U.S. patent application number 10/127691 was filed with the patent office on 2003-01-23 for recombinant c140 receptor, its agonists and antagonists, and nucleic acids encoding the receptor.
This patent application is currently assigned to COR THERAPEUTICS, INC.. Invention is credited to Scarborough, Robert M., Sundelin, Johan.
Application Number | 20030018184 10/127691 |
Document ID | / |
Family ID | 26793802 |
Filed Date | 2003-01-23 |
United States Patent
Application |
20030018184 |
Kind Code |
A1 |
Sundelin, Johan ; et
al. |
January 23, 2003 |
Recombinant C140 receptor, its agonists and antagonists, and
nucleic acids encoding the receptor
Abstract
Nucleic acid molecules encoding the C140 cell surface receptor
have been cloned and sequenced. The availability of C140 receptor
DNA permits the recombinant production of the C140 receptor which
can be produced on the surface of a cell, including an oocyte. The
nucleic acid molecules are useful in an assay for detecting a
substance which affects C140 receptor activity, either receptor
agonists or antagonists. Further, the elucidation of the structure
of the C140 receptor permits the design of agonist and antagonist
compounds which are useful in such assays. The availability of the
C140 receptor also permits production of antibodies specifically
immunoreactive with one or more antigenic epitopes of the C140
receptor.
Inventors: |
Sundelin, Johan; (Furulund,
SE) ; Scarborough, Robert M.; (Belmont, CA) |
Correspondence
Address: |
MORGAN LEWIS & BOCKIUS LLP
1111 PENNSYLVANIA AVENUE NW
WASHINGTON
DC
20004
US
|
Assignee: |
COR THERAPEUTICS, INC.
|
Family ID: |
26793802 |
Appl. No.: |
10/127691 |
Filed: |
April 23, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10127691 |
Apr 23, 2002 |
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08474414 |
Jun 7, 1995 |
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08474414 |
Jun 7, 1995 |
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08390301 |
Jan 25, 1995 |
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08390301 |
Jan 25, 1995 |
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08097938 |
Jul 26, 1993 |
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5629174 |
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Current U.S.
Class: |
536/23.5 ;
435/320.1; 435/325; 435/455; 435/69.1; 530/350 |
Current CPC
Class: |
C07K 7/08 20130101; C07K
14/705 20130101; A61K 48/00 20130101; A61K 38/00 20130101; C07K
14/723 20130101; G01N 2333/726 20130101; C07K 7/06 20130101; G01N
2500/00 20130101 |
Class at
Publication: |
536/23.5 ;
435/69.1; 435/320.1; 435/325; 530/350; 435/455 |
International
Class: |
C07K 014/715; C07H
021/04; C12P 021/02; C12N 005/06 |
Claims
1. A DNA molecule comprising an expression system capable, when
transformed into a recombinant host, of producing the C140 receptor
at the cell surface of the host, which expression system comprises
a nucleotide sequence encoding the C140 receptor operably linked to
a control sequence heterologous to said encoding nucleotide and
operable in said host cell.
2. A cell modified to contain the expression system of claim 1.
3. A method to produce cells that contain C140 receptor deployed at
their surface, which method comprises culturing the cells of claim
2 under conditions which effect the expression of the nucleotide
sequence encoding the C140 receptor to obtain said cells that
contain C140 receptor deployed at their surface.
4. A cRNA molecule that encodes the C140 receptor.
5. Cells which are oocytes modified to contain the cRNA of claim
4.
6. A method to produce cells which are oocytes that contain C140
receptor deployed at their surface, which method comprises
culturing the oocytes of claim 5 under conditions which effect the
expression of the cRNA encoding the C140 receptor to obtain said
cells that contain C140 receptor deployed at their surface.
7. A method to determine the C140 agonist activity of a candidate
substance, which method comprises: incubating the cells of claim 3
or 6 in the presence and absence of the substance, and detecting
the presence, absence or amount of activation of the C140 receptor
in the presence as compared to the absence of said substance
whereby an increase in said activation in the presence as compared
to the absence of said substance indicates agonist activity of the
substance.
8. A method to assess the ability of a candidate substance to
behave as a C140 antagonist, which method comprises: incubating the
cells of claim 3 or 6 in the presence of a C140 agonist and in the
presence and absence of said candidate, and measuring the
activation of the C140 receptor in the presence and absence of said
candidate, whereby a decrease in said activation in the presence of
the candidate indicates the antagonist activity of the
candidate.
9. A method to assess the ability of a candidate substance to bind
to C140 receptor, which method comprises: incubating the cells of
claim 3 or 6 in the presence of a C140 agonist or a known C140
antagonist and in the presence and absence of said candidate, and
measuring the binding of said C140 agonist or C140 antagonist to
the surface of said cells in the presence and absence of said
candidate, whereby a decrease in said binding in the presence of
the candidate indicates the ability of the candidate to bind
receptor.
10. An antibody composition specifically immunoreactive with an
extracellular region of the C140 receptor protein or a portion
thereof.
11. The antibody composition of claim 10 wherein said region is the
ligand-binding region, or which is specifically immunoreactive with
activated C140 receptor, or recognizes an epitope within the
receptor sequence SLIGRL, or is specifically reactive with the
cleaved activation peptide of the C140 receptor.
12. A method to localize activated C140 receptors in situ, which
method comprises: administering to a subject putatively harboring
activated C140 receptor an amount of antibody specific to said
activated receptor effective to bind to said activated receptor,
and detecting the location of said antibody.
13. A method for detecting the presence of activated C140 receptor
in a mammalian subject, which method comprises: contacting a sample
of the biological fluid of said subject with a detection system
which measures the presence, absence or amount of the cleaved
activation peptide of the C140 receptor; and detecting the
presence, absence or amount of said cleaved peptide.
14. An agonist peptide capable of activating C140 receptor, which
peptide is of the formula
AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-AA.sub.5-AA.sub.6-A- A.sub.7-Z
(1) wherein AA.sub.1 is a small amino acid or threonine; AA.sub.2
and AA.sub.3 are each independently neutral/nonpolar/large/nonar-
omatic amino acids; AA.sub.4 is a small amino acid; AA.sub.5 is a
basic amino acid; AA.sub.6 may be present or absent and, if
present, is a neutral/nonpolar/large/nonaromatic amino acid;
AA.sub.7 is absent if AA.sub.6 is absent and may be present or
absent if AA.sub.6 is present, and is an acidic amino acid; and Z
is a substituent that does not interfere with agonist activity.
15. The peptide of claim 14 wherein AA.sub.1 is ser, ala, gly, thr,
or 2,3-diamino-propionic (2,3-diaP); and/or wherein each of
AA.sub.2 and AA.sub.3 is independently selected from the group
consisting of ile, val, leu, and Cha; and/or wherein AA.sub.4 is
Gly; and/or wherein AA.sub.5 is Arg, Lys or Har; and/or wherein Z
comprises OR', or NR'R' wherein each R' is independently H or is a
straight or branched chain alkyl or 1-6C, wherein each R' may
optionally be substituted with one or more substituents selected
from the group consisting of --OR', --NR'R', and --NR'CNR'NR'R'
wherein each R' is H or is a straight or branched chain alkyl of
1-6C.
16. The peptide of claim 15 wherein AA.sub.1-AA.sub.2-AA.sub.3 is
selected from the group consisting of SLI, SLL, SChaI, SChaL,
(2,3-diaP)LI and (2,3-diaP)LL; and/or wherein Z includes additional
peptide sequence of 1-5 amino acids.
17. The peptide of claim 14 which is selected from the group
consisting of SLIGRLETQPPIT, SLIGRLETQPPI, SLIGRLETQPP, SLIGRLETQP,
SLIGRLETQ, SLIGRLET, SLIGRLE, SLIGRL, SLIGR, SLLGKVDGTSHVT,
SLLGKVDGTSHV, SLLGKVDGTSH, SLLGKVDGTS, SLLGKVDGT, SLLGKVDG,
SLLGKVD, SLLGKV, SLLGK, S(Cha)IGR, S(Cha)LGK, (2,3-diaP)-LIGR,
(2,3-diaP)LLGK, SLLGKR--NH.sub.2, SLIGRR--NH.sub.2,
S(Cha)LGKK--NH.sub.2, S(Cha)IGRK--NH.sub.2,
(2,3-diaP)-LIGRK--NH.sub.2, and (2,3-diaP)-LLGKK--NH.sub.2.
18. A peptide capable of inhibiting the function of the C140
receptor which peptide is of the formula
X-AA.sub.2-AA.sub.3-AA.sub.4-AA.sub.5-AA.- sub.6-AA.sub.7-Z wherein
X is an amino acid residue other than ser, ala, thr, cys, 2,3-diaP
or gly or is a desamino or acylated amino acid, wherein AA.sub.2
and AA.sub.3 are each independently
neutral/nonpolar/large/nonaromatic amino acids; AA.sub.4 is a small
amino acid; AA.sub.5 is a basic amino acid; AA.sub.6 may be present
or absent and, if present, is a neutral/nonpolar/large/nonaromatic
amino acid; AA.sub.7 is absent if AA.sub.6 is absent and may be
present or absent if AA.sub.6 is present, and is an acidic amino
acid; and Z is a substituent that does not interfere with agonist
activity.
19. The peptide of claim 18 wherein X is Mvl, Mpr, Mba, or SMeMpr;
and/or wherein each of AA.sub.2 and AA.sub.3 is independently
selected from the group consisting of ile, val, leu, Nle, Nva,
Cyclopentylalanine and Cha; and/or wherein AA.sub.4 is Gly; and/or
wherein AA.sub.5 is Arg, Lys, Orn or Har; and/or wherein Z
comprises OH or an ester or salt thereof, or NR'R' wherein each R'
is independently H or is a straight or branched chain alkyl of
1-6C, wherein each R' may optionally be substituted with one or
more substituents selected from the group consisting of --OR',
--NR'R', and --NR'CNR'NR'R' wherein each R' is H or is a straight
or branched chain alkyl of 1-6C.
20. The peptide of claim 19 wherein AA.sub.2-AA.sub.3 is selected
from the group consisting of LI, LL, ChaI, and ChaL; and/or wherein
Z includes a peptide extension of 1-5 amino acid residues.
21. The peptide of claim 18 which is selected from the group
consisting of Mpr-LLGK, Mpr-LIGR, Mpr-(Cha)LKG, Mpr-(Cha)IGR,
Mpr-LLGKK-NH.sub.2, Mpr-LIGRK-NH.sub.2, Mpr-LIGRKETQP-NH.sub.2,
Mpr-LLGKKDGTS-NH.sub.2,
(n-pentyl).sub.2-N-Leu-Ile-Gly-Arg-Lys-NH.sub.2 and
(Me-N-(n-pentyl)-Leu-Ile-Gly-Arg-Lys-NH2, and the amidated or
acylated forms thereof.
22. An isolated nucleic acid molecule which encodes a C140 receptor
polypeptide or which is complementary to a DNA or RNA molecule
encoding a C140 receptor polypeptide.
23. The nucleic acid molecule of claim 22 wherein said C140
receptor is the human C140 receptor.
24. A method to inhibit expression of C140 receptors in a cell
comprising providing to said cell an oligonucleotide molecule which
is antisense to, or forms a triple helix with, C140
receptor-encoding DNA or with DNA regulating expression of C140
receptor-encoding DNA, in an amount sufficient to inhibit
expression of said C140 receptors, thereby inhibiting said
expression.
25. A method to inhibit expression of C140 receptors in a subject,
comprising administering to said subject an oligonucleotide
molecule which is antisense to, or forms a triple helix with, C140
receptor-encoding DNA or with DNA regulating expression of C140
receptor-encoding DNA, in an amount sufficient to inhibit
expression of said C140 receptors in said subject, thereby
inhibiting said expression.
26. A pharmaceutical composition comprising an oligonucleotide
molecule of claim 25 together with a pharmaceutically acceptable
carrier or excipient.
Description
TECHNICAL FIELD
[0001] The invention relates to a newly discovered receptor which
is a member of the G-protein-coupled receptor superfamily. The
receptor is expressed in endothelial cells in blood vessels.
Avoidance of effects on this receptor is an essential element in
limiting side effects of drugs which are administered to stimulate
other receptors in this family. The invention also relates to
nucleic acid sequences encoding the receptor protein or
peptide.
BACKGROUND ART
[0002] Responses of animals to many therapeutic and prophylactic
drugs are mediated through receptors which reside on cell surfaces.
One class of such receptors comprises the G-protein-coupled
receptors, whose physiological effect is mediated by a
three-subunit protein complex, called G-proteins, that binds to
this type of receptor with the subsequent release of a subunit,
thus setting in motion additional intracellular events. Receptors
of this subclass include, among others, adrenergic receptors,
neuropeptide receptors, the thrombin receptor and the C140 receptor
which is the subject of the herein invention. This class of
receptor is characterized by the presence of seven transmembrane
regions which anchor the receptor within the cell surface.
[0003] It is the elusive goal of the designers of therapeutic
substances to effect a desired response in a subject in the absence
of side effects. Accordingly, pharmaceuticals designed to target a
specific receptor, such as the thrombin receptor, should react with
the thrombin receptor specifically and have no effect on related
receptors. The C140 receptor of the present invention may be
involved in controlling vascular pressure, and inadvertent
stimulation or blocking of this receptor would have unpredictable
and therefore undesirable results. It is therefore useful to
determine in advance whether therapeutic reagents designed to
target, for example, the thrombin receptor will or will not have
the undesired side effect of reactivity with the C140 receptor. By
providing the recombinant materials for the production of the C140
receptor in convenient assay systems, as well as agonist and
antagonist reagents for use in this assay, the invention makes
possible the prior determination of the presence or absence of the
side effect of reactivity with the C140 receptor in candidate
pharmaceuticals. This side effect will usually be undesired as it
is believed that the C140 receptor responds to enzymes such as
serine proteases associated with trauma and immune
disturbances.
DISCLOSURE OF THE INVENTION
[0004] The invention provides methods and materials useful in assay
systems to determine the propensity of candidate pharmaceuticals to
exert undesirable side effects. The isolation, recombinant
production and characterization of the C140 receptor permits the
design of assay systems using the receptor as a substrate and using
agonists and antagonists for the receptor as control reagents in
the assay.
[0005] Thus, in one aspect, the invention is directed to
recombinant materials associated with the production of C140
receptor. These include, for example, transfected cells which can
be cultured so as to display the C140 receptor on their surfaces,
and thus provide an assay system for the interaction of materials
with the native C140 receptor. In general, the limitations on the
host cells useful in these assay systems are that the cells have
the appropriate mechanism to display the receptor on their surfaces
and contain the G-protein as mediator to the intracellular
response. (However assays which merely assess binding do not
require the G-protein.) Most animal cells meet these
requirements.
[0006] In another aspect, the invention is directed to C140
receptor agonists which mimic the activated form of the
extracellular portion of the receptor protein. These agonists are
useful as control reagents in the above-mentioned assays to verify
the workability of the assay system. In addition, agonists for the
C140 receptor may exhibit hypotensive effects in vivo. Accordingly,
the agonists may be also, themselves, useful as
antihypertensives.
[0007] In still another aspect, the invention is directed to C140
receptor antagonists. These antagonists comprise modified forms of
the C140 receptor agonist peptides that lack the essential features
required for activation of the receptor. These antagonists bind to
receptor, do not activate it, and prevent receptor activation by
agonists and the native receptor-binding ligand.
[0008] A second group of antagonists includes antibodies designed
to bind specific portions of the receptor protein. In general,
these are monoclonal antibody preparations which are highly
specific for any desired region of the C140 receptor. The
antibodies of the invention are also useful in immunoassays for the
receptor protein, for example, in assessing successful expression
of the gene in recombinant systems.
[0009] Another aspect of the invention is to provide nucleic acids
encoding such a C140 receptor polypeptide and to use this nucleic
acid to produce the polypeptide in recombinant cell culture for
diagnostic use or for potential therapeutic use in hemostatic or
immune response regulation.
[0010] In still other aspects, the invention provides an isolated
nucleic acid molecule encoding a C140 receptor, labeled or
unlabeled, and a nucleic acid sequence that is complementary to, or
hybridizes under stringent conditions to, a nucleic acid sequence
encoding a C140 receptor. The isolated nucleic acid molecule of the
present invention excludes nucleic acid sequences which encode, or
are complementary to nucleic acid sequences encoding, other known G
protein-coupled receptors which are not C140 receptors, such as
adrenergic receptors, neuropeptide receptors, thrombin receptors,
and the like.
[0011] In addition, the invention provides a replicable vector
comprising a nucleic acid molecule encoding a C140 receptor
operably linked to control sequences recognized by a host
transformed by the vector; host cells transformed with the vector;
and a method of using a nucleic acid molecule encoding a C140
receptor to effect the production of a C140 receptor, comprising
expressing the nucleic acid molecule in a culture of the
transformed host cells and recovering a C140 receptor from the host
cell culture. The nucleic acid sequence is also useful in
hybridization assays for C140 receptor-encoding nucleic acid
molecules.
[0012] In still further embodiments, the invention provides a
method for producing C140 receptors comprising inserting into the
DNA of a cell containing the nucleic acid sequence encoding a C140
receptor a transcription modulatory element in sufficient proximity
and orientation to the C140 receptor coding sequence to influence
transcription thereof, with an optional further step comprising
culturing the cell containing the transcription modulatory element
and the C140 receptor-encoding nucleic acid sequence.
[0013] In still further embodiments, the invention provides a cell
comprising a nucleic acid sequence encoding a C140 receptor and an
exogenous transcription modulatory element in sufficient proximity
and orientation to the above coding sequence to influence
transcription thereof; and a host cell containing the nucleic acid
sequence encoding a C140 receptor operably linked to exogenous
control sequences recognized by the host cell.
[0014] Still further is provided a method for obtaining cells
having increased or decreased transcription of the nucleic acid
molecule encoding a C140 receptor, comprising:
[0015] (a) providing cells containing the nucleic acid
molecule;
[0016] (b) introducing into the cells a transcription modulating
element; and
[0017] (c) screening the cells for a cell in which the
transcription of the nucleic acid molecule is increased or
decreased.
[0018] In another aspect, the invention is related to assay systems
which utilize recombinant C140 receptor to screen for agonist and
antagonist activity of candidate drugs. This assay is especially
useful in assuring that these therapeutic agents do not have
undesired side effects caused by activation or inhibition of the
C140 receptor. In some cases agonist activity at this receptor
system may have therapeutic utility. Some of these assay systems
include the use of the agonist peptides as positive controls. The
assay can also be used to screen for antagonists which inhibit the
agonistic effect.
[0019] Another aspect of the invention relates to the diagnosis of
conditions characterized by activation of the C140 receptor by
detection in fluids, such as blood or urine, of the peptide cleaved
from the C140 receptor when the receptor is activated. Another
diagnostic method included in the invention is visualization of the
activated forms of receptor by localizing an imaging agent to
activated receptor in situ using antibodies specific to the
activated receptor.
[0020] Yet another aspect of this invention relates to the
therapeutic, prophylactic and research uses of various techniques
to block or modulate the expression of a C140 receptor by
interfering with the transcription of translation of a DNA or RNA
molecule encoding the C140 receptor. This includes a method to
inhibit or regulate expression of C140 receptors in a cell
comprising providing to the cell an oligonucleotide molecule which
is antisense to, or forms a triple helix with, C140
receptor-encoding DNA or with DNA regulating expression of C140
receptor-encoding DNA, in an amount sufficient to inhibit or
regulate expression of the C140 receptors, thereby inhibiting or
regulating their expression. Also included is a method to inhibit
or regulate expression of C140 receptors in a subject, comprising
administering to the subject an oligonucleotide molecule which is
antisense to, or forms a triple helix with, C140 receptor-encoding
DNA or with DNA regulating expression of C140 receptor-encoding
DNA, in an amount sufficient to inhibit or regulate expression of
the C140 receptors in the subject, thereby inhibiting or regulating
their expression. The antisense molecule or triple helix-forming
molecule in the above methods is preferably a DNA or RNA
oligonucleotide.
[0021] Additional aspects of the invention are directed to
pharmaceutical compositions containing the agonists and antagonists
of the invention. The agonists of the invention are
antihypertensives; conversely, the antagonists can elevate blood
pressure if desired. Other aspects of the invention include a
pharmaceutical composition useful for inhibiting or regulating C140
receptor expression in a cell or in a subject at the level of
transcription or translation, which composition comprises an
antisense or triple helix-forming molecule as described above which
corresponds to a portion of the sequence of the C140
receptor-coding nucleic acid.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIGS. 1A-1B show the DNA and deduced amino acid sequence of
murine C140 receptor.
[0023] FIGS. 2A-2B show the DNA and deduced amino acid sequence of
human C140 receptor.
[0024] FIG. 3 shows a comparison of amino acid sequences for the
human C140 receptor and murine C140 receptor.
[0025] FIG. 4 shows a proposed model of C140 receptor activation
based on the deduced amino acid sequence.
[0026] FIG. 5 shows a comparison of amino acid sequences for the
mouse C140 receptor and the human thrombin receptor.
[0027] FIG. 6 shows the results of Northern Blot to detect the
presence of mRNA encoding C140 receptor in various mouse
tissues.
[0028] FIG. 7 shows a trace of blood pressure demonstrating the in
vivo hypotensive effect of a C140 agonist peptide.
[0029] FIGS. 8a-8c show blood vessel dilation in rat femoral vein
induced by a C140 receptor agonist peptide.
[0030] FIG. 8a shows these results in the immobilized vein;
[0031] FIG. 8b shows these results for the immobilized vein
depleted of endothelial cells.
[0032] FIGS. 9a-9c show the results of an assay for activation of
the C140 receptor, expressed in frog oocytes, by plasmin,
kallikrein, or trypsin. FIG. 9a shows the results for plasmin; FIG.
9b shows the results for kallikrein; FIG. 9c shows the results for
trypsin.
[0033] FIGS. 10A-10B show the nucleotide sequence and deduced amino
acid sequence of a cDNA clone encoding murine C140 receptor.
[0034] FIGS. 11A-11B show the nucleotide sequence and deduced amino
acid sequence of a cDNA clone encoding human C140 receptor.
[0035] FIG. 12 shows the results of in situ hybridization of a
sectioned newborn mouse with mouse C140 receptor probes.
[0036] FIG. 13 shows a Northern blot of total RNA from human cell
lines hybridized to a human C140 receptor probe.
MODES OF CARRYING OUT THE INVENTION
[0037] The characteristics of the C140 receptor elucidated by the
invention herein are summarized in FIGS. 1A/1B-4. FIGS. 1A-1B shows
the complete DNA sequence of the clone encoding the murine
receptor, along with the deduced amino acid sequence. As used
herein, the "C140 receptor" refers to receptor in any animal
species corresponding to the murine receptor contained in clone
C140 described in Example 1 herein. Using the native DNA encoding
the murine form of this receptor, the corresponding receptors in
other species, including humans, as illustrated herein, may be
obtained. FIGS. 2A-2B shows the corresponding DNA and deduced amino
acid sequence of the human receptor.
[0038] The entire amino acid sequence of the murine receptor
contains 395 amino acids, including a 27 amino acid signal peptide
which, when cleaved, results in a 368 amino acid mature receptor
protein. Similarly, the human receptor is encoded by an open
reading frame corresponding to 398 amino acids including a probable
29 amino acid signal peptide sequence resulting in a 369 amino acid
mature receptor protein, as shown in FIGS. 2A-2B.
[0039] FIG. 3 shows a comparison of the human and murine amino acid
sequences; as shown, these sequences exhibit a high degree of
homology.
[0040] Hydrophobicity/hydrophilicity plots of the sequences shown
in FIGS. 1A-1B and 2A-2B indicate that the mature C140 receptor is
a member of the 7-transmembrane domain receptor family whose effect
on the cell is mediated by G-protein. The mature C140 receptor has
a relatively long extracellular amino acid extension containing
several consensus sites for asparagine-linked glycosylation. It
also contains a conserved asparagine in the first transmembrane
region, the motif Leu-Ala-X-X-Asp in the second transmembrane
region, a Trp in the fourth transmembrane region and a carboxy
terminal tail which contains multiple serine and threonine
residues. A proposed model of the in situ receptor is shown in FIG.
4.
[0041] Referring to FIG. 5, similarities to the thrombin receptor
are readily seen. FIG. 5 compares the amino acid sequence of murine
C140 with that of thrombin receptor. It is known that the thrombin
receptor is activated by proteolytic cleavage of the Arg-Ser bond
at positions 41 and 42, which releases an activation peptide that
permits refolding of the receptor and activation via the newly
created amino terminus. In an analogous manner, the C140 receptor
is activated by cleavage of the Arg-Ser bond at positions 34 and
35, also liberating an activation peptide extending from position 1
of the putative mature protein to the cleavage site. It is believed
that Arg-28 is the amino terminal amino acid residue of the mature
protein, so the activation peptide has the sequence RNNSKGR. This
peptide could thus be used as an index for activation of C140
receptor. In any event, the precise location of the N-terminus of
the mature protein is unimportant for the design of agonists or
antagonists. The activation peptide is likely to be freely filtered
by the kidney and possibly concentrated in the urine and can be
used as an index to activation of the C140 receptor.
[0042] Release of the activation peptide permits refolding of the
receptor protein to activate the receptor. This is shown
schematically in FIG. 4, which also shows that the conformational
changes resulting from the liberation of the activation peptide and
refolding results in an intracellular conformational change of the
receptor. This hypothesis is confirmed by the finding that the C140
receptor can be activated by a peptide mimicking the new amino
terminus created by the activation. Accordingly, mimics of the
N-terminus of the new amino terminus on the activated receptor
behave as agonists therefor. The importance of the first five amino
acids in the newly created amino terminus in the receptor for
receptor activation has also been confirmed hereinbelow.
[0043] Based on this information, and by analogy with the
mechanisms underlying trypsinogen activation to trypsin and
activation of the thrombin receptor, it appears that the positively
charged amino group on serine that is newly exposed when the ligand
cleaves the receptor plays an important role in receptor
activation. Peptides based on the agonist peptide sequence that
bind the C140 receptor, but which are modified to be lacking the
free .alpha.-amino group can function as antagonists of this
receptor. Thus, modifications of the agonist peptides which lack
the capacity for specific activating interaction serve as C140
receptor antagonists.
[0044] Ordinarily, the C140 receptors and analogs thereof claimed
herein will have an amino acid sequence having at least 75% amino
acid sequence identity with a "common" C140 receptor sequence (such
as that disclosed in FIGS. 1A-1B or FIGS. 2A-2B), more preferably
at least 80%, even more preferably at least 90%, and most
preferably at least 95%. Identity or homology with respect to a
common sequence is defined herein as the percentage of amino acid
residues in the candidate sequence that are identical with the
known C140 receptor, after aligning the sequences and introducing
gaps, if necessary, to achieve the maximum percent homology, and
not considering any conservative substitutions as part of the
sequence identity. None of N-terminal, C-terminal or internal
extensions, deletions, or insertions into the C140 receptor
sequence shall be construed as affecting homology.
[0045] Thus, the claimed C140 receptor and analog molecules that
are the subject of this invention include molecules having the C140
receptor amino acid sequence; fragments thereof having a
consecutive sequence of at least 10, 15, 20, 25, 30 or 40 amino
acid residues from a common C140 receptor sequence; amino acid
sequence variants of a common C140 receptor sequence wherein an
amino acid residue has been inserted N- or C-terminal to, or
within, the C140 receptor sequence or its fragments as defined
above; amino acid sequence variants of the common C140 receptor
sequence or its fragment as defined above which have been
substituted by another residue. C140 receptor polypeptides include
those containing predetermined mutations by, e.g., homologous
recombination, site-directed or PCR mutagenesis, and C140 receptor
polypeptides of other animal species, including but not limited to
rabbit, rat, murine, porcine, bovine, ovine, equine and non-human
primate species, and alleles or other naturally occurring variants
of the C140 receptor of the foregoing species and of human
sequences; derivatives of the commonly known C140 receptor or its
fragments wherein the C140 receptor or its fragments have been
covalently modified by substitution, chemical, enzymatic, or other
appropriate means with a moiety other than a naturally occurring
amino acid (for example a detectable moiety such as an enzyme or
radioisotope); glycosylation variants of C140 receptor (insertion
of a glycosylation site or deletion of any glycosylation site by
deletion, insertion or substitution of appropriate amino acid); and
soluble forms of C140.
[0046] The novel proteins and peptides of the present invention are
preferably those which share a common biological activity with the
C140 receptor, including but not limited to an effector or receptor
function or cross-reactive antigenicity. Such fragments and
variants exclude any C140 receptor polypeptide heretofore made
public, including any known protein or polypeptide of any animal
species, which is otherwise anticipatory under 35 U.S.C. .sctn.102
as well as polypeptides obvious over such known protein or
polypeptides under 35 U.S.C. .sctn.103. Specifically, the present
C140 receptor proteins, analogs, fragments and variants exclude
other known G protein-coupled receptors which are not C140
receptors, such as adrenergic receptors, neuropeptide receptors,
thrombin receptors, and the like.
COMPOUNDS OF THE INVENTION
[0047] The nomenclature used to describe the peptide compounds of
the invention follows the conventional practice where the
N-terminal amino group is assumed to be to the left and the carboxy
group to the right of each amino acid residue in the peptide. In
the formulas representing selected specific embodiments of the
present invention, the amino- and carboxy-terminal groups, although
often not specifically shown, will be understood to be in the form
they would assume at physiological pH values, unless otherwise
specified. Thus, the N-terminal H.sup.+.sub.2 and C-terminal
O.sup.- at physiological pH are understood to be present though not
necessarily specified and shown, either in specific examples or in
generic formulas. Free functional groups on the side chains of the
amino acid residues can also be modified by amidation, acylation or
other substitution, which can, for example, change the solubility
of the compounds without affecting their activity.
[0048] In the peptides shown, each gene-encoded residue, where
appropriate, is represented by a single letter designation,
corresponding to the trivial name of the amino acid, in accordance
with the following conventional list:
1 One-Letter Three-letter Amino Acid Symbol Symbol Alanine A Ala
Arginine R Arg Asparagine N Asn Aspartic acid D Asp Cysteine C Cys
Glutamine Q Gln Glutamic acid E Glu Glycine G Gly Histidine H His
Isoleucine I Ile Leucine L Leu Lysine K Lys Methionine M Met
Phenylalanine F Phe Proline P Pro Serine S Ser Threonine T Thr
Tryptophan W Trp Tyrosine Y Tyr Valine V Val
[0049] The amino acids not encoded genetically are abbreviated as
indicated in the discussion below.
[0050] In the specific peptides shown in the present application,
the L-form of any amino acid residue having an optical isomer is
intended unless the D-form is expressly indicated by a dagger
superscript (.dagger.).
[0051] The compounds of the invention are peptides which are
partially defined in terms of amino acid residues of designated
classes. Amino acid residues can be generally subclassified into
four major subclasses as follows:
[0052] Acidic: The residue has a negative charge due to loss of H
ion at physiological pH and the residue is attracted by aqueous
solution so as to seek the surface positions in the conformation of
a peptide in which it is contained when the peptide is in aqueous
medium at physiological pH.
[0053] Basic: The residue has a positive charge due to association
with H ion at physiological pH and the residue is attracted by
aqueous solution so as to seek the surface positions in the
conformation of a peptide in which it is contained when the peptide
is in aqueous medium at physiological pH.
[0054] Neutral/nonpolar: The residues are not charged at
physiological pH and the residue is repelled by aqueous solution so
as to seek the inner positions in the conformation of a peptide in
which it is contained when the peptide is in aqueous medium. These
residues are also designated "hydrophobic" herein.
[0055] Neutral/polar: The residues are not charged at physiological
pH, but the residue is attracted by aqueous solution so as to seek
the outer positions in the conformation of a peptide in which it is
contained when the peptide is in aqueous medium.
[0056] It is understood, of course, that in a statistical
collection of individual residue molecules some molecules will be
charged, and some not, and there will be an attraction for or
repulsion from an aqueous medium to a greater or lesser extent. To
fit the definition of "charged," a significant percentage (at least
approximately 25%) of the individual molecules are charged at
physiological pH. The degree of attraction or repulsion required
for classification as polar or nonpolar is arbitrary and,
therefore, amino acids specifically contemplated by the invention
have been classified as one or the other. Most amino acids not
specifically named can be classified on the basis of known
behavior.
[0057] Amino acid residues can be further subclassified as cyclic
or noncyclic, and aromatic or nonaromatic, self-explanatory
classifications with respect to the side chain substituent groups
of the residues, and as small or large. The residue is considered
small if it contains a total of 4 carbon atoms or less, inclusive
of the carboxyl carbon. Small residues are, of course, always
nonaromatic.
[0058] For the naturally occurring protein amino acids,
subclassification according to the foregoing scheme is as
follows.
[0059] Acidic: Aspartic acid and Glutamic acid;
[0060] Basic/noncyclic: Arginine, Lysine;
[0061] Basic/cyclic: Histidine;
[0062] Neutral/polar/small: Glycine, serine, cysteine;
[0063] Neutral/nonpolar/small: Alanine;
[0064] Neutral/polar/large/nonaromatic: Threonine, Asparagine,
Glutamine;
[0065] Neutral/polar/large aromatic: Tyrosine;
[0066] Neutral/nonpolar/large/nonaromatic: Valine, Isoleucine,
Leucine, Methionine;
[0067] Neutral/nonpolar/large/aromatic: Phenylalanine, and
Tryptophan
[0068] The gene-encoded secondary amino acid proline, although
technically within the group neutral/nonpolar/large/cyclic and
nonaromatic, is a special case due to its known effects on the
secondary conformation of peptide chains, and is not, therefore,
included in this defined group.
[0069] Certain commonly encountered amino acids, which are not
encoded by the genetic code, include, for example, beta-alanine
(beta-Ala), or other omega-amino acids, such as 3-amino propionic,
2,3-diamino propionic (2,3-diaP), 4-amino butyric and so forth,
alpha-aminisobutyric acid (Aib), sarcosine (Sar), ornithine (Orn),
citrulline (Cit), t-butylalanine (t-BuA), t-butylglycine (t-BuG),
N-methylisoleucine (N-MeIle), phenylglycine (Phg), and
cyclohexylalanine (Cha), norleucine (Nle), cysteic acid (Cya)
2-naphthylalanine (2-Nal); 1,2,3,4-tetrahydroisoquinol-
ine-3-carboxylic acid (Tic); .beta.-2-thienylalanine (Thi); and
methionine sulfoxide (MSO). These also fall conveniently into
particular categories.
[0070] Based on the above definitions,
[0071] Sar, beta-Ala, 2,3-diaP and Aib are
neutral/nonpolar/small;
[0072] t-BuA, t-BuG, N-MeIle, Nle, Mvl and Cha are
neutral/nonpolar/large/- nonaromatic;
[0073] Orn is basic/noncyclic;
[0074] Cya is acidic;
[0075] Cit, Acetyl Lys, and MSO are
neutral/polar/large/nonaromatic; and
[0076] Phg, Nal, Thi and Tic are
neutral/nonpolar/large/aromatic.
[0077] The various omega-amino acids are classified according to
size as neutral/nonpolar/small (beta-Ala, i.e., 3-aminopropionic,
4-aminobutyric) or large (all others).
[0078] Other amino acid substitutions of those encoded in the gene
can also be included in peptide compounds within the scope of the
invention and can be classified within this general scheme
according to their structure.
[0079] All of the compounds of the invention, when an amino acid
forms the C-terminus, may be in the form of the pharmaceutically
acceptable salts or esters. Salts may be, for example, Na.sup.+,
K.sup.+, Ca.sup.+2, Mg.sup.+2 and the like; the esters are
generally those of alcohols of 1-6C.
[0080] In all of the peptides of the invention, one or more amide
linkages (--CO--NH--) may optionally be replaced with another
linkage which is an isostere such as --CH.sub.2NH--, --CH.sub.2S--,
--CH.sub.2CH.sub.2, --CH.dbd.CH-- (cis and trans), --COCH.sub.2--,
--CH(OH)CH.sub.2-- and --CH.sub.2SO--. This replacement can be made
by methods known in the art. The following references describe
preparation of peptide analogs which include these
alternative-linking moieties: Spatola, A. F., Vega Data (March
1983), Vol. 1, Issue 3, "Peptide Backbone Modifications" (general
review); Spatola, A. F., in "Chemistry and Biochemistry of Amino
Acids Peptides and Proteins," B. Weinstein, eds., Marcel Dekker,
New York, p. 267 (1983) (general review); Morley, J. S., Trends
Pharm Sci (1980) pp. 463-468 (general review); Hudson, D., et al.,
Int J Pept Prot Res (1979) 14:177-185 (--CH.sub.2NH--,
--CH.sub.2CH.sub.2--); Spatola, A. F., et al., Life Sci (1986)
38:1243-1249 (--CH.sub.2--S); Hann, M. M., J Chem Soc Perkin Trans
I (1982) 307-314 (--CH--CH--, cis and trans); Almquist, R. G., et
al., J Med Chem (1980) 23:1392-1398 (--COCH.sub.2--);
Jennings-White, C., et al., Tetrahedron Lett (1982) 23:2533
(--COCH.sub.2--); Szelke, M., et al., European Application EP 45665
(1982) CA:97:39405 (1982) (--CH(OH)CH.sub.2--); Holladay, M. W., et
al., Tetrahedron Lett (1983) 24:4401-4404 (--C(OH)CH.sub.2--); and
Hruby, V. J., Life Sci (1982) 31:189-199 (--CH.sub.2--S--)
[0081] A. Agonists
[0082] The agonists of the invention comprise a series of peptides
of the formula
AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-AA.sub.5-AA.sub.6-AA.sub.7-Z
(1)
[0083] wherein AA.sub.1 is a small amino acid or threonine;
[0084] AA.sub.2 and AA.sub.3 are each independently
neutral/nonpolar/large/nonaromatic amino acids;
[0085] AA.sub.4 is a small amino acid;
[0086] AA.sub.5 is a basic amino acid;
[0087] AA.sub.6 may be present or absent and, if present, is a
neutral/nonpolar/large/nonaromatic amino acid;
[0088] AA.sub.7 is absent if AA.sub.6 is absent and may be present
or absent if AA.sub.6 is present, and is an acidic amino acid;
and
[0089] Z is a substituent that does not interfere with agonist
activity.
[0090] The peptide of formula 1 can be extended (shown as included
in Z) at the C-terminus (but not the N-terminus) by further amino
acid sequence to comprise a noninterfering substituent.
[0091] At the C-terminus of the compounds of formula 1, the
carboxyl group may be in the underivatized form or may be amidated
or may be an ester; in the underivatized form the carboxyl may be
as a free acid or a salt, preferably a pharmaceutically acceptable
salt.
[0092] If the C-terminus is amidated, the nitrogen atom of the
amido group, covalently bound to the carbonyl carbon at the
C-terminus, will be NR'R', wherein each R' is independently
hydrogen or is a straight or branched chain alkyl of 1-6C, such
alkyls are 1-6C straight- or branched-chain saturated hydrocarbyl
residues, such as methyl, ethyl, isopentyl, n-hexyl, and the like.
Representatives of such amido groups are: --NH.sub.2, --NHCH.sub.3,
--N(CH.sub.3).sub.2, --NHCH.sub.2CH.sub.3,
--NHCH.sub.2CH(CH.sub.3).sub.2, and
--NHCH.sub.2CH(CH.sub.3)CH.sub.2CH.su- b.3, among others.
Furthermore, either or both R' may in turn optionally be
substituted by one or more substituents such as, for example,
--OR', --NR'R', halo, --NR'CNR'NR'R' and the like, wherein each R'
is as independently defined above. Thus, Z may be --OH, or an ester
(OR') or salt forms thereof, or --NR'R' wherein R' is as above
defined.
[0093] Preferred embodiments of AA.sub.1 are Ser on
2,3-diaminopropionyl (2,3-diaP). Preferred embodiments of AA.sub.2
and AA.sub.3 are Val, Ile, Cha and Leu. Preferred embodiments for
the residues in the remainder of the compound of formula (1) are
those wherein AA.sub.4 is Gly, AA.sub.5 is Lys, Arg or Har,
AA.sub.6, if present, is Val, Ile, Cha or Leu, and AA.sub.7, if
present, is Asp or Glu. Particularly preferred are compounds of
formula (1) which are selected from the group consisting of
SLIGRLETQPPIT, SLIGRLETQPPI, SLIGRLETQPP, SLIGRLETQP, SLIGRLETQ,
SLIGRLET, SLIGRLE, SLIGRL, SLIGR, SLLGKVDGTSHVT, SLLGKVDGTSHV,
SLLGKVDGTSH, SLLGKVDGTS, SLLGKVDGT, SLLGKVDG, SLLGKVD, SLLGKV,
SLLGK, S(Cha)IGR, S(Cha)LGK, (2,3-diaP)-IGR, (2,3-diaP)LLGK,
SLLGKR-NH.sub.2, SLIGRR-NH.sub.2, S(Cha)LGKK-NH.sub.2,
S(Cha)IGRK-NH.sub.2, (2,3-diaP)-LIGRK-NH.sub.2,
(2,3-diaP)-LLGKK-NH.sub.2 and the amidated forms thereof.
[0094] B. Antagonists
[0095] Compounds of the invention which interfere with activities
mediated by the C140 receptor include modified agonist peptides
lacking the N-terminal serine residue; and antibodies which are
immunoreactive with various critical positions on the C140
receptor.
[0096] Peptide Antagonists
[0097] The antagonists of the first group--modified agonists--can
be represented by the formula:
X-AA.sub.2-AA.sub.3-AA.sub.4-AA.sub.5-AA.sub.6-AA.sub.7-Z
[0098] wherein X is an amino acid residue other than ser, ala, thr,
cys, 2,3-diaP or gly or is a desamino or alkylated or acylated
amino acid,
[0099] wherein AA.sub.2 and AA.sub.3 are each independently
neutral/nonpolar/large/nonaromatic amino acids;
[0100] AA.sub.4 is a small amino acid;
[0101] AA.sub.5 is a basic amino acid;
[0102] AA.sub.6 may be present or absent and, if present, is a
neutral/nonpolar/large/nonaromatic amino acid;
[0103] AA.sub.7 is absent if AA.sub.6 is absent and may be present
or absent if AA.sub.6 is present, and is an acidic amino acid;
and
[0104] Z is a substituent that does not interfere with agonist
activity.
[0105] Preferred acyl groups are of the formula RCO-- wherein R
represents a straight or branched chain alkyl of 1-6C. Acetyl is
particularly preferred.
[0106] Preferred embodiments of X include residues of
3-mercaptopropionic acid (Mpr), 3-mercaptovaleric acid (Mvl),
2-mercaptobenzoic acid (Mba) and S-methyl-3-mercaptopropionic acid
(SMeMpr). Preferred embodiments for AA.sub.2 through AA.sub.7 are
as described for the agonists above; Z is also as thus
described.
[0107] Particularly preferred among the antagonist peptides of this
class are those selected from the group consisting of Mpr-LLGK,
Mpr-LIGR, Mpr-(Cha)LKG, Mpr-(Cha)IGR, Mpr-LLGKK-NH.sub.2,
Mpr-LIGRK-NH.sub.2, Mpr-LIGRKETQP-NH.sub.2, Mpr-LLGKKDGTS-NH.sub.2,
(n-pentyl).sub.2-N-Leu-Il- e-Gly-Arg-Lys-NH.sub.2 and
(Me-N-(n-pentyl)-Leu-Ile-Gly-Arg-Lys-NH.sub.2.
[0108] Antibodies
[0109] Antagonists which are antibodies immunoreactive with
critical positions of the C140 receptor are obtained by
immunization of suitable mammalian subjects with peptides
containing as antigenic regions those portions of the C140 receptor
intended to be targeted by the antibodies. Critical regions include
the region of proteolytic cleavage, the segment of the
extracellular segment critical for activation (this includes the
cleavage site), and the portions of the sequence which form the
extracellular loops, in particular, that region which interacts
with the N-terminus of the activated receptor extracellular region.
The agonist peptides of the invention may be used as immunogens in
this case.
[0110] Thus, peptides which contain the proteolytic region, namely,
for example, SKGRSLIGRLET, the extracellular loops, such as those
including ISY HLHGNNWVYGEALC; QTIYIPALNITTCHDVLPEEVLVGDMFNYFL; and
HYFLIKTQRQSHVYA. The agonist peptides described below are also
useful as immunogens.
[0111] The antibodies are prepared by immunizing suitable mammalian
hosts in appropriate immunization protocols using the peptide
haptens alone, if they are of sufficient length, or, if desired, or
if required to enhance immunogenicity, conjugated to suitable
carriers. Methods for preparing immunogenic conjugates with
carriers such as BSA, KLH, or other carrier proteins are well known
in the art. In some circumstances, direct conjugation using, for
example, carbodiimide reagents may be effective; in other instances
linking reagents such as those supplied by Pierce Chemical Co.,
Rockford, Ill., may be desirable to provide accessibility to the
hapten. The hapten peptides can be extended at the amino or carboxy
terminus with a Cys residue or interspersed with cysteine residues,
for example, to facilitate linking to carrier. Administration of
the immunogens is conducted generally by injection over a suitable
time period and with use of suitable adjuvants, as is generally
understood in the art. During the immunization schedule, titers of
antibodies are taken to determine adequacy of antibody
formation.
[0112] While the polyclonal antisera produced in this way may be
satisfactory for some applications, for pharmaceutical
compositions, use of monoclonal preparations is preferred.
Immortalized cell lines which secrete the desired monoclonal
antibodies may be prepared using the standard method of Kohler and
Milstein or modifications which effect immortalization of
lymphocytes or spleen cells, as is generally known. The
immortalized cell lines secreting the desired antibodies are
screened by immunoassay in which the antigen is the peptide hapten
or is the C140 receptor itself displayed on a recombinant host
cell. When the appropriate immortalized cell culture secreting the
desired antibody is identified, the cells can be cultured either in
vitro or by production in ascites fluid.
[0113] The desired monoclonal antibodies are then recovered from
the culture supernatant or from the ascites supernatant. Fragments
of the monoclonals or the polyclonal antisera which contain the
immunologically significant portion can be used as antagonists, as
well as the intact antibodies. Use of immunologically reactive
fragments, such as the Fab, Fab', of F(ab').sub.2 fragments is
often preferable, especially in a therapeutic context, as these
fragments are generally less immunogenic than the whole
immunoglobulin.
[0114] The antibodies or fragments may also be produced, using
current technology, by recombinant means. Regions that bind
specifically to the desired regions of receptor can also be
produced in the context of chimeras with multiple species
origin.
[0115] The antibodies thus produced are useful not only as
potential antagonists for the receptor, filling the role of
antagonist in the assays of the invention, but are also useful in
immunoassays for detecting the activated receptor. As such these
antibodies can be coupled to imaging agents for administration to a
subject to allow detection of localized antibody to ascertain the
position of C140 receptors in either activated or unactivated form.
In addition, these reagents are useful in vitro to detect, for
example, the successful production of the C140 receptor deployed at
the surface of the recombinant host cells.
[0116] Preparation of Peptide Agonists and Antagonists
[0117] The peptide agonists and antagonists of the invention can be
prepared using standard solid phase (or solution phase) peptide
synthesis methods, as is known in the art. In addition, the DNA
encoding these peptides may be synthesized using commercially
available oligonucleotide synthesis instrumentation and produced
recombinantly using standard recombinant production systems. The
production using solid phase peptide synthesis is necessitated if
non-gene-encoded amino acids are to be included.
[0118] Preparation of C140 Receptor Nucleic Acids
[0119] C140 receptor "nucleic acid" is defined as RNA or DNA that
encodes a C140 receptor, or is complementary to nucleic acid
sequence encoding a C140 receptor, or hybridizes to such nucleic
acid and remains stably bound to it under stringent conditions, or
encodes a polypeptide sharing at least 75% sequence identity,
preferably at least 80%, and more preferably at least 85%, with the
translated amino acid sequences shown in FIGS. 3, 10A-10B or
11A-11B. It is typically at least about 10 nucleotides in length
and preferably has C140 receptor biological or immunological
activity, including the nucleic acid encoding an activation peptide
fragment having the nucleotide sequence shown in FIG. 4.
Specifically contemplated are genomic DNA, cDNA, mRNA and antisense
molecules, as well as nucleic acids based on alternative backbone
or including alternative bases whether derived from natural sources
or synthesized. Such hybridizing or complementary nucleic acid,
however, is defined further as being novel and unobvious over any
prior art nucleic acid including that which encodes, hybridizes
under stringent conditions, or is complementary to nucleic acid
encoding a known G protein-coupled receptor.
[0120] "Stringent conditions" are those that (1) employ low ionic
strength and high temperature for washing, for example, 0,015M
NaCl/0.0015M sodium titrate/0.1% NaDodSO4 at 50.degree. C., or (2)
employ during hybridization a denaturing agent such as formamide,
for example, 50% (vol/vol) formamide with 0.1% bovine serum
albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium
phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate
at 42.degree. C. Another example is use of 50% formamide,
5.times.SSC (0.75M NaCl, 0.075 M sodium citrate), 50 mM sodium
phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5.times.Denhardt's
solution, sonicated salmon sperm DNA (50 mu g/ml), 0.1% SDS, and
10% dextran sulfate at 42.degree. C., with washes at 42.degree. C.
in 0.2.times.SSC and 0.1% SDS.
[0121] "Isolated" nucleic acid will be nucleic acid that is
identified and separated from contaminant nucleic acid encoding
other polypeptides from the source of nucleic acid. The nucleic
acid may be labeled for diagnostic and probe purposes, using any
label known and described in the art as useful in connection with
diagnostic assays.
[0122] Of particular interest is a C140 receptor nucleic acid that
encodes a full-length molecule, including but not necessarily the
native signal sequence thereof. Nucleic acid encoding full-length
protein is obtained by screening selected cDNA (not kidney) or
genomic libraries using the deduced amino acid sequence disclosed
herein for the first time, and, if necessary, using conventional
primer extension procedures to secure DNA that is complete at its
5' coding end. Such a clone is readily identified by the presence
of a start codon in reading frame with the original sequence.
[0123] DNA encoding an amino acid sequence variant of a C140
receptor is prepared as described below or by a variety of methods
known in the art. These methods include, but are not limited to,
isolation from a natural source (in the case of naturally occurring
amino acid sequence variants) or preparation by
oligonucleotide-mediated (or site-directed) mutagenesis, PCR
mutagenesis, and cassette mutagenesis of an earlier prepared
variant or a non-variant version of a C140 receptor.
[0124] Techniques for isolating and manipulating nucleic acids are
disclosed for example by the following documents: U.S. Pat. No.
5,030,576, U.S. Pat. No. 5,030,576 and International Patent
Publications WO94/11504 and WO93/03162. See, also, Sambrook, J. et
al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold
Spring Harbor Press, Cold Spring Harbor, N.Y., 1989, and Ausubel,
F. M. et al. Current Protocols in Molecular Biology, Vol. 2,
Wiley--Interscience, New York, 1987. Disclosures of these documents
are expressly incorporated herein by reference in their
entireties.
[0125] Recombinant Production of C140 Receptor for Use in
Assays
[0126] The invention provides recombinant materials for the
production of C140 receptor for display on the surface of
recombinant cells. Production of the receptor using these
recombinant methods provides a useful reagent to determine the
ability of a candidate drug to bind to, to activate, or to
antagonize the C140 receptor. Determination of these properties is
essential in evaluating the specificity of drugs intended for
binding other related receptors.
[0127] For this recombinant production, a DNA sequence encoding the
C140 receptor, such as those set forth in FIGS. 1A-1B and 2A-2B, or
their substantial equivalents or their degenerate analogs, is
prepared either by retrieval of the native sequence, as set forth
below, or by using substantial portions of the known native
sequence as probe, or can be synthesized de novo using standard
procedures. The DNA is ligated into expression vectors suitable for
the desired host and transformed into compatible cells. The cells
are cultured under conditions which favor the expression of the
C140 receptor encoding gene and the cells displaying the receptor
on the surface are harvested for use in the assays.
[0128] The host cells are typically animal cells, most typically
mammalian cells. In order to be useful in the assays, the cells
must have intracellular mechanisms which permit the receptor to be
displayed on the cell surface in the configuration shown generally
in FIG. 4 herein. If the assay uses cellular response to activated
receptor as a detection system, the cells must also contain a
G-protein linked mechanism for response to activation of the
receptors. Most mammalian and other animal cells fulfill these
qualifications.
[0129] Particularly useful cells for use in the method of the
invention are Xenopus laevis frog oocytes, which typically utilize
cRNA rather than standard recombinant expression systems proceeding
from the DNA encoding the desired protein. Capped RNA (at the 5'
end) is typically produced from linearized vectors containing DNA
sequences encoding the receptor. The reaction is conducted using
RNA polymerase and standard reagents. cRNA is recovered, typically
using phenol/chloroform precipitation with ethanol and injected
into the oocytes.
[0130] The animal host cells expressing the DNA encoding the C140
receptor or the cRNA-injected oocytes are then cultured to effect
the expression of the encoding nucleic acids so as to produce the
C140 receptor displayed in a manner analogous to that shown in FIG.
4 on their surfaces. These cells then are used directly in assays
for assessment of a candidate drug to bind, antagonize, or activate
the receptor.
[0131] Assays
[0132] In one type of easily conducted assay, competition of the
candidate drug for binding to the receptor with either agonist or
known binding antagonist can be tested. In one method, the
competing agonist or antagonist may be labeled; the labeled
substance known to bind the receptor can, of course, be a synthetic
peptide. In one typical protocol, varying concentrations of the
candidate are supplied along with a constant concentration of
labeled agonist or antagonist and the inhibition of a binding of
label to the receptor can be evaluated using known techniques.
[0133] In a somewhat more sophisticated approach, the effect of
candidate compounds on agonist-induced responses can be measured in
the cells recombinantly expressing the C140 receptor as described
below. Assay systems for the effect of activation of receptor on
these cells include calcium mobilization and voltage clamp which
are described herein in further detail. These assays permit an
assessment of the effect of the candidate drug on the receptor
activity rather than simply ability to bind to the receptor.
[0134] Agonist-induced increases in .sup.45Ca release by oocytes
expressing cRNA encoding C140 receptor or other recombinant cells
producing C140 receptor are assessed by published techniques
(Williams, J. A., et al., Proc Natl Acad Sci USA (1988)
85:4939-4943). Briefly, intracellular calcium pools are labeled by
incubating groups of 30 oocytes in 300 .mu.l calcium-free modified
Barth's solution (MBSH) containing 50 .mu.Ci .sup.45CaCl.sub.2
(10-40 mCi/mg Ca; Amersham) for 4 hours at RT. The labeled oocytes
or cells are washed, then incubated in MBSH II without antibiotics
for 90 minutes. Groups of 5 oocytes are selected and placed in
individual wells in a 24-well tissue culture plate (Falcon 3047)
containing 0.5 ml/well MBSH II without antibiotics. This medium is
removed and replaced with fresh medium every 10 minutes; the
harvested medium is analyzed by scintillation counting to determine
.sup.45Ca released by the oocytes during each 10-minute incubation.
The 10-minute incubations are continued until a stable baseline of
.sup.45Ca release per unit time is achieved. Two additional
10-minute collections are obtained, then test medium including
agonist is added and agonist-induced .sup.45Ca release
determined.
[0135] Using the above assay, the ability of a candidate drug to
activate the receptor can be tested directly. In this case, the
agonists of the invention are used as controls. In addition, by
using the agonist of the invention to activate the recombinant
receptor, the effect of the candidate drug on this activation can
be tested directly. Recombinant cells expressing the nucleic acids
encoding the receptor are incubated in the assay in the presence of
agonist with and without the candidate compound. A diminution in
activation in the presence of the candidate will indicate an
antagonist effect. Conversely, the ability of a candidate drug to
reverse the antagonist effects of an antagonist of the invention
may also be tested.
[0136] In an alternative to measuring calcium mobilization, the
voltage clamp assay can be used as a measure for receptor
activation. Agonist-induced inward chloride currents are measured
in voltage-clamped oocytes expressing C140 receptor encoding cRNA
or cells expressing DNA from recombinant expressions systems
essentially as previously described (Julius, D., et al, Science
(1988) 241:558-563) except that the single electrode voltage-clamp
technique is employed.
[0137] Detection of Activated Receptors
[0138] In one embodiment, the availability of the recombinant C140
receptor protein permits production of antibodies which are
immunospecific to the activated form of the receptor which can then
be used for diagnostic imaging of activated receptors in vivo.
These antibodies are produced either to the activated form of the
receptor produced recombinantly, or to the peptide representing the
"new amino terminal" peptide described herein. The resulting
antibodies, or the immunospecific fragments thereof, such as the
Fab, Fab', Fab'.sub.2 fragments are then conjugated to labels which
are detected by known methods, such as radiolabels including
technetium.sup.99 and indium.sup.111 or other radioactive labels as
is known in the art. When injected in vivo, these antibodies home
to the sites of activated receptor, thus permitting localization of
areas containing activated receptors.
[0139] In another embodiment, the presence of the activation
peptide in body fluids or in culture media can be detected and
measured. Antibodies are made to the activation peptide as
described above and can be employed in standard ELISA or RIA assays
to detect excess amounts of the activation peptide in, for example,
urine.
[0140] Administration of Agonists and Antagonists as
Pharmaceuticals
[0141] The peptides of the invention which behave as agonists are
administered in conventional formulations for systemic
administration as is known in the art. Typical such formulations
may be found, for example, in Remington's Pharmaceutical Sciences,
Mack Publishing Co., Easton Pa., latest edition.
[0142] Preferred forms of systemic administration of peptides
include injection, typically-by intravenous injection. Other
injection routes, such as subcutaneous, intramuscular, or
intraperitoneal, can also be used. More recently, alternative means
for systemic administration of peptides have been devised which
include transmucosal and transdermal administration using
penetrants such as bile salts or fusidic acids or other detergents.
In addition, if properly formulated in enteric or encapsulated
formulations, oral administration may also be possible.
Administration of these compounds may also be topical and/or
localized, in the form of salves, pastes, gels and the like.
[0143] The dosage range required depends on the choice of peptide,
the route of administration, the nature of the formulation, the
nature of the patient's condition, and the judgment of the
attending physician. Suitable dosage ranges, however, are in the
range of 0.1-100 .mu.g/kg of subject. Wide variations in the needed
dosage, however, are to be expected in view of the variety of
peptides available and the differing efficiencies of various routes
of administration. For example, oral administration would be
expected to require higher dosages than administration by
intravenous injection. Variations in these dosage levels can be
adjusted using standard empirical routines for optimization as is
well understood in the art.
[0144] As shown hereinbelow, the agonists of the invention behave
as antihypotensives; antagonists have the opposite effect. Thus,
patients whose blood pressure needs to be raised or lowered benefit
by the administration of the suitable peptide.
[0145] In addition, the agonists have anti-inflammatory and wound
healing properties.
[0146] Antisense, Triple Helix and Gene Therapy Aspects
[0147] The constitutive expression of antisense RNA in cells has
been shown to inhibit the expression of about 20 different genes in
mammals and plants, and the list continually grows (Hambor, J. E.
et al., J. Exp. Med. 168:1237-1245 (1988); Holt, J. T. et al.,
Proc. Nat. Acad. Sci. 83:4794-4798 (1986); Izant, J. G. et al.,
Cell 36:1007-1015 (1984); Izant, J. G., et al., Science 229:345-352
(1985) and De Benedetti, A. et al., Proc. Nat. Acad. Sci.
84:658-662 (1987)). Possible mechanisms for the antisense effect
are the blockage of translation or prevention of splicing, both of
which have been observed in vitro. Interference with splicing
allows the use of intron sequences (Munroe, S. H., EMBO. J.
7:2523-2532 (1988) which should be less conserved and therefore
result in greater specificity in inhibiting expression of a protein
of one species but not its homologue in another species.
[0148] Therapeutic gene regulation is accomplished using the
"antisense" approach, in which the function of a target gene in a
cell or organism is blocked, by transfection of DNA, preferably an
oligonucleotide, encoding antisense RNA which acts specifically to
inhibit expression of the particular target gene. The sequence of
the antisense DNA is designed to result in a full or preferably
partial antisense RNA transcript which is substantially
complementary to a segment of the gene or mRNA which it is intended
to inhibit. The complementarity must be sufficient so that the
antisense RNA can hybridize to the target gene (or mRNA) and
inhibit the target gene's function, regardless of whether the
action is at the level of splicing, transcription or translation.
The degree of inhibition, readily discernible by one of ordinary
skill in the art without undue experimentation, must be sufficient
to inhibit, or render the cell incapable of expressing, the target
gene. One of ordinary skill in the art will recognize that the
antisense RNA approach is but one of a number of known mechanisms
which can be employed to block specific gene expression.
[0149] By the term "antisense" is intended an RNA sequence, as well
as a DNA sequence coding therefor, which is sufficiently
complementary to a particular mRNA molecule for which the antisense
RNA is specific to cause molecular hybridization between the
antisense RNA and the mRNA such that translation of the mRNA is
inhibited. Such hybridization must occur under in vivo conditions,
that is, inside the cell. The action of the antisense RNA results
in specific inhibition of gene expression in the cell. (See:
Albers, B. et al., MOLECULAR BIOLOGY OF THE CELL, 2nd Ed., Garland
Publishing, Inc., New York, N.Y. (1989), in particular, pages
195-196.
[0150] The antisense RNA of the present invention may be
hybridizable to any of several portions of a target mRNA, including
the coding sequence, a 3' or 5' untranslated region, or other
intronic sequences. A preferred antisense RNA is that complementary
to the human C140 receptor mRNA. As is readily discernible by one
of skill in the art, the minimal amount of homology required by the
present invention is that sufficient to result in hybridization to
the specific target mRNA and inhibition of its translation or
function while not affecting function of other mRNA molecules and
the expression of other genes.
[0151] Antisense RNA is delivered to a cell by transformation or
transfection with a vector into which has been placed DNA encoding
the antisense RNA with the appropriate regulatory sequences,
including a promoter, to result in expression of the antisense RNA
in a host cell.
[0152] "Triple helix" or "triplex" approaches involve production of
synthetic oligonucleotides which bind to the major groove of a
duplex DNA to form a colinear triplex. Such triplex formation can
regulate and inhibit cellular growth. See, for example: Hogan et
al., U.S. Pat. No. 5,176,996; Cohen, J. S. et al., Sci. Amer., Dec.
1994, p. 76-82; Helene, C., Anticancer Drug Design 6:569-584
(1991); Maher III, L. J. et al., Antisense Res. Devel. 1:227-281
(Fall 1991); Crook, S. T. et al. eds., ANTISENSE RESEARCH AND
APPLICATIONS, CRC Press, 1993. It is based in part on the discovery
that a DNA oligonucleotide can bind by triplex formation to a
duplex DNA target in a gene regulatory region, thereby repressing
transcription initiation (Cooney M. et. al. (1988) Science
241:456). The present invention utilizes methods such as those of
Hogan et al., supra (herein incorporated by reference in its
entirety), to designing oligonucleotides which will bind tightly
and specifically to a duplex DNA target comprising part of the C140
receptor-encoding DNA or a regulatory sequence thereof. Such
triplex oligonucleotides can therefore be used as a class of drug
molecules to selectively manipulate the expression of this
gene.
[0153] Thus the present invention is directed to providing to a
cell or administering to a subject a synthetic oligonucleotide in
sufficient quantity for cellular uptake and binding to a DNA duplex
of the target C140 receptor-coding DNA sequence or a regulatory
sequence thereof, such that the oligonucleotide binds to the DNA
duplex to form a colinear triplex. This method is used to inhibit
expression of the receptor on cells in vitro or in vivo. Preferably
the target sequence is positioned within the DNA domain adjacent to
the RNA transcription origin. This method can also be used to
inhibit growth of cells which is dependent on expression of this
receptor. The method may also be used to alter the relative amounts
or proportions of the C140 receptor expressed on cells or tissues
by administering such a triplex-forming synthetic
oligonucleotide.
[0154] The following examples are intended to illustrate but not to
limit the invention.
EXAMPLE 1
Isolation of the Gene Encoding Murine C140 Receptor
[0155] A mouse cosmid genomic library (obtained from Dr. R. A.
Wetsel, Washington University School of Medicine, St. Louis, Mo.
and described in Wetsel, R. A. et al., J Biol Chem (1990)
265:2435-2440) was screened with two .sup.32P-labeled
oligonucleotides corresponding to bp 190-249 and 742-801,
respectively, of the bovine substance K receptor cDNA (Masu, Y. et
al., Nature (1987) 329:836-838). The hybridization conditions are
5.times.SSC, 5.times.Denhardt's, 0.1% SDS, 0.1 mg/ml sperm DNA,
10.sup.6 cpm/ml of labeled oligonucleotides, 60.degree. C.
overnight, followed by washing with 1.times.SSC, 0.1% SDS at
60.degree. C.
[0156] In one of the clones isolated (C140) the hybridizing region
was localized to a 3.7 kb PstI fragment. This fragment was
subcloned into the commercially available pBluescript vector. The
hybridizing and adjacent regions were sequenced in both
orientations by the Sanger chain termination method. FIGS. 1A-1B
shows both the nucleotide sequence and the deduced amino acid
sequence of the mouse C140 receptor. The tentative signal sequence
(SP) and the seven transmembrane regions are overlined, potential
asparagine-linked glycosylation sites are marked with bold arrows,
and the putative protease receptor cleavage site at Arg34-Ser35 is
marked with an open arrow.
EXAMPLE 2
Isolation of the Gene Encoding Human C140 Receptor
[0157] The availability of genomic DNA encoding the mouse protease
C140 receptor permitted the retrieval of the corresponding human
gene. A human genomic library cloned in the vector EMBL3 was
screened at exactly the conditions in Example 1 using the entire
coding region of the murine clone as a probe. The recovered human
gene including the DNA sequence and the deduced amino acid sequence
are shown in FIGS. 2A-2B. Subsequent experiments indicated that the
human C140 gene is located in the same region of the long arm of
chromosome number 5 (5q12-5q13) as has been reported for the human
thrombin receptor gene.
[0158] In addition, a 1.1 kb genomic DNA fragment was obtained from
Genome Systems Inc., commercial screening service as was
PCR-positive with a primer pair that generates a fragment spanning
350-nucleotides of the human C140 protein coding region. A 1.1 kb
bamH1 fragment was subcloned and sequenced and found to contain
800-nucleotides of promoter sequence. The promoter lacks both a
TATA box and a CAAT box but is rich in G's and C's; features common
to promoters of many housekeeping genes. Two binding elements
specific for SP1 and AP2 were identified.
EXAMPLE 3
Comparison of Related G-Protein Receptors
[0159] As shown in FIG. 3, the deduced amino acid sequence of the
human protease C140 receptor shows extensive similarity (>90%)
to the mouse sequence.
[0160] FIG. 5 shows an amino acid sequence alignment between the
mouse C140 receptor and the related G-protein receptor human
thrombin receptor (Coughlin, S. Cell). The tentative signal
sequences (SP), transmembrane regions, and protease cleavage sites
are marked.
EXAMPLE 4
Recovery of Mouse C140 cDNA
[0161] A cDNA library from a mouse stomach was constructed in
.lambda. gt10 and screened with a probe encompassing the C1040
genomic DNA. A single phage clone was isolated and cut with EcoRI.
The insert was cloned into pBluescript and pSG5 and sequenced.
[0162] The isolated cDNA was 2732 nucleotides long including a 16
base polyA-stretch; 5' RACE resulted in the addition of only 27
bases to the 5' end. The 5' end of the apparent coding region
differs from the 5' end of the open reading frame of genomic DNA;
it is believed that the 5' end of the cDNA is correct. The complete
nucleotide sequence and deduced amino acid sequence of murine cDNA
encoding C140 is shown in FIGS. 10A-10B.
EXAMPLE 5
Recovery of Human cDNA Encoding C140
[0163] A human intestinal tumor cDNA library was subjected to PCR
using primers designed from the genomic clone of Example 2 and the
amplified fragment was cloned in pSG5 and sequenced. The nucleotide
sequence and deduced amino acid sequence are shown in FIGS.
11A-11B. There are four amino acid differences between the cDNA
encoded sequence and that encoded by the genomic DNA as is shown in
FIGS. 11A-11B.
EXAMPLE 6
Activation of Protease C140 Receptor in Oocytes
[0164] Both native and mutant C140 receptors were produced in
oocytes and activated with a peptide mimicking the new
amino-terminus", or by the proteolytic enzyme trypsin (which
cleaves the extracellular region). Native receptors were produced
by cloning the coding region of the receptor gene, using the
polymerase chain reaction, into the expression vector pSG-5 (Green,
S. et al., Nucleic Acid Res (1988) 16:369). The orientation and
integrity of the cloned coding region was verified by determining
the nucleotide sequence with the Sanger chain-termination method.
Site-directed mutagenesis was employed to construct mutant
receptors in the pSG-5. Three mutant receptors were made, in which
serine-35 was replaced with proline, arginine, and histidine,
respectively. The nucleotide sequences of the three mutants was
verified as above.
[0165] In order to produce the receptor at the surface of oocytes,
cRNA encoding the receptor was produced as follows. pSG-5 C140
plasmid DNA was made linear by digestion with XbaI, and capped cRNA
was produced in vitro using T7 RNA polymerase (Krieg and Melton,
Meth Enzymol (1987) 155:397-415, which reference is hereby
incorporateds by reference in its entirety).
[0166] Oocytes from Xenopus laevis were harvested and prepared
using published techniques (Coleman, A., in Hames, B. D., and
Higgins, S. J., eds, Transcription and Translation: A Practical
Approach, IRL Press, pp. 271-302; Williams, J. A., et al. Proc Natl
Acad Sci USA (1988) 85:4939-4943]. To remove follicular cells,
oocytes were incubated for 1.5 h with shaking in calcium-free
Barth's containing 2 mg/ml each of collagenase 1A and hyaluronidase
1S. The oocytes were then washed five times in regular Barth's and
incubated at 18.degree. C. in Barth's medium containing 100 U/ml
penicillin, 100 .mu.g/ml streptomycin, and 2.5 mM sodium pyruvate.
Stage V oocytes were selected and injected with 30 nl of cRNA (0.33
.mu.g/.mu.l water) or water alone, and then incubated with 0.25 ml
of medium in groups of four/well in a 96-well culture plate. After
36 hours the oocytes were incubated with .sup.45Ca (250 .mu.Ci/ml).
After 12 h incubation the oocytes were washed and 0.2 ml of medium
added and replaced every five minutes. The harvested medium was
analyzed by scintillation counting. After five replacements to
determine the baseline release of .sup.45Ca, test medium with the
agonist, e.g. SLIGRL, was added and the evoked .sup.45Ca-release
determined.
[0167] Oocytes were injected with capped cRNA (ca 10 ng) encoding
wild-type mouse C140 receptor (WT) or either of the three mutant
receptors 35Pro, 35Arg and 35His. After 36 hours, cRNA-injected and
control water-injected, oocytes were loaded with .sup.45Ca, and 12
hours thereafter peptide or trypsin-induced .sup.45Ca release were
determined as described above. The peptide SLIGRL was added at 100
.mu.M, and trypsin at 300 pM. The stimulation with the peptide was
done on the same group of oocytes after the stimulation with
trypsin. The data shown in Table 1 represent the mean of three
replicate determinations, and denotes the increase compared to
oocytes injected with water.
2 TABLE 1 Receptor Agonist Fold increase in .sup.45Ca WT Trypsin
6.6 35Pro Trypsin 0 35Arg Trypsin 0 35His Trypsin 0 WT SLIGRL 11
35Pro SLIGRL 23 35Arg SLIGRL 15 35His SLIGRL 23
[0168] As shown in Table 1, the agonist peptide SLIGRL was able to
activate both the wild-type and mutated receptors. On the other
hand, trypsin, which can activate only by cleavage of the
extracellular domain, is able only to activate the wild-type
receptor.
EXAMPLE 7
Activation of the C140 Receptor by Different Agonist Peptides
[0169] Various peptides were tested at 100 .mu.M in the assay above
using wild-type mouse C140 receptor, expressed in oocytes. The
results are shown in Table 2.
3 TABLE 2 Peptide Fold Increase in .sup.45Ca SLIGRL 15 SLIGRA 8.5
SLIGAL 0 SLIARL 4.3 SLAGRL 0 SAIGRL 0 ALIGRL 1.3 SFFLRW 1.7
[0170] The "native" peptide SLIGRL is most effective; replacing L
at position 6 with alanine lowers but does not destroy activity.
Positions 2 and 3 are more sensitive. Position 1 tolerates
substitution with alanine but decreases the activity by a factor of
10; the activity of this agonist is comparable to the analogous
thrombin receptor agonist SFFLRW.
EXAMPLE 8
Expression of C140 Receptor in Various Tissues
[0171] Poly(A)+RNA was prepared from mouse tissues, resolved on a
1.2% agarose gel containing 50% formamide and blotted onto Hybond C
extra membrane (Amersham). The blot was hybridized with a
.sup.32P-labeled "random priming probe" directed against the whole
coding region of murine C140 receptor. The probe was hybridized at
42.degree. C. for 48 hr then successively washed at 20.degree. C.
in 1.times.SSC, 0.1% SDS twice, 5 min each time, then at 65.degree.
C. in 1.times.SSC, again twice for 20 min each time, and then
0.1.times.SSC, 0.1% SDS twice for 20 min each time. The resulting
membrane was autoradiographed for 5 days at -80.degree. C. with an
intensifying screen.
[0172] The results, shown in FIG. 6 indicate that kidney and small
intestine, but not spleen, contain mRNA encoding C140. In FIG. 6,
where each lane contains 10 .mu.g RNA, lane A is derived from
spleen, lane B from kidney and lane C from small intestine.
EXAMPLE 9
Expression of C140 Transcripts in Mice
[0173] In situ hybridization using .sup.35S RNA probes was used to
localize C140 transcripts in mouse embryogenesis and in adult mouse
tissues. A strong signal was found in the gastrointestinal tract at
11.5 days; at 14 days there was strong hybridization to epithelial
structures in the nasopharynx, stomach-intestine, skin and
endothelial cells in larger vessels. There was some hybridization
in the liver and sclerotoma but no signal in muscle or CNS. At 17
days, the signals in the sclerotoma had disappeared and additional
epithelial structures showed hybridization including the esophagus,
kidney glomeruli, lung, hair follicles and epidermis.
[0174] In newborns, the signals found at 17 days were retained and
additional signals were found in the thymic medulla and kidney
medulla. Adults showed transcripts in the mucosa of stomach,
intestine and colon, white pulp of the spleen, thymus and kidney
medulla. Again, there were no signals in the CNS, liver, lung or
adrenal gland. FIG. 12 shows the results of in situ hybridization
in a sectioned newborn mouse using these probes.
EXAMPLE 10
Expression of C140 Transcripts in Human Tissues
[0175] FIG. 13 shows the results of a Northern blot of total RNA
from human cell lines hybridized to a human C140 receptor probe.
Ten mg of total RNA was used. Hybridization was obtained in RNA
from stomach (lane 1), Ca--Co-2 cells (lane 2); HT-29 cells (lane
3), A498 cells (lane 5), 5637 cells (lane 8); skin keratinocytes
(lane 12), and HUVEC (lanes 13 and 14). No hybridization was
detected in HuTu80 cells, J82 cells, MCF-7, HeLa or NCI 12 cells
(lanes 4, 6, 9 and 10).
EXAMPLE 11
Determination of Hypotensive Activity of C140 Agonists
[0176] The C140 agonist SLIGRL was injected in 0.2 ml buffer at
various-concentrations into rat femoral vein and the arterial
pressure was monitored. The results of various concentrations are
shown in FIG. 7.
[0177] The trace in FIG. 7 shows that even at 0.1 mM an appreciable
decrease in blood pressure occurred; larger decreases were observed
at 1 mM concentration.
[0178] This effect was also shown by observing vasodilation as a
result of stimulation of the rat femoral vein with the above
agonist. Adult Sprague-Dawley rats were killed by exsanguination
during diethylether anesthesia and the femoral vein was removed and
dissected free from fat and connective tissue. Circular
preparations of the vein were mounted in an organ bath (5 ml) on
two L-formed metal holders (0.2 mm diameter). One of the metal
holders was screwed into one of the levers of a Grass FTO C force
displacement transducer. The bathing liquid was Kreb's Ringer
solution containing 118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl.sub.2, 1.2
mM MgSO.sub.4, 24.8 mM NaHCO.sub.3, 1.2 mM KH.sub.2PO.sub.4 and 5.6
mM glucose. The bathing fluid was continuously treated with 88.5%
oxygen-11.5% CO.sub.2; the temperature was held at 37.degree. C.
The endothelium was removed by bubbling CO.sub.2 through the
vessels. The basal tension was between 7.5 and 12 mN. The
preparations were equilibrated for at least 1 hr before application
of agonist and control substances.
[0179] The results of these determinations are shown in FIGS. 8a
and 8b. As shown in FIG. 8a, contraction induced by application of
PGF.sub.2.alpha. at 3.times.10.sup.-5 M is relaxed by
administration of 10.sup.-5 M agonist. The results in FIG. 8a were
obtained using the vein with the endothelium still present.
[0180] In FIG. 8b, the endothelium has been removed. In an
analogous experiment, the contraction induced by 3.times.10.sup.-5
M PGF.sub.2.alpha. is not counteracted by 10.sup.-5 M agonist or by
10.sup.-5 M acetylcholine.
EXAMPLE 8
Activation of Recombinant C140 Receptor by Plasmin and
Kallikrein
[0181] FIGS. 9a and 9b show the ability of plasmin and kallikrein
respectively to activate oocytes injected with C140 cRNA (open
circles) or water (crosses) as control. FIG. 9c shows the ability
of trypsin to activate frog oocytes injected with C140 receptor
cRNA (filled circles) or substance K receptor cRNA (open circles).
Trypsin clearly has a differential effect on the C140
receptor-injected oocytes.
[0182] All references cited and mentioned above, including patents,
journal articles and texts, are all incorporated by reference
herein, whether expressly incorporated or not.
[0183] Having now fully described this invention, it will be
appreciated by those skilled in the art that the same can be
performed within a wide range of equivalent parameters,
concentrations, and conditions without departing from the spirit
and scope of the invention and without undue experimentation.
[0184] While this invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications. This application is intended to
cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth as follows in the scope of the appended
claims.
* * * * *