected into COS-7 cells by a
calcium-phosphate-mediated procedure.
[0262] The selective kappa agonist [.sup.3H]-U69593 did not bind to
the kappa .sub.1-74/delta.sub.65-372 chimera. The antagonist
[.sup.3H]-naloxone, which binds with high affinity to the wild-type
kappa but not delta receptor, bound to the chimera. The
[.sup.3H]-naloxone binding was potently inhibited by the kappa
selective antagonist nor-BNI, but not by the selective kappa
agonists U50, 488 or dynorphin. These findings indicated that the
amino-terminus of the kappa receptor likely has the antagonist
binding site but not the agonist binding site. The agonist binding
site, thus, likely resides in other regions of the receptor, such
as the third and fourth extracellular domains, which have different
amino acid sequences from the delta receptor. These data further
indicate that agonists and antagonists bind to clearly dissociated
regions of the kappa receptor.
[0263] The finding that the naloxone binding site is in the
amino-terminal region of the kappa receptor suggests that a limited
region of the kappa receptor may be similar to the mu receptor.
Naloxone potently binds to mu opioid receptors as well as kappa
receptors. These data indicate that screening cDNA libraries with
probes against the amino-terminus of the kappa receptor will
facilitate cloning of the mu receptor.
[0264] Both the selective delta agonist [.sup.3H]-DPDPE and the
selective delta antagonist [.sup.3H]-naltrindole potently bound to
the chimeric kappa.sub.1-74/delta.sub.65-372. These data indicate
that their binding sites are not in the N-terminus of the delta
receptor, because this chimera does not have an amino-terminus of
the delta receptor. Their binding sites likely reside in other
parts of the delta receptor.
[0265] XI. Ligand Binding and G Protein Coupling Domains of the
Kappa and Delta Opioid Receptors
[0266] A. Ligand Binding Domains
[0267] The kappa and delta opioid receptors exhibit distinct
pharmacological specificities. The N-terminal extracellular regions
of the two receptors are divergent in amino acid sequence.
Mutagenesis techniques are used to exchange the N-termini of each
receptor. The mutant cDNAs are transfected into suitable host cells
(e.g., COS cells) and the chimeric receptors tested for their
ability to bind kappa and delta subtype-selective agonists and
antagonists. For the mutagenesis, an SpeI restriction site in the
first transmembrane spanning region is engineered into the mouse
kappa receptor and delta receptor cDNAs using
oligonucleotide-directed mutagenesis. The site is engineered into
identical locations in both cDNAs thereby avoiding frameshifts
and/or deletions or additions of sequence. There are no naturally
occurring SpeI restriction sites in the coding regions of either
the kappa or delta opioid receptor cDNAs. Therefore, after cutting
at this newly engineered site in both cDNAs, it is possible to
isolate the fragments corresponding to the N-termini of each
receptor and ligate them to the purified cDNA corresponding to the
C-terminus of the opposite receptor to generate chimeric kappa
1-74/delta 65-372 and delta 1-64/kappa 75-380 receptors. Each
mutant DNA fragment is subcloned into a suitable mammalian
expression vector (e.g., pCMV6c) and either transiently transfected
into COS-7 cells or stably expressed in CHO cells.
[0268] The kappa 1-74/delta 65-372 chimera is tested for its
ability to be labeled with the delta receptor selective agonist
[.sup.3H]-DPDPE and the antagonist [.sup.3H]-naltrindole, which
bind potently to the wild-type delta receptor, and the kappa
selective agonist [.sup.3H]-U69,593 and the antagonist
[.sup.3H]-naloxone, which bind to the cloned kappa but not the
cloned delta opioid receptor. If the delta opioid receptor
radioligands do not bind to this chimeric receptor, but
[.sup.3H]-U69,593 and [.sup.3H]-naloxone do bind with high potency,
the ligand binding region of the both receptors is likely included
in the N-terminus. Similar pharmacological analysis of the delta
1-64/kappa 75-380 chimera serve to further establish whether the
ligand binding domains of both receptors are localized to their
N-termini.
[0269] Differences likely also exist in the ability of agonists and
antagonists to bind to the chimeric receptors. Such differences are
also examined using mutagenesis. By way of example, if the delta
selective agonist [.sup.3H]-DPDPE does not bind to the kappa
1-74/delta 65-372 chimera whereas [.sup.3H]-U69593 does, it is
likely that that [.sup.3H]-DPDPE would bind potently to the delta
1-64/ kappa 75-380 chimera, but [.sup.3H]-U69593 would not.
Conversely, if [.sup.3H]-naltrindole and [.sup.3H]-naloxone bind
similarly to the chimeric and wild-type receptors, then the results
would support the hypothesis that antagonists bind to different
regions of the opioid receptors than agonists.
[0270] To further identify and isolate the ligand binding domains
of the two receptors in the N-terminal regions, smaller regions of
the N-termini are exchanged and the mutant receptors tested for
their affinities for kappa or delta agonists or antagonists.
[0271] If the initial studies reveal that the N-termini do not
contain the ligand binding domains, it is likely that either the
third and fourth extracellular domains (the only two other
extracellular regions in the opioid receptors which differ
significantly in amino acid sequence) serve as ligand binding
domains. These regions correspond to residues 197-220 and 300-311
of the kappa receptor and residues 187-208 and 287-298 of the delta
opioid receptors. The third and fourth extracellular domains of the
receptors are exchanged between the two receptors and the mutant
receptors tested for their ability to bind kappa and delta receptor
agonists and antagonists.
[0272] Mouse kappa and delta opioid receptor cDNA are mutated using
the Altered Site.TM. in vitro Mutagenesis System (Promega Corp.
Madison Wis.). To engineer in the SpeI restriction site at residues
78-80 in the first transmembrane spanning regions of the kappa
receptor cDNA, the mouse kappa receptor cDNA is subcloned into the
phagemid pALTER.TM. and with the helper phage R408, single-stranded
template is produced. A 24-mer oligonucleotide
(GTGGGCAATTCACTAGTCATGTTT; SEQ ID NO:7) encoding the desired
mutation (TCTGGT to ACTAGT) is annealed to the single-stranded
template and elongated with T4 DNA polymerase. The heteroduplex DNA
is then used to transform the repair-minus E. coli strain BMH 71-18
mut S. Transformants are selected by growth in LB plates containing
125 ug/ml ampicillin. Double-stranded plasmid DNA is sequenced by
the Sanger dideoxy chain termination method and digested with Spel
to confirm the presence of the mutation. For the delta receptor
cDNA, a 24-mer oligonucleotide (CTGGGCAACGTACTAGTCATGTTT; SEQ ID
NO:8) encoding the desired mutation (GCTCGT to ACTAGT) is used and
similar procedures as described above for the kappa receptor cDNA
are employed.
[0273] Each mutated cDNA is excised from pALTER with EcoRI and SalI
in the case of the delta receptor cDNA and SalI and BamHI for the
kappa receptor cDNA and subcloned into the corresponding sites in a
suitable mammalian expression vector (e.g., pCMV6c). The 5' regions
of each cDNA corresponding to the N-terminal regions of each
receptor (residues 1-75 in the kappa receptor and 1-65 in the delta
receptor) are excised with EcoRI/Spel (delta receptor) and
SalI/Spel (kappa receptor) and gel purified. The N-terminal
fragment of the delta receptor is ligated to the C-terminal
fragment of the kappa receptor and the N-terminal fragment of the
kappa receptor is ligated to the C-terminal region of the delta
receptor. The inserts are excised from the vector and their size
determined by agarose gel electrophoresis to establish whether
appropriate ligation occurred.
[0274] The chimeric receptor cDNA is then transiently transfected
into COS-7 cells by a calcium- phosphate mediated procedure. The
chimeric receptors in which the third or fourth extracellular loops
are exchanged between the kappa and delta opioid receptors is
generated by PCR using a similar approach as described above.
[0275] For the receptor binding studies, chimeric receptors are
labeled with the radioligands [.sup.3H]-U69593, [.sup.3H]-naloxone,
[.sup.3H]-DPDPE and [.sup.3H]-naltrindole. Specific binding is
defined as naloxone-sensitive tissue binding. Competitive
inhibition studies are performed using a number of kappa ligands
such as U50488, U69593, nor-BNI and dynorphin. Stereospecificity of
binding is tested using the isomers of naloxone and by comparing
the potencies of levorphanol and dextorphan at inhibiting binding.
Delta receptor ligands such as DPDPE, DSLET, enkephalin, deltorphin
and BW373U86 and the antagonists naltrindole, NTB and BNTX are also
tested. Analysis of IC.sub.50 values is determined using the
computer curve fitting program PROPHET.
[0276] The effects of GTP.gamma.S on either radiolabeled agonist
binding or agonist inhibition of radiolabeled antagonist binding
are studied to determine whether the mutant receptors are G
protein-coupled. To investigate the functional activity of the
mutant receptors, the ability of the receptors to mediate agonist
inhibition of forskolin-stimulated cAMP formation is determined as
described using standard techniques.
[0277] B. G Protein Coupling Domains
[0278] G proteins couple the opioid receptors to various effector
systems and are therefore critical in mediating the cellular
actions of the opioids. The regions of the receptors involved in
associating with G proteins have not been previously identified.
For the adrenergic and muscarinic receptors, several different
intracellular domains have been identified as being involved in G
protein association (Dohlman et al., 1991). The third intracellular
loop of these receptors was first proposed to interact with G
proteins. The amino acid sequences of the third intracellular loops
of the kappa and delta opioid receptors are very similar (see
below). Therefore, exchanging the third intracellular loops of the
kappa and delta opioid receptors would be unlikely to provide any
significant information on whether these regions are G protein
coupling domains. However, the kappa and delta opioid receptors
have high amino acid sequence similarity with the somatostatin
receptor subtype SSTR1 with 40% amino acid identity overall.
Furthermore, the third intracellular loops of opioid receptors and
SSTR1 are identical in size (28 amino acids for each) but differ in
sequence (see below).
[0279] It has been shown that SSTR1 does not couple with G
proteins, nor does it mediate agonist inhibition of adenylyl
cyclase activity (Rens-Domiano et al., 1992; Yasuda et al., 1992).
As a result, the third intracellular loop of SSTR1 is not likely to
contain sequences required for G protein coupling. The third
intracellular loops of the kappa and delta receptors are exchanged
with the corresponding region of SSTR1 by site-directed mutagenesis
to determine if they are G protein coupling domains. If the third
intracellular loops of the opioid receptors is a G protein coupling
domain, the chimeric opioid receptors likely will lose their
ability to associate with G proteins. On the other hand, the
chimeric SSTR1 should gain an ability to couple to G proteins. G
protein association with the chimeric receptors is tested by the
effects of GTP.gamma.S on agonist binding to the receptor, the
effect of pertussis toxin treatment on agonist binding and on the
ability of the chimeric receptors to mediate agonist inhibition of
cAMP formation. Expression of the chimeric opioid receptors is
detected with both radiolabeled antagonist and agonist binding.
Chimeric SSTR1 expression is detected with [.sup.125I]-Tyr.sup.11
somatostatin binding as previously described (Raynor and Reisine,
1989).
4 Third Intracellular Loops (SEQ ID NO:13) SSTR1 Leu Ile Ile Ala
Lys Met Arg Met Val Ala Leu Lys Ala Gly Trp Gln Gln Arg Lys Arg Ser
Glu Arg Lys Ile Thr Leu Met. (SEQ ID NO:14) Kappa Leu Met Ile Leu
Arg Leu Lys Ser Val Arg Leu Leu Ser Gly Ser Arg Glu Lys Asp Arg Asn
Leu Arg Arg Ile Thr Lys Leu. (SEQ ID NO:15) Delta Leu Met Leu Leu
Arg Leu Arg Ser Val Arg Leu Leu Ser Gly Ser Lys Glu Lys Asp Arg Ser
Leu Arg Arg Ile Thr Arg Met. Second Intracellular Loops (SEQ ID
NO:16) SSTR1 Asp Arg Tyr Val Ala Val Val His Pro Ile Lys Ala Ala
Arg Tyr Arg Arg Pro. (SEQ ID NO:17) Kappa Asp Arg Tyr Ile Ala Val
Cys His Pro Val Lys Ala Leu Asp Phe Arg Thr Pro. (SEQ ID NO:17)
Delta Asp Arg Tyr Ile Ala Val Cys His Pro Val Lys Ala Leu Asp Phe
Arg Thr Pro.
[0280] The second intracellular loop of some receptors has also
been proposed to contribute to G protein coupling (Dohlman et al.,
1991). This region of the kappa and delta opioid receptors is
identical (See above). However, the sequences differ from those in
SSTR1. The second intracellular loop can contain a G protein
coupling domains of the opioid receptor. These regions are
exchanged with SSTR1 and the chimeric opioid receptors tested for
loss of G protein coupling and the chimeric SSTR1 tested for gain
of G protein association.
[0281] A third potential region of the opioid receptors that may be
involved in G protein coupling is the cytoplasmic tail. This is the
only intracellular domain that differs in amino acid sequence
between the two opioid receptors. While both opioid receptors
couple to pertussis toxin sensitive G proteins, the subtypes of G
proteins with which they associate are likely different. If these
receptors can interact with different G proteins, then the unique
sequences of the C-termini of the opioid receptors likely provides
the structural basis for their ability to interact with different G
proteins. These regions are exchanged with the corresponding region
of SSTR1 and the chimeric receptors tested for G protein
association using radioligand binding techniques and for their
ability to mediate agonist inhibition of adenylyl cyclase
activity.
[0282] The C-termini of the kappa and delta opioid receptors are
exchanged to determine whether the C-termini are involved in subtle
differences in the ability of the kappa and delta receptor to
associate with subtypes of G proteins. The chimeric and wild-type
receptors are then stably expressed in suitable host cells (e.g.,
CHO cells or PC12 cells). The chimeric receptors are then tested
for which G proteins they associate with using an
immunoprecipitation approach. Furthermore, the coupling of the
chimeric receptors to different effector systems, such as adenylyl
cyclase, Ca++ and K+ channels is also analyzed to determine whether
the C-termini direct the receptors to couple to selective G
proteins to regulate specific effector systems.
[0283] To construct the hybrid kappa receptor/SSTR1 or delta
receptor/SSTR1 mutants in which the third intracellular loop of
SSTR1 is exchanged with a similar region of the kappa and delta
receptor, PCR is employed. Three fragments, the N-termini,
C-termini and third intracellular loops of SSTR1, the delta and
kappa receptors are amplified from 10-50 ng of plasmid DNA under
the following conditions: 25-30 cycles consisting of 1 min at
95.degree. C., 1 min at 55.degree. C. and 1 min at 72.degree.
C.
[0284] N-terminal fragment: The N-terminal fragments is be
generated with a forward primer spanning a unique SalI site in the
cDNA for SSTR1, EcoR1 site of the delta receptor and SalI site for
the kappa receptor. The reverse primer is made to the 3' end of the
fifth membrane-spanning region of SSTR1, the delta and kappa
receptors. Digestion of the SSTR1, delta and kappa receptor
N-terminal products with SalI, EcoRI and SalI, respectively, yields
DNA fragments with 5' overhangs and 3' blunt ends. The forward (F)
and reverse (R) primers to be used in PCR amplification of the
N-terminal fragment (N-) include the following:
5 SSTR1 N-F=TATCTAGGTC GACGG; (SEQ ID NO:18), SSTR1 N-R=CATCTTAGCA
ATGAT; (SEQ ID NO:19), delta N-F=GTCGAGAATT CCCCG; (SEQ ID NO:20),
receptor delta N-R=CAGGCGCAGT AGCAT; (SEQ ID NO:21), receptor kappa
N-F=TAGGTCGACG GTATC (SEQ ID NO:22), and receptor kappa
N-R=CAGGCGCAGG ATCAT (SEQ ID NO:23). receptor
[0285] Third intracellular loop: The third intracellular loop
(3-i-loop) is amplified using a forward primer encoding the 5' end
of the 3-i-loop of SSTR1, delta and kappa receptors and a reverse
primer spanning the juncture between the 3-i-loop and the
C-terminal fragment. This primer incorporates the restriction site
MboI at identical positions within SSTR1, the delta and kappa
receptor cDNA. Digestion of SSTR1, delta and kappa receptor third
intracellular loop PCR fragments with MboI produces DNA with 5'
blunt ends and 3' MboI overhangs. Primers used in PCR amplification
of the 3-i-loop include the following:
6 SSTR1 3-i-loop-F=CGCATGGTGGCCCTC; (SEQ ID NO:24), SSTR1
3-i-loop-R=GGTGATCTTGCGCTC; (SEQ ID NO:25), delta receptor
3-i-loop-F=CGCAGCGTGCGTCTG; (SEQ ID NO:26), delta receptor
3-i-loop-R=CGTGATCCGCCGCAG; (SEQ ID NO:27), kappa receptor
3-i-loop-F=AAGAGTGTCCGGCTC; and (SEQ ID NO:28), kappa receptor
3-i-loop-R=GGTGATCCGGCGGAG; (SEQ ID NO:29).
[0286] C-terminal fragment: The C-terminal fragment is be generated
with a forward primer that spans the juncture between the 3-i-loop
and the C-terminal fragment and a reverse primer that encodes a
unique EcoRI site for SSTR1, SalI site for the delta receptor and
XbaI site for the kappa receptor. The forward primer encodes an
MboI site, just as the reverse primer of the 3-i-loop fragment
does. This provides directional ligation of the 3-i-loop with the
C-terminal fragment. Digestion of the C-terminal products of SSTR1,
the delta and kappa receptors with EcoRI, SalI and XbaI,
respectively, and MboI yields DNA fragments with 5' MboI overhangs
and their respective 3' overhangs. The following primers are used
in PCR amplification of the C-terminal fragment (C-):
7 SSTR1 C-F=GAGCGCAAGATCACC; (SEQ ID NO:30), SSTR1
C-R=TCGAGAATTCCCCGG; (SEQ ID NO:31), delta C-F=CTGCGGCGCGATCAC;
(SEQ ID NO:32), receptor delta C-R=TAGGTCGACGGTGTGG; (SEQ ID
NO:33), receptor kappa C-F=CTCCGGCGGATCACC; (SEQ ID NO:34), and
receptor kappa C-R=GGGTCGAGAACTAGT; (SEQ ID NO:35). receptor
[0287] After PCR amplification and digestions, the N- and
C-terminal fragments of SSTR1 are joined with the third
intracellular loop of the delta or kappa receptor and ligated into
pCMV-6b (that has been digested with SalI and EcoRI) in the
presence of T4 DNA ligase at 16.degree. C. for 24 hrs. Once the
hybrid is appropriately ligated into the expression vector, the
entire insert is sequenced using the Sanger dideoxy chain
termination method (Sequenase version 2.0, USB) as described by the
manufacturer. This procedure is repeated for the delta and kappa
receptor containing the third intracellular loop of SSTR1. It
should be noted that the C-terminal fragments of the delta and
kappa receptor have 1 and 2 endogenous MboI sites, respectively.
The inserts of the delta and kappa receptors have previously been
subcloned into the phagemid pALTER. By oligonucleotide-directed
mutagenesis (Altered Sites, Promega), these endogenous MboI sites
are destroyed by single nucleotide changes that do not alter amino
acid sequence. This is carried out prior to PCR amplification.
[0288] Exchange of the second intracellular loop is carried out in
analogous fashion to the third intracellular loop exchange. The
C-terminal exchanges are carried out in an analogous fashion as the
N-terminal exchanges in which a common restriction site is
engineered into the same corresponding site of SSTR1, the delta and
kappa receptor and the appropriate restriction enzyme are used to
digest the C-terminal fragment from each receptor and then the
C-terminal fragment of SSTR1 is ligated to either the remainder of
the kappa or delta receptor or the C-terminal fragment of the
opioid receptors is ligated to the remainder of SSTR1.
[0289] The chimeric receptors are stably expressed in suitable host
cells (e.g., CHO and PC12 cells) and tested for G protein coupling
by the ability of GTP analogs to reduce high affinity agonist
binding to each receptor. The kappa receptor is labeled with
[.sup.3H]-U69,593, the delta receptor is labeled with
[.sup.3H]-DPDPE and the chimeric SSTR1 labeled with
[.sup.125I]-Tyr.sup.11SRIF as previously described (Rens-Domiano et
al., 1992, Yasuda et al. 1992). The chimeric receptors are also
tested for their ability to mediate agonist inhibition of forskolin
stimulation of cAMP formation.
[0290] C. Ligand Binding of Delta Receptor
[0291] To investigate structural requirements of the delta opioid
receptor needed for ligand binding, an aspartate at residue 128 and
a histidine at residue 278 in the cloned mouse delta opioid
receptor were each converted independently to an asparagine by
site-directed mutagenesis (See Example 8, hereinafter). The
wild-type and mutant receptors were expressed in COS-7 cells and
tested for their affinities for opioid agonists and antagonists.
The receptors could be specifically labeled with the delta
receptor-selective antagonist .sup.3H-naltrindole. The wild-type
and mutant receptors bound the antagonists naltrindole, NTB and
diprenorphine with similar high affinities. In contrast, the
potencies of delta receptor-selective peptide agonists such as
D-Ala.sup.2 deltorphin II, DSLET, DPDPE and the non-peptide agonist
SIOM as well as non-selective opioid agonists such as
beta-endorphin, etorphine, bremazocine and levorphanol at binding
to the asparagine 128 mutant (D128N) mutant were greatly reduced
compared to their binding to the wild-type receptor. The reduced
affinity of the D128N mutant for agonists was not due to an
uncoupling of the receptor from G proteins since the D128N mutant
efficiently mediated the inhibition of forskolin-stimulated cAMP
formation induced by opioid agonists. However, consistent with the
binding results, the potency of DSLET and bremazocine to inhibit
forskolin-stimulated cAMP accumulation in cells expressing the
D128N mutant was diminished compared to the wild-type delta
receptor. The results of these studies indicate that the aspartate
at residue 128 in the mouse delta receptor is necessary for agonist
but not antagonist binding to the delta opioid receptor. This
negatively charged residue may serve as a counterion to the
positively charged nitrogen residues of opioid agonists. By
contrast, the mutant receptor with histidine 278 converted to an
asparagine (H278N) had higher affinity for agonists than the
wild-type receptor. This finding suggests that the aromatic
structure of this histidine is not essential for ligand binding to
the delta receptor and may in fact hinder agonist binding. The
alteration of agonist potencies but not antagonist potencies in
these two mutant receptors supports the hypothesis that agonists
and antagonists bind differently to the delta receptor, possibly by
interacting with distinct ligand binding domains.
[0292] XII. Identification of the G Proteins and Cellular Effector
Systems Coupled to the Kappa and Delta Opioid Receptors
[0293] A. G proteins Coupled to Opioid Receptors
[0294] Both kappa and delta opioid receptors couple to multiple
cellular effector systems. G proteins are necessary to link many
receptors to cellular effector systems. A biochemical approach to
directly determine which G proteins physically associate with the
opioid receptors has been developed by this laboratory. This
approach has been employed to identify the Gi and Go subtypes
associated with the SRIF receptors and the alpha2a adrenergic
receptors (Law et al., 1991, 1993; Law and Reisine, 1992; Okuma and
Reisine, 1992). Briefly, the approach involves (1) solubilizing the
receptors from tissue sources expressing the receptor with a mild
detergent CHAPS to maintain receptor/G protein association, (2)
centrifuging at high speed to remove unsolubilized material, and
immunoprecipitating the receptor/G protein complex with
peptide-directed antisera against either Gi.alpha.1 (3646),
Gi.alpha.2 (1521), Gioc3 (1518), Go.alpha.1 or Go.alpha.2. The
antisera have been generated and provided by Dr. D. Manning (Dept.
Pharmacology, Univ. PA) and are directed against internal sequences
of the alpha subtypes. The antisera are selective for each alpha
subunit, based on their specificities determined with recombinant
forms of the alpha subunits, and the antisera are equally effective
at immunoprecipitating the alpha subunits as determined by there
ability to immunoprecipitate alpha subunits metabolically labeled
with [.sup.35S]-methionine. Following immunoprecipitation, the
immunoprecipitate is separated from the supernatant and high
affinity agonist binding to either the immunoprecipitated receptor
or the solubilized receptor remaining in the supernatant can be
performed. Specificity of the immunoprecipitation is determined
using the peptides to which the antisera where generated to block
the immunoprecipitation. The delta opioid receptor is labelled
using [.sup.125I]-beta-endorphin because it is one of the most
potent agonist available at binding to the delta receptor and its
high specific activity facilitates detection of the receptor.
Furthermore, labeling the receptor with agonists assures that the
receptor detected is G protein coupled since the affinity of the G
protein uncoupled receptor for agonists is low. The solubilized and
immunoprecipitated delta receptor is also labelled with the
antagonist [.sup.3H]-naltrindole. To label the kappa receptor, the
high affinity agonist [.sup.3H]-U69,593 and the antagonist
[.sup.3H]-naloxone are employed. For tissue sources, both the delta
and kappa opioid receptors have been stably expressed in CHO cells
and PC12 cells. These studies can determine which G proteins
physically associate with the cloned delta and kappa opioid
receptors.
[0295] For these studies, a similar methodology to that employed to
study G protein coupling to SRIF and alpha2 adrenergic receptors
was utilized (see Law et al., 1991, 1993: Okuma and Reisine, 1992).
Either CHO (DG44) or PC 12 cells stably expressing either the
cloned delta or kappa receptors are solubilized with a buffer
containing the non-ionic detergent CHAPS (20 mM CHAPS, 20%
glycerol, 250 mM PMSF and buffer A which consists of 50 mM Tris-HCl
(pH 7.8), 1 mM EGTA, 5 mM MgCl2, 10 .mu.g leupeptin, 2 .mu.g
pepstatin and 200 .mu.g bacitracin. Following solubilization, the
solution is centrifuged at 100,000.times.g for 60 min at 4.degree.
C. and the supernatant removed and diluted 1:5 in 7.5% glycerol,
0.5 .mu.g/ml aprotinin in buffer A. The sample is then concentrated
using an Amicon 8050 ultrafiltration device. To immunoprecipitate
opioid receptor/G protein complexes, the solubilized receptors is
incubated with an aliquot of G protein specific antisera, the
samples are placed in a rotator at 4.degree. C. for 4-6 hrs. 100
.mu.l of 50% (w/v) protein A sepharose beads are then added to the
samples and incubated overnight. Another aliquot of antisera is
subsequently added bringing the total antisera dilution to 1:20
which is the optimal concentration of antisera that
immunoprecipitates somatostatin and alpha2 receptor/G proteins
complexes. The samples are incubated for 3 hrs and then centrifuged
at 10,000 rpm for 2 min in an Eppendorp microcentrifuge. The
supernatant is removed and tested for the presence of opioid
receptor using a binding assay described below. The
immunoprecipitate is resuspended in buffer A and centrifuged again.
The supernatant is removed and the immunoprecipitate resuspended in
buffer A and the presence of opioid receptor detected with a
binding assay described below.
[0296] Solubilized opioid receptors are detected by radioligand
binding assay. For the delta receptor, [.sup.125I]-beta-endorphin
is typically used to label the receptor. Specific binding is
determined by DSLET (1 .mu.M) or naltrindole (1 .mu.M) displaceable
binding. The binding reaction is at 25.degree. C. and is terminated
by adding 9 ml of cold Tris-HCl buffer (pH 7.8) to the reaction
mixture and filtering the samples under vacuum. The bound
radioactivity is analyzed using a gamma counter. Parallel studies
are conducted using [.sup.3H]-naltrindole. [.sup.3H]-naltrindole is
used to determine the total amount of solubilized delta receptor
present, since its binding is not dependent on G protein coupling.
In contrast, [.sup.125I]-beta-endorphin only detects the presence
of G protein coupled receptor. To detect immunoprecipitated delta
opioid receptors, the immunoprecipitated receptor is resuspended in
Tris-HCl (pH 7.8) buffer and similar binding assays as described
above are performed. To detect solubilized and immunoprecipitated
kappa opioid receptors, the agonist [.sup.3 H]-U69,593 is used to
detect kappa receptor/G protein complexes and [.sup.3H]-naloxone is
used to detect total kappa receptor present.
[0297] The G protein-directed antisera used are the same employed
previously to study SRIF and alpha2 adrenergic receptor/G protein
coupling. The antiserum 8730 is directed against the C-terminus of
Gi.alpha. and recognizes all forms of Gi.alpha.. The Gi.alpha.
subtype selective antisera used are 3646 (Gi.alpha.1), 1521
(Gi.alpha.2) and 1518 (Gi.alpha.3). These antisera are directed
against internal regions of Gia. Their selectivity has been
established on their specificity towards recombinant forms of the
Gi.alpha. subtypes. The antisera 9072 and 2353 are directed against
the C-terminus and an internal region of Go.alpha., respectively.
They selectively interact with Go.alpha.. The Go.alpha.1 and
Go.alpha.2 antisera used to distinguish which splice variant of
Go.alpha. the opioid receptor interact with have been generated
against the peptides Glu Tyr Pro Gly Ser Asn Thr Tyr Glu Asp (SEQ
ID NO:36) and Glu Tyr Thr Gly Pro Ser Ala Phe Thr Glu (SEQ ID
NO:37) which correspond to residues 290-299 of the Go.alpha.
subtypes (Law et al., 1993).
[0298] XIII. The Molecular Basis of Agonist Regulation of Opioid
Receptors and their mRNA
[0299] While acute stimulation of opioid receptors can induce
analgesia, chronic exposure of the receptors to agonists can induce
tolerance (Koob and Bloom, 1992). The specific neurochemical
mechanisms involved in these behavioral phenomena are not known.
However, a number of studies have linked tolerance development to
opioid receptor desensitization (Nestler, 1993, Loh and Smith,
1990; Childers, 1988). Delta opioid receptors in cell lines and in
animals have been reported to desensitize following chronic
exposure to opioid agonists (Law et al., 1983-85). Similarly, the
cloned delta opioid receptor expressed in COS or CHO cells
desensitizes following agonist pretreatment (unpublished results).
Studies in rodents have also suggested that kappa receptors can be
modulated by chronic opioid treatment. As expected, cloned kappa
receptors expressed in COS cells are desensitized following agonist
pretreatment.
[0300] While short-term opioid treatment can induce opioid receptor
desensitization, prolonged exposure of cells in culture to opioids
or long-term treatment of animals with opioids causes opioid
receptor downregulation. Downregulation involves an inactivation of
the receptor due to its internalization or degradation. This has
been most clearly established for delta opioid receptors expressed
in NG-108 cells (Law et al., 1984, 1985). For many hormone and
neurotransmitter receptors, receptor desensitization and
downregulation are linked, both in a temporal and molecular manner
(Hausdorff et al., 1992).
[0301] For many neurotransmitters and hormones, receptor
down-regulation can cause a number of long-term adaptive cellular
responses. One of the most clear-cut changes is modification in the
expression of genes encoding the receptors that are downregulated.
While chronic opioid treatments have been reported to cause opioid
receptor down-regulation, little is known about the adaptive
cellular responses following chronic opioid use.
[0302] A. Molecular Basis of Opioid Receptor Desensitization
[0303] Pretreatment of COS cells expressing the cloned kappa
receptor to agonists desensitizes the kappa receptor. The enzyme
BARK is involved in kappa receptor desensitization because in cells
coexpressing the kappa receptor and a dominant negative BARK
mutant, agonist pretreatment did not cause kappa receptor
desensitization. BARK catalyzes the phosphorylation of a number of
agonist occupied receptors and the phosphorylation has been linked
to the agonist induced desensitization of those receptors, since
phosphorylation has been shown to uncouple receptors from G
proteins and effector systems (Hausdorff et al., 1992; Benovic et
al., 1989). Peptides directed antisera against the kappa receptor
are generated and used to test whether the kappa receptor becomes
phosphorylated-during desensitization and whether BARK is involved
in catalyzing the phosphorylation. The present invention discloses
two synthetic peptides which correspond to the C-terminus (Thr Val
Gln Asp Pro Ala Ser Met Arg Asp Val Gly; SEQ ID NO:38, residues 367
to 378) and N-terminus (Ser Pro Ile Gln Ile Phe Arg Gly Asp Pro Gly
Pro Thr Cys Ser; SEQ ID NO:39, residues 3 to 17) of the kappa
receptor. These sequences are unique regions of the kappa receptor,
and do not correspond to any other sequences available in the
Genbank database. The peptides are used to generate antisera using
the same approach employed to generate peptide directed antisera
against the SRIF receptors. The antisera are tested for their
ability to immunoprecipitate solubilized kappa receptors, detected
using radioligand binding techniques, and for their ability to
immunoprecipitate kappa receptors in transfected COS cells
metabolically labeled with [.sup.35S]-methionine. Specificity of
the antisera is determined by the ability of the peptides to which
they were generated to block the ability of the antisera to
immunoprecipitate the receptors. The antisera is also tested for
their ability to selectively detect the cloned kappa receptor by
immunoblotting using COS or CHO cells transiently or stably
expressing the kappa receptor, respectively.
[0304] Once the specificity of the antisera have been
characterized, they are used to determine whether the kappa opioid
receptor becomes phosphorylated during desensitization. For these
studies, either COS or CHO cells expressing the kappa receptor are
preloaded with [.sup.32P]-orthophosphate. The cells are treated for
varying times (1, 5, 10, 15, 30, 45 min and 1, 2 and 4 hrs) with
U50,488, the treatment stopped and the cells solubilized and the
kappa receptors immunoprecipitated. The immunoprecipitated
receptors are then subjected to SDS-PAGE and autoradiography to
determine whether they are phosphorylated. The N-terminal directed
antisera should be able to recognize both the phosphorylated and
non-phosphorylated receptors equally well since its epitope is in
an extracellular domain that is not accessible to intracellular
kinases and therefore should not be obstructed by phosphate groups.
The C-terminal directed antisera are also used to immunoprecipitate
the receptor. It may be affected by phosphorylation, if
phosphorylated residues are near the antiseras epitope. If it is
unable to immunoprecipitate the phosphorylated receptor whereas the
N-terminal directed does, then the results suggest that regions of
the C-terminus are phosphorylated. If the receptor becomes
phosphorylated, the specificity of the reaction is tested by
determining whether nor-BNI can block the agonist induced
phosphorylation, just as it can block agonist induced
desensitization. The role of BARK in the phosphorylation is tested
by determining whether the BARK dominant negative mutant prevents
the receptor from becoming phosphorylated, just as it prevents
kappa receptor desensitization.
[0305] If the BARK dominant negative mutant blocks agonist induced
kappa receptor phosphorylation, regions within the kappa receptor
that are phosphorylated and involved in kappa receptor
desensitization are identified using standard techniques.
Phosphorylation likely occurs at intracellular domains of the
receptor since these are regions that would be accessible to BARK.
Exchange mutagenesis is used to localization regions within the
kappa receptor that may be phosphorylated and involved in
desensitization. Previous studies in this laboratory have shown
that the SRIF receptor SSTR1 does not desensitize following chronic
agonist treatment and therefore would not be expected to be
phosphorylated in an agonist dependent manner (Rens-Domiano et al.,
1992). This receptor has 40% identity in amino acid sequence with
the kappa receptor. Previously generated series of kappa
receptor/SSTR1 exchange mutants, the second and third intracellular
loops and the cytoplasmic tail are the major intracellular domains
of the two receptors and are the regions of the kappa receptor
likely to be phosphorylated since they contain multiple serine and
threonine residues, which are acceptors of BARK catalyzed
phosphorylation. Treatment of CHO cells expressing the chimeric
kappa receptor/SSTR1 with kappa agonists (U50,488) is used to test
phosphorylation of the receptors. For these studies, CHO cells
stably expressing the mutant receptors are preloaded with
[.sup.32P]-orthophosphate and following the agonist treatments, the
cells are solubilized and the chimeric receptors immunoprecipitated
with antisera directed against the N-terminal region of the kappa
receptor, which is an epitope that should not be disturbed by the
receptor mutagenesis. Once chimeras that are not phosphorylated
following agonist pretreatment are identified, single point
mutations of the serines and threonines in the wild-type kappa
receptor are induced in those regions that had been exchanged in
chimeric receptors. The mutant receptors are tested for their
ability to desensitize following chronic agonist treatment and
whether they become phosphorylated in response to agonist
stimulation.
[0306] The peptides Ser Pro Ile Gln Ile Phe Arg Gly Asp Pro Gly Pro
Thr Cys Ser (SEQ ID NO:39), and Thr Val Gln Asp Pro Ala Ser Met Arg
Asp Val Gly (SEQ ID NO:38), which correspond to unique sequences in
the N- and C-terminus of the kappa receptor were synthesized by Dr.
S. Khan of the peptide synthesis facility of the Wistar Inst.
Philadelphia, Pa. The peptides are covalently linked to Keyhole
Limpet Hemocyanin (KLH) protein as a carrier using a bifunctional
coupling reagent, glutaraldehyde. Peptide-KLH conjugates are
emulsified in the presence of Freund's Complete Adjuvant for the
first injection, followed by incomplete Adjuvant for the next
injections. New Zealand rabbits receive subcutaneous injections
every four weeks and are bled 10 days after each immunization.
[0307] Membranes from either CHO cells or COS cells expressing the
cloned kappa receptor and control cells are subjected to 8%
SDS-PAGE, the proteins transferred to nitroscreen membrane, and the
membranes saturated at 37.degree. C. for 2 hr with 5% defatted
milk, 0.02% azide and PBS. Varying dilutions of the antisera (1:10
to 1:10,000) in 5% milk/PBS are incubated with the membranes over
night at 4.degree. C. under continuous shaking, the nitrocellulose
membranes are then washed and the complexed antibodies detected
with a phosphatase alkaline labeled anti-rabbit antibody kit.
Non-specific reactions are determined by specific peptide blockade.
Preimmune sera is also used as a control for specificity.
[0308] Kappa receptors in COS or CHO cells are solubilized and
incubated overnight in the presence of the antisera precoated
Protein A-Sepharose beads (20 .mu.l serum for 20 .mu.l of a 50%
protein A-Sepharose beads/50% PBS solution) at 4.degree. C. The
supernatants and immunoprecipitates are analyzed for the presence
of high affinity [.sup.3H]-U69593 and [.sup.3H]-naloxone binding.
In addition, the receptor is metabolically labelled with
[.sup.35S]-methionine, as described in Theveniau et al., 1992 and
immunoprecipitated with antisera. For these studies, COS or CHO
cells expressing the cloned kappa receptor are incubated overnight
in methionine-free medium containing 0.5 mCi of
[.sup.35S]-methionine. The cells are washed with PBS, and the
proteins solubilized in RIPA buffer. The receptor is
immunoprecipitated by an overnight incubation with antibody-coated
protein A beads. The immunoprecipitate is boiled in sample buffer
and subjected to 10% SDS-PAGE and autoradiography.
[0309] Either COS or CHO cells expressing the kappa receptor or the
chimeric kappa receptor/SSTR1 are incubated with 0.3 mCi of
[.sup.32P]-orthophosphate for 24 hrs to determine which receptor is
phosphorylated during desensitization. The cells are then
stimulated with U50488 for varying times (0, 5, 15, 30, 45, 60, 90,
120, or 240 min). The reaction is stopped, the cells washed with
cold PBS, the membranes solubilized as described above and the
receptor immunoprecipitated with the peptide directed antisera. The
immunoprecipitate is subjected to SDS-PAGE and autoradiography to
determine whether the receptor is phosphorylated. Specificity of
the immunoprecipitation is demonstrated by blocking with the
peptide to which the antisera whether generated and the lack of
phosphorylation of the kappa receptor in control, non-treated
cells.
[0310] B. Molecular Basis of Delta opioid receptor desensitization
Like kappa receptors, delta opioid receptors desensitize following
chronic agonist pretreatment. The cloned delta receptor stably
expressed in CHO cells desensitizes following chronic agonist
pretreatment. The desensitization is characterized as a decrease in
affinity of the receptors for agonists, an uncoupling of the
receptors from G proteins and a diminished ability of the delta
receptor to mediate agonist inhibition of cAMP formation. Studies
similar to those described above for the kappa receptor are
performed to test whether the delta receptor expressed in COS cells
become desensitized following agonist treatment.
[0311] For these studies, peptide-directed antisera against the
cloned delta receptor are generated using the peptides (Ser Asp Ala
Phe Pro Ser Ala Phe Pro Ser Ala Gly Ala; SEQ ID NO:40), and (Ala
Thr Thr Arg Glu Arg Val Thr Ala Cys Thr Pro Ser; SEQ ID NO:41),
which correspond to the residues 20 to 32 and 367 to 379 in the N-
and C- termini respectively. The antisera are then used to
determine whether the delta receptor becomes phosphorylated during
desensitization. For these studies the cloned delta receptor is
expressed in COS and CHO cells, the cells are preloaded with
32P-orthophosphate and stimulated for varying times [1, 5, 10, 15,
30 and 60 min) with DSLET (1 .mu.M)]. The cells are then
solubilized and the delta receptor immunoprecipitated with the
antisera. The immunoprecipitate is subjected to SDS-PAGE and
autoradiography to determine whether the receptor becomes
phosphorylated. Similar studies are performed on COS cells
cotransfected with the delta receptor and the BARK dominant
negative mutant to determine whether BARK is involved in the
phosphorylation of the delta receptor.
[0312] To determine regions of the delta opioid receptor that
become phosphorylated during desensitization, similar approaches as
described for the kappa receptor are used with delta opioid
receptor/SSTR1 chimeric receptors. Where chimeras do not become
phosphorylated, point mutations are induced in the wild-type
receptor to convert the serines and threonines in the corresponding
region that was exchanged with SSTR1. The mutated delta receptor is
then tested for its ability to be desensitized following agonist
pretreatment and whether it becomes phosphorylated.
[0313] XIV. Expression of the Opioid Receptor Genes
[0314] Chronic opioid treatment induces a number of adaptive
cellular responses. For some neurotransmitters and hormones,
chronic exposure of target cells or tissues to agonists can induce
long-term changes in the expression of receptor genes. There is no
information available to date concerning the long-term effects of
opioid treatment on the expression of the delta and kappa opioid
receptor genes.
[0315] NG-108 cells, which endogenously express delta opioid
receptors (Law et al., 1983), are treated with delta agonists to
desensitize and downregulate the receptor and determine whether
accompanying changes occur in the expression of the delta opioid
receptor gene. Changes in delta receptor gene expression are
measured by Northern analysis employing delta receptor specific
probes. NG-108 cells are treated for varying times (5, 15, 45, 60
min, 2, 4, 8, 16 and 24 hrs) with the delta selective agonist DPDPE
(1-100 nM). Where DPDPE treatment alters delta receptor mRNA
levels, the ability of other agonists (DSLET, deltorphin and
bremazocine) to induce this effect are studied. These studies test
whether a cellular response to chronic delta receptor agonist
treatment is a change in delta receptor gene expression.
[0316] To investigate whether chronic treatment with delta opioid
selective agonists modifies delta receptor gene expression in vivo,
post-mortem, frozen brains of rats and mice that have been
chronically treated with DPDPE and made tolerant (antinociception)
to this agonist are obtained. The procedures used and scheduling of
the drug administrations are the same as previously described
(Cowan and Murray, 1990; Heyman et al., 1988). In selected brain
regions (cerebral cortex, striatum, hippocampus, and cerebellum)
and spinal cord of saline treated controls and DPDPE treated
animals, changes in delta opioid receptor mRNA are quantified by
Northern analysis. On the same blots, kappa receptor mRNA is
reprobed to determine the selectivity of the changes in opioid
receptor gene expression. In addition, in other groups of control
and treated animals, relative levels of delta opioid receptor mRNA
are measured by semi-quantitative in situ hybridization
histochemistry. The advantage of the use of in situ hybridization
histochemistry to detect changes in delta receptor mRNA as a
consequence of chronic delta agonist treatment is the superior
anatomical resolution at the regional (film autoradiography) and
cellular level (emulsion autoradiography). This is particularly
important for analyzing changes in delta receptor mRNA in small
nuclei such as the locus coerleus and other brainstem nuclei in
which delta receptors have important roles and in which tolerance
to delta agonists have been demonstrated (Nestler, 1993). For the
in situ hybridization, the sections are processed and applied to
film and the optical density of the autoradiograms in selected
regions, such as the locus coerleus, substantia nigra, striatum,
nucleus accumbens, hippocampus, amygdala, hypothalamus and central
grey analyzed. These regions express delta opioid receptor mRNA in
mouse brain and have been shown in autoradiographic studies to
express delta receptors in rat brain (Herz, 1993). After exposure
to film, the sections are dipped in photographic emulsion and the
autoradiographic signal determined at the single cell level to
confirm the anatomic specificity of the labeling. Quantitation at
the single cell level is performed in brain regions where labeling
on films is not optimal due to scattering of the labeled cells.
Single cell analysis also complements optical density measurements
if microscopic analysis suggests heterogeneous effects on
subpopulations of neurons in a given regions. Parallel studies are
performed to determine whether kappa receptor mRNA is modified in
the brains of animals made tolerant to DPDPE to determine the
specificity of the effect on delta receptor gene expression. The
delta receptor agonist used for these treatments, DPDPE, does not
bind to the kappa receptor, nor any other opioid receptor.
[0317] A. Selective Changes in Kappa Receptor Gene Expression
[0318] There are no cell lines that endogenously express kappa
receptors. Furthermore, the COS and CHO cells which stably express
the cloned kappa receptor are transfected with the mouse cDNA under
a CMV promoter. Therefore, the cDNA is not under the normal control
of regulatory regions and factors that would modulate kappa
receptor gene expression. Therefore, chronic treatment of rodents
with kappa agonists determines if such agonists can induce changes
in kappa receptor gene expression. For these studies, frozen
post-mortem brains are obtained from rats and mice treated with
U50,488 to induce behavioral tolerance to the antinociceptive
actions of kappa agonists using previously described procedures
(Cowan and Murray, 1990). Modified kappa receptor gene expression
in selective brain regions is made using Northern analysis and by
in situ hybridization histochemistry employing kappa receptor
selective RNA probes. Results from brain sections of the treated
animals are be compared to levels of kappa receptor mRNA detected
in brain sections from control, saline treated animals. In adjacent
sections, delta opioid receptor mRNA levels are detected to
determine whether the treatment selectively effects kappa receptor
gene expression. U50,488 does not bind to delta opioid receptors
nor any other receptor besides kappa receptors. Therefore, if
U50,488 treatments induce selective changes in kappa receptor mRNA
levels but not delta receptor mRNA levels, then the changes in
kappa receptor gene expression are likely directly linked to
activation and modulation of kappa receptors.
[0319] B. Effects of Morphine on Opioid Receptor Gene
Expression
[0320] Morphine binds potently to mu receptors with nM IC.sub.50
values. However it is impotent at the cloned kappa receptor
(IC.sub.51 .mu.M) and does not inhibit binding to the cloned delta
receptor at 10 uM. Its selective high affinity for mu receptors
suggests that it may not affect kappa or delta opioid receptor gene
expression, if changes in expression of the genes is due solely to
activation of kappa or delta receptors.
[0321] The effects of morphine on kappa and delta opioid receptor
mRNA levels in brains sections are studied using in situ
hybridization histochemistry and in brain regions by Northern
analysis. Frozen post-mortem brains from rats and mice made
tolerant to the antinociceptive actions of morphine are obtained
using previously described procedures (Tortella et al., 1981; Cowan
and Murray, 1990).
[0322] NG-108 cells will be exposed to DPDPE (1 uM) for varying
times (0, 5, 15, 45 mins, 1, 2, 4, 8, 16 and 24 hrs). The cells are
washed with PBS, detached from flasks and RNA extracted with the
guanidinium isothiocyanate-cesium chloride procedure, denatured
with glyoxal, fractionated on a 1% agarose gel and transferred to a
nylon membranes. The blots are probed with a [.sup.32P]-labeled
fragment of the cloned mouse delta opioid receptor cDNA
corresponding to the initial 350 bp of the coding region of the
cDNA. After hybridization, the blot is washed at room temperature
in 2.times.SSC and 0.05% SDS at room temperature and then at
48.degree. C. in 0.1.times.SSC and 0. 1% SDS for 30 min. The blot
is then exposed to X-ray film in the presence of an intensifying
screen at -75.degree. C. As an internal control to account for
differences in total RNA per lane, the blot is reprobed with a
probe for beta-actin mRNA. Relative levels of delta receptor mRNA
are quantitated by densitometry and by excising the bands on the
gel containing the mRNA and determining radioactive content by
scintillation spectroscopy. If levels of delta receptor mRNA in
NG-108 cells are too low to be detected by Northern analysis,
reverse-transcriptase PCR issued to measure the mRNA levels.
Northern analysis for delta opioid receptor mRNA in different rat
and mouse brain regions are conducted using similar procedures as
described above. Similar procedures are used to detect kappa
receptor mRNA using the Pst1/EcoR1 fragment of the mouse kappa
receptor cDNA which corresponds to the initial 375 bp of the cDNA
as described by Yasuda et al.
[0323] In situ hybridization histochemistry is performed with
35S-radiolabeled RNA probes as previously described (Chesselet et
al., 1987). For these studies, brain sections are kept at
-70.degree. C., brought to room temperature, acetylated, incubated
in Tris/glycine 0.1 M, pH 7.0 and dehydrated in graded ethanol.
Hybridization is conducted at 50.degree. C. for 3.5 hr in humid
chambers. The hybridization buffer contains 40% formamide,
4.times.SSC (1.times.SCC in 15 mM sodium citrate and 150 mM NaCl,
pH 7.2), 10% dextran sulfate, 10 mM DTT, tRNA, herring sperm DNA,
Denhardt's solution and labeled probe. Quantitative differences in
the level of mRNAs can be reliably detected under these conditions
(Weiss-Wunder and Chesselet, 1991). For these studies brain
sections from saline treated control animals and the corresponding
brain section from the treated animal are processed together.
Post-hybridization washes are in 50% formamide/2.times.SSC at
52.degree. C., for 5, 20 and 25 min. Between the second and third
washes, the sections are rinsed in 2.times.SSC and treated with
RNAse A (100 mg/ml) in 2.times.SSC at 37.degree. C. for 30 min. The
sections are rinsed overnight in 2.times.SSC/Triton X-100 (0.0%),
dehydrated in graded ethanol containing 300 mM ammonium acetate and
processed for autoradiography. Autoradiograms are quantified as
previously described (Soghomomian et al., 1992). Controls include
hybridization with sense probes, and verification of the anatomical
pattern of hybridization with non-overlapping antisense probes.
[0324] For single cell analysis with the Morphon Image analysis
system, cells are observed under brightfield illumination with a
100.times. or a 40.times. objective and the image magnified and
transferred onto a videoscreen. Autoradiographic grains within a
defined region are analyzed as previously described (Weiss-Wunder
and Chesselet, 1991).
[0325] The exact procedures used to treat animals will vary
depending upon the animal model. By way of example, male ICR mice
(20-25 g, Hilltop Inc., Pennsylvania) are housed eight per cage
with food and water freely available. A 12 hr light/12 hr dark
daily cycle is maintained. Groups of 8 mice receive s.c. injections
of U50,488, morphine or distilled water. DPDPE is injected into the
left lateral cerebroventricle. The animals are lightly anesthetized
with ether and then each mouse receives a small incision in the
scalp. By using a 10 .mu.l microsyringe fitted with a 27-gauge
needle, 5 .mu.l of DPDPE or distilled water is delivered 2 mm
lateral and caudal to bregma at a depth of 3 mm. The wound is
closed with a stainless steel clip and subsequent icv injections
are made through the same hole in the skull. A typical injection
schedule has been previously described (Cowan and Murray, 1990;
Mattia et al., 1991). Groups of mice are injected with either
U50,488 (s.c), morphine (s.c.), DPDPE (icv) and distilled water
(n-64, s.c. or n=32, icv) at 1 PM on day 1 at appropriate doses.
Antinociception is assessed at 0, 10, 20, and 30 min using the
latency to tail-flick with 50.degree. C. warm water as the
nociceptive stimulus and calculated as 100.times.(test
latency-control latency)/(15 or 30 - control latency). A cut-off
point of 15 or 30 sec is typically chosen depending on the initial
latencies. Control mice receiving the distilled water are also
measured for tail-flick latency. Regression lines, A50 values and
95% confidence limits are determined from individual data points
using procedure 8 in the computer program of Tallarida and Murray
(1987). The mice are injected with agonist or distilled water
according to an injection schedule and then re-run in the
antinociceptive assay. Pharmacological tolerance is reflected by
the rightward (and possibly downward) displacement of initial
dose-response curves. Four hr after the last injection, each animal
is decapitated and the whole brain dissected out over crushed ice
and immediately stored at -80.degree. C. For some animals,
following decapitation, the brains are dissected and cerebral
cortex, hippocampus, cerebellum, medulla, midbrain, hypothalamus
and striatum, collected, immediately frozen at -80.degree. C. and
used for Northern analysis.
[0326] For studies on rats, male S.D. albino rats are housed five
per cage with a 12 hr light/12 hr dark daily cycle. The rats
receive s.c. injections of U50,488, morphine or distilled water.
DPDPE (5 .mu.l) is injected into rats previously implanted with
PE10 cannula in the left lateral cerebral ventricle (Tortella et
al., 1981). The rats are injected as previously described (Heyman
et al., 1988; Cowan and Murray, 1990). Groups of rats are injected
with U50,488 (s.c.), morphine (s.c.), DPDPE (icv) or distilled
water (n=64 s.c. or n=32 ICV) at 1 PM on day 1. Antinociception is
assessed at 0, 10, 20, and 30 min using the latency to hind-paw
lick on the 50.degree. C. hot plate as the nociceptive stimulus and
calculated as 100.times.(test latency-control latency)/(30, 45, or
60-control latency). A cut-off point of 30, 45, or 60 sec is chosen
depending on the initial latencies. Calculations, injection
schedules and data analysis are the same as described for mice.
[0327] XV. Assay Kits.
[0328] In another aspect, the present invention contemplates
diagnostic assay kits for detecting the presence of opioid receptor
polypeptides in biological samples, where the kits comprise a first
container containing a first antibody capable of immunoreacting
with opioid receptor polypeptides, with the first antibody present
in an amount sufficient to perform at least one assay. Preferably,
the assay kits of the invention further comprise a second container
containing a second antibody that immunoreacts with the first
antibody. More preferably, the antibodies used in the assay kits of
the present invention are monoclonal antibodies. Even more
preferably, the first antibody is affixed to a solid support. More
preferably still, the first and second antibodies comprise an
indicator, and, preferably, the indicator is a radioactive label or
an enzyme.
[0329] The present invention also contemplates a diagnostic kit for
screening agents. Such a kit can contain an opioid receptor of the
present invention. The kit can contain reagents for detecting an
interaction between an agent and a receptor of the present
invention. The provided reagent can be radiolabelled. The kit can
contain a known radiolabelled agent capable of binding or
interacting with a receptor of the present invention.
[0330] It is further contemplated that the kit can contain a
secondary polypeptide. The secondary polypeptide can be a
G-protein. The secondary polypeptide can also be an effector
protein. When a secondary polypeptide is included in a kit,
reagents for detecting an interaction between the receptor and the
secondary polypeptide can be provided. As a specific example, an
antibody capable of detecting a receptor/G-protein complex can be
provided. As another specific example, an antibody capable of
detecting a G-protein/effector complex can be provided. Reagents
for the detection of the effector can be provided. For example, if
the effector provided is adenylyl cyclase, reagents for detecting
the activity of adenylyl cyclase can be provided. The identity of
such agents is within the knowledge of those skilled in the
relevant art.
[0331] In an alternative aspect, the present invention provides
diagnostic assay kits for detecting the presence, in biological
samples, of a polynucleotide that encode receptor polypeptides, the
kits comprising a first container that contains a second
polynucleotide identical or complementary to a segment of at least
10 contiguous nucleotide bases of SEQ ID NO: 1, SEQ ID NO: 3, SEQ
ID NO: 5, or SEQ ID NO: 11.
[0332] In another embodiment, the present invention contemplates
diagnostic assay kits for detecting the presence, in a biological
sample, of antibodies immunoreactive with opioid receptor
polypeptides, the kits comprising a first container containing an
opioid receptor polypeptide that immunoreacts with the antibodies,
with the polypeptides present in an amount sufficient to perform at
least one assay. The reagents of the kit can be provided as a
liquid solution, attached to a solid support or as a dried powder.
Preferably, when the reagent is provided in a liquid solution, the
liquid solution is an aqueous solution. Preferably, when the
reagent provided is attached to a solid support, the solid support
can be chromatograph media or a microscope slide. When the reagent
provided is a dry powder, the powder can be reconstituted by the
addition of a suitable solvent. The solvent can be provided.
EXAMPLES
[0333] Examples have been included to illustrate preferred modes of
the invention. Certain aspects of the following examples are
described in terms of techniques and procedures found or
contemplated by the present inventors to work well in the practice
of the invention. These examples are exemplified through the use of
standard laboratory practices of the inventor. In light of the
present disclosure and the general level of skill in the art, those
of skill will appreciate that the following examples are intended
to be exemplary only and that numerous changes, modifications and
alterations can be employed without departing from the spirit and
scope of the invention.
Example 1
Isolation of cDNA Clones
[0334]
8 Two Degenerate Oligonucleotides, SSTR-D1, 5'-
ACCAA(T/C)(G/A)TCTA(T/C)AT(T/C)AT(T/C)CTIAACCTGGC-3'; SEQ ID NO:9
and SSTR-D2, 5'-ACIGGTCAG(G/A)CAG(A/T)A(G/T)AT(G/A- )CTGGTGAA-3'
SEQ ID NO:10
[0335] were selected using conserved sequences present in the
second and third transmembrane domains of the somatostatin (SRIF)
receptor subtypes, SSTR1, SSTR2 and SSTR3 (Yasuda, et al. 1992;
Yamada, et al. 1992). Amplification using the polymerase chain
reaction (PCR) was carried using an aliquot
(.apprxeq.1.times.10.sup.6 pfu) of a mouse brain cDNA library
(Clontech, Palo Alto, Calif.; catalogue no. ML1036a) as a
template.
[0336] The cycle conditions were: 10 cycles of denaturation at
94.degree. C. for 1 min, annealing at 37.degree. C. for 1 min and
extension at 72.degree. C. for 2 min, followed by 35 cycles of
denaturation at 94.degree. C. for 1 min, annealing at 55.degree. C.
for 1 min and extension at 72.degree. C. for 2 min. The PCR
products were separated on a 3% low melting temperature agarose
gel, and DNA fragments between 150 and 200 bp were isolated, cloned
into M13 mp18 and sequenced.
[0337] Two PCR products encoding novel SRIF receptor-like sequences
were identified, termed msl-1 (SD3) and msl-2 (SD15). These were
.sup.32P-labeled by nick translation and used to screen the mouse
brain cDNA library by hybridization using standard conditions with
a final post-hybridization wash in 0.1.times.SSC and 0.1% SDS at
50.degree. C. before exposure to X-ray film.
[0338] Mouse brain cDNAs encoding SRIF receptor-related sequences
were amplified using PCR and degenerate oligonucleotide primers as
set forth above. PCR products of 150-200 bp were cloned and
sequenced. Of the 33 clones characterized, two encoded mSSTR1, two
SSTR2, nine mSSTR3, four were identical and encoded a new member of
the G protein-coupled receptor superfamily designated msl-1, and
one encoded a second new receptor-like sequence termed msl-2. The
sequences of the remaining 15 clones were unrelated to those of G
protein-coupled receptors or of any other sequences in the GenBank
data base. The clones msl-1 and msl-2 were used as probes to screen
a mouse brain cDNA library and to isolate .lambda.msl-1 and Xmsl-2
having inserts of 3.1 and 2.3 kb, respectively. msl-1 was renamed
as mouse kappa opioid receptor, and msl-2 was renamed as mouse
delta opioid receptor.
[0339] The sequences of the inserts in .lambda.msl-.lambda. and
.lambda.msl-2 (deposited in the GenBank database with accession
numbers L11065 and L11064, respectively) were determined and shown
to encode polypeptides of 380 and 372 amino acids, respectively
(FIG. 1). The sequences of msl-1 and msl-2 were most closely
related to those of members of the recently described SRIF receptor
family with 35% identity with the sequence of mSSTR1.
[0340] The sequences of msl-1 and msl-2 share many features
conserved among members of the G polypeptide receptor superfamily
including the sequence Asp-Arg-Tyr (DRY) in the NH.sub.2-terminal
end of the second intracellular loop and cysteine residues in the
first and second extracellular loops that can form a disulfide
bond. There are also potential sites for N-linked glycosylation in
the putative NH2-terminal domain and several potential
phosphorylation sites for cAMP-dependent protein kinase and protein
kinase C (Kennelly & Krebs, 1991) in intracellular loops and in
the COOH-terminal domain (See FIG. 1).
[0341] Alignment of the amino acid sequences of msl-1 and msl-2
showed that they have 61% amino acid identity and 71% similarity.
As noted previously in other comparisons of closely-related G
protein-coupled receptors (Probst, et al., 1992), the sequences of
the putative membrane-spanning segments are more highly conserved
than those of the NH.sub.2- and COOH-terminal domains. It is
notable that the sequences of the intracellular loops, including
the short third intracellular loop which is believed to be critical
for G protein coupling (Kobilka, et al., 1988), are highly
conserved between msl-1 and msl-2, suggesting that they can couple
to the same G proteins. Both msl-1 and msl-2 have a conserved Asp
residue in the second transmembrane domain. This Asp has been
proposed to mediate sodium inhibition of agonist binding in the
adrenergic (Horstman, et al., 1990) and somatostatin receptors.
Example 2
Expression and Binding Results
[0342] A 1.2 kb Pst I fragment of the mouse kappa opioid receptor
cDNA clone .lambda.msl-1, and 1.3 kb Eco RI-Sac I fragment of the
delta opioid receptor cDNA clone .lambda.msl-2, were cloned into
the CMV promoter-based expression vectors pCMV-6b and pCMV-6c
(obtained from Dr. Barbara Chapman, Chiron Corp., Emeryville,
Calif.), respectively. The resulting constructs, pCMV-msl-1 and
pCMV-msl-2, were used to transfect COS-1 cells as described
previously (Yasuda, et al. 1992).
[0343] Binding studies using membranes prepared from COS-1 cells
transiently expressing msl-1 and msl-2 were carried out 72 h
post-transfection. Briefly, cells were harvested in 50 mM Tris-HCl
(pH 7.8), 1 mM ethylene glycol bis(.beta.-aminoethyl
ether)-N,N'-tetraacetic acid, 5 mM MgCl.sub.2, 10 .mu.g/ml
leupeptin, 10 .mu.g/ml pepstatin, 200 .mu.g/ml bacitracin, and 0.5
.mu.g/ml aprotinin (Buffer 1) using a Polytron (Brinkmann, setting
2.5, 30 sec). The homogenate was then centrifuged at 48,000.times.g
for 20 min at 4.degree. C. The pellet was re-suspended in Buffer 1
and this membrane preparation was used for radioligand binding
studies.
[0344] Cell membranes (20-30 .mu.g total protein) were incubated
with [.sup.3H]U69,593 (1 nM, specific activity 37.2 Ci/mmol) or
[.sup.3H]dextromethorphan (1 nM, specific activity 82.7 Ci/mmol),
[.sup.3H]DTG (1 mM, specific activity 37.2 Ci/mmol) or
[.sup.3H]DAMGO (1 nM, specific activity 55 Ci/mmol) (Dupont NEN,
Boston, Mass.) in a final volume of 200 .mu.l for 40 min at
25.degree. C. in the presence or absence of competing agents.
Nonspecific binding was defined as the radioactivity remaining
bound in the presence of 10 .mu.M naloxone for all radioligands
except [.sup.3H]dextromethorphan and [.sup.3H]DTG for which 10
.mu.M haloperidol or carbetapentane citrate were used.
[0345] The binding reaction was terminated by the addition of
ice-cold 50 mM Tris-HCl (pH 7.8) and rapid filtration over Whatman
GF/B glass fiber filters that were pre-treated with 0.5%
polyethylimine and 0.1% bovine serum albumin for at least 1 h. The
filters were then washed with 12 ml of ice-cold 50 mM Tris-HCl (pH
7.8) and the bound radioactivity determined using a liquid
scintillation counter. Data from radioligand binding studies were
used to generate inhibition curves. IC.sub.50 values were obtained
by curve-fitting performed by the mathematical modeling program
FITCOMP available on the NIH-sponsored PROPHET system.
[0346] The homology between msl-1 and msl-2 and the SRIF receptors
suggested that they might be new members of the SRIF receptor
family. However, membranes from COS-1 cells transiently expressing
msl-1 and msl-2 did not show specific [.sup.125I-Tyr.sup.11] SRIF
binding demonstrating that msl-1 and msl-2 were not SRIF receptors.
Since the SRIF agonist SMS 201-995 has been reported to bind to
SRIF and mu opioid receptors, it was possible that msl-1 and msl-2
might be opioid receptors. While studies were in progress to test
this hypothesis, two groups reported the cloning of a mouse
delta-opioid receptor from NG 108-15 cells. The sequence of their
receptor was identical to msl-2.
[0347] Binding studies using agonists selective for delta, kappa,
mu and sigma opioid receptors confirmed that msl-2 was a delta
opioid receptor (mORD1, SEQ ID NO:4) and showed that msl-1 was a
kappa receptor (mORK1, SEQ ID NO:2). The binding properties of
membranes prepared from COS-1 cells expressing msl-1 and msl-2 are
summarized in Table 2.
9TABLE 2 BINDING POTENCY OF OPIOID LIGANDS FOR CLONED MOUSE OPIOID
RECEPTORS EXPRESSED IN COS-1 CELLS IC.sub.50(nM) [.sup.3H]U-69,693
[.sup.3H]Naltrindole msl-1 (mORK1) msl-2 (mORD1) Endogenous opioid
ligands Dynorphin A (1-17) 0.4 >100 Dynorphin A (1-8) 0.2
>100 Dynorphin B 0.1 >100 .alpha.-Neoendorphin 0.1 10
.beta.-Endorphin (human) 42 15.3 Leu-enkephalin >1000 79
Met-enkephalin >1000 41 Kappa-selective ligands Dynorphin
(1-17)NH.sub.2 0.2 >100 [D-Ala.sup.2, F.sub.5Phe.sup.4]Dynorhpin
0.2 >100 (1-17)NH.sub.2 Bremazocine 0.3 19 [Met.sup.5]Dynorphin
(1-17) 0.6 >100 U-62,066 1.0 >1000 Ethylketocyclazocine 1.1
611 U-50,488 1.1 >1000 nor-BNI 1.2 197 U-69,593 2.6 >1000 ICI
204,448 6.6 >1000 [D-Ala.sup.2, F.sub.5Phe.sup.4]Dynorphin 19
>100 (1-13)NH.sub.2 Nalbuphine 36 >1000 Dynorphin (7-17)
>1000 >1000 Delta-selective ligands Naltridole 37 1.9 DADL
>1000 20 DSLET >1000 21 DPDPE >1000 122 Other Naltrexone
0.66 368 (-)-Naloxone 4.9 565 (+)-Naloxone >1000 >1000
Levorphanol 5.3 103 Dextrorphan >1000 >1000 DAMGO >1000
>1000 Haloperidol >1000 >1000 DTG >1000 >1000
Dextromethorphan >1000 >1000 Carbetapentane citrate >1000
>1000 SRIF >1000 >1000 SMS 201-995 >1000 >1000
[0348] That msl-1 is a kappa type receptor is indicated by the high
affinity of the receptor for U-50,488 and U-69,593 which bind
potently and specifically to kappa.sub.1 receptors but not to any
other receptor (Zukin, et al., 1988, Clark, et al., 1989). Also
consistent with msl-1 being a kappa receptor is its high affinity
for dynorphin A and its much lower affinity for .beta.-endorphin
and the enkephalins. Furthermore, msl-1 exhibited very low affinity
for mu, delta or sigma specific ligands. Agonist and antagonist
binding to msl-1 was stereospecific, as expected for an opioid
receptor.
[0349] Both msl-1 and msl-2 are coupled to G proteins since GppNHp
(100 .mu.M) decreased agonist binding to msl-1 by 44% and to msl-2
by 20%. Moreover, 90 mM NaCl decreased agonist binding to msl-1 and
msl-2 by 95% and 60%, respectively, confirming the sodium
dependence of opioid agonist binding noted using membranes prepared
from brain (Pert & Snyder, 1974; Ott, et al., 1988).
[0350] Inhibition of forskolin-stimulated cyclic AMP accumulation
was observed in COS-1 cells transiently expressing msl-1 and msl-2
for 72 h. Briefly, cells cultured in 12 well Costar tissue culture
plates were incubated with 1 ml of DMEM medium containing 10% fetal
bovine serum and 500 nM 3-isobutyl-1-methylxanthine for 30 min. The
medium was removed, the cells were washed and replaced with similar
medium containing 10 .mu.M forskolin alone or with 1 .mu.M opioid
agonists and/or antagonists. After 30 min, the medium was removed
and 0.5 ml of 1 N HCl added to the cells which were then sonicated
for 10 sec. The HCl was removed by evaporation in a SpeedVac and
the c-AMP content of the samples determined using a
radioimmunoassay kit (NEN/Dupont).
[0351] The two opioid receptors mediate opioid inhibition of
adenylyl cyclase activity. The kappa-specific agonists U-50,448 and
ethylketocyclazocine inhibited forkolin-stimulated cAMP
accumulation in COS-1 cells transiently expressing msl-1 by 50% and
this effect was completely reversed by naloxone (See FIG. 2A and
FIG. 2B). The delta-specific agonists DPDPE and DSLET inhibited
forskolin-stimulated cAMP formation in COS-1 cells expressing msl-2
by 70% and this effect could also be blocked by naloxone. These
results show that both msl-1 and msl-2 are able to mediate
subtype-specific agonist induced inhibition of adenylyl cyclase
activity in COS-1 cells.
Example 3
Tissue Distribution of Kappa Opioid Receptor (msl-1) mRNA
[0352] For Northern blot analysis, a mouse multiple tissue Northern
blot (Clontech) was hybridized with a .sup.32P-labeled 376 bp Pst
I-EcoRI fragment of .lambda.msl-1, corresponding to nucleotides
172-548, according to the manufacturer's recommendations. After
hybridization, the blot was washed at room temperature in
2.times.SSC and 0.05% SDS at room temperature and then at
48.degree. C. in 0.1.times.SSC and 0.1% SDS for 30 min. The blot
was exposed to X-ray in the presence of an intensifying screen at
-75.degree. C. for 7 days. For Southern blot analysis, 10 .mu.g of
mouse and human DNA was digested with EcoRI, separated on a 1%
agarose gel, and transferred to a nitrocellulose filter. The blot
was hybridized with a .sup.32P-labeled 1.2 kb Pst I fragment of
.lambda.msl-1, nucleotides 172-1408 using standard conditions. The
blot was washed at 48.degree. C. in 0.1.times.SSC and 0.1% SDS for
30 min before exposure to X-ray film for 6 days.
[0353] In situ hybridization using brain sections prepared from
adult male BALB/c mice was carried out as described previously
(Breder, et al., 1992) using .sup.35S-labeled antisense and sense
riboprobes transcribed from a plasmid containing the 376 bp
Pst-I-EcoRI fragment of .lambda.msl-1 described above. After
hybridization and washing, the sections were dipped in NTB2
photographic emulsion and exposed for 4 weeks. Slides were
developed with D-19 developer and then counterstained for 3 min in
thionin before viewing using darkfield microscopy.
[0354] RNA blotting showed a single transcript of 5 kb encoding the
kappa opioid receptor msl-1 mRNA in adult mouse brain. No
hybridization signal was seen in heart, spleen, liver, lung,
skeletal muscle, kidney or testes. The distribution of kappa opioid
receptor mRNA in the central nervous system of the adult mouse was
studied by in situ hybridization. There are high levels of
expression in the neocortex, piriform cortex, hippocampus,
amygdala, medial habenula, hypothalamus (arcuate and
paraventricular nuclei), locus ceruleus and parabrachial
nucleus.
[0355] The hybridization of .sup.32P-labeled msl-1 cDNA to
EcoRI-digested mouse and human DNAs showed intense labeling of two
mouse DNA fragments of 18 and 3.4 kb whereas this probe hybridized
to multiple fragments of human DNA: strongly to fragments of 8.0,
6.0 and 2.5 kb, and faintly to fragments of 9.5, 5.1, 4.8 and 3.1
kb. The molecular basis for the multiple bands seen in these blots
needs to be established. The presence of an internal Eco RI site in
the msl-1 cDNA sequence can account for hybridization to two mouse
fragments. Moreover, this result suggests that there can only be a
single kappa receptor gene in the mouse genome.
[0356] The hybridization to multiple DNA fragments in the human
blot is more difficult to interpret. The partial sequence of the
human kappa opioid receptor gene indicates that there are at least
two introns in this gene located in codons corresponding to amino
acids Arg.sup.86 and Asp.sup.184 of the mouse sequence and this
result can explain, at least in part, the multiple bands seen in
the Southern blot of human DNA.
[0357] Recent reports (Xie, et al., 1992) disclose the expression
cloning of a putative opioid receptor cDNA from human placenta, a
rich source of kappa receptors. Cells expressing this cloned
receptor bound opioid ligands with only moderate affinity, although
in a stereospecific manner, but did not show the expected kappa
receptor selectivity. The sequence of this clone also showed
greater sequence identity with the human neuromedin K receptor than
to the cloned mouse delta or kappa opioid receptors further
confounding its relationship with these latter receptors which
exhibit affinity and selectivity expected for bona-fide opioid
receptors.
[0358] The recent cloning of a delta opioid receptor and the data
presented herein describing the cloning of a kappa-type opioid
receptor strongly suggest that the different opioid receptor
classes represent distinct gene products. However, the molecular
basis for the different subtypes within some classes, e.g., kappa,,
kappa.sub.2 and kappa.sub.3, remains to be determined. The
pharmacological characterization of msl-1 as expressed in COS-1
cells suggests that it is a kappal receptor. The other kappa
subtypes could be the products of other genes or arise by
differential glycosylation or other post-translational modification
of a common polypeptide or represent G protein coupled and
uncoupled states an identical molecule (Frielle, et al., 1988).
Alternatively, if there are introns in the kappa opioid receptor
gene as the preliminary analysis of the human gene indicates, then
perhaps alternative splicing could generate kappa subtypes with
slightly different pharmacological properties.
[0359] The comparison of the amino acid sequences of the mouse
delta and kappa opioid receptors showed the sequences of the
putative membrane spanning segments and connecting loops were more
highly conserved than the NH-.sub.2 and COOH-termini. The sequence
conservation included the third intracellular loop. This is the
region where other G protein-coupled receptors bind to G proteins
which suggests that perhaps these two receptors interact with
similar G proteins. The availability of these two cloned receptors
with very distinct pharmacological properties will permit the
localization of the ligand binding site(s) by comparing the binding
properties of chimeric polypeptides as has been done for the
adrenergic (Frielle, et al. 1988) and tachykinin receptors (Yokota,
et al. 1992).
[0360] The different classes of opioid receptors are believed to
subserve different physiological functions (Olson, et al., 1989;
Simon 1991; Lutz & Pfister 1992). The distribution of kappa
opioid receptor mRNA in the mouse brain suggests that the kappa
receptor can be involved in the regulation of arousal,
neuroendocrine and autonomic functions, as well as processing of
sensory information. Preliminary RNA blotting studies suggest that
there can be differences in the distribution of kappa opioid
receptor mRNA among species. For example, the in situ hybridization
show high levels of mRNA in the cortex and very low levels in the
striatum, whereas Northern blotting studies using RNA prepared from
different regions of the rat brain suggest that mRNA levels are
higher in the striatum than in the cerebral cortex, a result
consistent with ligand binding studies in rat brain (Mansour, et
al., 1987; Nock, et al., 1988; Unterwald, et al., 1991). The
functional consequences of such differences are unknown but imply
that results of studies using kappa selective agonists in one
species cannot be extrapolated to other species. Indeed, of the
three opioid receptor classes, the kappa type shows the most
divergent distribution among species. Preliminary in situ
hybridization studies show that delta opioid receptor mRNA has a
similar but distinct distribution compared with that of the kappa
receptor in the mouse brain including expression in the cerebral
cortex, hippocampus, amygdala and hypothalamus.
[0361] The availability of the cloned opioid receptors will permit
direct studies of their functions in vivo. They will also greatly
facilitate the development of more selective agonists and
antagonists for clinical applications. This will be particularly
important in the future for kappa receptors since agonists for this
class of opioid receptor induce analgesia but have limited abuse
potential (Unterwald, et al. 1987) and fewer side effects on
respiratory function (Shook, et al. 1990). Similarly,
identification of other members of the kappa opioid receptor family
can lead to the development of selective ligands that induce
analgesia but have few of the sedative or psychomimetic
side-effects of kappa agonists (Pfeiffer, et al. 1986) or instead
selectively antagonize these unfavorable side-effects.
Example 4
Diagnostic/Therapeutic Applications
[0362] Given the isolation and purification of distinct opioid
receptor polypeptides, it is possible to utilize these polypeptides
in methods designed to screen candidate substances such as
candidate agonists and antagonists with potentially preferential
properties for use in diagnostic and therapeutic applications.
[0363] For instance, as noted recently by (Dohlman, et al. 1991)
with the growing number of receptor sub-types, G proteins, and
effectors, characterization of ligand binding and G protein
recognition properties of receptors is an important challenge for
the diagnostic and therapeutic industries. As noted therein,
reconstitution experiments were the first to show that receptors
can, with varying degrees of specificity, couple to multiple (and
in some cases functionally distinct) G proteins (Kanaho, et al.
1984)
[0364] For instance, cloning and over-production of the muscarinic
and .alpha..sub.2-adrenergic receptors led to the demonstration
that a single receptor sub-type, when expressed at high levels in
the cell, will couple to more than one type of G protein. For each
of these receptors, agonist treatment led to both inhibition of
adenylyl cyclase and stimulation of phosphoinositide metabolism.
Finally, individual G protein species have been shown to stimulate
more than one effector, G.sub.s, for example, has been reported to
regulate calcium channels, in addition to adenylyl cyclase. These
authors note that given this complexity and apparent degeneracy of
function, a question of fundamental importance is how, and under
what circumstances, can G proteins organize signals from multiple
receptors and direct them to the appropriate effectors?
[0365] The traditional approach has been to reconstitute the
purified receptor and G protein components in vitro. Unfortunately,
as noted by these authors, purification schemes have been
successful for only a very limited number of receptor sub-types and
there cognate G-proteins. Alternatively, and as here enabled by the
cloning and sequencing of the opioid receptors identified thus far,
heterologous expression systems can be of more general usefulness
in the characterization of cloned receptors and in elucidating
receptor-G protein coupling specificity.
[0366] One such system has been recently developed in yeast cells,
in which genes for a mammalian .beta..sub.2-adrenergic and G.sub.s
.alpha.-subunit were coexpressed (King, et al. 1990). Expression of
the .beta..sub.2-adrenergic to levels several hundred-fold higher
than any human tissue was attained, and ligand binding was shown to
be of the appropriate affinity, specificity, and stereoselectivity.
Moreover, a .beta..sub.2-adrenergic-mediated activation of the
pheromone signal transduction pathway was demonstrated by several
criteria, including altered growth rates, morphological changes,
and induction of a pheromone-responsive promoter (FUSI) fused to
the Escherichia coli lacZ gene (encoding .beta.-galactosidase).
[0367] The ability to control the yeast pheromone response pathway
by expression of the .beta..sub.2-adrenergic and G.sub.s .alpha.
has the potential to greatly facilitate structural and functional
characterization of such receptors. By scoring for growth rates or
.beta.-galactosidase induction, the properties of mutant receptors
can be tested rapidly. In addition, isolated recombinant opioid
receptors as enabled herein should be capable of discriminating
candidate substances with the desirable properties of opioids,
which however lack the undesirable properties of opioids.
Furthermore, it should be possible using systems such as that
described above to identify candidate substances having selective
ability to interact with one or more of the opioid receptor
polypeptides enabled by the present application over others in the
same family of opioid receptors.
[0368] Thus, for instance, it will be possible to utilize a battery
of opioid receptors cloned and expressed in a particular common
cell line and to expose such a battery of receptor polypeptides to
a variety of candidate substances. The results of such a screening
assay should be capable of identifying a candidate substance
capable of, for instance, interacting with a delta, kappa, mu or
sigma opioid receptor.
[0369] Furthermore, it should be possible then to investigate the
structure-activity relationships of opioids when compared to the
isolated recombinant opioid receptors enabled by the present
application. Such studies would include not only binding studies to
identify candidate substances such as agonists and antagonists
which will bind each individual opioid receptor, but will also
include studies to identify those candidate substances which
stimulate an activity in the opioid receptor apart from the binding
of the same to the receptor.
[0370] Moreover, as noted by Dohlman, et al. 1991, as additional
genes for the putative G-protein, coupled receptors, such as those
enabled by the present application, are isolated, a series of
ligands can be conveniently screened to identify those with
activity toward the unidentified gene product. As noted by these
authors as well, expression of a single receptor in the absence of
other related sub-types is often impossible to achieve in native
mammalian cells. Thus, expression in a microorganism, or in an
isolated eukaryotic cell that has no such endogenous receptors can
be useful for screening and evaluating sub-type-selective drugs
(Marullo, et al. 1988; Payette, et al. 1990; and King, et al.
1990).
Example 5
Human Opioid Receptors
[0371] Human opioid receptor polypeptides are isolated and
identified from human gene sequences that encode such receptor
polypeptides. A partial genomic sequence containing both introns
and exons of a human kappa opioid receptor is shown in FIG. 3. FIG.
4A and FIG. 4B compare the partial amino acid sequences of human
kappa opioid receptor with the mouse kappa opioid receptor. The
mouse sequence begins with amino acid residue 1 and the human
sequence begins with amino acid residue 87.
[0372] A cDNA library was constructed from the hippocampus of a
human brain and screened with a polynucleotide probe from the mouse
kappa opioid receptor. Briefly, cDNA molecules were ligated with
Eco R1 linkers. The vector .lambda.gt10 was digested with Eco R1 to
create linear vector. The cDNA molecules with the Eco R1 linkers
were ligated into the linear vector. The host cell for library
construction was E. Coli strain LE 392.
[0373] The amino acid sequences of the human and mouse kappa opioid
receptors are highly homologous. As can be seen in FIG. 3, of the
293 amino acids, 292 are identical or similar. 281 residues are
identical and 6 residues involve conservative substitutions.
Residues 232, 284, 285, 328, and 348 are substitutions which
involve leucine, isoleucine or valine. As is appreciated by skilled
artisans, substitutions involving leucine, isoleucine and valine
are conservative substitutions. Residue 218 is a change from
glutamic acid to aspartic acid, and residue 274 is a change from
lysine to arginine. As is well known in the art, the hydropathic
index of glutamic acid and aspartic acid are identical at -3.5.
Furthermore, lysine and arginine are the two least hydropathic
amino acids with an index of -3.9 and -4.5, respectively. Thus the
amino acid changes at positions 218 and 274 are conservative
substitutions. In addition there are 4 amino acids in the human
kappa opioid receptor at positions, 255, 267, 351, and 355 which
have not yet been identified because the complete nucleotide
sequences have not yet been ascertained. However, it is noted that
there is only one nucleotide missing from the sequences that encode
for residues 351 and 355. It is likely that when these two
nucleotides are identified, amino acid residues 351 and 355 will be
homologous. Residues 255 and 267 are not presently identified
because two nucleotides that encode for the residues are missing
from the nucleotide sequence. The only significant difference
between the human and mouse kappa opioid receptor is found in
residue 358 in which a serine is replace by an asparagine.
[0374] The human kappa opioid receptor shown in FIG. 4A and FIG. 4B
is a partial sequence in which the amino terminus of the human
kappa opioid receptor is not presented. The gene sequence encoding
the amino terminus of the human kappa opioid receptor is to be
identified by screening a genomic or a cDNA library with a
polynucleotide of the human or mouse kappa opioid receptor.
Preferably a polynucleotide of the human kappa opioid receptor of
FIG. 3 is the probe. Human opioid receptor subtypes are identified
by screening with a human opioid receptor probe.
[0375] Further, human cDNA that encodes an opioid receptor
polypeptide is transfected into a suitable host cells using
techniques set forth hereinbefore and the opioid receptor
polypeptide is expressed. The expressed human polypeptide is
screened using agonists and antagonists to identify the opioid
receptor subtype.
Example 6
Stable Transfection of Mammalian Cells
[0376] A. Isolation of Stable Transformants
[0377] PC-12 cells were grown in RPMI medium with 10% horse serum
and 5% bovine serum in 5% CO.sub.2 at 37.degree. C. to 50%
confluency. The cells were transfected by the lipofection method
(Muller et al., 1990) with 7 .mu.g of the 1.2-kilobasc Pst I
fragment of the mouse K receptor cDNA cloned into the CMV
promoter-based expression vector pCMV-6c as previously described
(Yasuda et al., 1993). The cells were selected and maintained in a
similar medium containing 200 .mu.g/ml G418. The generation of the
CHO-DC44 cell line stably expressing the mouse 6 receptor was
accomplished as previously described (Rens-Domiano et al., 1992).
Briefly, a 1.3-kilobase EcoRI-Sac I fragment of the mouse 6 opioid
receptor cDNA was inserted into the expression vector pCMV-6c and
contransfected with pSV2noo into CHO cells and stable transfectants
were selected and grown as previously described (Yasuda et al.,
1993 and Rens-Domiano et al., 1992). The rat .mu. receptor was
expressed transiently in COS-7 cells, as previously described (Chen
et al., 1993 and Kong et al., 1993).
[0378] B. Pharmacological Properties
[0379] Receptor binding assays were performed using membranes from
either PC12 cells stably expressing the cloned mouse K receptor,
CHO-DG44 cells stably expressing the mouse 8 receptor, or COS-7
cells transiently expressing the rat .mu. receptor 48-72 hours
after transfection as previously described (4,10). For radioligand
binding assays, cells were harvested in 50 mM Tris-HCl (pH 7.8)
containing 1 mM ethylene glycol bis(.beta.-aminoethyl
ether)-N,N'-tetraacctic acid, 5 mM MgCl.sub.2, 10 .mu.g/ml
leupeptin, 10 .mu.g/ml pepstatin 200 .mu.g/ml bacitracin and 0.5
.mu.g/ml aprotinin (buffer 1) and centrifuged at 24,000 K g for 7
min at 4.degree. C. The pellet was homogenized in buffer 1 using a
Polytron (Brinkmann, setting 2.5 30 sec). The Homogenate was then
centrifuged at 48,000 .kappa. g for 20 min at 4.degree. C. The
pellet was homogenized in buffer 1 and this membrane preparation
was used for the radioligand binding studies. For inhibition
studies, cell membranes (10-20 .mu.g protein) were incubated with
[.sup.3H]U-69,593 (2 nM, specific activity 47.4 Ci/mmol),
[.sup.311]naltrindote (1 nM, specific activity 31.2 Ci/mrnmol), or
[.sup.3 HDAMGO (I nM, specific activity 55 Ci/mmol) (NEN/Dupont,
Wilmington, Del.) in a final volume of 200 .mu.L for 40 min at
25.degree. C. in the presence or absence of competing agents. For
saturation experiments, cell membranes were incubated with
increasing concentrations of the radioligands. Nonspecific binding
was defined as the radioactivity remaining bound in the presence of
10 .mu.M naloxone for all radioligands. The binding reaction was
terminated by the addition of ice-cold 50 mM Tris-HCl buffer (pH
7.8) and rapid filtration over Whatman GF/B glass fiber filters
which were pretreated with 0.5% polyethyleneimine/0.1% BSA for at
least 1 hour. The filters were then washed with 12 mL of ice-cold
Tris-HCl buffer and the bound radioactivity counted in a
scintillation counter. Data from radioligand binding studies were
used to generate inhibition curves. IC.sub.50 values were obtained
from curve-fitting performed by the mathematical modeling program
FITCOMP (Perry and McGonigle, 1988) and saturation data was
analyzed using FITSAT (McGonigle et al., 1988) available on the
National Institutes of Health-sponsored PROPHET system. The
inhibitory binding constant (K.sub.1) was calculated from the
IC.sub.50 values using the Chong-Prusoff equation (Cheng and
Prusoff, 1973).
[0380] Cloned cDNAs encoding .kappa., .delta., and .mu. receptors
were expressed stably in PC12 (.kappa.) or CHO-D644 cells (.delta.)
or transiently in COS-7 cells (.mu.). The K, .delta., and .mu.
opioid receptors were labelled with the selective opioid
radioligands [.sup.3H]U-69,593, [.sup.3H]naltrindole, or
[.sup.3H]DAMGO, respectively. The binding of these radioligands is
saturable and of high affinity (FIG. 5A and FIG. 5B). The
saturation experiments demonstrated that [.sup.3H]U-69,593 binds to
the cloned .kappa. receptor with a K.sub.D of 2.8 nM and a
B.sub.max of 3346 fmol/mg protein. Similarly, [.sup.3H]naltrindole
binding to the cloned 6 receptor is of high affinity and binds with
a K.sub.D of 0.18 nM and a B.sub.max of 633 fmol/mg protein. The
K.sub.D for [.sup.3H]DAMGO binding to the cloned .mu. receptor is
0.57 nM and the B.sub.max is 444 fmol/mg protein. All data were
best fit by a single-site analysis. No specific radioligand binding
was detectable in appropriate nontransfected control cells.
[0381] A battery of opioid ligands were used to identify the
pharmacological specificities of the cloned .kappa., .delta., and
.mu. opioid receptors (Table 3). These include both peptide and
nonpeptide compounds previously characterized as selective and
nonselective agents for opioid receptors (Lutz and Pfister, 1992;
Goldstein and Naidu, 1989; Schiller, 1993; Portoghese, 1993; and
Corbett et al., 1993). The endogenous opioid peptide dynorphin A is
selective for the .kappa. receptor, whereas .beta.-endorphin, Leu-
and Met-enkephalin are selective for the .mu. and .delta. receptors
as they either did not bind to the .kappa. receptor, as for Lcu-
and Mcl-enkephalin, or bound with low potency, as for
.beta.-endorphin. Des-Tyr.sup.1-.beta.-endorphin did not bind to
any of the opioid receptors. The binding to each receptor is
stereoselective, being inhibited by (-)nuloxone and levorphanol but
not by their respective isomers (+)naloxone or dextrorphan. Other
relatively nonselective compounds tested were (.+-.)bremazocine,
ethylketocyclzocine, etorphine, pentazocine, and diprenorphine.
Each of these compounds is relatively non-selective for .mu. vs.
.kappa. and displayed higher affinities for these receptors than
for the .delta. receptor. Analogous results were found with
.beta.-FNA and .beta.-CNA, although the values given are not true
Ki's due to the covalent nature of these ligands. Furthermore,
naltrexone, nalbuphine, and nalorphine were also relatively
selective for .kappa., .mu., only binding to the .delta. receptor
at much higher concentrations.
10TABLE 3 BINDING POTENCIES (K.sub.i-nM) OF LIGANDS FOR THE CLONED
.kappa., .delta. AND .mu. OPIOID RECEPTORS .kappa. RECEPTOR .delta.
RECEPTOR .mu. RECEPTOR [.sup.3H]U-69,593 [.sup.3H]naltrindole
[.sup.3H]DAMGO NON-SELECTIVE COMPOUNDS dynorphin A 0.5 >1000 32
Leu-enkephalin >1000 >1000 3.4 Met-enkephalin >1000 4.0
3.4 .beta.-endorphin 52 1.0 1.0 des-Tyr.sup.1-.beta.-endorphin
>1000 >1000 >1000 (-)naloxone 2.3 17 0.93 (+)naloxone
>1000 >1000 >1000 levorphano 6.5 5.0 0.086 dextrorphan
>1000 >1000 >1000 (.+-.)bremazocine 0.089 2.3 0.75
ethylketocyclazocine 0.40 101 3.1 etorphine 0.13 1.4 0.23
pentazocine 7.2 31 5.7 diprenorphine 0.017 0.23 0.072 .beta.-CNA
0.083 115 0.90 NON-SELECTIVE COMPOUNDS- .beta.-FNA 2.8 48 0.33
naltrexone 3.9 149 1.0 nalbuphine 39 >1000 11 nalorphine 1.1 148
0.97 MU-SELECTIVE COMPOUNDS CTOP >1000 >1000 0.18 dermorphin
>1000 >1000 0.33 methadone >1000 >1000 0.72 DAMGO
>1000 >1000 2.0 PLO17 >1000 >1000 30 morphiceptin
>1000 >1000 56 codeine >1000 >1000 79 fentanyl 255
>1000 0.39 sufentanil 75 50 0.15 lofentanil 5.9 5.5 0.68
naloxonazine 11 8.6 0.054 morphine 538 >1000 14 KAPPA-SELECTIVE
COMPOUNDS norBNI 0.027 65 2.2 spiradoline 0.036 >1000 21
U-50,488 0.12 >1000 >1000 U-69,593 0.59 >1000 >1000 ICI
204,488 0.71 >1000 >1000 DELTA-SELECTIVE COMPOUNDS DPDPE
>1000 14 >1000 D-Ala.sup.2-deltorphin II >1000 3.3
>1000 DSLET >1000 4.8 39 BW 3734 17 0.013 26 DADL >1000
0.74 16 SIOM >1000 1.7 33 naltrindole 66 0.02 64 NTB 13 0.013 12
BNTX 55 0.66 18
[0382] Compounds which have been previously characterized as
.mu.-selective including both peptide and non-peptide agonists and
antagonists were also utilized. As expected, most of these
compounds bound to the cloned .mu. receptor with K.sub.1 values in
the low nM range (Table 3). Exceptions include morphine, codeine,
morphiceptin and PL017, which bind with affinities in the 10-100 nM
range. The majority of the ligands tested are selective for the
.mu. receptor and did not bind to the .kappa. or .delta. receptors.
Of the ligands which showed crossreactivity, fentanyl binds to the
.mu. receptor with high selectivity but its derivatives lofentanil
and sufentanil were less selective, interacting with both .delta.
and .kappa. receptors, albeit with lower affinity than with the
.mu. receptor. Similar crossreactivity was found with the compound
naloxonazine, which has been suggested to discriminate between
subtypes of .mu. receptors, having high affinity for the .mu.l
receptor (Pasternack and Wood, 1986). The high affinity of the
cloned .mu. receptor for naloxonazine, a compound possessing
subtype selectivity (Pasternack and Wood, 1986), suggests that the
cloned .mu. receptor corresponds to the endogenously expressed
.mu..sub.1 receptor subtype.
[0383] Results with the K- selective ligands U-50,488, U-69,593,
ICI 204488 and spiradoline (Table 3) confirmed previous results
showing their K selectively (Lutz and Pfister, 1992; Goldstein and
Naidu, 1989; Schiller, 1993; Portoghese, 1993; and Corbett et al.,
1993). The .kappa. antagonist norBNI was also selective for the
.kappa. receptor, but less so than the agonists tested. These
results indicate that the cloned .kappa. receptor corresponds
pharmacologically to the .kappa..sub.1 receptor previously
characterized in heterogenous tissues (Clark et al., 1989).
[0384] Various peptide and non-peptide agonists and antagonists at
the .delta. receptor (Lutz and Pfister, 1992; Goldstein and Naidu,
1989; Schiller, 1993; Portoghese, 1993; and Corbett et al., 1993)
were tested and results confirmed the .delta.-selectivity of these
compounds (Table 3). Thus, the peptide agonists DPDPE and
D-Ala.sup.2-deltorphin II are highly selective for the .delta.
receptor, whereas DSLET and DADL are less selective. The recently
developed nonpeptide agonists BW 3734 (Lee et al., 1992) and SIOM
(Portoghese et al., 1993) were also examined. BW3734 is highly
.delta.-selective. Compounds which have been proposed to
distinguish between .delta..sub.1 and .delta..sub.2 receptor
subtypes were tested. These agents bound to the cloned .delta.
receptor with differing affinities. The agonists DSLET and
D-Ala.sup.2 deltorphin II, which have been proposed as
.delta..sub.2 ligands, were found to be more potent than DPDPE,
which is .delta..sub.1-selective. Furthermore, the antagonists
naltrindole and NTB were more potent than BNTX at binding to the
cloned .delta. receptor. The pharmacological profile of the cloned
.delta. opioid receptor differs from .delta. opioid receptors
previously characterized in diverse tissues. The existence of
subtypes of .delta. receptors has been suggested based on
behavioral data employing compounds such as DPDPE and BNTX, which
interact with .delta..sub.1 receptors, and DSLET,
D-Ala.sup.2-deltorphin II, and NTB which interact with
.delta..sub.2 receptors (Sofuglu et al., 1991; Portoghese et al.,
1992; and Sofuglu et al., 1991). The demonstration of the existence
of .delta. receptor subtypes based on results of radioligand
binding studies has been more ambiguous, perhaps due to the lack of
sufficiently selective radioligands. These results suggest that the
pharmacological profile of the cloned .delta. opioid receptor
matches that of the .delta..sub.2 receptor subtype.
[0385] Correlational analyses comparing the K.sub.1 values obtained
in this laboratory with those reported in the literature were
performed (Goldstein and Naidu, 1989; Schiller, 1993; Portoghese,
1993; and Corbett et al., 1993). To determine whether the
pharmacological profiles of the cloned opioid receptors were
similar to those previously reported for receptors expressed in
vivo in biological tissues containing multiple opioid receptor
subtypes. Compounds for which literature values were not available
or which did not bind to a given receptor are not included in the
analysis. The correlation coefficients obtained for both the .mu.
(FIG. 6A) and .kappa. (FIG. 6B) receptors are very high with r
values of 0.954 (n=25) and 0.879 (n=16), respectively. In contrast,
the correlation for the .delta. receptor is low (not shown), with
an r value of 0.185 (n=17), indicating that the cloned .delta.
receptor differs pharmacologically from those characterized in
diverse tissues.
[0386] Interestingly, the endogenous opioid peptides
.beta.-endorphin, Leu- and Met-enkephalin were selective for the
.mu. and .delta. receptors vs. the .kappa. receptor. In fact, the
.kappa..sub.1 values for these peptides were comparable at the .mu.
and .delta. receptors. Because the potencies of the enkephalins to
bind to the .mu. and .delta. receptors are within the physiological
concentrations, these peptides may be endogenous ligands for both
these receptor subtypes.
[0387] Our results indicate that opioid agents with abuse
liabilities possess high affinities for the .mu. receptor. Thus,
the addictive compounds morphine, fentanyl, and methadone have high
affinities for the cloned .mu. receptor, but little or no affinity
for the .delta. or .kappa. receptors. Furthermore, etorphine,
sufentanil, levorphanol, nalbuphine, and codeine, which have been
shown to possess abuse liability (Jaffe and Martin, 1990) have in
common relatively high affinity for the .mu. receptor. Development
of analgesic agents which are .kappa.- or .delta.-selective may
obviate this limitation of .mu.-selective analgesics.
[0388] The ability to individually study the pharmacological
properties of the cloned opioid receptor subtypes will allow for
identification of structural features of ligands which permit
selective interactions. Identification of the pharmacological
interactions of drugs which the individual opioid receptors could
lead to the identification of therapeutic agents less burdened with
the potential to produce undesirable side effects.
Example 7
Chimeric Opioid Receptors
[0389] Opioids such as morphine are used for the management of
chronic pain (Jaffe and Martin, 1990). However, the use of opioids
has undesirable side effects including respiratory depression,
decreased gastrointestinal motility, sedation, nausea, and mood
changes. Other major limitations include abuse potential,
tolerance, and dependence. Morphine and the endogenous opioid
peptides, the enkephalins, endorphins, and dynorphins, exert their
physiological effects through membrane-bound receptors expressed in
the central and peripheral nervous systems and target tissues.
[0390] The three major-types of opioid receptors, .delta., .kappa.
and .mu., that have been cloned and functionally characterized
(Evans et al., 1993; Kieffer et al., 1992; Yasuda et al., 1993; and
Chen et al., 1993) belong to the DRY-containing subfamily of seven
transmembrane-spanning receptors. There is 60% amino acid identity
among the sequences of the .delta., .kappa. and .mu. opioid
receptors. The sequences of the putative membrane-spanning segments
(TM I-VII) and the three intracellular loops connecting these
segments are highly conserved whereas the sequences of the
extracellular NH-.sub.2-termini segments, second and third
extracellular loops and the intracellular COOH-termini are
divergent. These divergent extracellular regions are likely to be
responsible for the distinct ligand binding profiles of the
.delta., .kappa. and .mu. receptors. The present invention
describes the preparation and characterization of chimeric opioid
receptors. The chimeric receptors include
.kappa..sub.1-78/.delta..sub.70-372, .delta..sub.1-69/.kappa..sub-
.79-380, .kappa..sub.1-74/.delta..sub.65-372 and
.delta..sub.1-64/.kappa..- sub.75-380. In the notation for chimeric
receptors, the amino terminus is designated first and the carboxy
terminus is designated second. Thus for
.kappa..sub.1-78/.delta..sub.70-372, the amino terminus of the
chimera is composed of amino acid residues 1-78 of the kappa
receptor and the carboxy terminus is composed of amino residues
70-372 of the delta receptor. FIG. 7C and FIG. 7D shows a pictorial
representation of chimeras .kappa..sub.1-78/.delta..sub.70-372 and
.delta..sub.1-69/.kappa.- .sub.79-380, respectively. The agonist
and antagonist binding properties of these chimeras as well as the
chimera's ability to mediate inhibition of adenylyl cyclase
activity are also described.
[0391] Generation of Chimeras:
[0392] To exchange NH.sub.2-termini between the mouse .delta. and
.kappa. opioid receptors, a common restriction site. Spe I, was
generated at an equivalent position in the cDNAs in the region
encoding the first transmembrane domain without otherwise altering
the amino acid sequence of either receptor. Site-directed
mutagenesis was carried out using the Altered Sites.TM. In vitro
Mutagenesis System (Promega, Madison, Wis.) and 27-mer
oligonucleotides containing the Spe I site, .delta. receptor
oligonucleotide-CTGGGCAACGTACTAGTCATGTTTGGC (SEQ ID NO:42) and
.kappa. receptor oligonucleotide-GTGGGCAATTCACTAGTCATGTTTGTC (SEQ
ID NO:43). After digestion with Spe I and the appropriate 5' and/or
3' enzymes, the cDNA fragments encoding the NH.sub.2- and
COOH-termini of .delta. and .kappa. were isolated from a 1.2 % low
melting point agarose gel. Fragments encoding the NH.sub.2-terminus
of .delta. receptor and the COOH-terminus of .kappa. receptor and
vice versa were ligated together and cloned into the mammalian
expression vector pCMV-6c. Truncated .delta. and .kappa. receptors
were generated by ligating the fragments encoding the COOH-termini
directly into the expression vector; translation of the receptor
sequences in these constructs was predicted to begin at a conserved
ATG just distal to the Spe I site.
[0393] As shown previously (Evans et al., 1993; Kieffer et al.,
1992; Yasuda et al., 1993; and Chen et al., 1993), the wild-type K
receptor can be labeled with the K-selective agonist
[.sup.3H]U-69,593 and the antagonist [.sup.3H]naloxone, and the
wild-type .delta. receptor can be labeled with the
.delta.-selective agonist, [.sup.3H][D-Pen.sup.2,
D-Pen.sup.5]-enkephalin (DPDPE) and with the antagonist,
[.sup.3H]naltrindole. The .kappa.-selective and .delta.-selective
ligands have minimal cross reactivity. The
.kappa..sub.1-78/.delta..sub.70-372 and
.delta..sub.1-69/.kappa..sub.79-380 chimeric opioid receptors show
unique agonist and antagonist binding properties. The
.kappa..sub.1-78/.delta..sub.70-372 receptor binds the antagonist,
[.sup.3H]naloxone (which poorly labels the wild-type .delta.
receptor), and the .delta.-selective agonist and antagonist,
[.sup.3H]DPDPE and [.sup.3H]naltrindole, respectively (FIG. 8). In
contrast, the .delta..sub.1-69/.kappa..sub.79-380 receptor binds
only the K-selective agonist [.sup.3H]U-69,593, although at lower
levels when compared to the wild-type .kappa.-receptor which binds
at 46 fmol/mg protein. These results show that agonist and
antagonist binding domains of the .kappa. receptor are separable
and located in different regions of the protein. The antagonist
binding domain of .kappa. is localized to the region of amino acids
1-78 which includes the NH-.sub.2-terminal extracellular domain. In
contrast, the antagonist binding domain of the .delta. receptor is
not located in the corresponding region of this receptor.
[0394] Radioligand Binding Assay:
[0395] For receptor binding studies, COS-7 cells expressing the
receptors are harvested 72 hours after transfection in 50 nM
Tris-HCl (pH 7.8) containing 1 mM EGTA, 5mM MgCl.sub.2, 10 .mu.g/ml
leupeptin, 10 .mu.g/ml pepstatin, 200 &82 g/ml bacitracin, and
0.5 .mu.g/ml aprotinin (Buffer 1) and centrifuged at 24,000.times.g
for 7 min at 4.degree. C. and the pellet resuspended in Buffer 1
using a polytron. The homogenate is centrifuged at 48,000.times.g
for 20 min at 4.degree. C. and the pellet resuspended in Buffer 1
and used in the radioligand binding assay. Cell membranes (10-20
.mu.g of protein) were incubated with [.sup.3H]U69,593 (2 nM,
specific activity 47.4 Ci/mmol), [.sup.3H]naloxone (6 nM, specific
activity 72.1 Ci/mmol), [.sup.3H]DPDPE (2 nM, specific activity
34.3 Ci/mmol), or [.sup.3H]naltrindole (1 nM, specific activity
31.2 Ci/mmol) in a final volume of 200 .mu.l for 40 min at
25.degree. C. in the presence or absence of competing agents. All
radioligands were obtained from NEN/Dupont (Boston, Mass.).
Nonspecific binding is defined as radioactivity remaining bound in
the presence of 1 .mu.M naltrindole or naloxone for .delta.- and
.kappa.-selective ligands, respectively. The binding reaction is
terminated by the addition of ice-cold 50 mM Tris-HCL (pH 7.8) and
rapid filtration over Whatman GF/B glass fiber filters that were
pretreated with 0.5% polyethleneimine and 0.1% bovine serum
albumin. The filters were washed with 12 ml of ice-cold buffer and
soaked overnight in scintillation fluid. The bound radioactivity
was determined using a scintillation counter. IC.sub.50 values were
obtained using the curve-fitting program FITCOMP on the NIH-based
Prophet system (H. Perry and P. McGonigle in PHOPHET Public
Procedure Notebook. (Bolt, Berabek, and Newman, Inc., Cambrige,
Mass., 1988), pp. 187-197.
[0396] The binding properties of the
.kappa..sub.1-78/.delta..sub.70-372 chimera were further examined
by inhibition studies. As shown in FIG. 9 [.sup.3H]naloxone binding
to the .kappa..sub.1-78/.delta..sub.70-372 chimera was not
inhibited by the .kappa.-selective agonist U-50,488. Dynorphin, the
endogenous ligand for the .kappa. receptor, inhibited
[.sup.3H]naloxone binding to the
.kappa..sub.1-78/.delta..sub.70-372 chimera with an IC.sub.50 value
of 40 nM, which is approximately 500-fold less potent than the
binding observed for wild-type .kappa. receptor (2).
[.sup.3H]naloxone binding was dose-dependent and was potently
inhibited by the antagonist naloxone with an IC.sub.50 value of 14
nM (FIG. *) and was also inhibited by the K-selective antagonist
nor-binaltorphimine (nor-BNI) with an IC.sub.50 value of 0.14 nM.
[.sup.3H]Naltrindole binding to this chimera was inhibited in a
dose-dependent manner by the potent 6-selective agonists
[D-Ser.sup.2]-Leu-enkephalin-Thr (DSLET) and DPDPE and the
.delta.-selective antagonist naltrindole. This results show that
the agonist and antagonist binding sites in the .delta. receptor
are contained within residues 70-372.
[0397] Cyclic AMP Accumulation Assays:
[0398] cAMP accumulation in COS-7 cells expressing the wild-type or
mutant receptors is measured as previously described (Yasuda et
al., 1992). Briefly, COS-7 cells were subcultured in 12-well
culture plates. The cells were transfected 72 hours prior to the
cAMP experiments. Culture medium was removed from the wells and
replaced with 500 .mu.l of fresh medium containing 0.5 mM
isobutylmethylxanthine (IBMX). Cells were incubated for 20 min at
37.degree. C. Medium was removed and replaced with fresh medium
containing 0.5 mM IBMX, with or without 10 .mu.M forskolin and
various opioid agonists and antagonists. The cells were incubated
for 30 min at 37.degree. C. Medium was removed and cells sonicated
in the wells in 500 .mu.l of 1 N HCl. HCl was removed under vacuum
and the cAMP quantified using a radioimmunoassay kit from
DuPont-New England Nuclear.
[0399] As shown in FIG. 10A and FIG. 10B,
.kappa..sub.1-78/.delta..sub.70-- 372 and
.delta..sub.1-69/.kappa..sub.79-380 chimeras were functionally
active and can mediate selective agonist inhibition of
forskolin-stimulated cyclic AMP (cAMP) accumulation (Yasuda et al.,
1992). Inhibition of cAMP accumulation by U-50,488 via the
.delta..sub.1-69/.kappa..sub.79-380 chimera was not blocked by
naloxone. The potency of U-50,488 to inhibit cAMP formation was
approximately 1 nM which is similar to its potency at interacting
with wild-type .kappa. receptor. Furthermore, dynorphin was able to
inhibit cAMP formation via the .delta..sub.1-69/.kappa..sub.79-380
chimera and its effects were not blocked by naloxone. Thus it is
likely that the naloxone binding site resides in the
NH.sub.2-terminus of the .kappa. receptor. Expression of a
truncated version of the .kappa. receptor, .kappa..sub.79-380, in
which the extracellular NH.sub.2-terminal domain is missing also
shows that the naloxone binding site resides in the amino terminus.
Cells transfected with a construct encoding this truncated .kappa.
receptor showed little specific [.sup.3H]-U69,593 binding but were
able to mediate U-50,488 inhibition of forskolin-stimulated cAMP
formation (FIG. 10A). This effect was not blocked by the
.kappa.-selective antagonist naloxone, consistent with residues
1-78 not being involved in agonist recognition but necessary for
antagonism by naloxone. Furthermore, the 6-selective agonist DSLET
had no effect on cAMP formation in cells expressing the truncated
.kappa. receptor, as with the .delta..sub.1-69/.kappa..sub.79-3- 80
chimera.
[0400] Expression in COS-7 cells of the chimeric
.kappa..sub.1-78/.delta..- sub.70-372 or the truncated .delta.
receptor, .delta..sub.70-372, conferred functional properties
indistinguishable from the wild-type .delta. receptor (FIG. 9B).
FIG. 10 shows that the .delta.-selective agonist DSLET was
inhibited forskolin-stimulated cAMP formation which was blocked by
naltrindole. This result demonstrates that the agonist and
antagonist binding domains of the .delta. receptor is localized to
residues 70-372. The .kappa.-selective agonist U-50,488 did not
have any effect on the functional properties of the
.kappa..sub.1-78/.delta..sub.7- 0-372 receptor or the truncated
.delta. receptor, .delta..sub.70-372.
[0401] The present invention demonstrates an unexpected difference
between the .kappa. and .delta. receptors with respect to the
locations of agonist and antagonist binding domains and the
important role played by the NH.sub.2-terminal 78 residues of the
.kappa. receptor in antagonist binding. The demonstration that
agonists and antagonists bind to different regions of the .kappa.
receptor should facilitate development of more selective .kappa.
ligands. This is an area of considerable interest Because .kappa.
receptor-selective agents have limited abuse potential and
respiratory depressant effects development of .kappa. selective
ligands is of considerable interest. The structural analysis of the
ligand binding domains of the opioid receptors will provide the
basis for the rational design of a new generation of
therapeutically useful analgesics with limited side effects.
Example 8
Mutant Delta Opioid Receptor Polypeptide
[0402] The recent cloning of the opioid receptors (Evans, 1992;
Kieffer et al, 1992; Yasuda et al., 1993; Chen et al., 1993) has
allowed for analysis of the amino acid residues and domains of the
receptors involved in ligand binding. Previous studies with the
beta-adrenergic receptor have suggested that aspartates in the
second and third transmembrane-spanning region are critical for
ligand binding (Strader et al., 1987). Mutation of aspartate 113 in
the third transmembrane-spanning region in the
beta.sub.2-adrenergic receptor to an asparagine greatly reduces the
potency of antagonist binding to the receptor and increases the
Kact of agonists to stimulate adenylyl cyclase activity (Strader et
al., 1987). The carboxyl group of aspartate 113 likely serves as a
counterion to the cationic group of beta-adrenergic agonists and
antagonists (Strader et al., 1987). Mutation of aspartate 79 in the
second transmembrane spanning region to an asparagine diminishes
the affinity of the receptor for agonists but not antagonists. This
aspartate is thus likely selectively involved in agonist binding to
the beta-adrenergic receptor and that agonist and antagonist
binding domains of this receptor are distinct but overlapping.
[0403] In recent studies, it has been shown that mutation of
aspartate 95 in the second transmembrane-spanning domain of the
mouse delta opioid receptor to an asparagine reduces the affinity
of the receptor for delta-selective agonists but not antagonists
(Kong et al., 1993). The mutant receptor expressed similar affinity
as the wild-type delta receptor for non-selective opioid agonists,
indicating that selective delta opioid agonists bind differently to
the delta receptor than do either non-selective agonists or
antagonists.
[0404] In addition to having a positively-charged nitrogen, all
opioids possesses an aromatic ring structure that is essential for
high affinity ligand binding (Gilman et al., 1990; Simon, 1991).
Recent studies with the neurokinin-1 receptor have shown that the
aromatic ring structure of a histidine at residue 197 interacts
with antagonists and the histidine is a critical residue in the
receptor needed for tachykinin antagonist binding (Fong et al.,
1993; Gether et al., 1993). A histidine at residue 278 in the sixth
transmembrane-spanning domain of the delta receptor has been
mutated to an asparagine and tested for its interaction with
opioids.
[0405] The results show that aspartate 128 is necessary for opioid
agonists to bind to the delta receptor with high affinity but is
not involved in antagonist binding and the histidine 278 is not
essential for ligand binding. The combined results with the D128N
and H278N mutants demonstrate that agonists and antagonists bind
differently to the cloned delta receptor, possibly by interacting
with distinct ligand binding domains.
[0406] [D-Pen.sup.2, D-Pen.sup.5]enkephalin (DPDPE), [D-Ser.sup.2,
Leu.sup.5] enkephalin-Thr.sup.6 (DSLET), D-Ala.sup.2 deltorphin II
and beta-endorphin were obtained from Peninsula Labs. (Belmont,
Calif.) and the agonists levorphanol and bremazocine and the
antagonists diprenorphine and naltrindole were obtained from
Research Biochemicals Inc, (Natick, Mass.).,
7-spiroindino-oxymorphone (SIOM), 7-Benyllidenenaltrexone (BNTX)
and the benzofuran analog of naltrindole (NTB) were provided by Dr.
P. Portoghese (Univ. Minnesota). .sup.3H-naltrindole (specific
activity 28.8 Ci/mmol) was obtained from Dupont/NEN (Boston,
Mass.).
[0407] Mutagenesis of the Cloned Mouse Delta Opioid Receptor
[0408] The mouse delta opioid receptor cDNA was mutated using the
Altered Site.TM. in vitro Mutagenesis system (Promega Corp. Madison
Wis.). To mutate aspartic acid 128 and histidine 278 to an
asparagine, the delta receptor cDNA was subcloned into the phagemid
pALTER.TM. and with the helper phage R408, single-stranded template
was produced. For the mutation of the aspartic acid 128, the 21-mer
oligonucleotide (GCTCTCCATTAACTACTACAA) (SEQ ID NO:44) containing
the desired mutation (GAC to AAC) was annealed to the
single-stranded template and elongated with T4 DNA polymerase. For
the mutation of the histidine 278, the 21-mer oligonucleotide
(GGCGGCCCATCAACATCTTCGT) (SEQ ID NO:45) containing the desired
mutant (CAC to AAC) was annealed to the single-stranded template
and elongated. For each, the heteroduplex DNA was used to transform
the repair-minus E. coli BMH 71-18 mut S. Transformants were
selected by growth on LB plates containing 125 ug/ml ampicillin.
The mutation was confirmed by DNA sequencing. The cDNA was excised
from pALTER with EcoR1 and Sal I and subcloned into the
corresponding sites in the mammalian expression vector pCMV6c
(Yasuda et al., 1929).
[0409] Expression of the Mouse Delta Opioid Receptor cDNA in COS-7
Cells:
[0410] The mutated and wild-type cDNA were transfected into COS-7
cells by a calcium-phosphate-mediated procedure. For the receptor
binding studies, COS-7 cells expressing the delta receptor were
harvested 48 hrs. after transfection in 50 mM Tris-HCl (pH 7.8)
containing 1 mM EGTA, 5 mM MgCl.sub.2. 10 .mu.g/ml leupeptin, 10
.mu.g/ml pepstatin, 200 .mu.g/ml bacitracin and 0.5 .mu.g/ml
aprotinin (Buffer 1) and centrifuged at 24,000.times.g for 7 min.
at 4.degree. C. The pellet was homogenized in Buffer 1 using a
polytron. The homogenate was centrifuged at 48,000.times.g for 20
min at 4.degree. C. and the pellet resuspended in Buffer 1 and used
in the radioligand binding assay. Cell membranes (20-30 ug protein)
were incubated with the delta-selective antagonist
.sup.3H-naltrindole (Simon, 1991; Sofuglu et al., 1991; Portoghese
et al., 1992) (1 nM) in a final volume of 200 .mu.l for 40 min at
25.degree. C. in the presence of absence of competing agents.
Non-specific binding was defined as radioactivity remaining bound
in the presence of 10 .mu.M naloxone. The binding reaction was
terminated by the addition of ice-cold 50 mM Tris-HCl (pH 7.8) and
rapid filtration over Whatman GF/B glass fiber filters that were
pretreated with 0.5% polyethyleneimine and 0.1% bovine serum
albumin. The filters were washed with 12 ml of ice-cold buffer and
the bound radioactivity determined using a liquid scintillation
counter. Data from radioligand binding studies were used to
generate inhibition curves. IC.sub.50 values were obtained by
curve-fitting performed by the program FITCOMP (Kong et al., 1993;
Yasuda et al., 1992) and converted to K.sub.1 values using the
equation K.sup.2=IC.sub.500/(1+L/Kd).
[0411] cAMP Accumulation
[0412] Briefly, COS-7 cells were subcultured in 12-well culture
plates. The cells were transfected 48 hrs. prior to the cAMP
experiments. Culture medium was removed from the wells and replaced
with 500 .mu.l of fresh medium containing 0.5 mM
isobutylmethylxanthine (IBMX). Cells were incubated for 20 min at
37.degree. C. Medium was removed and replaced with fresh medium
containing 0.5 mM IBMX, with or without 10 .mu.M forskolin and
various opioid agonists. The cells were incubated for 30 min at
37.degree. C. Medium was removed and cells sonicated in the wells
in 500 .mu.l of 1N HCl. The HCl was evaporated off in a Speed-Vac
and the cAMP analyzed using a radioimmunoassay kit from NEN/Dupont
(Wilmington, Del.).
[0413] Wild-type delta opioid receptors and the D128N and H278N
mutant receptors were transiently expressed in COS-7 cells. The
delta receptor selective antagonist.sup.3H-naltrindole specifically
bound to all three receptors. The binding of .sup.3H-naltrindole to
all three receptors in COS-7 cell membranes was saturable and of
high affinity. The saturable binding of .sup.3H-naltrindole is
illustrated graphically in FIG. 11A, FIG. 11B, and FIG. 11C. FIG.
11A shows the binding of .sup.3-H-naltrindole to the wild-type
receptor (open squares). FIG. 11B shows the binding of
.sup.3-H-naltrindole to the D128N mutant (filled circles), and FIG.
11C shows the binding of .sup.3-H-naltrindole to the H278N mutant
(open circles). The K.sub.d and B.sub.max values are presented in
Table 4, below.
11TABLE 4 The K.sub.d and B.sub.max values of .sup.3H-naltrindole
binding to the wild-type and D128N and H278N mutant delta opioid
receptors. Receptor Kd (nM) Bmax (pmol/mg protein) Wild-type 0.6
.+-. 0.2 45.4 .+-. 9 D128 mutant 1.3 .+-. 0.6 12.2 .+-. 4 H278N
mutant 0.2 .+-. 0.1 0.7 .+-. 0.4 These are the mean .+-. SEMs of
results from three separate experiments.
[0414] The potency of the delta receptor-selective antagonists
naltrindole and NTB and the non-selective opioid antagonist
diprenorphine to inhibit .sup.3H-naltrindole binding to the
wild-type receptor, D128N mutant and H278 mutant are shown in FIG.
12A, FIG. 12B, and FIG. 12C. FIG. 12A shows .sup.3H-Naltrindole
binding to membranes from COS-7 cells expressing the wild-type
receptor (open squares), D128N (filled circles), and H278N (opened
circles) in the presence of delta-selective antagonist NTB. All
three receptors, wild type, D128N, and H278N were inhibited by
delta-selective antagonist NTB. FIG. 12B shows .sup.3H-Naltrindole
binding to membranes from COS-7 cells expressing the wild-type
receptor (open squares), D128N (filled circles), and H278N (opened
circles) in the presence of delta-selective antagonist DPDPE. All
three receptors, wild type, D128N, and H278N were inhibited by
delta-selective antagonist DPDPE. FIG. 12C shows 3H-Naltrindole
binding to membranes from COS-7 cells expressing the wild-type
receptor (open squares), D128N (filled circles), and H278N (opened
circles) in the presence of non-selective agonist levorphanol. All
three receptors, wild type, D128N, and H278N were inhibited by
non-selective agonist DPDPE. The K.sub.1 values from these
experiments are presented in Tables 5 and 6.
12TABLE 5 Potencies of Antagonist binding to the Wild-type and
Mutant Delta Opioid Receptors. D 128N mutant (K.sub.i values Drug
Wild-type in nM) H278N mutant Naltrindole 1.4 5.6 0.5 NTB 0.04 0.07
0.03 Diprenorphine 5.0 16 1.4 Values are the means of at least
three different determination for each drug. The SEM for each drug
is less than 15% of the means.
[0415] In contrast, the potencies of peptide (DPDPE, DSLET and
D-Ala.sup.2 deltorphin II) and non-peptide (SIOM) delta receptor
selective agonists and the non-selective opioid agonists etorphine,
levorphanol, beta-endorphin and bremazocine to inhibit
.sup.3H-naltrindole binding to the D128N mutant receptor were less
than their binding to the wild-type receptor (Table 6). The
potencies of DPDPE, DSLET and beta-endorphin were over 100-fold
less at binding to the D128N mutant than to the wild-type
receptor.
13TABLE 6 Potencies of Agonists to Bind to the Wild-type and Mutant
Delta opioid receptors. D128N mutant (K.sub.i values H278N Drug
Wild-type in nM) mutant Delta selective DPDPE 116 39181 15 DSLET 49
15000 0.5 Deltorphin II 32 1938 4.0 SIOM 46 1216 1.8 Non-selective
Etorphine 73 2323 0.3 Levorphanol 187 4378 2.8 Bremazocine 58 2694
1.7 Beta-endorphin 26 15806 8.1 K.sub.1 values are the averaged
results of at least three different determinations. The SEM is less
than 10% of the means for each drug.
[0416] The reduced affinity of the D128N mutant receptor for
agonists was not due to an uncoupling of the receptor from G
proteins since the D128N mutant receptor mediated agonist
inhibition of forskolin-stimulated cAMP formation, a response
requiring the coupling of the receptor to G proteins. In COS-7
cells expressing the D128N mutant, the delta agonist DSLET
maximally inhibited forskolin-stimulated cAMP formation to the same
extent as in cells expressing either the wild-type or H278N
receptors. However, the potencies of DSLET and bremazocine to
inhibit forskolin-stimulated cAMP formation in COS-7 cells
expressing the D128N mutant were less than in cells expressing the
wild-type receptor (Table 7). FIG. 13 shows the inhibition of
Forskolin-stimulated cAMP accumulation by the delta agonist DSLET
in COS-7 cells expressing the wild-type and mutant delta opioid
receptors. cAMP accumulation was measured in COS-7 cells expressing
the wild-type (open bars), D128N mutant (dark bars) and H278N
mutant (hatched bars). Basal levels and levels stimulated by 10 uM
forskolin in the absence (FORSKOLIN) or presence of 1 uM DSLET
(DSLET) or 1 uM DSLET together with 1 uM naltrindole
(DSLET+NALTRINDOLE) were assessed.
14TABLE 7 Potencies of Agonists to inhibit Forskolin-stimulated
cAMP Accumulation in COS-7 cells expressing either the Wild-type or
Mutant Delta Opioid Receptors. D 1278N mutant (EC.sub.50 values
H278N Drug Wild-type in nM) mutant Bremazocine 7.4 80 1.5 DSLET 1.5
30 3.8 Values are the averaged results of three different
experiments in which the SEM was less than 20% of the mean
values.
[0417] These results are consistent with the results of the ligand
binding studies which show a reduced affinity of the D128N mutant
for agonists. Therefore, the reduced affinity of the D128N mutant
receptor for agonists was not due to gross conformational changes
resulting in G protein uncoupling but instead is due to the
essential role played by aspartate 128 in agonist binding to the
delta opioid receptor.
[0418] The affinity of the H278N mutant receptor for agonists was
greater than the wild-type delta opioid receptor (Table 6). In
contrast, the potencies of antagonists to bind to the H278N mutant
and wild-type receptors were relatively similar (Table 5). The
H278N mutant, like the wild-type delta receptor, was functionally
active since in cells expressing the receptor delta agonists
inhibited forskolin-stimulated cAMP accumulation (Table 7).
[0419] Pharmacological studies indicate that all potent opioid
agonists contain a cationic nitrogen in close proximity to a
hydrophobic aromatic group (Gilman et al., 1990; Simon, 1991). The
positively charged amino group has been proposed to associate with
negatively charged residues in opioid receptors and this
electrostatic interaction is believed to be essential for the
specificity and high affinity of binding of opioids to their
receptors. Two aspartates in the mouse delta opioid receptor affect
agonist binding and may provide the counterion to the cationic
nitrogen of opioid agonists. As set forth hereinbefore, aspartate
95 in the second transmembrane spanning region of the delta
receptor was necessary for the high affinity binding of selective
agonists to the delta receptor. As shown herein, aspartate 128 is
essential for the high affinity binding of all opioid agonists to
the delta receptor.
[0420] The distinct roles played by aspartate 95 and aspartate 128
in ligand binding suggest that selective and non-selective agonists
bind differently to the delta opioid receptor. The mechanism by
which the D128N mutant has reduced affinity for agonists while
maintaining functional coupling to adenylyl cyclase and G proteins
could be due to a selective increase in the rate of dissociation of
agonists from the receptor, without a reduction of the rate of
association of agonists. This would explain the reduced potency of
agonists to interact with the mutant receptor as detected in the
binding studies but the maintained ability of the agonists to
inhibit cAMP accumulation via this receptor since the functional
response is less dependent on rates of dissociation of agonists
than the binding assay. Furthermore, if Aspartate 128 in the delta
opioid receptor serves as a counterion to stabilize agonist
binding, then removal of the charge of this residue, as occurs
following this mutation to an asparagine, would be expected to
destabilize agonist binding and increase the rate of dissociation
of agonists. A similar proposal has been made to explain the
reduced affinity of a D95N mutant delta receptor for selective
agonists (Kong, 1993). Neither .sup.3H-DPDPE nor 125I-beta
endorphin were able to specifically bind to the D128N mutant,
whereas they bind potently and specifically to the wild-type delta
receptor (data not shown). This is consistent with their greatly
reduced potencies to inhibit .sup.3H-naltrindole binding to the
D128N mutant.
[0421] Mutations of the aspartate at residue 95 or 128 in the delta
receptor did not clearly affect antagonist binding. This implies
that either the cationic nitrogen of opioid antagonists is not
essential for ligand binding or the amino acid residues in the
receptor providing the negative charge for the electrostatic
interaction are at some other position in the receptor. The
non-essential role of aspartate 128 in the delta receptor for
antagonist binding contrasts with the results obtained with the
beta-adrenergic receptor. Strader et al. reported that mutation of
aspartate 113 in the third transmembrane spanning region of the
beta.sub.2-adrenergic receptor to an asparagine resulted in a
receptor with reduced affinity for antagonists and agonists
(Strader et al., 1987). These authors proposed that this residue is
an essential recognition site of the ligand binding domain of the
beta-adrenergic receptor for catecholamine agonists and
antagonists. The fundamentally different results obtained with a
similar mutation of a conserved aspartate in the delta and
beta-adrenergic receptors indicates that ligands bind differently
to these receptors.
[0422] Recent site-directed mutagenesis studies on the neurokinin
receptor indicate that a histidine residue in the fifth
transmembrane spanning region is necessary for high affinity
antagonist binding (Fong et al., 1993). The results of those
studies indicate that the aromatic ring structure of the histidine
residue interacts with aromatic ring structures of neurokinin
antagonists. Since an aromatic ring is present in all opioid
ligands and has been proposed to be essential for the high affinity
binding of opioids to their receptors (Gilman et al., 1990; Simon,
1991) histidine was mutated at residue 278 to an asparagine,
because it is the only histidine in an analogous region of the
delta receptor compared to the neurokinin receptor. The levels of
expression of this mutant receptor were less than either the
wild-type or D128N mutant. However, the affinity of the receptor
for agonists was higher than the affinity of the wild-type delta
receptor for these compounds. Antagonists bound with similar
potency to both receptors although the H278N mutant bound
.sup.3H-naltrindole and other opioid antagonists with slightly
higher affinities than the wild-type receptor. The finding that
replacement of the histidine with an asparagine did not reduce the
affinity of the receptor for agonists and antagonists indicates
that this residue is not essential for ligand binding to the delta
opioid receptors. The improved affinity of the mutant receptor for
agonists could be due to subtle conformational changes in the
receptor induced by its mutation or may indicate that the histidine
normally hinders agonist binding.
[0423] The selective large increase in affinity of the H278N mutant
for agonists combined with the selective loss in affinity of the
D128N mutant for agonists is consistent with our (Kong et al.,
1993) previous finding with a D95N mutant delta receptor that
agonists and antagonists bind differently to delta receptors,
possibly by interacting with distinct ligand binding domains.
Furthermore the finding that mutations of aspartate 95 and 128 and
histidine 278 to asparagines did not reduce antagonist 5 binding to
delta opioid receptor differs from results obtained with similar
mutations of the beta-adrenergic (Strader et al., 1987) and
tachykinin receptors (Fong et al., 1993) and suggests that opioid
antagonists interact with the delta receptor in a manner different
from antagonist binding to these other neurotransmitter
receptors.
Example 9
Analysis of MOP2
[0424] MOP2 is a mouse receptor with pharmacological properties
which are dissimilar to the pharmacological properties of classic
opioid receptors. MOP2 is likely to be an opioid receptor with
unusual ligand binding properties. The antagonists 3H-Naloxone,
.sup.3H-naltrindole and .sup.3H-diprenorphine did not bind
specifically to this receptor (MOP2) nor did the agonists
.sup.3H-U69,593, .sup.3H-DPDE, 3H-DAGO, .sup.3H-EKC,
.sup.125I-beta-endorphin nor the sigma ligand .sup.3H-pentazocine.
For the analysis of MOP2 (SEQ ID NO:6), the cDNA (SEQ ID NO:5) was
expressed in COS-7 cells and initially the expressed protein was
tested for binding of opioid radioligands.
[0425] To test for potential functional activity, the ability of a
number of opioid ligands to inhibit forskolin stimulated cAMP
accumulation in COS-7 cells expressing MOP2 was tested. Two
opioids, etorphine and lofentanil, at a concentration of 1 .mu.M
inhibited forskolin stimulated cAMP formation by 63.+-.11% and
52.+-.7% (N=5), respectively.
[0426] These compounds did not inhibit stimulated cAMP accumulation
m cells transfected with vector alone. The effects of these two
opioids were concentation dependent with half-maximal effects
occurring at 100 nM. At a concentration of 100 nM, the effects of
these two opioids were completely blocked the opioid antagonist
naloxonazine (1 .mu.m) and partially blocked by the antagonists
naloxone and B-FNA.
[0427] Furthermore, pretreatment of COS-7 expressing MOP2 with
pertussis toxin, which uncouples inhibitory G proteins form
adenylyl cyclase, completely blocked the inhibition of cAMP
formation by etorphine and lofentanil. In addition to lofentanil,
in one experiment, the analog fentanyl reduced forskolin stimulated
cAMP formation by 40%. In contrast, morphine, methadone, codeine,
EKC, levorphanol, bremazocine and beta-endorphin at a concentration
of 1 uM had no effect on forskolin-stimulated cAMP formation in
COS-7 cells expressing MOP2.
[0428] The high amino acid sequence similarity of MOP2 with cloned
opioid receptors and the ability of this unusual receptor to
mediate etorphine and lofentanil inhibition of cAMP formation in a
naloxonazine and naloxone sensitive manner suggests that this
receptor may be a novel opioid receptor. The novelty of this
receptor is further suggested by the inability of a number of
opioid agonists to interact with the receptor.
[0429] Etorphine is one of the most potent analgesics available. In
fact, its potent ability to induce respirator depression and its
high abuse potential are some of t he reasons that etorphine is a
Schedule 1 drug and is not used clinically. Fentanyl derivatives
such as lofentanil have extremely high abuse potential. Of the
derivative of fentanyl that have been made, lofentanil is the most
potent and effective analgesic. The finding that drugs of extremely
high abuse potential and analgesic potency and efficacy selectively
interact with MOP2 suggests that this receptor may be an important
site for the development of drugs that could be useful in treating
addiction.
Example 10
The Discovery That Physically Separate Extracellular Domains of the
Kappa Receptor are Necessary for Agonist and Antagonist Binding
Allows for Assaying Receptor Specific Agonists
[0430] The cloned .kappa. and .delta. receptors display unique
pharmacological profiles. Yet, the amino acid sequences of the
kappa and delta receptors are about 60% identical. The areas of the
receptors most divergent are the NH.sub.2- and COOH-termini as well
as the extacellular loops. The inventors hypothesized that these
extracellular domains have a role in ligand recognition and/or
binding. To test this hypothesis, the inventors constructed
chimeric receptors of .kappa. and .delta. in which the
NH.sub.2-termini as well as the second extracellular loops have
been exchanged. These results have been discussed in detail in the
section on screening assays. Studies of a chimera containing the
NH.sub.2-terminus of the .kappa. receptor and the remainder of the
.delta. receptor (.kappa..delta.) revealed that agonist and
antagonist binding to the .kappa. receptor are on separate domains.
This chimera bound .kappa.-selective antagonists but not
.kappa.-selective agonists.
[0431] These pharmacological and functional results suggest that
the NH.sub.2-terminus of the .kappa. receptor is responsible for
antagonist recognition and binding, whereas selective agonists bind
to the second extracellular loop. Selective agonists such as
U50,488 and its derivatives such as U69,593 and spiradoline as well
as the endogenous transmitters at the kappa receptor, dynorphin and
its analogs dynorphin (1-8), dynorphin (1-17), did not bind to the
chimera. These .kappa.-selective agonists could not bind to the
second extracellular loop of the .delta. receptor. The abilities of
.kappa.-selective agonists at the .kappa./.delta.2eloop chimera to
inhibit forskolin-stimulated cAMP accumulation was lost.
[0432] In contrast, less selective agonists such as
ethylketocyclazocine (EKC), bremazocine and levorphanol bound to
the .kappa./.delta.2eloop chimera and the wild-type receptors with
similar affinities. Furthermore, these non-selective agonists
inhibited cAMP formation in COS-7 cells expressing the chimeric
receptor and the wild-type receptor to a similar extent.
[0433] Antagonist binding to the .kappa./.delta.2eloop chimera and
the wild-type kappa receptor were similar, indicating that the
chimera was expressed at similar levels as the wild-type and that
the second extracellular loop is not necessary for antagonist
binding.
[0434] These results clearly demonstrate that .kappa.-selective
agonists and antagonists bind to physically distinct regions of the
.kappa. receptor. The second extacellular loop of the .kappa.
receptor contains a binding domain for .kappa.-selective agonists.
Non-selective agonists such as EKC bind to other regions of the
.kappa. receptor. These findings are the first identification of an
agonist binding domain of an opioid receptor and the first
demonstration that selective and non-selective agonists can bind to
different regions of the same receptor and cause agonism.
[0435] These findings have important pharmacological implications.
Non-selective agonists, such as EKC, are known to induce dysphoria
and psychosis whereas selective agonists such as dynorphin and the
endogenous transmitters at the kappa receptor do not Therefore,
these findings provide the basis for methods allowing the
development and screening of agonists of the dynorphin receptor
that are specific, that are therapeutically useful and may avoid
the side-effects of available non-specific kappa agonists.
[0436] Methods and conditions for screening candidate agonist
substances are discussed in detail above, in the section on
Screening Assays. Once provided with the teachings of this
application with regards to the different binding sites of
kappa-specific and non-specific opioid receptor agonists, those of
skill in the art will understand that these methods can be used to
screen for kappa receptor specific agonists.
[0437] Screening assays involve obtaining an opioid receptor
polypeptide, obtaining a candidate specific kappa opioid receptor
agonist, and assaying the ability of the candidate substance to
interact with the opioid receptor. Those of skill in the art will
recognize that the ability of the candidate substance to interact
with the kappa receptor may be assayed in any number of ways,
including, but not limited to, those describe in detail in the
Detailed Description of the Invention section of the
application.
[0438] The opioid receptor polypeptide used in the screening assay
should contain at least a portion of a kappa opioid receptor
polypeptide. More specifically, the screening polypeptide should
contain a portion of a second extracellular loop of the kappa
opioid receptor polypeptide, which has been shown to have a binding
site for kappa receptor-specific agonists. It is expected that
opioid receptor polypeptides comprising a negatively charged region
of the second extracellular loop of the kappa opioid receptor will
be particularly preferred for use in these screening procedures,
since kappa receptor specific agonist-kappa receptor binding
appears to be based, at least in part, on charge interactions
between the negatively-charged portions of the second extracellular
loop and positively charged portions of the agonists.
[0439] Chimeric opioid receptor polypeptides will be usable in the
above-described assays. In fact, the studies that led to the
elucidation of these assays were carried out with chimeric
receptors. As a class, the opioid receptors comprise extracellular
loops, transmembrane regions, intracellular loops, and an
extracellular amino terminus. The inventors have shown that the
extracellular portions of the receptors: the second and third
extracellular loops and the amino terminus, serve as the binding
sites for opioid receptor ligands. For example, with regards to the
kappa receptor, it has been shown that kappa-specific agonists bind
to the second extracellular loop while antagonists bind to the
amino terminus. The inventors strongly suspect that non-specific
agonists bind to the third extracellular loop of the kappa
receptor, and studies are in progress that should prove this. With
this knowledge, it is possible to design chimeras that are very
useful as specific screening tools.
[0440] In preferred embodiments, the chimeric receptor comprises
the second extracellular loop of the kappa opioid receptor. The
kappa second extracellular loop is located between amino acid
residues 167-228 of the kappa opioid receptor polypeptide. Results
of studies of the inventor have shown that residues 1-78 of the
Kappa receptor specifically bound antagonists. Residues 167-228 of
the Kappa receptor bind selective agonists. Non-selective agonists,
such as ethylketin cyclazocine, do not bind to either of these
regions. The only extracellular domain of the Kappa receptor which
has a unique amino acid sequence is the third extracellular loop
(residues 271-318). This is a region likely to bind non-selective
agonists. A chimera Kappa receptor consisting of the third
extracellular loop of the delta receptor would be expected to only
bind selective Kappa agonists and not non-selective agonists
(chimera is Kappa 1-270/delta 258-300/K319-380). This chimera could
be used to screen to selective Kappa agonists. Furthermore, chimera
Delta 149/Kappa 79-270/delta 258-306/K319-380 would be expected to
bind only selective agonists. If one wishes to screen for
kappa-specific agonists, a chimera having the second extracellular
loop of the kappa receptor should be used. Further, a chimera
having the second extracellular loop of the kappa receptor but
lacking the third extracellular loop could have the advantage of
detecting kappa specific agonists without any fear of detecting
non-specific agonists. Of course, a chimera having all of the
regions of the kappa receptor except the second extracellular loop
can be used as a negative control in assays designed to screen for
kappa-specific agonists. Other preferred chimeras will have the
second extracellular loop of the kappa receptor, but lack the third
extracellular loop. Since the third extracellular loop contains the
putative non-specific agonist binding region, a chimera lacking
this region will be expected to not be able to detect non-specific
agonist activity. Therefore, any agonism seen for such a chimera
will have to be the result of a kappa-specific agonist binding to
the second extracellular loop. Chimeras lacking the second
extracellular loop will be useful as negative controls.
[0441] When provided with the teachings of this specification,
those of skill will be able to formulate chimeras and controlled
screening strategies that allow for the screening of all forms of
opioid receptor agonists and antagonists. The inventors have
constructed many such chimeras, and are in the process of
constructing more. It is possible to create an almost endless array
of chimeras using standard genetic manipulations and the knowledge
that the inventors have derived concerning the ligand binding sites
of the opioid receptors. All such chimeras, the polynucleotides
encoding them, and methods of using them in assays are contemplated
within the scope of the invention. Specific examples of chimeric
opioid receptors are useful in screening assays are:
.kappa..sub.1-78.delta..sub.70-372,
.delta..sub.1-69/.kappa..sub.79-380,
.kappa..sub.1-74/.delta..sub.65-372 or
.delta..sub.1-64/.kappa..sub.75-38- 0.
[0442] Truncated opioid receptor polypeptide will be useful in the
above-described candidate screening assays. Short polypeptides that
exhibit kappa receptor-specific agonist binding will have certain
advantages over longer polypeptide. Preferably, the truncated
opioid receptor polypeptide is a n ed kappa opioid receptor
polypeptide that includes at least the agonist-binding portion of
the second extracellular loop. For example, a truncated opioid
receptor polypeptide comprising amino acid residues 79 to 380 of a
kappa opioid receptor polypeptide is expected to be useful in this
regard. Truncated kappa opioid receptors comprising the second
extracellular loop of the receptor will be useful in these assays.
For example, a truncated kappa receptor comprising amino acid
residues 167 to 228 will be useful in the invention.
[0443] Potential kappa receptor-specific agonists can be
prescreened prior to being tested with the described assays by
determining whether the candidate has a positive charge. Charge
relationships influence the kappa receptor-specific agonist binding
mechanism, with the negatively charged binding region binding
positively charged agonists. Of course, it is possible for an
effective agonist not to be positively charged, however, the
assessment of charge will provide one mechanism for narrowing of
the range of agonists to be tested.
[0444] Those of skill in the art will, once provided with the
teaching of this specification, be able to practice the
invention.
[0445] The development of specific kappa agonists is aided by
knowledge of the unique nature of the second extracellular loop of
the kappa receptor. The second extracellular loop of the kappa
receptor is highly negatively charged with seven aspartates and
glutamates. In contrast, the delta and mu receptors have only
negatively charged residue in the corresponding loop. Because
dynophin is positively charged, and the second extracellular loop
of the kappa receptor has multiple negative charges, the inventors
are mutating the negatively charged to neutral, conserved residues
to determine the extent to which charge is critical for the
selective binding of dynorphin analogs to this receptor. This work
is in progress, and results from it should provide further
direction to those seeking to elucidate kappa-specific
agonists.
Example 11
Cloned Kappa and Mu Opioid Receptors Couple to an Inward Rectifier
Potassium Current When Expressed in Mouse AtT-20 Cells
[0446] The cloning of the kappa, delta, and mu opioid receptors
allows new techniques to be used to study cellular mechanisms of
action of these receptors. Experiments focusing on endogenous
opioid receptors have shown that regulation of ionic conductances
is an important mechanism by which these receptors mediate cellular
events. The inventors observed that the cloned kappa opioid
receptor stably expressed in PC-12 cells is able to modulate an
N-type calcium current. To further examine opioid receptor-ion
channel coupling, the inventors generated AtT-20 cell lines which
stably express kappa and mu receptors. Whole cell patch clamp
recordings demonstrate that both the kappa and mu receptor are able
to modulate the activation of an inward rectifier potassium current
that has previously been described in this cell line. These effects
are selective as both kappa- and mu-mediated activation is blocked
by selective antagonists. Having both receptors expressed
separately in the same cell line will allow the inventors to
compare functional properties of the two receptors, including G
protein coupling and desensitization.
Example 12
The Third Intracellular Loop of the Delta Opioid Receptor is
Involved in Coupling to Adenylyl Cyclase
[0447] To investigate the role of the third intracellular loop in
coupling to adenylyl cyclase, chimeric receptors between the delta
opioid receptor and the somatostatin receptor SSTR1 were generated.
SSTR1 is a somatostatin receptor that does not couple to adenylyl
cyclase. Chimeric receptors were generated in which the third
intracellular loop of SSTR1 was replaced with that of the delta
receptor, and also in which the third intracellular loop of the
delta receptor was replaced with that of SSTR1. Although wild type
SSTR1 showed no coupling to adenylyl cyclase in an cAMP
accumulation assay, the SSTR1-delta chimeric receptor was able to
mediate inhibition of a forskolin-stimulated increase in cAMP in
response to agonist The cAMP inhibition by agonist was
dose-dependent. Conversely, while the wild type delta receptor
mediated an inhibition of adenylyl cyclase activity, the
delta-SSTR1 chimera demonstrated a reduced ability to inhibit cAMP
accumulation with agonist. These results indicate that the third
intracellular loop of the delta receptor is involved in coupling of
the receptor to G proteins.
Example 13
Development of a Peptide-Directed Antiserum Against the Delta
Opioid Receptor
[0448] A peptide-directed polyclonal antiserum was developed
against the C-terminus of the cloned delta opioid receptor. In
order to generate the antiserum, rabbits were injected with the
unique peptide Ala-Thr-Thr-Arg-Glu-Arg-Val-Thr-Ala-Cys-Thr-Pro-Ser,
(SEQ ID NO: 46) corresponding to a thirteen residue stretch of
amino acids in the C-terminus of the cloned delta opioid receptor.
The antiserum was then partially purified and tested for its
ability to specifically recognize the delta receptor. The antiserum
recognizes an approximately 70 kDa protein in CHO cells stably
expressing the delta receptor. This 70 kDa protein can be
.sup.35S-methionine labeled and immunoprecipitated by the
peptide-directed antiserum. Immunoprecipitation of the 70 kDa
protein can be specifically blocked by an excess of the peptide
against which the antiserum was raised. The size of this protein is
in agreement with results of crosslinking experiments on the cloned
delta receptor using .sup.125I-.beta. endorphin. The
peptide-directed antiserum was able to immunoprecipitate specific
.sup.125I-.beta. endorphin binding activity from CHO cells stably
expressing the date receptor. Binding of .sup.125I-.beta. endorphin
to the immunoprecipitate was inhibited by the delta-selective
agonist DPDPE. The preimmune serum was unable to immunoprecipitate
binding activity, and immunoprecipitation of binding activity by
the antiserum was blocked by an excess of peptide. The antiserum
was unable to recognize the delta receptor by immunoblotting,
suggesting that the epitope that it recognizes may become denatured
or is otherwise rendered unrecognizable to the antibodies. This
antiserum will be useful in investigating the physical properties
of the delta receptor and the post-translational modifications it
may undergo.
Example 14
Mutagenesis of Conserved Residues in the Delta, Kappa, and Mu
Opioid Receptors
[0449] To investigate the role of aspartate (D) residues in the
putative second and third transmembrane-spanning domains (TM2 and
TM3) in the binding of opioid ligands to these receptors, the
inventors mutated aspartates 95, 105, and 114 in TM2, and 128, 138,
and 147 in TM3 to asparagines (N) for .delta., .kappa., and .mu.,
respectively. As the inventors previously demonstrated for the
delta TM2 mutant (Kong, 1993, J. Biol. Chem.), mutation of D's in
TM2 of each receptor had dramatic effects on the binding of
selective agonists, with generally greater than 100-fold reductions
in affinities of these compounds for their respective receptors. In
addition, this mutation abolished Na.sup.+ regulation of agonist
binding. Mutations of D's in TM3 of these receptors also
dramatically reduced selective agonist binding to these receptors,
and also decreased the affinities of non-selective drugs.
Interestingly, mutation of the histidine residues 278, 291, and 297
conserved in TM6 produced divergent effects on the receptors.
Whereas no effect was observed for the .delta. TM6 mutant, a
reduced affinity of some peptide agonists for the mutant .mu.
receptor was observed. None of these mutations had dramatic effects
on the binding of antagonists, with the notable exception of
peptide antagonists for the .mu. receptor, such as CTOP and SMS
201-995, which displayed decreased affinities for the .mu. mutants.
together, these studies identify residues important in the binding
of opioid agonists to each of the cloned opioid receptors and
demonstrate that agonists and antagonists bind to these receptors
in discernible manners.
Example 15
Characterization of the Cloned Human Mu Opioid Receptor
[0450] The clinical use of opioids in humans is marred by a
constellation of undesirable side effects, including respiratory
depression, miosis, decreased gastrointestinal motility, sedation,
nausea and vomiting (Jaffe and Martin, 1990). Other liabilities
concerning opioid administration are the potentials for tolerance,
dependence, and abuse. Because the effects of opioids are mediated
via a variety of receptors, one receptor (sub)type may mediate the
therapeutic effects whereas a different receptor (sub)type may
precipitate the undesirable side effects (Pasternack, 1993).
Therefore, with the use of more selective agents for the
therapeutically relevant receptor, the undesirable side effects
could be minimized or eliminated. Most of the clinically employed
opioids, including morphine, methadone, codeine, and fentanyl,
selectively interact with the .mu. receptor. This was shown
directly in studies on the recently cloned rat .mu. receptor
(Raynor et al., 1994). To gain better insight into the mechanism of
actions of these compounds in humans, the inventors examined the
pharmacological properties of the cloned human .mu. opioid receptor
and the distribution of message encoding the .mu. receptor in human
brain.
[0451] Materials and Methods
[0452] Abbreviations:
[0453] .beta.-FNA .beta.-funaltrexamine CTOP
D-Phe-Cys-Tyr-D-Trp-Om-Thr-Pe- n-Thr-NH2 DAMGO
[D-Ala2,MePhe4,Gly-ol5]enkephalin GTPgSguanosine-5'-O-3-th-
iotriphosphate) IBMX isobutylmethylxanthine
[0454] PTX pertussis toxin
[0455] Cloning: To clone the human .mu. opioid receptor, a cDNA
library was constructed from human caudate nucleus mRNA was
screened under reduced stringency with the rat .mu. opioid receptor
cDNA (Chen et al., 1993) and complete sequence analysis of one cDNA
revealed an open reading frame of 1200 bp, predicting a protein of
400 amino acids. For receptor expression, the cDNA containing the
open reading frame of the receptor was cloned downstream of the
human cytomegalovirus promoter in the mammalian expression vector
pcDNA3 (Invitrogen). Details concerning the isolation of the human
.mu. opioid receptor cDNA will be reported elsewhere (Mestek et
al., submitted). The cDNA sequence has been submitted to GenBank
(accession number L29301).
[0456] Radioligand Binding Studies: Receptor binding assays were
performed using membranes from COS-7 cells transiently expressing
the human .mu. receptor 48 hours after transfection as previously
described (Raynor et al., 1994). For radioligand binding assays,
cells were harvested in 50 mM Tris-HCl (pH 7.8) containing 1 mM
ethylene glycol bis(b-aminoethyl ether)-N,N'-tetraacetic acid, 5 mM
MgCl.sub.2, 10 mg/ml leupeptin, 10 mg/ml pepstatin, 200 mg/ml
bacitracin and 0.5 mg/ml aprotinin (buffer 1) and centrifuged at
24,000.times.g for 7 min at 4.degree. C. The pellet was homogenized
in buffer 1 using a Polytron (Brinkmann, setting 2.5, 30 sec). The
homogenate was then centrifuged at 48,000.times.g for 20 min at
4.degree. C. The pellet was homogenized in buffer 1 and this
membrane preparation was used for the radioligand binding studies.
Cell membranes (10-20 mg protein) were incubated with the .mu.
agonist [3H]DAMGO (2 nM, specific activity 55 Ci/mmol) or the
antagonist [3H]naloxone (4 nM, specific activity 55
Ci/mmol)(NEN/Dupont, Wilmington, Del.) in a final volume of 200 mL
for 40 min at 25.degree. C. in the presence or absence of competing
agents. For saturation experiments, cell membranes were incubated
with increasing concentrations (0.25-15 nM) of [3H]DAMGO.
Nonspecific binding was defined as the radioactivity remaining
bound in the presence of 1 mM naloxone. The binding reaction was
terminated by the addition of ice-cold 50 mM Tris-HCl buffer (pH
7.8) and rapid filtration over Whatman GF/B glass fiber filters
which were pretreated with 0.5% polyethyleneimine/0.1% BSA for at
least 1 hour. The filters were then washed with 12 mL of ice-cold
Tris-HCl buffer and the bound radioactivity counted in a
scintillation counter. Data from radioligand binding 10 studies
were used to generate inhibition curves. IC50 values were obtained
from curve-fitting performed by the mathematical modeling program
FITCOMP (Perry and McGonigle, 1988) and saturation data was
analyzed using FITSAT (McGonigle et al., 1988) available on the
National Institutes of Health-sponsored PROPHET system. The
inhibitory binding constant (K.sub.i) was calculated from the IC50
values using the Cheng-Prusoff equation (Cheng and Prusoff,
1973).
[0457] The effect of pretreatment of cells expressing the human
.mu. receptor with morphine or with pertussis toxin on subsequent
agonist binding to membranes was also investigated. Cells were
treated with either control medium, 1 mM morphine for 4 hr, or 100
ng/ml pertussis toxin for 18 hr prior to radioligand binding
studies.
[0458] cAMP Accumulation Studies: Studies examining the abilities
of compounds to inhibit forskolin-stimulated adenylyl cyclase
activity were performed as previously described (Kong et al., 1993,
J. Biol. Chem.). Briefly, cells used for cAMP accumulation studies
were subcultured in 12-well culture plates. The following day,
cells were transfected and cAMP experiments were conducted 48 hr
subsequently. Culture medium was removed from wells and replaced
with 500 mL fresh medium containing 0.5 mM isobutylmethylxanthine
(IBMX). Cells were incubated for 20 min at 37.degree. C. Medium was
then removed and replaced with fresh medium containing 0.5 mM IBMX,
with or without 10 mM forskolin and various concentrations of
drugs. Cells were incubated for 30 min at 37.degree. C. Medium was
then removed and cells sonicated in the wells in 250 mL 1M HCl and
frozen for subsequent determination of cAMP content by RIA. Samples
were thawed and diluted in cAMP RIA buffer before analysis of cAMP
content using the commercially available assay kit from NEN/Dupont
(Wilmington, Del.).
[0459] RNA blotting analysis: RNA blotting analysis was performed
as previously described (Kong et al., 1994, Neuroscience, Delfs et
al., in press). The human brain RNA blot was obtained from CLONTECH
laboratories (Palo Alto, Calif.). Each lane contained 2 mg of poly
A-selected mRNA. The blot was hybridized at 42.degree. C. for 24
hours with random-primed 32P-labelled DNA (Prime-It, Stratagene)
corresponding to a 1.6 kilobase (kB) fragment isolated after
digestion with EcoR V and Xba I. This fragment includes the entire
coding region of the human .mu. opioid receptor. The blots were
washed at 65.degree. C. in 2.times.SSC/0.5% SDS (0.3 M sodium
chloride/0.03 M sodium citrate) for 20 minutes and in
0.2.times.SSC/0.2% SDS for 20 minutes before exposure to X-ray film
for 5-7 days to detect signal.
[0460] Discussion
[0461] To characterize pharmacologically the cloned human .mu.
receptor, the inventors transiently expressed the cDNA encoding
this receptor in COS-7 cells as previously described (Yasuda et
al., 1993, Proc. Natl. Acad. Sci. USA, 90:6736; Kong et al., 1993,
J. Biol. Chem.; Raynor et al., 1994). For comparative purposes, the
rat .mu. receptor was also expressed in parallel experiments. The
binding of [3H]DAMGO to the human .mu. receptor was saturable and
of high affinity (FIG. 14). Scatchard analysis of the saturation
experiments demonstrated that [3H]DAMGO bound to the cloned human
.mu. receptors with a KD of 1.0 nM and a B.sub.max of 232 fmol/mg.
All data were best fit by single-site analysis. The inventors
previously reported that [3H]DAMGO bound to the cloned rat .mu.
receptor with a KD of 0.57 nM and a B.sub.max of 444 fmol/mg
protein (Raynor et al., 1994). No specific radioligand binding was
detectable in nontransfected or vector-transfected COS-7 cells.
[0462] To identify the pharmacological profile of the cloned human
.mu. opioid receptor, a number of opioid ligands were tested for
their abilities to inhibit [3H]DAMGO binding to this receptor
(Table 8).
15TABLE 8 Binding potencies (K.sub.i-nM) of ligands for the cloned
human .mu. oplold receptor .mu. RECEPTOR [.sup.3H]DAMGO
Leu-enkephalin 6.6 (1.2) .beta.-endorphin 0.94 (0.06)
des-Tyr.sup.1-.beta.-endorphin >1000 (-)naloxone 1.4 (0.4)
(+)naloxone >1000 (-)buprenorphine 0.51 (0.09) (+)buprenorphine
>1000 lovorphanol 1.9 (0.6) dextrorphan >1000 DAMGO 1.4
(0.04) morphine 2.0 (0.6) methadone 5.6 (0.4) codeine 65 (13)
fentanyl 1.9 (0.4) sufentanil 0.3 (0.08) CTOP 3.9 (0.4) SMS 201-995
12 (3) etorphine 0.18 (0.04) .beta.-FNA 0.29 (0.02) nalorphine 6.6
(1.2) (.+-.)bremazocine 1.4 (0.3) naltrexone 1.5 (0.05)
diprenorphine 0.18 (0.04)
[0463] These ligands included a variety of compounds which have
been previously characterized as .mu.-selective including both
peptide and non-peptide agonists and antagonists (Lutz and Pfister,
1992, Goldstein and Naidu, 1989; Raynor et al., 1994). As expected,
most of these compounds bound to the cloned .mu. receptor with Ki
values in the low nM range (Table 8). The endogenous opioid
peptides leu-enkephalin and .beta.-endorphin bound potently to .mu.
receptors whereas des-Tyrl-.beta.-endorphin did not bind. The
binding was stereoselective, being inhibited by (-)naloxone,
(-)buprenorphine, and levorphanol but not by their respective
isomers (+)naloxone, (+)buprenorphine, or dextrorphan. The
.mu.-selective compounds DAMGO, morphine, methadone, fentanyl, and
sufentanil bound with affinities in the low nanomolar range,
whereas the affinity of codeine was somewhat lower. The
.mu.-selective peptide antagonists CTOP and SMS 201-995 also bound
with high affinities. Other relatively nonselective compounds
tested were etorphine, .beta.-FNA, nalorphine, (+)bremazocine,
naltrexone, and diprenorphine, and all bound with high affinities.
The .delta.-selective agonists DPDPE and D-Ala2 deltorphin II and
the .kappa.-selective compounds U-50,488 and U-69,593 did not bind
to the human .delta. receptor at concentrations as high as 1 mM.
Comparisons of the affinities of all of these ligands for the human
and rat .mu. receptors showed that most, but not all, of these
drugs bind to these receptors with similar affinities. The
affinities of morphine, methadone, and codeine were significantly
higher for the human .mu. receptor than for the rat .mu. receptor
(Table 9). All other drugs tested demonstrated indistinguishable
affinities for the human and rat .mu. receptors, as exemplified in
Table 9.
16TABLE 9 Binding potencies (K.sub.i-nM) of ligands for the cloned
human and rat .mu. opioid receptor [.sup.3H]DAMGO human rat
morphine 2.0 (0.6) 22 (6.8) methadone 5.6 (0.4) 19 (1.4) codeine 65
(13) 168 (4) fentanyl 1.9 (0.4) 1.3 (0.5) etorphine 0.18 (0.04)
0.27 (0.6) .beta.-endorphin 0.94 (0.06) 1.7 (0.4) (-)buprenorphine
0.51 (0.09) 0.42 (0.03)
[0464] To investigate the association of the human .mu. receptor
with guanine-nucleotide binding proteins (G proteins), the
inventors examined the effects of nonhydrolyzable analogues of GTP
and of pertussis toxin treatment of COS-7 cells transiently
expressing the receptor on the binding of radiolabelled agonist to
the receptor. As shown in FIG. 15, inclusion of 100 mM GTPgS in the
[3H]DAMGO binding assay decreased specific labelling of the human
and rat .mu. receptors by 65.+-.1.5% and by 55.+-.7%, respectively.
In addition, PTX-pretreatment of cells expressing the receptor
substantially decreased [3H]DAMGO labelling of human and rat .mu.
receptors by 79.+-.8% and by 42.+-.5%, respectively. These results
are consistent with coupling of both human and rat .mu. receptors
to G-proteins.
[0465] The cloned rat .mu. receptor functionally couples to the
inhibition of adenylyl cyclase (Chen et al., 1993). To determine
whether the human p receptor is also coupled to adenylyl cyclase,
the effects of agonists to decrease cAMP accumulation in cells
expressing the receptor were examined (FIG. 16).
Forskolin-stimulated cAMP accumulation was significantly reduced by
leu-enkephalin and the effect was antagonized by (-)naloxone. The
effect was stereoselective in that levorphanol also decreased cAMP
accumulation, but dextrorphan was without effect.
[0466] A potential cellular mechanism of tolerance to opioids could
be related to desensitization/down-regulation of specific receptors
for these drugs. To determine whether agonist causes regulation of
the .mu. receptor, cells expressing the human and rat .mu.
receptors were exposed to 1 .mu.M morphine for four hours. The
inventors have previously demonstrated that the cloned mouse
.delta. and .kappa. receptors undergo significant desensitization
and/or downregulation after four hour exposures to high
concentrations of selective agonists (Raynor et al., submitted; K.
R. and T. R., unpublished observations). As shown in FIG. 17, no
significant changes in either radiolabelled agonist or antagonist
binding were detectable. These results suggest that the .mu.
receptor is not as readily regulated by agonist exposure as are the
.delta. and .kappa. receptors.
[0467] RNA blotting using a probe against the fill length coding
region of the human .mu. opioid receptor detected multiple
transcripts (FIG. 18) including a prominent mRNA of approximately
13.5 kB. This is of similar size to .mu. opioid receptor mRNA that
the inventors and others have reported for the rat .mu. receptor
mRNA (Fukuda et al., 1993; Delis et al., 1994). Smaller size bands
of 11, 4.3, and 2.8 were also detected. The highest levels of .mu.
opioid receptor mRNA in human brain were detected in the
hypothalamus, thalamus and subthalamic nuclei (FIG. 18, lanes 5, 7,
8). High levels were also detected in the amygdala and caudate
nucleus (FIG. 18, lanes 1, 2). Much lower levels were detected in
the hippocampus, corpus callosum and substantia nigra (FIG. 18,
lanes 3, 4, 6). The 11 kB RNA was most abundant in the amygdala and
subthalamic nucleus, whereas the 4.3 kB RNA was found in high
abundance also in the corpus callosum.
[0468] In the present example, the pharmacological profile,
regulation and cellular effector coupling of the cloned human .mu.
receptor are examined. The characteristics of the receptor are very
similar to those of the cloned rat .mu. receptor, consistent with
the high degree of structural homology found between the receptors
in these species. The pharmacological profile of the human .mu.
receptor is similar to that which the inventors have previously
reported for the rat .mu. receptor (Chen et al., 1993) with the
notable exceptions of the affinities of several clinically-employed
opioids such as morphine, methadone, and codeine. These compounds
bound to the human .mu. receptor with higher affinities than to the
rat .mu. receptor. The human and rat receptors are most divergent
in the N-terminus, and these amino acid substitutions may
contribute to the differing pharmacological properties of the rat
and human .mu. receptors. Interestingly, the endogenous opioid
peptides .beta.-endorphin and leu-enkephalin bound with high
affinities to the .mu. receptor, suggesting these peptides may be
act at this receptor under physiological conditions. Likewise, as
the inventors had found for the rat .mu. receptor, the present
findings indicate that opioid agents with abuse liabilities, such
as morphine, fentanyl, and methadone, possess high affinities for
the human .mu. receptor, whereas they demonstrate little or no
affinity for the mouse .delta. or .kappa. receptors (Raynor et al.,
1994). Development of analgesic agents which are .kappa.- or
.delta.-selective may obviate this concern of .mu.-selective
analgesics, as well as other serious side effects including
respiratory depression.
[0469] Another problem associated with the chronic use of opioids
is the development of tolerance to these agents. While
desensitization/downregul- ation of the opioid receptor(s) has been
suggested as a potentially causal underlying mechanism of this
phenomenon, a large body of evidence suggests that this is not the
case for the .mu. opioid receptor with chronic in vivo exposures
(reviewed in Zulkin et al., 1993). These present results with the
human .mu. opioid receptor expressed in cultured cells also suggest
that down-regulation at the receptor level does not readily occur,
as it does for the cloned .kappa. and .delta. receptors (Raynor et
al., submitted; K. R. and T. R, unpublished observations), and that
other mechanisms must be involved in tolerance development to
.mu.-selective opioids.
[0470] In general, the distribution of the .mu. opioid receptor
mRNA was similar in rat and human brain with highest levels
detected in thalamic regions and lower levels in the striatum. The
high levels of mRNA expression in the subthalamic region is unusual
and suggests that this important relay nuclei involved in motor
control may have high .mu. opioid receptor expression.
[0471] The RNA blotting revealed multiple .mu. receptor transcripts
expressed in human brain The size of the largest transcript
(.about.13.5 kB) is similar to that reported for rat .mu. opioid
receptor mRNA (Fukuda et al., 1993; Delfs et al., 1994). However,
the smaller discrete RNA species detected in human brain differ
from that detected in rat tissues. The identity of the multiple RNA
species detected by RNA blot is not clear. They could represent the
same RNA with different polyA+ tails or processing intermediaries.
Pharmacological evidence suggests that subtypes of .mu. receptors
are expressed in the nervous system. One intriguing possibility is
that some of the distinct transcripts encode .mu. receptor
subtypes.
[0472] The ability to study individually the pharmacological
properties of the cloned opioid receptor subtypes will allow for
identification of structural features of ligands which permit
selective interactions. Identification of the pharmacological
interactions of drugs with the individual opioid receptors could
lead to the identification of therapeutic agents less burdened with
the potential to produce undesirable side effects.
[0473] Examples have been included to illustrate preferred modes of
the invention. Certain aspects of the following examples are
described in terms of techniques and procedures found or
contemplated by the present inventors to work well in the practice
of the invention. These examples are exemplified through the use of
standard laboratory practices of the inventor. In light of the
present disclosure and the general level of skill in the art, those
of skill will appreciate that the following examples are intended
to be exemplary only and that numerous changes, modifications and
alterations can be employed without departing from the spirit and
scope of the invention.
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Sequence CWU 1
1
232 1 8 PRT Artificial Sequence Indolicidin Analogue 1 Arg Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 1 5 2 8 PRT Artificial Sequence Indolicidin
Analogue 2 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 3 10 PRT Artificial
Sequence Indolicidin Analogue 3 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 10 4 11 PRT Artificial Sequence Indolicidin Analogue 4 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 5 17 PRT Artificial
Sequence Indolicidin Analogue 5 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Met Ile Leu Xaa Xaa Ala Gly 1 5 10 15 Ser 6 18 PRT Artificial
Sequence Indolicidin Analogue 6 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Met Ile Leu Xaa Xaa Ala 1 5 10 15 Gly Ser 7 18 PRT Artificial
Sequence Indolicidin Analogue 7 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Met Ile Leu Xaa Xaa Ala 1 5 10 15 Gly Ser 8 19 PRT Artificial
Sequence Indolicidin Analogue 8 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Met Ile Leu Xaa Xaa 1 5 10 15 Ala Gly Ser 9 10 PRT
Artificial Sequence Indolicidin Analogue 9 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Met 1 5 10 10 11 PRT Artificial Sequence Indolicidin
Analogue 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Met 1 5 10 11 8
PRT Artificial Sequence Indolicidin Analogue 11 Leu Xaa Xaa Xaa Xaa
Xaa Arg Lys 1 5 12 9 PRT Artificial Sequence Indolicidin Analogue
12 Leu Xaa Xaa Xaa Xaa Xaa Xaa Arg Lys 1 5 13 10 PRT Artificial
Sequence Indolicidin Analogue 13 Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Arg Lys 1 5 10 14 11 PRT Artificial Sequence Indolicidin Analogue
14 Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Lys 1 5 10 15 9 PRT
Artificial Sequence Indolicidin Analogue 15 Leu Xaa Xaa Xaa Xaa Xaa
Xaa Arg Lys 1 5 16 10 PRT Artificial Sequence Indolicidin Analogue
16 Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Lys 1 5 10 17 10 PRT
Artificial Sequence Indolicidin Analogue 17 Leu Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Arg Lys 1 5 10 18 9 PRT Artificial Sequence Indolicidin
Analogue 18 Leu Xaa Xaa Xaa Xaa Xaa Xaa Arg Lys 1 5 19 10 PRT
Artificial Sequence Indolicidin Analogue 19 Leu Xaa Xaa Xaa Xaa Xaa
Xaa Arg Arg Lys 1 5 10 20 11 PRT Artificial Sequence Indolicidin
Analogue 20 Leu Lys Xaa Xaa Xaa Xaa Xaa Xaa Arg Arg Lys 1 5 10 21
11 PRT Artificial Sequence Indolicidin Analogue 21 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 22 11 PRT Artificial Sequence
Indolicidin Analogue 22 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 23 12 PRT Artificial Sequence Indolicidin Analogue 23 Ile
Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys 1 5 10 24 20 PRT
Artificial Sequence Indolicidin Analogue 24 Ile Leu Arg Trp Pro Trp
Trp Pro Trp Arg Arg Lys Met Ile Leu Lys 1 5 10 15 Lys Ala Gly Ser
20 25 13 PRT Artificial Sequence Indolicidin Analogue 25 Lys Arg
Arg Trp Pro Trp Trp Pro Trp Lys Lys Leu Ile 1 5 10 26 13 PRT
Artificial Sequence Indolicidin Analogue 26 Trp Arg Ile Trp Lys Pro
Lys Trp Arg Leu Pro Lys Trp 1 5 10 27 12 PRT Artificial Sequence
Indolicidin Analogue 27 Ile Leu Arg Trp Val Trp Trp Val Trp Arg Arg
Lys 1 5 10 28 13 PRT Artificial Sequence Indolicidin Analogue 28
Ile Leu Arg Arg Trp Val Trp Trp Val Trp Arg Arg Lys 1 5 10 29 10
PRT Artificial Sequence Indolicidin Analogue 29 Leu Arg Trp Trp Trp
Pro Trp Arg Arg Lys 1 5 10 30 12 PRT Artificial Sequence
Indolicidin Analogue 30 Ala Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg
Lys 1 5 10 31 12 PRT Artificial Sequence Indolicidin Analogue 31
Ile Leu Arg Trp Ala Trp Trp Pro Trp Arg Arg Lys 1 5 10 32 13 PRT
Artificial Sequence Indolicidin Analogue 32 Trp Arg Trp Trp Lys Pro
Lys Trp Arg Trp Pro Lys Trp 1 5 10 33 13 PRT Artificial Sequence
Indolicidin Analogue 33 Ile Leu Lys Lys Ile Pro Ile Ile Pro Ile Arg
Arg Lys 1 5 10 34 13 PRT Artificial Sequence Indolicidin Analogue
34 Ile Leu Lys Lys Tyr Pro Tyr Tyr Pro Tyr Arg Arg Lys 1 5 10 35 13
PRT Artificial Sequence Indolicidin Analogue 35 Ile Leu Lys Lys Tyr
Pro Trp Tyr Pro Trp Arg Arg Lys 1 5 10 36 13 PRT Artificial
Sequence Indolicidin Analogue 36 Ile Leu Lys Lys Phe Pro Trp Phe
Pro Trp Arg Arg Lys 1 5 10 37 13 PRT Artificial Sequence
Indolicidin Analogue 37 Ile Leu Lys Lys Phe Pro Phe Trp Pro Trp Arg
Arg Lys 1 5 10 38 12 PRT Artificial Sequence Indolicidin Analogue
38 Ile Leu Arg Tyr Val Tyr Tyr Val Tyr Arg Arg Lys 1 5 10 39 15 PRT
Artificial Sequence Indolicidin Analogue 39 Ile Leu Arg Trp Pro Trp
Trp Pro Trp Trp Pro Trp Arg Arg Lys 1 5 10 15 40 12 PRT Artificial
Sequence Indolicidin Analogue 40 Trp Trp Arg Trp Pro Trp Trp Pro
Trp Arg Arg Lys 1 5 10 41 13 PRT Artificial Sequence Indolicidin
Analogue 41 Ile Leu Arg Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys 1 5
10 42 12 PRT Artificial Sequence Indolicidin Analogue 42 Ile Leu
Arg Arg Trp Pro Trp Trp Pro Trp Arg Lys 1 5 10 43 12 PRT Artificial
Sequence Indolicidin Analogue 43 Ile Leu Lys Trp Pro Trp Trp Pro
Trp Arg Arg Lys 1 5 10 44 12 PRT Artificial Sequence Indolicidin
Analogue 44 Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg Lys 1 5 10
45 11 PRT Artificial Sequence Indolicidin Analogue 45 Ile Leu Lys
Trp Pro Trp Trp Pro Trp Arg Lys 1 5 10 46 12 PRT Artificial
Sequence Indolicidin Analogue 46 Lys Arg Arg Trp Pro Trp Trp Pro
Trp Arg Leu Ile 1 5 10 47 21 PRT Artificial Sequence Indolicidin
Analogue 47 Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys Ile Met
Ile Leu 1 5 10 15 Lys Lys Ala Gly Ser 20 48 21 PRT Artificial
Sequence Indolicidin Analogue 48 Ile Leu Arg Trp Pro Trp Trp Pro
Trp Arg Arg Lys Asp Met Ile Leu 1 5 10 15 Lys Lys Ala Gly Ser 20 49
14 PRT Artificial Sequence Indolicidin Analogue 49 Ile Leu Arg Trp
Pro Trp Arg Arg Trp Pro Trp Arg Arg Lys 1 5 10 50 28 PRT Artificial
Sequence Indolicidin Analogue 50 Ile Leu Arg Trp Pro Trp Trp Pro
Trp Arg Arg Lys Met Ile Leu Arg 1 5 10 15 Trp Pro Trp Trp Pro Trp
Arg Arg Lys Met Ala Ala 20 25 51 20 PRT Artificial Sequence
Indolicidin Analogue 51 Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg
Arg Met Ile Leu Lys 1 5 10 15 Lys Ala Gly Ser 20 52 21 PRT
Artificial Sequence Indolicidin Analogue 52 Ile Leu Lys Lys Trp Pro
Trp Trp Pro Trp Arg Arg Ile Met Ile Leu 1 5 10 15 Lys Lys Ala Gly
Ser 20 53 13 PRT Artificial Sequence Indolicidin Analogue 53 Ile
Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg Arg Met 1 5 10 54 14 PRT
Artificial Sequence Indolicidin Analogue 54 Ile Leu Lys Lys Trp Pro
Trp Trp Pro Trp Arg Arg Ile Met 1 5 10 55 11 PRT Artificial
Sequence Indolicidin Analogue 55 Ile Leu Lys Lys Trp Trp Trp Pro
Trp Arg Lys 1 5 10 56 11 PRT Artificial Sequence Indolicidin
Analogue 56 Ile Leu Lys Lys Trp Pro Trp Trp Trp Arg Lys 1 5 10 57
13 PRT Artificial Sequence Indolicidin Analogue 57 Ile Leu Lys Lys
Trp Val Trp Trp Val Trp Arg Arg Lys 1 5 10 58 13 PRT Artificial
Sequence Indolicidin Analogue 58 Ile Leu Lys Lys Trp Pro Trp Trp
Val Trp Arg Arg Lys 1 5 10 59 13 PRT Artificial Sequence
Indolicidin Analogue 59 Ile Leu Lys Lys Trp Val Trp Trp Pro Trp Arg
Arg Lys 1 5 10 60 12 PRT Artificial Sequence Indolicidin Analogue
60 Lys Arg Arg Trp Val Trp Trp Val Trp Arg Leu Ile 1 5 10 61 14 PRT
Artificial Sequence Indolicidin Analogue 61 Ile Leu Arg Trp Trp Val
Trp Trp Val Trp Trp Arg Arg Lys 1 5 10 62 8 PRT Artificial Sequence
Indolicidin Analogue 62 Leu Arg Trp Pro Trp Trp Pro Trp 1 5 63 9
PRT Artificial Sequence Indolicidin Analogue 63 Arg Trp Trp Trp Pro
Trp Arg Arg Lys 1 5 64 13 PRT Artificial Sequence Indolicidin
Analogue 64 Arg Arg Ile Trp Lys Pro Lys Trp Arg Leu Pro Lys Arg 1 5
10 65 12 PRT Artificial Sequence Indolicidin Analogue 65 Ile Leu
Lys Lys Trp Pro Trp Pro Trp Arg Arg Lys 1 5 10 66 11 PRT Artificial
Sequence Indolicidin Analogue 66 Ile Leu Trp Pro Trp Trp Pro Trp
Arg Arg Lys 1 5 10 67 12 PRT Artificial Sequence Indolicidin
Analogue 67 Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg Arg Lys 1 5 10
68 8 PRT Artificial Sequence Indolicidin Analogue 68 Pro Trp Trp
Pro Trp Arg Arg Lys 1 5 69 21 PRT Artificial Sequence Indolicidin
Analogue 69 Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg Arg Lys Met
Ile Leu 1 5 10 15 Lys Lys Ala Gly Ser 20 70 7 PRT Artificial
Sequence Indolicidin Analogue 70 Trp Trp Pro Trp Arg Arg Lys 1 5 71
7 PRT Artificial Sequence Indolicidin Analogue 71 Ile Leu Lys Lys
Trp Pro Trp 1 5 72 14 PRT Artificial Sequence Indolicidin Analogue
72 Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg Arg Lys Met 1 5 10
73 12 PRT Artificial Sequence Indolicidin Analogue 73 Ile Lys Lys
Trp Pro Trp Trp Pro Trp Arg Arg Lys 1 5 10 74 12 PRT Artificial
Sequence Indolicidin Analogue 74 Ile Leu Lys Lys Pro Trp Trp Pro
Trp Arg Arg Lys 1 5 10 75 12 PRT Artificial Sequence Indolicidin
Analogue 75 Ile Leu Lys Lys Trp Trp Trp Pro Trp Arg Arg Lys 1 5 10
76 12 PRT Artificial Sequence Indolicidin Analogue 76 Ile Leu Lys
Lys Trp Pro Trp Trp Trp Arg Arg Lys 1 5 10 77 12 PRT Artificial
Sequence Indolicidin Analogue 77 Ile Leu Lys Lys Trp Pro Trp Trp
Pro Arg Arg Lys 1 5 10 78 11 PRT Artificial Sequence Indolicidin
Analogue 78 Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Lys 1 5 10 79
11 PRT Artificial Sequence Indolicidin Analogue 79 Ile Leu Lys Lys
Trp Pro Trp Trp Pro Trp Arg 1 5 10 80 9 PRT Artificial Sequence
Indolicidin Analogue 80 Trp Pro Trp Trp Pro Trp Arg Arg Lys 1 5 81
10 PRT Artificial Sequence Indolicidin Analogue 81 Leu Trp Pro Trp
Trp Pro Trp Arg Arg Lys 1 5 10 82 12 PRT Artificial Sequence
Indolicidin Analogue 82 Ile Ala Arg Trp Pro Trp Trp Pro Trp Arg Arg
Lys 1 5 10 83 12 PRT Artificial Sequence Indolicidin Analogue 83
Ile Leu Ala Trp Pro Trp Trp Pro Trp Arg Arg Lys 1 5 10 84 12 PRT
Artificial Sequence Indolicidin Analogue 84 Ile Leu Arg Ala Pro Trp
Trp Pro Trp Arg Arg Lys 1 5 10 85 12 PRT Artificial Sequence
Indolicidin Analogue 85 Ile Leu Arg Trp Pro Ala Trp Pro Trp Arg Arg
Lys 1 5 10 86 12 PRT Artificial Sequence Indolicidin Analogue 86
Ile Leu Arg Trp Pro Trp Ala Pro Trp Arg Arg Lys 1 5 10 87 12 PRT
Artificial Sequence Indolicidin Analogue 87 Ile Leu Arg Trp Pro Trp
Trp Ala Trp Arg Arg Lys 1 5 10 88 12 PRT Artificial Sequence
Indolicidin Analogue 88 Ile Leu Arg Trp Pro Trp Trp Pro Ala Arg Arg
Lys 1 5 10 89 12 PRT Artificial Sequence Indolicidin Analogue 89
Ile Leu Arg Trp Pro Trp Trp Pro Trp Ala Arg Lys 1 5 10 90 12 PRT
Artificial Sequence Indolicidin Analogue 90 Ile Leu Arg Trp Pro Trp
Trp Pro Trp Arg Ala Lys 1 5 10 91 12 PRT Artificial Sequence
Indolicidin Analogue 91 Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg
Ala 1 5 10 92 4 PRT Artificial Sequence Octomeric branched lysine
core peptide 92 Lys Lys Lys Ala 1 93 13 PRT Artificial Sequence
Cationic Peptide Analogue 93 Ile Leu Lys Lys Phe Pro Phe Phe Pro
Phe Arg Arg Lys 1 5 10 94 13 PRT Artificial Sequence Cationic
Peptide Analogue 94 Ile Leu Arg Arg Trp Pro Trp Trp Pro Trp Arg Arg
Arg 1 5 10 95 13 PRT Artificial Sequence Cationic Peptide Analogue
95 Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg Arg Lys 1 5 10 96 18
PRT Artificial Sequence Cationic Peptide Analogue 96 Gly Asn Asn
Arg Pro Val Tyr Ile Pro Gln Pro Arg Pro Pro His Pro 1 5 10 15 Arg
Ile 97 25 PRT Artificial Sequence Cationic Peptide Analogue 97 Lys
Lys Ala Ala Ala Lys Ala Ala Ala Ala Ala Lys Ala Ala Trp Ala 1 5 10
15 Ala Lys Ala Ala Ala Lys Lys Lys Lys 20 25 98 13 PRT Artificial
Sequence Cationic Peptide Analogue 98 Ile Leu Pro Trp Lys Trp Pro
Trp Trp Pro Trp Arg Arg 1 5 10 99 13 PRT Artificial Sequence
Cationic Peptide Analogue 99 Ile Leu Lys Lys Trp Pro Trp Trp Pro
Trp Arg Arg Lys 1 5 10 100 13 PRT Artificial Sequence Cationic
Peptide Analogue 100 Ile Leu Lys Lys Phe Pro Phe Phe Pro Phe Arg
Arg Lys 1 5 10 101 11 PRT Artificial Sequence Cationic Peptide
Analogue 101 Ile Leu Trp Pro Trp Trp Pro Trp Arg Arg Lys 1 5 10 102
13 PRT Artificial Sequence Cationic Peptide Analogue 102 Ile Leu
Arg Arg Trp Pro Trp Trp Pro Trp Arg Arg Arg 1 5 10 103 13 PRT
Artificial Sequence Cationic Peptide Analogue 103 Ile Leu Lys Lys
Trp Pro Trp Trp Pro Trp Lys Lys Lys 1 5 10 104 28 PRT Artificial
Sequence Cationic Peptide Analogue 104 Ile Leu Arg Trp Pro Trp Trp
Pro Trp Arg Arg Lys Ile Leu Met Arg 1 5 10 15 Trp Pro Trp Trp Pro
Trp Arg Arg Lys Met Ala Ala 20 25 105 13 PRT Artificial Sequence
Cationic Peptide Analogue 105 Ile Leu Lys Lys Trp Ala Trp Trp Pro
Trp Arg Arg Lys 1 5 10 106 13 PRT Artificial Sequence Cationic
Peptide Analogue 106 Ile Leu Lys Lys Trp Pro Trp Trp Ala Trp Arg
Arg Lys 1 5 10 107 13 PRT Artificial Sequence Cationic Peptide
Analogue 107 Trp Trp Lys Lys Trp Pro Trp Trp Pro Trp Arg Arg Lys 1
5 10 108 14 PRT Artificial Sequence Cationic Peptide Analogue 108
Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg Arg Lys Met 1 5 10 109
13 PRT Artificial Sequence Cationic Peptide Analogue 109 Ile Leu
Lys Lys Trp Pro Trp Trp Pro Trp Arg Arg Met 1 5 10 110 14 PRT
Artificial Sequence Cationic Peptide Analogue 110 Ile Leu Lys Lys
Trp Pro Trp Trp Pro Trp Arg Arg Ile Met 1 5 10 111 12 PRT
Artificial Sequence Cationic Peptide Analogue 111 Cys Leu Arg Trp
Pro Trp Trp Pro Trp Arg Arg Lys 1 5 10 112 12 PRT Artificial
Sequence Cationic Peptide Analogue 112 Ile Leu Lys Lys Trp Pro Trp
Trp Pro Trp Arg Arg 1 5 10 113 11 PRT Artificial Sequence Cationic
Peptide Analogue 113 Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Lys 1
5 10 114 11 PRT Artificial Sequence Cationic Peptide Analogue 114
Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg 1 5 10 115 24 PRT
Artificial Sequence Cationic Peptide Analogue 115 Lys Lys Trp Trp
Arg Arg Val Leu Ser Gly Leu Lys Thr Ala Gly Pro 1 5 10 15 Ala Ile
Gln Ser Val Leu Asn Lys 20 116 24 PRT Artificial Sequence Cationic
Peptide Analogue 116 Lys Lys Trp Trp Arg Arg Ala Leu Gln Gly Leu
Lys Thr Ala Gly Pro 1 5 10 15 Ala Ile Gln Ser Val Leu Asn Lys 20
117 20 PRT Artificial Sequence Cationic Peptide Analogue 117 Lys
Lys Trp Trp Arg Arg Val Leu Lys Gly Leu Ser Ser Gly Pro Ala 1 5 10
15 Leu Ser Asn Val 20 118 20 PRT Artificial Sequence Cationic
Peptide Analogue 118 Lys Lys Trp Trp Arg Arg Ala Leu Gln Ala Leu
Lys Asn Gly Leu Pro 1 5 10 15 Ala Leu Ile Ser 20 119 26 PRT
Artificial Sequence Cationic Peptide Analogue 119 Lys Trp Lys Ser
Phe Ile Lys Lys Leu Thr Ser Ala Ala Lys Lys Val 1 5 10 15 Val Thr
Thr Ala Lys Pro Leu Ile Ser Ser 20 25 120 26 PRT Artificial
Sequence Cationic Peptide Analogue 120 Lys Trp Lys Leu Phe Lys Lys
Ile Gly Ile Gly Ala Val Leu Lys Val 1 5 10 15 Leu Thr Thr Gly Leu
Pro Ala Leu Ile Ser 20 25 121 28 PRT Artificial Sequence Cationic
Peptide Analogue 121 Lys Trp Lys Leu Phe Lys Lys Ile Gly Ile Gly
Ala Val Leu Lys Val 1 5 10 15 Leu Thr Thr Gly Leu Pro Ala Leu Lys
Leu Thr Lys 20 25 122 26 PRT Artificial Sequence Cationic Peptide
Analogue 122 Lys Trp Lys Ser Phe Ile Lys Lys Leu Thr Thr Ala Val
Lys Lys Val 1 5 10 15 Leu Thr Thr Gly Leu Pro Ala Leu Ile Ser 20 25
123 26 PRT Artificial Sequence Cationic Peptide Analogue 123 Lys
Trp Lys Ser Phe Ile Lys Asn Leu Thr Lys Val Leu Lys Lys Val 1 5 10
15 Val Thr Thr Ala Leu Pro Ala Leu Ile Ser 20 25 124 26 PRT
Artificial Sequence Cationic Peptide Analogue 124 Lys Trp Lys Ser
Phe Ile Lys Lys Leu Thr Ser Ala Ala Lys Lys Val 1 5 10 15 Leu Thr
Thr Gly Leu Pro Ala Leu Ile Ser 20 25 125 26 PRT Artificial
Sequence Cationic Peptide Analogue 125 Lys Trp Lys Leu Phe Ile Lys
Lys Leu Thr Pro Ala Val Lys Lys Val 1 5 10 15 Leu Leu Thr Gly Leu
Pro Ala Leu Ile Ser 20 25 126 18 PRT Artificial Sequence Cationic
Peptide Analogue 126 Gly Lys Pro Arg Pro Tyr Ser Pro Ile Pro Thr
Ser Pro Arg Pro Ile 1 5 10 15 Arg Tyr 127 12 PRT Artificial
Sequence Cationic Peptide Analogue 127 Arg Leu Ala Arg Ile Val Val
Ile Arg Val Ala Arg 1 5 10 128 26 PRT Artificial Sequence Fusion
Peptides 128 Lys Trp Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Val 1 5 10 15 Leu Thr Thr Gly Leu Pro Ala Leu Ile Ser 20 25
129 26 PRT Artificial Sequence Fusion Peptides 129 Lys Trp Lys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val 1 5 10 15 Val Thr
Thr Ala Lys Pro Leu Ile Ser Ser 20 25 130 26 PRT Artificial
Sequence Fusion Peptides 130 Lys Trp Lys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Ile 1 5 10 15 Leu Thr Thr Gly Leu Pro Ala
Leu Ile Ser 20 25 131 26 PRT Artificial Sequence Fusion Peptides
131 Lys Trp Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly
1 5 10 15 Gly Leu Leu Ser Asn Ile Val Thr Ser Leu 20 25 132 26 PRT
Artificial Sequence Fusion Peptides 132 Lys Trp Lys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly 1 5 10 15 Pro Ile Leu Ala Asn
Leu Val Ser Ile Val 20 25 133 20 PRT Artificial Sequence Fusion
Peptides 133 Lys Lys Trp Trp Arg Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Gly Pro Ala 1 5 10 15 Leu Ser Asn Val 20 134 30 PRT Artificial
Sequence Fusion Peptides 134 Lys Lys Trp Trp Arg Arg Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 135 29 PRT Artificial Sequence
Fusion Peptides 135 Lys Lys Trp Trp Arg Arg Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 20 25 136 28 PRT Artificial Sequence Fusion Peptides
136 Lys Lys Trp Trp Arg Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 137
27 PRT Artificial Sequence Fusion Peptides 137 Lys Lys Trp Trp Arg
Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 138 26 PRT Artificial
Sequence Fusion Peptides 138 Lys Lys Trp Trp Arg Arg Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 20 25 139 25 PRT Artificial Sequence Fusion Peptides
139 Lys Lys Trp Trp Arg Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 140 24 PRT
Artificial Sequence Fusion Peptides 140 Lys Lys Trp Trp Arg Arg Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 20 141 23 PRT Artificial Sequence Fusion Peptides 141
Lys Lys Trp Trp Arg Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 142 22 PRT Artificial Sequence
Fusion Peptides 142 Lys Lys Trp Trp Arg Arg Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa 20 143 21 PRT
Artificial Sequence Fusion Peptides 143 Lys Lys Trp Trp Arg Arg Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa
20 144 20 PRT Artificial Sequence Fusion Peptides 144 Lys Lys Trp
Trp Arg Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa
Xaa Xaa Xaa 20 145 29 PRT Artificial Sequence Fusion Peptides 145
Lys Lys Trp Trp Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 146
28 PRT Artificial Sequence Fusion Peptides 146 Lys Lys Trp Trp Lys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 147 27 PRT Artificial
Sequence Fusion Peptides 147 Lys Lys Trp Trp Lys Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 20 25 148 26 PRT Artificial Sequence Fusion
Peptides 148 Lys Lys Trp Trp Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25
149 25 PRT Artificial Sequence Fusion Peptides 149 Lys Lys Trp Trp
Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 150 24 PRT Artificial Sequence
Fusion Peptides 150 Lys Lys Trp Trp Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 151 23
PRT Artificial Sequence Fusion Peptides 151 Lys Lys Trp Trp Lys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 20 152 22 PRT Artificial Sequence Fusion Peptides 152
Lys Lys Trp Trp Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa Xaa 20 153 21 PRT Artificial Sequence
Fusion Peptides 153 Lys Lys Trp Trp Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa 20 154 20 PRT
Artificial Sequence Fusion Peptides 154 Lys Lys Trp Trp Lys Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa 20
155 19 PRT Artificial Sequence Fusion Peptides 155 Lys Lys Trp Trp
Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa
Xaa 156 34 PRT Apis mellifera 156 Tyr Val Pro Leu Pro Asn Val Pro
Gln Pro Gly Arg Arg Pro Phe Pro 1 5 10 15 Thr Phe Pro Gly Gln Gly
Pro Phe Asn Pro Lys Ile Lys Trp Pro Gln 20 25 30 Gly Tyr 157 34 PRT
Drosophila melanogaster 157 Val Phe Ile Asp Ile Leu Asp Lys Val Glu
Asn Ala Ile His Asn Ala 1 5 10 15 Ala Gln Val Gly Ile Gly Phe Ala
Lys Pro Phe Glu Lys Leu Ile Asn 20 25 30 Pro Lys 158 18 PRT Apis
mellifera 158 Gly Asn Asn Arg Pro Val Tyr Ile Pro Gln Pro Arg Pro
Pro His Pro 1 5 10 15 Arg Ile 159 18 PRT Apis mellifera 159 Gly Asn
Asn Arg Pro Val Tyr Ile Pro Gln Pro Arg Pro Pro His Pro 1 5 10 15
Arg Leu 160 18 PRT Apis mellifera 160 Gly Asn Asn Arg Pro Ile Tyr
Ile Pro Gln Pro Arg Pro Pro His Pro 1 5 10 15 Arg Leu 161 12 PRT
Bos taurus 161 Arg Leu Cys Arg Ile Val Val Ile Arg Val Cys Arg 1 5
10 162 42 PRT Bos taurus 162 Arg Phe Arg Pro Pro Ile Arg Arg Pro
Pro Ile Arg Pro Pro Phe Tyr 1 5 10 15 Pro Pro Phe Arg Pro Pro Ile
Arg Pro Pro Ile Phe Pro Pro Ile Arg 20 25 30 Pro Pro Phe Arg Pro
Pro Leu Arg Phe Pro 35 40 163 59 PRT Bos taurus 163 Arg Arg Ile Arg
Pro Arg Pro Pro Arg Leu Pro Arg Pro Arg Pro Arg 1 5 10 15 Pro Leu
Pro Phe Pro Arg Pro Gly Pro Arg Pro Ile Pro Arg Pro Leu 20 25 30
Pro Phe Pro Arg Pro Gly Pro Arg Pro Ile Pro Arg Pro Leu Pro Phe 35
40 45 Pro Arg Pro Gly Pro Arg Pro Ile Pro Arg Pro 50 55 164 37 PRT
Manduca sexta 164 Trp Asn Pro Phe Lys Glu Leu Glu Arg Ala Gly Gln
Arg Val Arg Asp 1 5 10 15 Ala Val Ile Ser Ala Ala Pro Ala Val Ala
Thr Val Gly Gln Ala Ala 20 25 30 Ala Ile Ala Arg Gly 35 165 37 PRT
Manduca sexta 165 Trp Asn Pro Phe Lys Glu Leu Glu Arg Ala Gly Gln
Arg Val Arg Asp 1 5 10 15 Ala Ile Ile Ser Ala Gly Pro Ala Val Ala
Thr Val Gly Gln Ala Ala 20 25 30 Ala Ile Ala Arg Gly 35 166 37 PRT
Manduca sexta 166 Trp Asn Pro Phe Lys Glu Leu Glu Arg Ala Gly Gln
Arg Val Arg Asp 1 5 10 15 Ala Ile Ile Ser Ala Ala Pro Ala Val Ala
Thr Val Gly Gln Ala Ala 20 25 30 Ala Ile Ala Arg Gly 35 167 37 PRT
Manduca sexta 167 Trp Asn Pro Phe Lys Glu Leu Glu Arg Ala Gly Gln
Arg Val Arg Asp 1 5 10 15 Ala Val Ile Ser Ala Ala Ala Val Ala Thr
Val Gly Gln Ala Ala Ala 20 25 30 Ile Ala Arg Gly Gly 35 168 24 PRT
Bombina variegata 168 Gly Ile Gly Ala Leu Ser Ala Lys Gly Ala Leu
Lys Gly Leu Ala Lys 1 5 10 15 Gly Leu Ala Glx His Phe Ala Asn 20
169 27 PRT Bombina orientalis 169 Gly Ile Gly Ala Ser Ile Leu Ser
Ala Gly Lys Ser Ala Leu Lys Gly 1 5 10 15 Leu Ala Lys Gly Leu Ala
Glu His Phe Ala Asn 20 25 170 27 PRT Bombina orientalis 170 Gly Ile
Gly Ser Ala Ile Leu Ser Ala Gly Lys Ser Ala Leu Lys Gly 1 5 10 15
Leu Ala Lys Gly Leu Ala Glu His Phe Ala Asn 20 25 171 17 PRT
Megabombus pennsylvanicus 171 Ile Lys Ile Thr Thr Met Leu Ala Lys
Leu Gly Lys Val Leu Ala His 1 5 10 15 Val 172 17 PRT Megabombus
pennsylvanicus 172 Ser Lys Ile Thr Asp Ile Leu Ala Lys Leu Gly Lys
Val Leu Ala His 1 5 10 15 Val 173 58 PRT Bos taurus 173 Arg Pro Asp
Phe Cys Leu Glu Pro Pro Tyr Thr Gly Pro Cys Lys Ala 1 5 10 15 Arg
Ile Ile Arg Tyr Phe Tyr Asn Ala Lys Ala Gly Leu Cys Gln Thr 20 25
30 Phe Val Tyr Gly Gly Cys Arg Ala Lys Arg Asn Asn Phe Lys Ser Ala
35 40 45 Glu Asp Cys Met Arg Thr Cys Gly Gly Ala 50 55 174 24 PRT
Rana esculenta 174 Phe Leu Pro Leu Leu Ala Gly Leu Ala Ala Asn Phe
Leu Pro Lys Ile 1 5 10 15 Phe Cys Lys Ile Thr Arg Lys Cys 20 175 33
PRT Rana esculenta 175 Gly Ile Met Asp Thr Leu Lys Asn Leu Ala Lys
Thr Ala Gly Lys Gly 1 5 10 15 Ala Leu Gln Ser Leu Leu Asn Lys Ala
Ser Cys Lys Leu Ser Gly Gln 20 25 30 Cys 176 37 PRT Hyalophora
cecropia 176 Lys Trp Lys Leu Phe Lys Lys Ile Glu Lys Val Gly Gln
Asn Ile Arg 1 5 10 15 Asp Gly Ile Ile Lys Ala Gly Pro Ala Val Ala
Val Val Gly Gln Ala 20 25 30 Thr Gln Ile Ala Lys 35 177 35 PRT
Hyalophora cecropia 177 Lys Trp Lys Val Phe Lys Lys Ile Glu Lys Met
Gly Arg Asn Ile Arg 1 5 10 15 Asn Gly Ile Val Lys Ala Gly Pro Ala
Ile Ala Val Leu Gly Glu Ala 20 25 30 Lys Ala Leu 35 178 40 PRT
Drosophila melanogaster 178 Gly Trp Leu Lys Lys Leu Gly Lys Arg Ile
Glu Arg Ile Gly Gln His 1 5 10 15 Thr Arg Asp Ala Thr Ile Gln Gly
Leu Gly Ile Ala Gln Gln Ala Ala 20 25 30 Asn Val Ala Ala Thr Ala
Arg Gly 35 40 179 36 PRT Hyalophora cecropia 179 Trp Asn Pro Phe
Lys Glu Leu Glu Lys Val Gly Gln Arg Val Arg Asp 1 5 10 15 Ala Val
Ile Ser Ala Gly Pro Ala Val Ala Thr Val Ala Gln Ala Thr 20 25 30
Ala Leu Ala Lys 35 180 31 PRT Sus scrofa 180 Ser Trp Leu Ser Lys
Thr Ala Lys Lys Leu Glu Asn Ser Ala Lys Lys 1 5 10 15 Arg Ile Ser
Glu Gly Ile Ala Ile Ala Ile Gln Gly Gly Pro Arg 20 25 30 181 37 PRT
Leiurus quin-questriatus hebraeus 181 Glx Phe Thr Asn Val Ser Cys
Thr Thr Ser Lys Glu Cys Trp Ser Val 1 5 10 15 Cys Gln Arg Leu His
Asn Thr Ser Arg Gly Lys Cys Met Asn Lys Lys 20 25 30 Cys Arg Cys
Tyr Ser 35 182 13 PRT Vespa crabo 182 Phe Leu Pro Leu Ile Leu Arg
Lys Ile Val Thr Ala Leu 1 5 10 183 35 PRT Mus musculus 183 Leu Arg
Asp Leu Val Cys Tyr Cys Arg Ser Arg Gly Cys Lys Gly Arg 1 5 10 15
Glu Arg Met Asn Gly Thr Cys Arg Lys Gly His Leu Leu Tyr Thr Leu 20
25 30 Cys Cys Arg 35 184 35 PRT Mus musculus 184 Leu Arg Asp Leu
Val Cys Tyr Cys Arg Thr Arg Gly Cys Lys Arg Arg 1 5 10 15 Glu Arg
Met Asn Gly Thr Cys Arg Lys Gly His Leu Met Tyr Thr Leu 20 25 30
Cys Cys Arg 35 185 33 PRT Oryctolagus cuniculus 185 Val Val Cys Ala
Cys Arg Arg Ala Leu Cys Leu Pro Arg Glu Arg Arg 1 5 10 15 Ala Gly
Phe Cys Arg Ile Arg Gly Arg Ile His Pro Leu Cys Cys Arg 20 25 30
Arg 186 33 PRT Oryctolagus cuniculus 186 Val Val Cys Ala Cys Arg
Arg Ala Leu Cys Leu Pro Leu Glu Arg Arg 1 5 10 15 Ala Gly Phe Cys
Arg Ile Arg Gly Arg Ile His Pro Leu Cys Cys Arg 20 25 30 Arg 187 31
PRT Cavia cutteri 187 Arg Arg Cys Ile Cys Thr Thr Arg Thr Cys Arg
Phe Pro Tyr Arg Arg 1 5 10 15 Leu Gly Thr Cys Ile Phe Gln Asn Arg
Val Tyr Thr Phe Cys Cys 20 25 30 188 31 PRT Cavia cutteri 188 Arg
Arg Cys Ile Cys Thr Thr Arg Thr Cys Arg Phe Pro Tyr Arg Arg 1 5 10
15 Leu Gly Thr Cys Leu Phe Gln Asn Arg Val Tyr Thr Phe Cys Cys 20
25 30 189 30 PRT Homo Sapien 189 Ala Cys Tyr Cys Arg Ile Pro Ala
Cys Ile Ala Gly Glu Arg Arg Tyr 1 5 10 15 Gly Thr Cys Ile Tyr Gln
Gly Arg Leu Trp Ala Phe Cys Cys 20 25 30 190 29 PRT Homo Sapien 190
Cys Tyr Cys Arg Ile Pro Ala Cys Ile Ala Gly Glu Arg Arg Tyr Gly 1 5
10 15 Thr Cys Ile Tyr Gln Gly Arg Leu Trp Ala Phe Cys Cys 20 25 191
33 PRT Oryctolagus cuniculus 191 Val Val Cys Ala Cys Arg Arg Ala
Leu Cys Leu Pro Arg Glu Arg Arg 1 5 10 15 Ala Gly Phe Cys Arg Ile
Arg Gly Arg Ile His Pro Leu Cys Cys Arg 20 25 30 Arg 192 33 PRT
Oryctolagus cuniculus 192 Val Val Cys Ala Cys Arg Arg Ala Leu Cys
Leu Pro Leu Glu Arg Arg 1 5 10 15 Ala Gly Phe Cys Arg Ile Arg Gly
Arg
Ile His Pro Leu Cys Cys Arg 20 25 30 Arg 193 32 PRT Rattus
norvegicus 193 Val Thr Cys Tyr Cys Arg Arg Thr Arg Cys Gly Phe Arg
Glu Arg Leu 1 5 10 15 Ser Gly Ala Cys Gly Tyr Arg Gly Arg Ile Tyr
Arg Leu Cys Cys Arg 20 25 30 194 32 PRT Rattus norvegicus 194 Val
Thr Cys Tyr Cys Arg Ser Thr Arg Cys Gly Phe Arg Glu Arg Leu 1 5 10
15 Ser Gly Ala Cys Gly Tyr Arg Gly Arg Ile Tyr Arg Leu Cys Cys Arg
20 25 30 195 38 PRT Bos taurus 195 Asp Phe Ala Ser Cys His Thr Asn
Gly Gly Ile Cys Leu Pro Asn Arg 1 5 10 15 Cys Pro Gly His Met Ile
Gln Ile Gly Ile Cys Phe Arg Pro Arg Val 20 25 30 Lys Cys Cys Arg
Ser Trp 35 196 40 PRT Bos taurus 196 Val Arg Asn His Val Thr Cys
Arg Ile Asn Arg Gly Phe Cys Val Pro 1 5 10 15 Ile Arg Cys Pro Gly
Arg Thr Arg Gln Ile Gly Thr Cys Phe Gly Pro 20 25 30 Arg Ile Lys
Cys Cys Arg Ser Trp 35 40 197 38 PRT Bos taurus 197 Asn Pro Val Ser
Cys Val Arg Asn Lys Gly Ile Cys Val Pro Ile Arg 1 5 10 15 Cys Pro
Gly Ser Met Lys Gln Ile Gly Thr Cys Val Gly Arg Ala Val 20 25 30
Lys Cys Cys Arg Lys Lys 35 198 40 PRT Sacrophaga peregrina 198 Ala
Thr Cys Asp Leu Leu Ser Gly Thr Gly Ile Asn His Ser Ala Cys 1 5 10
15 Ala Ala His Cys Leu Leu Arg Gly Asn Arg Gly Gly Tyr Cys Asn Gly
20 25 30 Lys Ala Val Cys Val Cys Arg Asn 35 40 199 38 PRT Aeschna
cyanea 199 Gly Phe Gly Cys Pro Leu Asp Gln Met Gln Cys His Arg His
Cys Gln 1 5 10 15 Thr Ile Thr Gly Arg Ser Gly Gly Tyr Cys Ser Gly
Pro Leu Lys Leu 20 25 30 Thr Cys Thr Cys Tyr Arg 35 200 38 PRT
Leiurus quinquestriatus 200 Gly Phe Gly Cys Pro Leu Asn Gln Gly Ala
Cys His Arg His Cys Arg 1 5 10 15 Ser Ile Arg Arg Arg Gly Gly Tyr
Cys Ala Gly Phe Phe Lys Gln Thr 20 25 30 Cys Thr Cys Tyr Arg Asn 35
201 32 PRT Phyllomedusa sauvagii 201 Ala Leu Trp Lys Thr Met Leu
Lys Lys Leu Gly Thr Met Ala Leu His 1 5 10 15 Ala Gly Lys Ala Ala
Leu Gly Ala Ala Asp Thr Ile Ser Gln Thr Gln 20 25 30 202 19 PRT
Drosophila melanogaster 202 Gly Lys Pro Arg Pro Tyr Ser Pro Arg Pro
Thr Ser His Pro Arg Pro 1 5 10 15 Ile Arg Val 203 46 PRT Rana
esculenta 203 Gly Ile Phe Ser Lys Leu Gly Arg Lys Lys Ile Lys Asn
Leu Leu Ile 1 5 10 15 Ser Gly Leu Lys Asn Val Gly Lys Glu Val Gly
Met Asp Val Val Arg 20 25 30 Thr Gly Ile Asp Ile Ala Gly Cys Lys
Ile Lys Gly Glu Cys 35 40 45 204 13 PRT Bos taurus 204 Ile Leu Pro
Trp Lys Trp Pro Trp Trp Pro Trp Arg Arg 1 5 10 205 25 PRT Bos
taurus 205 Phe Lys Cys Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly
Ala Pro 1 5 10 15 Ser Ile Thr Cys Val Arg Arg Ala Phe 20 25 206 34
PRT Lactococcus lactis 206 Ile Thr Ser Ile Ser Leu Cys Thr Pro Gly
Cys Lys Thr Gly Ala Leu 1 5 10 15 Met Gly Cys Asn Met Lys Thr Ala
Thr Cys His Cys Ser Ile His Val 20 25 30 Ser Lys 207 34 PRT
Staphylococcus epidermidis 207 Thr Ala Gly Pro Ala Ile Arg Ala Ser
Val Lys Gln Cys Gln Lys Thr 1 5 10 15 Leu Lys Ala Thr Arg Leu Phe
Thr Val Ser Cys Lys Gly Lys Asn Gly 20 25 30 Cys Lys 208 56 PRT
Bacillus subtilis 208 Met Ser Lys Phe Asp Asp Phe Asp Leu Asp Val
Val Lys Val Ser Lys 1 5 10 15 Gln Asp Ser Lys Ile Thr Pro Gln Trp
Lys Ser Glu Ser Leu Cys Thr 20 25 30 Pro Gly Cys Val Thr Gly Ala
Leu Gln Thr Cys Phe Leu Gln Thr Leu 35 40 45 Thr Cys Asn Cys Lys
Ile Ser Lys 50 55 209 37 PRT Leuconostoc gelidum 209 Lys Tyr Tyr
Gly Asn Gly Val His Cys Thr Lys Ser Gly Cys Ser Val 1 5 10 15 Asn
Trp Gly Glu Ala Phe Ser Ala Gly Val His Arg Leu Ala Asn Gly 20 25
30 Gly Asn Gly Phe Trp 35 210 23 PRT Xenopus laevis 210 Gly Ile Gly
Lys Phe Leu His Ser Ala Gly Lys Phe Gly Lys Ala Phe 1 5 10 15 Val
Gly Glu Ile Met Lys Ser 20 211 23 PRT Xenopus laevis 211 Gly Ile
Gly Lys Phe Leu His Ser Ala Lys Lys Phe Gly Lys Ala Phe 1 5 10 15
Val Gly Glu Ile Met Asn Ser 20 212 21 PRT Xenopus laevis 212 Gly
Met Ala Ser Lys Ala Gly Ala Ile Ala Gly Lys Ile Ala Lys Val 1 5 10
15 Ala Leu Lys Ala Leu 20 213 24 PRT Xenopus laevis 213 Gly Val Leu
Ser Asn Val Ile Gly Tyr Leu Lys Lys Leu Gly Thr Gly 1 5 10 15 Ala
Leu Asn Ala Val Leu Lys Gln 20 214 25 PRT Xenopus laevis 214 Gly
Trp Ala Ser Lys Ile Gly Gln Thr Leu Gly Lys Ile Ala Lys Val 1 5 10
15 Gly Leu Lys Glu Leu Ile Gln Pro Lys 20 25 215 14 PRT Vespula
lewisii 215 Ile Asn Leu Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu
1 5 10 216 26 PRT Apis mellifera 216 Gly Ile Gly Ala Val Leu Lys
Val Leu Thr Thr Gly Leu Pro Ala Leu 1 5 10 15 Ile Ser Trp Ile Lys
Arg Lys Arg Gln Gln 20 25 217 40 PRT Phormia terronovae 217 Ala Thr
Cys Asp Leu Leu Ser Gly Thr Gly Ile Asn His Ser Ala Cys 1 5 10 15
Ala Ala His Cys Leu Leu Arg Gly Asn Arg Gly Gly Tyr Cys Asn Gly 20
25 30 Lys Gly Val Cys Val Cys Arg Asn 35 40 218 39 PRT Phormia
terronovae 218 Ala Thr Cys Asp Leu Leu Ser Gly Thr Gly Ile Asn His
Ser Ala Cys 1 5 10 15 Ala Ala His Cys Leu Leu Arg Gly Asn Arg Gly
Gly Tyr Cys Asn Arg 20 25 30 Lys Gly Val Cys Val Arg Asn 35 219 18
PRT Limulus polyphemus 219 Arg Arg Trp Cys Phe Arg Val Cys Tyr Arg
Gly Phe Cys Tyr Arg Lys 1 5 10 15 Cys Arg 220 18 PRT Limulus
polyphemus 220 Arg Arg Trp Cys Phe Arg Val Cys Tyr Lys Gly Phe Cys
Tyr Arg Lys 1 5 10 15 Cys Arg 221 18 PRT Sus scrofa 221 Arg Gly Gly
Arg Leu Cys Tyr Cys Arg Arg Arg Phe Cys Val Cys Val 1 5 10 15 Gly
Arg 222 16 PRT Sus scrofa 222 Arg Gly Gly Arg Leu Cys Tyr Cys Arg
Arg Arg Phe Cys Ile Cys Val 1 5 10 15 223 18 PRT Sus scrofa 223 Arg
Gly Gly Gly Leu Cys Tyr Cys Arg Arg Arg Phe Cys Val Cys Val 1 5 10
15 Gly Arg 224 51 PRT Apis mellifera 224 Val Thr Cys Asp Leu Leu
Ser Phe Lys Gly Gln Val Asn Asp Ser Ala 1 5 10 15 Cys Ala Ala Asn
Cys Leu Ser Leu Gly Lys Ala Gly Gly His Cys Glu 20 25 30 Lys Gly
Val Cys Ile Cys Arg Lys Thr Ser Phe Lys Asp Leu Trp Asp 35 40 45
Lys Tyr Phe 50 225 39 PRT Sacrophaga peregrina 225 Gly Trp Leu Lys
Lys Ile Gly Lys Lys Ile Glu Arg Val Gly Gln His 1 5 10 15 Thr Arg
Asp Ala Thr Ile Gln Gly Leu Gly Ile Ala Gln Gln Ala Ala 20 25 30
Asn Val Ala Ala Thr Ala Arg 35 226 39 PRT Sacrophaga peregrina 226
Gly Trp Leu Lys Lys Ile Gly Lys Lys Ile Glu Arg Val Gly Gln His 1 5
10 15 Thr Arg Asp Ala Thr Ile Gln Val Ile Gly Val Ala Gln Gln Ala
Ala 20 25 30 Asn Val Ala Ala Thr Ala Arg 35 227 47 PRT Bos taurus
227 Ser Asp Glu Lys Ala Ser Pro Asp Lys His His Arg Phe Ser Leu Ser
1 5 10 15 Arg Tyr Ala Lys Leu Ala Asn Arg Leu Ala Asn Pro Lys Leu
Leu Glu 20 25 30 Thr Phe Leu Ser Lys Trp Ile Gly Asp Arg Gly Asn
Arg Ser Val 35 40 45 228 17 PRT Tachypleus tridentatus 228 Lys Trp
Cys Phe Arg Val Cys Tyr Arg Gly Ile Cys Tyr Arg Arg Cys 1 5 10 15
Arg 229 17 PRT Tachypleus tridentatus 229 Arg Trp Cys Phe Arg Val
Cys Tyr Arg Gly Ile Cys Tyr Arg Lys Cys 1 5 10 15 Arg 230 46 PRT
Hordeum vulgare 230 Lys Ser Cys Cys Lys Asp Thr Leu Ala Arg Asn Cys
Tyr Asn Thr Cys 1 5 10 15 Arg Phe Ala Gly Gly Ser Arg Pro Val Cys
Ala Gly Ala Cys Arg Cys 20 25 30 Lys Ile Ile Ser Gly Pro Lys Cys
Pro Ser Asp Tyr Pro Lys 35 40 45 231 23 PRT Trimeresurus wagleri
231 Gly Gly Lys Pro Asp Leu Arg Pro Cys Ile Ile Pro Pro Cys His Tyr
1 5 10 15 Ile Pro Arg Pro Lys Pro Arg 20 232 63 PRT Androctonus
australis hector 232 Val Lys Asp Gly Tyr Ile Val Asp Asp Val Asn
Cys Thr Tyr Phe Cys 1 5 10 15 Gly Arg Asn Ala Tyr Cys Asn Glu Glu
Cys Thr Lys Leu Lys Gly Glu 20 25 30 Ser Gly Tyr Cys Gln Trp Ala
Ser Pro Tyr Gly Asn Ala Cys Tyr Cys 35 40 45 Lys Leu Pro Asp His
Val Arg Thr Lys Gly Pro Gly Arg Cys His 50 55 60
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