U.S. patent application number 12/032029 was filed with the patent office on 2008-10-02 for drosophila g protein coupled receptors, nucleic acids, and mehtods related to the same.
This patent application is currently assigned to PHARMACIA & UPJOHN COMPANY. Invention is credited to Teresa M. Kubiak, Martha J. Larsen, David E. Lowery, Valdin G. Smith.
Application Number | 20080241961 12/032029 |
Document ID | / |
Family ID | 23687571 |
Filed Date | 2008-10-02 |
United States Patent
Application |
20080241961 |
Kind Code |
A1 |
Lowery; David E. ; et
al. |
October 2, 2008 |
DROSOPHILA G PROTEIN COUPLED RECEPTORS, NUCLEIC ACIDS, AND MEHTODS
RELATED TO THE SAME
Abstract
The present invention provides a Drosophila melanogaster GPCR
(DmGPCR) polypeptides and polynucleotides which identify and encode
such a DmGPCR. In addition, the invention provides expression
vectors, host cells and methods for its production. The invention
also provides methods for the identification of homologs in other
animals, and of DmGPCR agonists/antagonists, useful for the
treatment of diseases in animals and for the control of insects
that are injurious or harmful to plants or animals.
Inventors: |
Lowery; David E.; (Portage,
MI) ; Smith; Valdin G.; (Kalamazoo, MI) ;
Kubiak; Teresa M.; (Richland, MI) ; Larsen; Martha
J.; (Kalamazoo, MI) |
Correspondence
Address: |
Pepper Hamilton LLP
400 Berwyn Park, 899 Cassatt Road
Berwyn
PA
19312-1183
US
|
Assignee: |
PHARMACIA & UPJOHN
COMPANY
KALAMAZOO
MI
|
Family ID: |
23687571 |
Appl. No.: |
12/032029 |
Filed: |
February 15, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10736048 |
Dec 15, 2003 |
7354724 |
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12032029 |
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09693746 |
Oct 20, 2000 |
6835546 |
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10736048 |
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09425676 |
Oct 22, 1999 |
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09693746 |
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Current U.S.
Class: |
436/501 |
Current CPC
Class: |
A61P 25/00 20180101;
A61P 25/10 20180101; A61P 19/02 20180101; A61P 31/18 20180101; A61P
9/02 20180101; A61P 9/00 20180101; A61P 31/12 20180101; A61P 37/06
20180101; A61P 35/00 20180101; C12N 2799/021 20130101; A61P 13/12
20180101; A61P 25/18 20180101; A61P 3/10 20180101; A61P 25/16
20180101; A61P 9/10 20180101; A61P 9/12 20180101; C07K 14/705
20130101; A61P 25/24 20180101; A61P 3/04 20180101; A61P 29/00
20180101; A61P 25/14 20180101; A61P 25/04 20180101; A61P 7/02
20180101 |
Class at
Publication: |
436/501 |
International
Class: |
G01N 33/566 20060101
G01N033/566 |
Claims
1.-62. (canceled)
63. A method for identifying a modulator of binding between a
DmGPCR and an allatostatin, comprising the steps of: (a) contacting
an allatostatin and a composition comprising a DmGPCR in the
presence and in the absence of a putative modulator compound; (b)
detecting binding between the allatostatin and the DmGPCR; and (c)
determining whether binding in the presence of said putative
modulator is increased or decreased compared to binding in the
absence of said putative modulator compound, whereby putative
modulator compounds that increase or decrease binding are
identified as binding modulators; wherein the DmGPCR is DmGPCR4
that binds to an allatostatin and has a sequence with at least 90%
sequence homology to SEQ ID NO:8; and wherein the allatostatin is a
peptide having a sequence selected from the group consisting of SEQ
ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, and SEQ ID NO:37.
64. The method of claim 63 wherein the allatostatin is a peptide
having a sequence of SEQ ID NO:34.
65. The method of claim 63 wherein the allatostatin is a peptide
having a sequence of SEQ ID NO:35.
66. The method of claim 63 wherein the allatostatin is a peptide
having a sequence of SEQ ID NO:36.
67. The method of claim 63 wherein the allatostatin is a peptide
having a sequence of SEQ ID NO:37.
68. The method of claim 63 wherein the DmGPCR4 has a sequence with
at least 95% sequence homology to SEQ ID NO:8.
69. The method of claim 63 wherein the DmGPCR4 has a sequence with
at least 99% sequence homology to SEQ ID NO:8.
70. The method of claim 63 wherein modulation of binding is
determined by a gel-shift assay.
71. The method of claim 63 wherein modulation of binding is
determined by a protein binding assay.
72. The method of claim 63 further comprising characterizing one or
more properties of the binding modulator.
73. The method of claim 72 wherein the one or more properties of
the binding modulator are physical, biological or biochemical
properties.
74. The method of claim 63 wherein the DmGPCR4 has a sequence of at
least 95% sequence homology to SEQ ID NO:8.
75. The method of claim 63 wherein the DmGPCR4 has a sequence of at
least 99% sequence homology to SEQ ID NO:8.
76. A method for identifying a modulator of binding between a
DmGPCR and an allatostatin, comprising the steps of: (a) contacting
an allatostatin and a composition comprising a DmGPCR in the
presence and in the absence of a putative modulator compound; (b)
detecting binding between the allatostatin and the DmGPCR; and (c)
determining whether binding in the presence of said putative
modulator is increased or decreased compared to binding in the
absence of said putative modulator compound, whereby putative
modulator compounds that increase or decrease binding are
identified as binding modulators; wherein the DmGPCR is DmGPCR4
that has the sequence of SEQ ID NO:8; and wherein the allatostatin
is a peptide having the sequence selected from the group consisting
of SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO:36, and SEQ ID NO:37
77. The method of claim 76 wherein modulation of binding is
determined by a protein binding assay.
78. The method of claim 76 further comprising characterizing one or
more properties of the binding modulator.
79. The method of claim 78 wherein the one or more properties of
the binding modulator are physical, biological or biochemical
properties.
80. The method of claim 76 wherein modulation of binding is
determined by a gel-shift assay.
Description
FIELD OF THE INVENTION
[0001] The present invention is directed, in part, to nucleic acid
molecules encoding novel Drosophila melanogaster G protein coupled
receptors (DmGPCRs), novel polypeptides, and assays for screening
compounds that bind to GPCR and/or modulate the activity of
GPCR.
BACKGROUND OF THE INVENTION
[0002] Humans and other life forms are comprised of living cells.
Among the mechanisms through which the cells of an organism
communicate with each other and obtain information and stimuli from
their environment is through cell membrane receptor molecules
expressed on the cell surface. Many such receptors have been
identified, characterized, and sometimes classified into major
receptor superfamilies based on structural motifs and signal
transduction features. Such families include (but are not limited
to) ligand-gated ion channel receptors, voltage-dependent ion
channel receptors, receptor tyrosine kinases, receptor protein
tyrosine phosphatases, and G protein-coupled receptors. The
receptors are a first essential link for translating an
extracellular signal into a cellular physiological response.
[0003] G protein-coupled receptors (i.e., GPCRs) form a vast
superfamily of cell surface receptors which are characterized by an
amino-terminal extracellular domain, a carboxy-terminal
intracellular domain, and a serpentine structure that passes
through the cell membrane seven times. Hence, such receptors are
sometimes also referred to as seven transmembrane (7TM) receptors.
These seven transmembrane domains define three extracellular loops
and three intracellular loops, in addition to the amino- and
carboxy-terminal domains. The extracellular portions of the
receptor have a role in recognizing and binding one or more
extracellular binding partners (e.g., ligands), whereas the
intracellular portions have a role in recognizing and communicating
with downstream effector molecules.
[0004] The GPCRs bind a variety of ligands including calcium ions,
hormones, chemokines, neuropeptides, neurotransmitters,
nucleotides, lipids, odorants, and even photons. Not surprisingly,
GPCRs are important in the normal (and sometimes the aberrant)
function of many cell types. See generally Strosberg, Eur. J.
Biochem., 1991, 196, 1-10 and Bohm et al., Biochem J., 1997, 322,
1-18. When a specific ligand binds to its corresponding receptor,
the ligand typically stimulates the receptor to activate a specific
heterotrimeric guanine nucleotide-binding regulatory protein (G
protein) that is coupled to the intracellular portion or region of
the receptor. The G protein, in turn, transmits a signal to an
effector molecule within the cell by either stimulating or
inhibiting the activity of that effector molecule. These effector
molecules include adenylate cyclase, phospholipases and ion
channels. Adenylate cyclase and phospholipases are enzymes that are
involved in the production of the second messenger molecules cAMP,
inositol triphosphate and diacyglycerol. It is through this
sequence of events that an extracellular ligand stimulus exerts
intracellular changes through a G protein-coupled receptor. Each
such receptor has its own characteristic primary structure,
expression pattern, ligand binding profile, and intracellular
effector system.
[0005] Because of the vital role of G protein-coupled receptors in
the communication between cells and their environment, such
receptors are attractive targets for therapeutic intervention, for
example by activating or antagonizing such receptors. For receptors
having a known ligand, the identification of agonists or
antagonists may be sought specifically to enhance or inhibit the
action of the ligand. Some G protein-coupled receptors have roles
in disease pathogenesis (e.g., certain chemokine receptors that act
as HIV co-receptors may have a role in AIDS pathogenesis), and are
attractive targets for therapeutic intervention even in the absence
of knowledge of the natural ligand of the receptor. Other receptors
are attractive targets for therapeutic intervention by virtue of
their expression pattern in tissues or cell types that are
themselves attractive targets for therapeutic intervention.
Examples of this latter category of receptors include receptors
expressed in immune cells, which can be targeted to either inhibit
autoimmune responses or to enhance immune responses to fight
pathogens or cancer; and receptors expressed in the brain or other
neural organs and tissues, which are likely targets in the
treatment of schizophrenia, depression, bipolar disease, or other
neurological disorders. This latter category of receptor is also
useful as a marker for identifying and/or purifying (e.g., via
fluorescence-activated cell sorting) cellular subtypes that express
the receptor. Unfortunately, only a limited number of G protein
receptors from the central nervous system (CNS) are known. Thus, a
need exists for G protein-coupled receptors that have been
identified and show promise as targets for therapeutic intervention
in a variety of animals, including humans.
[0006] Insects are recognized as major pests in agriculture and in
human domestic environments. Insects also parasitize animals and
humans, being denoted as ectoparasites in such cases, causing
morbidity and mortality. Insects also serve as vectors for the
transmission of viral and parasitic diseases to plants, animals and
humans. Thus, there is a continuing and compelling need to discover
new methods for controlling insect populations and for repelling
and/or killing pathogenic or pestiferous species. One way to
control insect populations by killing or paralyzing insects is
through the use of chemical agents, denoted as insecticides, that
are selectively toxic to insects and potentially other
invertebrates. Currently, insecticides have enormous value for the
control of insects that are damaging to agricultural products,
including crops and livestock. Insecticides are also used in human
domestic situations, for the control of lawn and garden pests as
well as insects that are damaging or annoying to humans, including
stinging or biting insects, flies and cockroaches. Insecticides
also have enormous value for the treatment or prevention of disease
states caused by ectoparasites in livestock animals and pets,
including fleas, lice, ticks, mites and biting flies. However,
current chemicals used as insecticide are not optimal. Some have
demonstrable toxicity for mammals, while resistance to some of them
has arisen in certain target species. Therefore, there exists a
need for new selective insecticides that have novel mechanisms of
action.
[0007] Examples of insect GPCRs that have neuropeptide ligands are
known (Li, et al., EMBO Journal, 1991, 10, 3221-3229; Li, et al.,
J. Biol. Chem. 1992, 267, 9-12; Monnier, et al., J. Biol. Chem.,
1992, 267, 1298-1302; Vanden Broeck, et al., Int. Rev. Cytology,
1996, 164, 189-268; Guerrero, Peptides, 1997, 18, 1-5; Hauser, et
al., J. Biol. Chem., 1997, 272, 1002-1010; Birgul et al., EMBO J.
1999, 18, 5892-5900; Torfs et al., J. Neurochem. 2000, 74,
2182-2189; and Hauser et al. Biochem. Biophys. Res. Comm. 1998,
249, 822-828), though none has yet been publicly reported as having
been exploited for insecticide discovery.
[0008] A large family of peptides generally 4-12 amino acids in
length typically found in invertebrate animals (e.g. insects) is a
class of neuropeptides known as FMRFamide related peptides (i.e.,
FaRPs). The prototypical FMRFamide peptides are so named because of
the "FMRF" consensus amino acid sequence at their C-termini,
consisting generally of (F,Y)(M,V,I,L)R(F,Y)NH2. As neuropeptides,
these molecules are involved in vital biological processes
requiring controlled neuromuscular activity. Although some
neurotransmitters and neuromodulators (including neuropeptides)
have been shown to function as ligands for receptors, to date there
has been no identification of a FaRP neuropeptide as a ligand of a
GPCR.
[0009] The allatostatins are an important group of insect
neurohormones controlling diverse functions including the synthesis
of juvenile hormones known to play a central role in metamorphosis
and reproduction in various insect species. The very first
Drosophila allatostatin, Ser-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH2 (i.e.,
drostatin-3), was isolated from Drosophila head extracts (Birgul et
al., The EMBO J., 1999, 18, 5892-5900). Recently, a Drosophila
allatostatin preprophormone gene has been cloned which encodes four
Drosophila allatostatins: Val-Glu-Arg-Tyr-Ala-Phe-Gly-Leu-NH2
(drostatin-1), Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu-NH2 (drostatin-2),
Ser-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH2 (drostatin-3) and
Thr-Thr-Arg-Pro-Gln-Pro-Phe-Asn-Phe-Gly-Leu-NH2 (drostatin-4) (Lenz
et al., Biochem. Biophys. Res. Comm. 2000, 273, 1126-1131). The
first Drosophila allatostatin receptor was cloned by Birgul et al.
and shown to be functionally activated by drostatin-3 via Gi/Go
pathways (Birgul et al., EMBO J. 1999, 18, 5892-5900). A second
putative Drosophila allatostatin receptor (i.e., DARII). has been
recently cloned (Lenz et al., Biochem. Biophys. Res. Comm. 2000,
273, 571-577). The DARII receptor cDNA (accession No. AF253526)
codes for a protein that is strongly related to the first
Drosophila allatostatin receptor. However, to date no functional
activation of DARII by allatostatins has been reported.
[0010] The sulfakinins are a family of insect Tyr-sulfated
neuropeptides. They show sequence and functional (myotropic
effects, stimulation of digestive enzyme release) similarity to the
vertebrate peptides gastrin and cholecystokinin. A gene encoding
two sulfakinins-(also called drosulfakinins), DSKI
[Phe-Asp-Asp-Tyr(SO3H)-Gly-His-Met-Arg-Phe-amide] <SEQ ID NO:
160> and DSKII
[Gly-Gly-Asp-Asp-Gln-Phe-Asp-Asp-Tyr(SO3H)-Gly-His-Met-Arg-Phe-amide]
<SEQ ID NO: 161>, has been identified in Drosophila
melanogaster (Nichols, (Mol. Cell Neuroscience, 1992, 3, 342-347;
Nichols et al., J. Biol. Chem. 1988, 263, 12167-12170). The
C-terminal heptapeptide sequence,
Asp-Tyr(SO3H)-Gly-His-Met-Arg-Phe-amide <SEQ ID NO: 162>, is
identical in all sulfakinin identified so far from insects that are
widely separated in evolutionary terms. The conservation of the
heptapeptide sequence, including the presence of the sulfated Tyr
residue, in widely divergent insect taxa presumably reflects
functional significance of this myotropic "active core" (Nachman
& Holman, in Insect Neuropeptides; chemistry, biology and
action, Menn, Kelly & Massler, Eds., 1991, pp. 194-214,
American Chemical Society, Washington, D.C.). To our knowledge, to
date no receptors for insect sulfakinins have been identified.
SUMMARY OF THE INVENTION
[0011] The present invention involves the surprising discovery of
novel polypeptides in Drosophila melanogaster, designated herein
DmGPCRs (Drosophila melanogaster G Protein-Coupled Receptors),
which exhibit varying degrees of homology to other neuropeptide
GPCRs. The present invention provides genes encoding these
heretofore unknown G protein-coupled receptors, the DmGPCR
polypeptides encoded by the genes; antibodies to the polypeptides;
kits employing the polynucleotides and polypeptides, and methods of
making and using all of the foregoing. The DmGPCRs may play a role
as a key component, for example, in regulating neuropeptide binding
and/or signaling. DmGPCRs are thus useful in the search for novel
agents that can modify and/or control binding and/or signaling by
neuropeptides or other agents. The DmGPCRs of the present invention
are also useful in the search for human homologs which bind
neuropeptides, and which may lead to eventual treatment regimens.
Exemplary diseases, and conditions, amenable to such treatment
include, but are not limited to, infections, such as viral
infections caused by HIV-1 or HIV-2, pain; cancers, Parkinson's
disease, hypotension, hypertension, diabetes, obesity,
atherosclerosis, thrombosis, stroke, renal failure, inflammation,
rheumatoid arthritis, autoimmune disorders, and psychotic and
neurological disorders, including anxiety, schizophrenia, manic
depression, delirium, dementia, severe mental retardation and
dyskinesias, such as Huntington's disease or Tourette's Syndrome,
among others. These and other aspects of the invention are
described below.
[0012] In one embodiment, the invention provides purified and
isolated DmGPCR polypeptides comprising the amino acid sequence set
forth in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,
or 24, or a fragment thereof comprising an epitope specific to the
DmGPCR. By "epitope specific to" is meant a portion of the DmGPCR
receptor that is recognizable by an antibody that is specific for
the DmGPCR, as defined in detail below. Preferred embodiments
comprise purified and isolated polypeptides comprising the complete
amino acid sequences set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, 22, or 24, found in Table 4 below. These amino acid
sequences were deduced from polynucleotide sequences encoding
DmGPCR (SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23,
found in Table 4 below). The term "DmGPCR" as used herein in
singular form is intended to encompass each of the ten amino acid
sequences exemplified below, encoded by the respective
polynucleotide sequences.
[0013] Although the sequences provided are particular drosophila
sequences, the invention is intended to include within its scope
other allelic variants and other vertebrate forms of DmGPCR.
[0014] It will be appreciated that extracellular epitopes are
particularly useful for generating and screening for antibodies and
other binding compounds that bind to receptors such as DmGPCR.
Thus, in another preferred embodiment, the invention provides a
purified and isolated polypeptide comprising at least one
extracellular domain (e.g., the N-terminal extracellular domain or
one of the three extracellular loops) of DmGPCR. A purified and
isolated polypeptide comprising the N-terminal extracellular domain
of DmGPCR is highly preferred. Also preferred is a purified and
isolated polypeptide comprising a DmGPCR fragment selected from the
group consisting of the N-terminal extracellular domain of DmGPCR,
transmembrane domains of DmGPCR, an extracellular loop connecting
transmembrane domains of DmGPCR, an intracellular loop connecting
transmembrane domains of DmGPCR, the C-terminal cytoplasmic region
of DmGPCR, and fusions thereof. Such fragments may be continuous
portions of the native receptor. However, it will also be
appreciated that knowledge of the DmGPCR gene and protein sequences
as provided herein permits recombining of various domains that are
not contiguous in the native protein.
[0015] In another embodiment, the invention provides purified and
isolated polynucleotides (e.g., cDNA, genomic DNA, synthetic DNA,
RNA, or combinations thereof, whether single- or double-stranded)
that comprise a nucleotide sequence encoding the amino acid
sequence of the polypeptides of the invention. Such polynucleotides
are useful for recombinantly expressing the receptor and also for
detecting expression of the receptor in cells (e.g., using Northern
hybridization and in situ hybridization assays. Such
polynucleotides also are useful in the design of antisense and
other molecules for the suppression of the expression of DmGPCR in
a cultured cell, a tissue, or an animal; for therapeutic purposes;
or to provide a model for diseases or conditions characterized by
aberrant DmGPCR expression. Specifically excluded from the
definition of polynucleotides of the invention are entire isolated,
non-recombinant native chromosomes of host cells. A preferred
polynucleotide has the sequence of any sequence set forth in SEQ ID
NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23, which correspond
to naturally occurring DmGPCR sequences. It will be appreciated
that numerous other polynucleotide sequences exist that also encode
the DmGPCR having the sequence set forth in any of SEQ ID NOs: 2,
4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 due to the well-known
degeneracy of the universal genetic code.
[0016] The invention also provides a purified and isolated
polynucleotide comprising a nucleotide sequence that encodes a
mammalian polypeptide, wherein the polynucleotide hybridizes to a
polynucleotide having the sequence set forth in any of SEQ ID NOs:
1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23 or the non-coding
strand complementary thereto, under the following hybridization
conditions:
[0017] (a) hybridization for 16 hours at 42.degree. C. in a
hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl,
10% dextran sulfate; and
[0018] (b) washing 2 times for 30 minutes each at 60.degree. C. in
a wash solution comprising 0.1% SSC, 1% SDS.
[0019] In a related embodiment, the invention provides vectors
comprising a polynucleotide of the invention. Such vectors are
useful, e.g., for amplifying the polynucleotides in host cells to
create useful quantities thereof. In preferred embodiments, the
vector is an expression vector wherein the polynucleotide of the
invention is operatively linked to a polynucleotide comprising an
expression control sequence. Such vectors are useful for
recombinant production of polypeptides of the invention.
[0020] In another related embodiment, the invention provides host
cells that are transformed or transfected (stably or transiently)
with polynucleotides of the invention or vectors of the invention.
As stated above, such host cells are useful for amplifying the
polynucleotides and also for expressing the DmGPCR polypeptide or
fragment thereof encoded by the polynucleotide.
[0021] In still another related embodiment, the invention provides
a method for producing a DmGPCR polypeptide (or fragment thereof)
comprising the steps of growing a host cell of the invention in a
nutrient medium and isolating the polypeptide or variant thereof
from the cell or the medium. Because DmGPCR is a seven
transmembrane receptor, it will be appreciated that, for some
applications, such as certain activity assays, the preferable
isolation may involve isolation of cell membranes containing the
polypeptide embedded therein, whereas for other applications a more
complete isolation may be preferable.
[0022] In still another embodiment, the invention provides an
antibody that is specific for the DmGPCR of the invention. Antibody
specificity is described in greater detail below. However, it
should be emphasized that antibodies that can be generated from
polypeptides that have previously been described in the literature
and that are capable of fortuitously cross-reacting with DmGPCR
(e.g., due to the fortuitous existence of a similar epitope in both
polypeptides) are considered "cross-reactive" antibodies. Such
cross-reactive antibodies are not antibodies that are "specific"
for DmGPCR. The determination of whether an antibody is specific
for DmGPCR or is cross-reactive with another known receptor is made
using any of several assays, such as Western blotting assays, that
are well known in the art. For identifying cells that express
DmGPCR and also for modulating DmGPCR-ligand binding activity,
antibodies that specifically bind to an extracellular epitope of
the DmGPCR are preferred.
[0023] In one preferred variation, the invention provides
monoclonal antibodies. Hybridomas that produce such antibodies also
are intended as aspects of the invention. In yet another variation,
the invention provides a humanized antibody. Humanized antibodies
are useful for in vivo therapeutic indications.
[0024] In another variation, the invention provides a cell-free
composition comprising polyclonal antibodies, wherein at least one
of the antibodies is an antibody of the invention specific for
DmGPCR. Antisera isolated from an animal is an exemplary
composition, as is a composition comprising an antibody fraction of
an antisera that has been resuspended in water or in another
diluent, excipient, or carrier.
[0025] In still another related embodiment, the invention provides
an anti-idiotypic antibody specific for an antibody that is
specific for DmGPCR.
[0026] It is well known that antibodies contain relatively small
antigen binding domains that can be isolated chemically or by
recombinant techniques. Such domains are useful DmGPCR binding
molecules themselves, and also may be reintroduced into human
antibodies, or fused to toxins or other polypeptides. Thus, in
still another embodiment, the invention provides a polypeptide
comprising a fragment of a DmGPCR-specific antibody, wherein the
fragment and the polypeptide bind to the DmGPCR. By way of
non-limiting example, the invention provides polypeptides that are
single chain antibodies and CDR-grafted antibodies.
[0027] Also within the scope of the invention are compositions
comprising polypeptides, polynucleotides, or antibodies of the
invention that have been formulated with, e.g., a pharmaceutically
acceptable carrier.
[0028] The invention also provides methods of using antibodies of
the invention. For example, the invention provides a method for
modulating ligand binding of a DmGPCR comprising the step of
contacting the DmGPCR with an antibody specific for the DmGPCR,
under conditions wherein the antibody binds the receptor.
[0029] Mammalian homologs of DmGPCRs that are expressed in the
brain provide an indication that aberrant DmGPCR signaling activity
may correlate with one or more neurological or psychological
disorders. The invention also provides a method for treating a
neurological or psychiatric disorder comprising the step of
administering to a mammal in need of such treatment an amount of an
antibody-like polypeptide of the invention that is sufficient to
modulate ligand binding to a DmGPCR in neurons of the mammal.
Mammalian homologs of DmGPCR may also be expressed in other
tissues, including but not limited to pancreas (and particularly
pancreatic islet tissue), pituitary, skeletal muscle, adipose
tissue, liver, and thyroid.
[0030] The invention also provides assays to identify compounds
that bind a DmGPCR. One such assay comprises the steps of: (a)
contacting a composition comprising a DmGPCR with a compound
suspected of binding DmGPCR; and (b) measuring binding between the
compound and DmGPCR. In one variation, the composition comprises a
cell expressing DmGPCR on its surface. In another variation,
isolated DmGPCR or cell membranes comprising DmGPCR are employed.
The binding may be measured directly, e.g., by using a labeled
compound, or may be measured indirectly by several techniques,
including measuring intracellular signaling of DmGPCR induced by
the compound (or measuring changes in the level of DmGPCR
signaling).
[0031] The invention also provides a method for identifying a
modulator of binding between a DmGPCR and a DmGPCR binding partner,
comprising the steps of: (a) contacting a DmGPCR binding partner
and a composition comprising a DmGPCR in the presence and in the
absence of a putative modulator compound; (b) detecting binding
between the binding partner and the DmGPCR; and (c) identifying a
putative modulator compound or a modulator compound in view of
decreased or increased binding between the binding partner and the
DmGPCR in the presence of the putative modulator, as compared to
binding in the absence of the putative modulator.
[0032] DmGPCR binding partners that stimulate DmGPCR activity are
useful as agonists in disease states or conditions characterized by
insufficient DmGPCR signaling (e.g., as a result of insufficient
activity of a DmGPCR ligand). DmGPCR binding partners that block
ligand-mediated DmGPCR signaling are useful as DmGPCR antagonists
to treat disease states or conditions characterized by excessive
DmGPCR signaling. In addition DmGPCR modulators in general, as well
as DmGPCR polynucleotides and polypeptides, are useful in
diagnostic assays for such diseases or conditions.
[0033] In another aspect, the invention provides methods for
treating a disease or abnormal condition by administering to a
patient in need of such treatment a substance that modulates the
activity or expression of a polypeptide selected from the group
consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,
or 24.
[0034] In another aspect, the invention features methods for
detection of a polypeptide in a sample as a diagnostic tool for
diseases or disorders, wherein the method comprises the steps of:
(a) contacting the sample with a nucleic acid probe which
hybridizes under hybridization assay conditions to a nucleic acid
target region of a polypeptide selected from the group consisting
of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24, said
probe comprising the nucleic acid sequence encoding the
polypeptide, fragments thereof, and the complements of the
sequences and fragments; and (b) detecting the presence or amount
of the probe:target region hybrid as an indication of the
disease.
[0035] In preferred embodiments of the invention, the disease is
selected from the group consisting of metabolic disorders,
rheumatoid arthritis, artherosclerosis, autoimmune disorders, organ
transplantation, myocardial infarction, cardiomyopathies, stroke,
renal failure, oxidative stress-related neurodegenerative disorders
and cancer.
[0036] Substances useful for treatment of disorders or diseases
preferably show positive results in one or more in vitro assays for
an activity corresponding to treatment of the disease or disorder
in question. Substances that modulate the activity of the
polypeptides preferably include, but are not limited to, antisense
oligonucleotides, agonists and antagonists, and inhibitors of
protein kinases.
[0037] Hybridization conditions should be such that hybridization
occurs only with the genes in the presence of other nucleic acid
molecules. Under stringent hybridization conditions only highly
complementary nucleic acid sequences hybridize. Preferably, such
conditions prevent hybridization of nucleic acids having 1 or 2
mismatches out of 20 contiguous nucleotides. Such conditions are
defined supra.
[0038] The diseases for which detection of genes in a sample could
be diagnostic include diseases in which nucleic acid (DNA and/or
RNA) is amplified in comparison to normal cells. By "amplification"
is meant increased numbers of DNA or RNA in a cell compared with
normal cells.
[0039] The diseases that could be diagnosed by detection of nucleic
acid in a sample preferably include central nervous system and
metabolic diseases. The test samples suitable for nucleic acid
probing methods of the present invention include, for example,
cells or nucleic acid extracts of cells, or biological fluids. The
samples used in the above-described methods will vary based on the
assay format, the detection method and the nature of the tissues,
cells or extracts to be assayed. Methods for preparing nucleic acid
extracts of cells are well known in the art and can be readily
adapted in order to obtain a sample that is compatible with the
method utilized.
[0040] Additional features and variations of the invention will be
apparent to those skilled in the art from the entirety of this
application, including the detailed description, and all such
features are intended as aspects of the invention. Likewise,
features of the invention described herein can be re-combined into
additional embodiments that also are intended as aspects of the
invention, irrespective of whether the combination of features is
specifically mentioned above as an aspect or embodiment of the
invention. Also, only such limitations which are described herein
as critical to the invention should be viewed as such; variations
of the invention lacking limitations which have not been described
herein as critical are intended as aspects of the invention.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0041] The present invention provides, inter alia, isolated and
purified polynucleotides that encode D. melanogaster G protein
coupled receptor (DmGPCR) or a portion thereof, vectors containing
these polynucleotides, host cells transformed with these vectors,
processes of making DmGPCR, methods of using the above
polynucleotides and vectors, isolated and purified DmGPCR, methods
of screening compounds which modulate DmGPCR activity, and methods
of identifying mammalian or other invertebrate homologs of
DmGPCR.
[0042] Various definitions are made throughout this document. Most
words have the meaning that would be attributed to those words by
one skilled in the art. Words specifically defined either below or
elsewhere in this document have the meaning provided in the context
of the present invention as a whole and as are typically understood
by those skilled in the art. "Synthesized" as used herein and
understood in the art, refers to polynucleotides produced by purely
chemical, as opposed to enzymatic, methods. "Wholly" synthesized
DNA sequences are therefore produced entirely by chemical means,
and "partially" synthesized DNAs embrace those wherein only
portions of the resulting DNA were produced by chemical means. By
the term "region" is meant a physically contiguous portion of the
primary structure of a biomolecule. In the case of proteins, a
region is defined by a contiguous portion of the amino acid
sequence of that protein. The term "domain" is herein defined as
referring to a structural part of a biomolecule that contributes to
a known or suspected function of the biomolecule. Domains may be
co-extensive with regions or portions thereof; domains may also
incorporate a portion of a biomolecule that is distinct from a
particular region, in addition to all or part of that region.
Examples of GPCR protein domains include, but are not limited to,
the extracellular (i.e., N-terminal), transmembrane and cytoplasmic
(i.e., C-terminal) domains, which are co-extensive with like-named
regions of GPCRs; each of the seven transmembrane segments of a
GPCR; and each of the loop segments (both extracellular and
intracellular loops) connecting adjacent transmembrane
segments.
[0043] As used herein, the term "activity" refers to a variety of
measurable indicia suggesting or revealing binding, either direct
or indirect; affecting a response, i.e. having a measurable affect
in response to some exposure or stimulus, including, for example,
the affinity of a compound for directly binding a polypeptide or
polynucleotide of the invention, or, for example, measurement of
amounts of upstream or downstream proteins or other similar
functions after some stimulus or event.
[0044] As used herein, the term "antibody" is meant to refer to
complete, intact antibodies, and Fab, Fab', F(ab)2, and other
fragments thereof. Complete, intact antibodies include monoclonal
antibodies such as murine monoclonal antibodies, chimeric
antibodies and humanized antibodies.
[0045] As used herein, the term "binding" means the physical or
chemical interaction between two proteins or compounds or
associated proteins or compounds or combinations thereof. Binding
includes ionic, non-ionic, Hydrogen bonds, Van der Waals,
hydrophobic interactions, etc. The physical interaction, the
binding, can be either direct or indirect, indirect being through
or due to the effects of another protein or compound. Direct
binding refers to interactions that do not take place through or
due to the effect of another protein or compound but instead are
without other substantial chemical intermediates.
[0046] As used herein, the term "compound" means any identifiable
chemical or molecule, including, but not limited to, small
molecule, peptide, protein, sugar, nucleotide, or nucleic acid, and
such compound can be natural or synthetic.
[0047] As used herein, the term "complementary" refers to
Watson-Crick basepairing between nucleotide units of a nucleic acid
molecule.
[0048] As used herein, the term "contacting" means bringing
together, either directly or indirectly, a compound into physical
proximity to a polypeptide or polynucleotide of the invention. The
polypeptide or polynucleotide can be in any number of buffers,
salts, solutions etc. Contacting includes, for example, placing the
compound into a beaker, microtiter plate, cell culture flask, or a
microarray, such as a gene chip, or the like, which contains the
nucleic acid molecule, or polypeptide encoding the GPCR or fragment
thereof.
[0049] As used herein, the phrase "homologous nucleotide sequence,"
or "homologous amino acid sequence," or variations thereof, refers
to sequences characterised by a homology, at the nucleotide level
or amino acid level, of at least the specified percentage.
Homologous nucleotide sequences include those sequences coding for
isoforms of proteins. Such isoforms can be expressed in different
tissues of the same organism as a result of, for example,
alternative splicing of RNA. Alternatively, isoforms can be encoded
by different genes. Homologous nucleotide sequences include
nucleotide sequences encoding for a protein of a species other than
insects, including, but not limited to, mammals. Homologous
nucleotide sequences also include, but are not limited to,
naturally occurring allelic variations and mutations of the
nucleotide sequences set forth herein. A homologous nucleotide
sequence does not, however, include the nucleotide sequence
encoding other known GPCRs. Homologous amino acid sequences include
those amino acid sequences which encode conservative amino acid
substitutions, as well as polypeptides having neuropeptide binding
and/or signalling activity. A homologous amino acid sequence does
not, however, include the amino acid sequence encoding other known
GPCRs. Percent homology can be determined by, for example, the Gap
program (Wisconsin Sequence Analysis Package, Version 8 for Unix,
Genetics Computer Group, University Research Park, Madison Wis.),
using the default settings, which uses the algorithm of Smith and
Waterman (Adv. Appl. Math., 1981, 2, 482-489, which is incorporated
herein by reference in its entirety).
[0050] As used herein, the term "isolated" nucleic acid molecule
refers to a nucleic acid molecule (DNA or RNA) that has been
removed from its native environment. Examples of isolated nucleic
acid molecules include, but are not limited to, recombinant DNA
molecules contained in a vector, recombinant DNA molecules
maintained in a heterologous host cell, partially or substantially
purified nucleic acid molecules, and synthetic DNA or RNA
molecules.
[0051] As used herein, the terms "modulates" or "modifies" means an
increase or decrease in the amount, quality, or effect of a
particular activity or protein.
[0052] As used herein, the term "oligonucleotide" refers to a
series of linked nucleotide residues which has a sufficient number
of bases to be used in a polymerase chain reaction (PCR). This
short sequence is based on (or designed from) a genomic or cDNA
sequence and is used to amplify, confirm, or reveal the presence of
an identical, similar or complementary DNA or RNA in a particular
cell or tissue. Oligonucleotides comprise portions of a DNA
sequence having at least about 10 nucleotides and as many as about
50 nucleotides, preferably about 15 to 30 nucleotides. They are
chemically synthesized and may be used as probes.
[0053] As used herein, the term "probe" refers to nucleic acid
sequences of variable length, preferably between at least about 10
and as many as about 6,000 nucleotides, depending on use. They are
used in the detection of identical, similar, or complementary
nucleic acid sequences. Longer length probes are usually obtained
from a natural or recombinant source, are highly specific and much
slower to hybridize than oligomers. They may be single- or
double-stranded and carefully designed to have specificity in PCR,
hybridization membrane-based, or ELISA-like technologies.
[0054] The term "preventing" refers to decreasing the probability
that an organism contracts or develops an abnormal condition.
[0055] The term "treating" refers to having a therapeutic effect
and at least partially alleviating or abrogating an abnormal
condition in the organism.
[0056] The term "therapeutic effect" refers to the inhibition or
activation factors causing or contributing to the abnormal
condition. A therapeutic effect relieves to some extent one or more
of the symptoms of the abnormal condition. In reference to the
treatment of abnormal conditions, a therapeutic effect can refer to
one or more of the following: (a) an increase in the proliferation,
growth, and/or differentiation of cells; (b) inhibition (i.e.,
slowing or stopping) of cell death; (c) inhibition of degeneration;
(d) relieving to some extent one or more of the symptoms associated
with the abnormal condition; and (e) enhancing the function of the
affected population of cells. Compounds demonstrating efficacy
against abnormal conditions can be identified as described
herein.
[0057] The term "abnormal condition" refers to a function in the
cells or tissues of an organism that deviates from their normal
functions in that organism. An abnormal condition can relate to
cell proliferation, cell differentiation, cell signalling, or cell
survival. An abnormal condition may also include obesity, diabetic
complications such as retinal degeneration, and irregularities in
glucose uptake and metabolism, and fatty acid uptake and
metabolism.
[0058] Abnormal cell proliferative conditions include cancers such
as fibrotic and mesangial disorders, abnormal angiogenesis and
vasculogenesis, wound healing, psoriasis, diabetes mellitus, and
inflammation.
[0059] Abnormal differentiation conditions include, but are not
limited to, neurodegenerative disorders, slow wound healing rates,
and slow tissue grafting healing rates.
[0060] Abnormal cell signalling conditions include, but are not
limited to, psychiatric disorders involving excess neurotransmitter
activity.
[0061] Abnormal cell survival conditions may also relate to
conditions in which programmed cell death (apoptosis) pathways are
activated or abrogated. A number of protein kinases are associated
with the apoptosis pathways. Aberrations in the function of any one
of the protein kinases could lead to cell immortality or premature
cell death.
[0062] The term "administering" relates to a method of
incorporating a compound into cells or tissues of an organism. The
abnormal condition can be prevented or treated when the cells or
tissues of the organism exist within the organism or outside of the
organism. Cells existing outside the organism can be maintained or
grown in cell culture dishes. For cells harbored within the
organism, many techniques exist in the art to administer compounds,
including (but not limited to) oral, parenteral, dermal, injection,
and aerosol applications. For cells outside of the organism,
multiple techniques exist in the art to administer the compounds,
including (but not limited to) cell microinjection techniques,
transformation techniques and carrier techniques.
[0063] The abnormal condition can also be prevented or treated by
administering a compound to a group of cells having an aberration
in a signal transduction pathway to an organism. The effect of
administering a compound on organism function can then be
monitored. The organism is preferably a mouse, rat, rabbit, guinea
pig or goat, more preferably a monkey or ape, and most preferably a
human.
[0064] By "amplification" it is meant increased numbers of DNA or
RNA in a cell compared with normal cells. "Amplification" as it
refers to RNA can be the detectable presence of RNA in cells, since
in some normal cells there is no basal expression of RNA. In other
normal cells, a basal level of expression exists, therefore in
these cases amplification is the detection of at least 1-2-fold,
and preferably more, compared to the basal level.
[0065] As used herein, the phrase "stringent hybridization
conditions" or "stringent conditions" refers to conditions under
which a probe, primer, or oligonucleotide will hybridize to its
target sequence, but to no other sequences. Stringent conditions
are sequence-dependent and will be different in different
circumstances. Longer sequences hybridize specifically at higher
temperatures. Generally, stringent conditions are selected to be
about 5.degree. C. lower than the thermal melting point (Tm) for
the specific sequence at a defined ionic strength and pH. The Tm is
the temperature (under defined ionic strength, pH and nucleic acid
concentration) at which 50% of the probes complementary to the
target sequence hybridize to the target sequence at equilibrium.
Since the target sequences are generally present in excess, at Tm,
50% of the probes are occupied at equilibrium. Typically, stringent
conditions will be those in which the salt concentration is less
than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium
ion (or other salts) at pH 7.0 to 8.3 and the temperature is at
least about 30.degree. C. for short probes, primers or
oligonucleotides (e.g. 10 to 50 nucleotides) and at least about
60.degree. C. for longer probes, primers or oligonucleotides.
Stringent conditions may also be achieved with the addition of
destabilizing agents, such as formamide.
[0066] The amino acid sequences are presented in the amino to
carboxy direction, from left to right. The amino and carboxy groups
are not presented in the sequence. The nucleotide sequences are
presented by single strand only, in the 5' to 3' direction, from
left to right. Nucleotides and amino acids are represented in the
manner recommended by the IUPAC-ITUB Biochemical Nomenclature
Commission, or (for amino acids) by three letters code.
[0067] Genomic DNA of the invention comprises the protein coding
region for a polypeptide of the invention and is also intended to
include allelic variants thereof. It is widely understood that, for
many genes, genomic DNA is transcribed into RNA transcripts that
undergo one or more splicing events wherein intron (i.e.,
non-coding regions) of the transcripts are removed, or "spliced
out." RNA transcripts that can be spliced by alternative
mechanisms, and therefore be subject to removal of different RNA
sequences but still encode a DmGPCR polypeptide, are referred to in
the art as splice variants which are embraced by the invention.
Splice variants comprehended by the invention therefore are encoded
by the same original genomic DNA sequences but arise from distinct
mRNA transcripts. Allelic variants are modified forms of a
wild-type gene sequence, the modification resulting from
recombination during chromosomal segregation or exposure to
conditions which give rise to genetic mutation. Allelic variants,
like wild type genes, are naturally occurring sequences (as opposed
to non-naturally occurring variants which arise from in vitro
manipulation).
[0068] The invention also comprehends cDNA that is obtained through
reverse transcription of an RNA polynucleotide encoding DmGPCR
(conventionally followed by second strand synthesis of a
complementary strand to provide a double-stranded DNA).
[0069] A preferred DNA sequence encoding a DmGPCR polypeptide is
set out in any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,
21, or 23. A preferred DNA of the invention comprises a double
stranded molecule along with the complementary molecule (the
"non-coding strand" or "complement") having a sequence
unambiguously deducible from the coding strand according to
Watson-Crick base-pairing rules for DNA. Also preferred are other
polynucleotides encoding any of the particular DmGPCR polypeptides
of the invention which differ in sequence from the particular
polynucleotides described herein by virtue of the well-known
degeneracy of the universal nuclear genetic code.
[0070] The invention further embraces species, preferably
mammalian, homologs of the DmGPCR DNA. Species homologs, sometimes
referred to as "orthologs," in general, share at least 35%, at
least 40%, at least 45%, at least 50%, at least 60%, at least 65%,
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 98%, or at least 99% homology with DNA
of the invention. Generally, percent sequence "homology" with
respect to polynucleotides of the invention may be calculated as
the percentage of nucleotide bases in the candidate sequence that
are identical to nucleotides in the DmGPCR sequence set forth in a
particular polynucleotide sequence, after aligning the sequences
and introducing gaps, if necessary, to achieve the maximum percent
sequence identity.
[0071] The polynucleotide sequence information provided by the
invention makes possible large-scale expression of the encoded
polypeptide by techniques well known and routinely practiced in the
art. Polynucleotides of the invention also permit identification
and isolation of polynucleotides encoding related DmGPCR
polypeptides, such as allelic variants and species homologs, by
well-known techniques including Southern and/or Northern
hybridization, and polymerase chain reaction (PCR). Examples of
related polynucleotides include genomic sequences, including
allelic variants, as well as polynucleotides encoding polypeptides
homologous to DmGPCR and structurally related polypeptides sharing
one or more biological, immunological, and/or physical properties
of DmGPCR. Genes encoding proteins homologous to DmGPCR can also be
identified by Southern and/or PCR analysis and are useful in animal
models for GPCR disorders. Knowledge of the sequence of a DmGPCR
DNA also makes possible through use of Southern hybridization or
polymerase chain reaction (PCR) the identification of genomic DNA
sequences encoding DmGPCR expression control regulatory sequences
such as promoters, operators, enhancers, repressors, and the like.
Polynucleotides of the invention are also useful in hybridization
assays to detect the capacity of cells to express DmGPCR.
Polynucleotides of the invention may also provide a basis for
diagnostic methods useful for identifying a genetic alteration(s)
in a DmGPCR locus that underlies a disease state or states, which
information is useful both for diagnosis and for selection of
therapeutic strategies.
[0072] The disclosure herein of a full-length polynucleotide
encoding a DmGPCR polypeptide makes readily available to the worker
of ordinary skill in the art every possible fragment of the full
length polynucleotide. The invention therefore provides fragments
of DmGPCR-encoding polynucleotides comprising at least 14, and
preferably at least 16, 18, 20, 25, 50, or 75 consecutive
nucleotides of a polynucleotide encoding DmGPCR. Preferably,
fragment polynucleotides of the invention comprise sequences unique
to the DmGPCR-encoding polynucleotide sequence, and therefore
hybridize under highly stringent or moderately stringent conditions
only (i.e., "specifically") to polynucleotides encoding DmGPCR (or
fragments thereof). Polynucleotide fragments of genomic sequences
of the invention comprise not only sequences unique to the coding
region, but also include fragments of the full-length sequence
derived from introns, regulatory regions, and/or other
non-translated sequences. Sequences unique to polynucleotides of
the invention are recognizable through sequence comparison to other
known polynucleotides, and can be identified through use of
alignment programs routinely utilized in the art, e.g., those made
available in public sequence databases. Such sequences also are
recognizable from Southern hybridization analyses to determine the
number of fragments of genomic DNA to which a polynucleotide will
hybridize. Polynucleotides of the invention can be labeled in a
manner that permits their detection, including radioactive,
fluorescent, and enzymatic labeling.
[0073] Fragment polynucleotides are particularly useful as probes
for detection of full-length or fragment DmGPCR polynucleotides.
One or more polynucleotides can be included in kits that are used
to detect the presence of a polynucleotide encoding DmGPCR, or used
to detect variations in a polynucleotide sequence encoding
DmGPCR.
[0074] The invention also embraces DNAs encoding DmGPCR
polypeptides that hybridize under moderately stringent or high
stringency conditions to the non-coding strand, or complement, of
the polynucleotides in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, or 23.
[0075] Exemplary highly stringent hybridization conditions are as
follows: hybridization at 42.degree. C. in a hybridization solution
comprising 50% formamide, 1% SDS, 1 M NaCl, 10% Dextran sulfate,
and washing twice for 30 minutes at 60.degree. C. in a wash
solution comprising 0.1.times.SSC and 1% SDS. It is understood in
the art that conditions of equivalent stringency can be achieved
through variation of temperature and buffer, or salt concentration
as described Ausubel, et al. (Eds.), Protocols in Molecular
Biology, John Wiley & Sons (1994), pp. 6.0.3 to 6.4.10.
Modifications in hybridization conditions can be empirically
determined or precisely calculated based on the length and the
percentage of guanosine/cytosine (GC) base pairing of the probe.
The hybridization conditions can be calculated as described in
Sambrook, et al., (Eds.), Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.
(1989), pp. 9.47 to 9.51.
[0076] Autonomously replicating recombinant expression constructs
such as plasmid and viral DNA vectors incorporating polynucleotides
of the invention are also provided. Expression constructs wherein
DmGPCR-encoding polynucleotides are operatively linked to an
endogenous or exogenous expression control DNA sequence and a
transcription terminator are also provided. Expression control DNA
sequences include promoters, enhancers, operators, and regulatory
element binding sites generally, and are typically selected based
on the expression systems in which the expression construct is to
be utilized. Preferred promoter and enhancer sequences are
generally selected for the ability to increase gene expression,
while operator sequences are generally selected for the ability to
regulate gene expression. Expression constructs of the invention
may also include sequences encoding one or more selectable markers
that permit identification of host cells bearing the construct.
Expression constructs may also include sequences that facilitate,
and preferably promote, homologous recombination in a host cell.
Preferred constructs of the invention also include sequences
necessary for replication in a host cell.
[0077] Expression constructs are preferably utilized for production
of an encoded protein, but may also be utilized simply to amplify a
DmGPCR-encoding polynucleotide sequence.
[0078] According to another aspect of the invention, host cells are
provided, including prokaryotic and eukaryotic cells, comprising a
polynucleotide of the invention (or vector of the invention) in a
manner which permits expression of the encoded DmGPCR polypeptide.
Polynucleotides of the invention may be introduced into the host
cell as part of a circular plasmid, or as linear DNA comprising an
isolated protein coding region or a viral vector. Methods for
introducing DNA into the host cell that are well known and
routinely practiced in the art include transformation,
transfection, electroporation, nuclear injection, or fusion with
carriers such as liposomes, micelles, ghost cells, and protoplasts.
Expression systems of the invention include bacterial, yeast,
fungal, plant, insect, invertebrate, vertebrate, and mammalian
cells systems.
[0079] Host cells of the invention are a valuable source of
immunogen for development of antibodies specifically immunoreactive
with DmGPCR. Host cells of the invention are also useful in methods
for the large-scale production of DmGPCR polypeptides wherein the
cells are grown in a suitable culture medium and the desired
polypeptide products are isolated from the cells, or from the
medium in which the cells are grown, by purification methods known
in the art, e.g., conventional chromatographic methods including
immunoaffinity chromatography, receptor affinity chromatography,
hydrophobic interaction chromatography, lectin affinity
chromatography, size exclusion filtration, cation or anion exchange
chromatography, high pressure liquid chromatography (HPLC), reverse
phase HPLC, and the like. Still other methods of purification
include those methods wherein the desired protein is expressed and
purified as a fusion protein having a specific tag, label, or
chelating moiety that is recognized by a specific binding partner
or agent. The purified protein can be cleaved to yield the desired
protein, or can be left as an intact fusion protein. Cleavage of
the fusion component may produce a form of the desired protein
having additional amino acid residues as a result of the cleavage
process.
[0080] Knowledge of DmGPCR DNA sequences allows for modification of
cells to per nit, or increase, expression of endogenous DmGPCR.
Cells can be modified (e.g., by homologous recombination) to
provide increased expression by replacing, in whole or in part, the
naturally occurring DmGPCR promoter with all or part of a
heterologous promoter so that the cells express DmGPCR at higher
levels. The heterologous promoter is inserted in such a manner that
it is operatively linked to endogenous DmGPCR encoding sequences.
(See, for example, PCT International Publication No. WO 94/12650,
PCT International Publication No. WO 92/20808, and PCT
International Publication No. WO 91/09955.) It is also contemplated
that, in addition to heterologous promoter DNA, amplifiable marker
DNA (e.g., ada, dhfr, and the multifunctional CAD gene which
encodes carbamoyl phosphate synthase, aspartate transcarbamylase,
and dihydroorotase) and/or intron DNA may be inserted along with
the heterologous promoter DNA. If linked to the DmGPCR coding
sequence, amplification of the marker DNA by standard selection
methods results in co-amplification of the DmGPCR coding sequences
in the cells.
[0081] The DNA sequence information provided by the present
invention also makes possible the development (e.g., by homologous
recombination or "knock-out" strategies; see Capecchi, Science
244:1288-1292 (1989)) of animals that fail to express functional
DmGPCR or that express a variant of DmGPCR. Such animals
(especially small laboratory animals such as rats, rabbits, and
mice) are useful as models for studying the in vivo activities of
DmGPCR and modulators of DmGPCR.
[0082] Also made available by the invention are anti-sense
polynucleotides which recognize and hybridize to polynucleotides
encoding DmGPCR. Full-length and fragment anti-sense
polynucleotides are provided. Fragment antisense molecules of the
invention include (i) those which specifically recognize and
hybridize to DmGPCR RNA (as determined by sequence comparison of
DNA encoding DmGPCR to DNA encoding other known molecules).
Identification of sequences unique to DmGPCR-encoding
polynucleotides, can be deduced through use of any publicly
available sequence database, and/or through use of commercially
available sequence comparison programs. After identification of the
desired sequences, isolation through restriction digestion or
amplification using any of the various polymerase chain reaction
techniques well known in the art can be performed. Anti-sense
polynucleotides are particularly relevant to regulating expression
of DmGPCR by those cells expressing DmGPCR mRNA.
[0083] Antisense nucleic acids (preferably 10 to 20 base-pair
oligonucleotides) capable of specifically binding to DmGPCR
expression control sequences or DmGPCR RNA are introduced into
cells (e.g., by a viral vector or colloidal dispersion system such
as a liposome). The antisense nucleic acid binds to the DmGPCR
target nucleotide sequence in the cell and prevents transcription
and/or translation of the target sequence. Phosphorothioate and
methylphosphonate antisense oligonucleotides are specifically
contemplated for therapeutic use by the invention. The antisense
oligonucleotides may be further modified by poly-L-lysine,
transferrin polylysine, or cholesterol moieties at their 5' end.
Suppression of DmGPCR expression at either the transcriptional or
translational level is useful to generate cellular or animal models
for diseases/conditions characterized by aberrant DmGPCR
expression.
[0084] The DmGPCR sequences taught in the present invention
facilitate the design of novel transcription factors for modulating
DmGPCR expression in native cells and animals, and cells
transformed or transfected with DmGPCR polynucleotides. For
example, the Cys.sub.2-His.sub.2 zinc finger proteins, which bind
DNA via their zinc finger domains, have been shown to be amenable
to structural changes that lead to the recognition of different
target sequences. These artificial zinc finger proteins recognize
specific target sites with high affinity and low dissociation
constants, and are able to act as gene switches to modulate gene
expression. Knowledge of the particular DmGPCR target sequence of
the present invention facilitates the engineering of zinc finger
proteins specific for the target sequence using known methods such
as a combination of structure-based modeling and screening of phage
display libraries (Segal et al., Proc. Natl. Acad. Sci. (USA)
96:2758-2763 (1999); Liu et al., Proc. Natl. Acad. Sci. (USA)
94:5525-5530 (1997); Greisman et al., Science 275:657-661 (1997);
Choo et al., J. Mol. Biol. 273:525-532 (1997)). Each zinc finger
domain usually recognizes three or more base pairs. Since a
recognition sequence of 18 base pairs is generally sufficient in
length to render it unique in any known genome, a zinc finger
protein consisting of 6 tandem repeats of zinc fingers would be
expected to ensure specificity for a particular sequence (Segal et
al.) The artificial zinc finger repeats, designed based on DmGPCR
sequences, are fused to activation or repression domains to promote
or suppress DmGPCR expression (Liu et al.) Alternatively, the zinc
finger domains can be fused to the TATA box-binding factor (TBP)
with varying lengths of linker region between the zinc finger
peptide and the TBP to create either transcriptional activators or
repressors (Kim et al., Proc. Natl. Acad. Sci. (USA) 94:3616-3620
(1997). Such proteins, and polynucleotides that encode them, have
utility for modulating DmGPCR expression in vivo. The novel
transcription factor can be delivered to the target cells by
transfecting constructs that express the transcription factor (gene
therapy), or by introducing the protein. Engineered zinc finger
proteins can also be designed to bind RNA sequences for use in
therapeutics as alternatives to antisense or catalytic RNA methods
(McColl et al., Proc. Natl. Acad. Sci. (USA) 96:9521-9526 (1997);
Wu et al., Proc. Natl. Acad. Sci. (USA) 92:344-348 (1995)). The
present invention contemplates methods of designing such
transcription factors based on the gene sequence of the invention,
as well as customized zinc finger proteins, that are useful to
modulate DmGPCR expression in cells (native or transformed) whose
genetic complement includes these sequences.
[0085] The invention also provides purified and isolated mammalian
DmGPCR polypeptides encoded by a polynucleotide of the invention.
Presently preferred is a DmGPCR polypeptide comprising the amino
acid sequence set out in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,
16, 18, 20, 22, or 24.
[0086] The invention also embraces polypeptides that have at least
99%, at least 95%, at least 90%, at least 85%, at least 80%, at
least 75%, at least 70%, at least 65%, at least 60%, at least 55%
or at least 50% identity and/or homology to the preferred
polypeptide of the invention. Percent amino acid sequence
"identity" with respect to the preferred polypeptide of the
invention is defined herein as the percentage of amino acid
residues in the candidate sequence that are identical with the
residues in the DmGPCR sequence after aligning both sequences and
introducing gaps, if necessary, to achieve the maximum percent
sequence identity, and not considering any conservative
substitutions as part of the sequence identity. Percent sequence
"homology" with respect to the preferred polypeptide of the
invention is defined herein as the percentage of amino acid
residues in the candidate sequence that are identical with the
residues in the DmGPCR sequence after aligning the sequences and
introducing gaps, if necessary, to achieve the maximum percent
sequence identity, and also considering any conservative
substitutions as part of the sequence identity.
[0087] In one aspect, percent homology is calculated as the
percentage of amino acid residues in the smaller of two sequences
which align with identical amino acid residue in the sequence being
compared, when four gaps in a length of 100 amino acids may be
introduced to maximize alignment (Dayhoff, in Atlas of Protein
Sequence and Structure, Vol. 5, p. 124, National Biochemical
Research Foundation, Washington, D.C. (1972), incorporated herein
by reference).
[0088] Polypeptides of the invention may be isolated from natural
cell sources or may be chemically synthesized, but are preferably
produced by recombinant procedures involving host cells of the
invention. Use of mammalian host cells is expected to provide for
such post-translational modifications (e.g., glycosylation,
truncation, lipidation, and phosphorylation) as may be needed to
confer optimal biological activity on recombinant expression
products of the invention. Glycosylated and non-glycosylated forms
of DmGPCR polypeptides are embraced by the invention.
[0089] The invention also embraces variant (or analog) DmGPCR
polypeptides. In one example, insertion variants are provided
wherein one or more amino acid residues supplement a DmGPCR amino
acid sequence. Insertions may be located at either or both termini
of the protein, or may be positioned within internal regions of the
DmGPCR amino acid sequence. Insertional variants with additional
residues at either or both termini can include, for example, fusion
proteins and proteins including amino acid tags or labels.
[0090] Insertion variants include DmGPCR polypeptides wherein one
or more amino acid residues are added to a DmGPCR acid sequence, or
to a biologically active fragment thereof.
[0091] Variant products of the invention also include mature DmGPCR
products, i.e., DmGPCR products wherein leader or signal sequences
are removed, with additional amino terminal residues. The
additional amino terminal residues may be derived from another
protein, or may include one or more residues that are not
identifiable as being derived from specific proteins. DmGPCR
products with an additional methionine residue at position -1
(Met.sup.-1-DmGPCR) are contemplated, as are variants with
additional methionine and lysine residues at positions -2 and -1
(Met.sup.-2-Lys.sup.-1-DmGPCR). Variants of DmGPCR with additional
Met, Met-Lys, Lys residues (or one or more basic residues in
general) are particularly useful for enhanced recombinant protein
production in bacterial host cells.
[0092] The invention also embraces DmGPCR variants having
additional amino acid residues which result from use of specific
expression systems. For example, use of commercially available
vectors that express a desired polypeptide as part of a
glutathione-S-transferase (GST) fusion product provides the desired
polypeptide having an additional glycine residue at position -1
after cleavage of the GST component from the desired polypeptide.
Variants which result from expression in other vector systems are
also contemplated.
[0093] Insertional variants also include fusion proteins wherein
the amino terminus and/or the carboxy terminus of DmGPCR is/are
fused to another polypeptide.
[0094] In another aspect, the invention provides deletion variants
wherein one or more amino acid residues in a DmGPCR polypeptide are
removed. Deletions can be effected at one or both termini of the
DmGPCR polypeptide, or with removal of one or more non-terminal
amino acid residues of DmGPCR. Deletion variants, therefore,
include all fragments of a DmGPCR polypeptide.
[0095] The invention also embraces polypeptide fragments of the
sequence set out in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22, or 24 wherein the fragments maintain biological (e.g.,
ligand binding and/or intracellular signaling) immunological
properties of a DmGPCR polypeptide. Fragments comprising at least
5, 10, 15, 20, 25, 30, 35, or 40 consecutive amino acids of any of
the polypeptides described herein are comprehended by the
invention. Preferred polypeptide fragments display antigenic
properties unique to, or specific for, DmGPCR and its allelic and
species homologs. Fragments of the invention having the desired
biological and immunological properties can be prepared by any of
the methods well known and routinely practiced in the art.
[0096] In still another aspect, the invention provides substitution
variants of DmGPCR polypeptides. Substitution variants include
those polypeptides wherein one or more amino acid residues of a
DmGPCR polypeptide are removed and replaced with alternative
residues. In one aspect, the substitutions are conservative in
nature; however, the invention embraces substitutions that are also
non-conservative. Conservative substitutions for this purpose may
be defined as set out in Tables 1, 2, or 3 below.
[0097] Variant polypeptides include those wherein conservative
substitutions have been introduced by modification of
polynucleotides encoding polypeptides of the invention. Amino acids
can be classified according to physical properties and contribution
to secondary and tertiary protein structure. A conservative
substitution is recognized in the art as a substitution of one
amino acid for another amino acid that has similar properties.
Exemplary conservative substitutions are set out in Table 1 (from
WO 97/09433, page 10, published Mar. 13, 1997 (PCT/GB96/02197,
filed Sep. 6, 1996), immediately below.
TABLE-US-00001 TABLE 1 Conservative Substitutions I SIDE CHAIN
CHARACTERISTIC AMINO ACID Aliphatic Non-polar G A P I L V Polar -
uncharged C S T M N Q Polar - charged D E K R Aromatic H F W Y
Other N Q D E
Alternatively, conservative amino acids can be grouped as described
in Lehninger, (Biochemistry, Second Edition; Worth Publishers, Inc.
NY, N.Y. (1975), pp. 71-77) as set out in Table 2, immediately
below.
TABLE-US-00002 TABLE 2 Conservative Substitutions II SIDE CHAIN
CHARACTERISTIC AMINO ACID Non-polar (hydrophobic) A. Aliphatic: A L
I V P B. Aromatic: F W C. Sulfur-containing: M D. Borderline: G
Uncharged-polar A. Hydroxyl: S T Y B. Amides: N Q C. Sulfhydryl: C
D. Borderline: G Positively Charged (Basic): K R H Negatively
Charged (Acidic): D E
As still another alternative, exemplary conservative substitutions
are set out in Table 3, below.
TABLE-US-00003 TABLE 3 Conservative Substitutions III Original
Residue Exemplary Substitution Ala (A) Val, Leu, Ile Arg (R) Lys,
Gln, Asn Asn (N) Gln, His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q)
Asn Glu (E) Asp His (H) Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met,
Ala, Phe, Leu (L) Ile, Val, Met, Ala, Phe Lys (K) Arg, Gln, Asn Met
(M) Leu, Phe, Ile Phe (F) Leu, Val, Ile, Ala Pro (P) Gly Ser (S)
Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp, Phe, Thr, Ser Val (V) Ile,
Leu, Met, Phe, Ala
[0098] It should be understood that the definition of polypeptides
of the invention is intended to include polypeptides bearing
modifications other than insertion, deletion, or substitution of
amino acid residues. By way of example, the modifications may be
covalent in nature, and include for example, chemical bonding with
polymers, lipids, other organic, and inorganic moieties. Such
derivatives may be prepared to increase circulating half-life of a
polypeptide, or may be designed to improve the targeting capacity
of the polypeptide for desired cells, tissues, or organs.
Similarly, the invention further embraces DmGPCR polypeptides that
have been covalently modified to include one or more water-soluble
polymer attachments such as polyethylene glycol, polyoxyethylene
glycol, or polypropylene glycol. Variants that display ligand
binding properties of native DmGPCR and are expressed at higher
levels, as well as variants that provide for constitutively active
receptors, are particularly useful in assays of the invention; the
variants are also useful in providing cellular, tissue and animal
models of diseases/conditions characterized by aberrant DmGPCR
activity.
[0099] In a related embodiment, the present invention provides
compositions comprising purified polypeptides of the invention.
Preferred compositions comprise, in addition to the polypeptide of
the invention, a pharmaceutically acceptable (i.e., sterile and
non-toxic) liquid, semisolid, or solid diluent that serves as a
pharmaceutical vehicle, excipient, or medium. Any diluent known in
the art may be used. Exemplary diluents include, but are not
limited to, water, saline solutions, polyoxyethylene sorbitan
monolaurate, magnesium stearate, methyl- and propylhydroxybenzoate,
talc, alginates, starches, lactose, sucrose, dextrose, sorbitol,
mannitol, glycerol, calcium phosphate, mineral oil, and cocoa
butter.
[0100] Variants that display ligand binding properties of native
DmGPCR and are expressed at higher levels, as well as variants that
provide for constitutively active receptors, are particularly
useful in assays of the invention; the variants are also useful in
assays of the invention and in providing cellular, tissue and
animal models of diseases/conditions characterized by aberrant
DmGPCR activity.
[0101] With the knowledge of the nucleotide sequence information
disclosed in the present invention, one skilled in the art can
identify and obtain nucleotide sequences which encode DmGPCRs from
different sources (i.e., different tissues or different organisms)
through a variety of means well known to the skilled artisan and as
disclosed by, for example, Sambrook et al., "Molecular cloning: a
laboratory manual", Second Edition, Cold Spring Harbor Press, Cold
Spring Harbor, N.Y. (1989), which is incorporated herein by
reference in its entirety.
[0102] For example, DNA that encodes DmGPCR may be obtained by
screening of mRNA, cDNA, or genomic DNA with oligonucleotide probes
generated from the DmGPCR gene sequence information provided
herein. Probes may be labeled with a detectable group, such as a
fluorescent group, a radioactive atom or a chemiluminescent group
in accordance with procedures known to the skilled artisan and used
in conventional hybridization assays, as described by, for example,
Sambrook et al.
[0103] A nucleic acid molecule comprising any of the DmGPCR
nucleotide sequences described above can alternatively be
synthesized by use of the polymerase chain reaction (PCR)
procedure, with the PCR oligonucleotide primers produced from the
nucleotide sequences provided herein. See U.S. Pat. Nos. 4,683,195
to Mullis et al. and 4,683,202 to Mullis. The PCR reaction provides
a method for selectively increasing the concentration of a
particular nucleic acid sequence even when that sequence has not
been previously purified and is present only in a single copy in a
particular sample. The method can be used to amplify either single-
or double-stranded DNA. The essence of the method involves the use
of two oligonucleotides probes to serve as primers for the
template-dependent, polymerase mediated replication of a desired
nucleic acid molecule.
[0104] A wide variety of alternative cloning and in vitro
amplification methodologies are well known to those skilled in the
art. Examples of these techniques are found in, for example, Berger
et al., Guide to Molecular Cloning Techniques, Methods in
Enzymology 152 Academic Press, Inc., San Diego, Calif. (Berger),
which is incorporated herein by reference in its entirety.
[0105] The nucleic acid molecules of the present invention, and
fragments derived therefrom, are useful for screening for
restriction fragment length polymorphism (RFLP) associated with
certain disorders, as well as for genetic mapping.
[0106] Antisense oligonucleotides, or fragments of a nucleotide
sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5,
7, 9, 11, 13, 15, 17, 19, 21, or 23, or sequences complementary or
homologous thereto, derived from the nucleotide sequences of the
present invention encoding DmGPCR are useful as diagnostic tools
for probing gene expression in various tissues. For example, tissue
can be probed in situ with oligonucleotide probes carrying
detectable groups by conventional autoradiography techniques to
investigate native expression of this enzyme or pathological
conditions relating thereto. Antisense oligonucleotides are
preferably directed to regulatory regions of a nucleotide sequence
selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9,
11, 13, 15, 17, 19, 21, or 23, or mRNA corresponding thereto,
including, but not limited to, the initiation codon, TATA box,
enhancer sequences, and the like.
[0107] Automated sequencing methods can be used to obtain or verify
the nucleotide sequence of DmGPCR. The DmGPCR nucleotide sequences
of the present invention are believed to be 100% accurate. However,
as is known in the art, nucleotide sequence obtained by automated
methods may contain some errors. Nucleotide sequences determined by
automation are typically at least about 90%, more typically at
least about 95% to at least about 99.9% identical, to the actual
nucleotide sequence of a given nucleic acid molecule. The actual
sequence may be more precisely determined using manual sequencing
methods, which are well known in the art. An error in a sequence
which results in an insertion or deletion of one or more
nucleotides may result in a frame shift in translation such that
the predicted amino acid sequence will differ from that which would
be predicted from the actual nucleotide sequence of the nucleic
acid molecule, starting at the point of the mutation.
[0108] Another aspect of the present invention is directed to
vectors, or recombinant expression vectors, comprising any of the
nucleic acid molecules described above. Vectors are used herein
either to amplify DNA or RNA encoding DmGPCR and/or to express DNA
which encodes DmGPCR. Preferred vectors include, but are not
limited to, plasmids, phages, cosmids, episomes, viral particles or
viruses, and integratable DNA fragments (i.e., fragments
integratable into the host genome by homologous recombination).
Preferred viral particles include, but are not limited to,
adenoviruses, baculoviruses, parvoviruses, herpesviruses,
poxviruses, adeno-associated viruses, Semliki Forest viruses,
vaccinia viruses, and retroviruses. Preferred expression vectors
include, but are not limited to, pcDNA3 (Invitrogen) and pSVL
(Pharmacia Biotech). Other expression vectors include, but are not
limited to, pSPORT vectors, pGEM vectors (Promega), pPROEXvectors
(LTI, Bethesda, Md.), Bluescript vectors (Stratagene), pQE vectors
(Qiagen), pSE420 (Invitrogen), and pYES2 (Invitrogen).
[0109] Preferred expression vectors are replicable DNA constructs
in which a DNA sequence encoding DmGPCR is operably linked or
connected to suitable control sequences capable of effecting the
expression of the DmGPCR in a suitable host. DNA regions are
operably linked or connected when they are functionally related to
each other. For example, a promoter is operably linked or connected
to a coding sequence if it controls the transcription of the
sequence. Amplification vectors do not require expression control
domains, but rather need only the ability to replicate in a host,
usually conferred by an origin of replication, and a selection gene
to facilitate recognition of transformants. The need for control
sequences in the expression vector will vary depending upon the
host selected and the transformation method chosen. Generally,
control sequences include a transcriptional promoter, an optional
operator sequence to control transcription, a sequence encoding
suitable mRNA ribosomal binding, and sequences which control the
termination of transcription and translation.
[0110] Preferred vectors preferably contain a promoter that is
recognised by the host organism. The promoter sequences of the
present invention may be prokaryotic, eukaryotic or viral. Examples
of suitable prokaryotic sequences include the P.sub.R and P.sub.L
promoters of bacteriophage lambda (The bacteriophage Lambda,
Hershey, A. D., Ed., Cold Spring Harbor Press, Cold Spring Harbor,
N.Y. (1973), which is incorporated herein by reference in its
entirety; Lambda II, Hendrix, R. W., Ed., Cold Spring Harbor Press,
Cold Spring Harbor, N.Y. (1980), which is incorporated herein by
reference in its entirety); the trp, recA, heat shock, and lacZ
promoters of E. coli and the SV40 early promoter (Benoist, et al.
Nature, 1981, 290, 304-310, which is incorporated herein by
reference in its entirety). Additional promoters include, but are
not limited to, mouse mammary tumor virus, long terminal repeat of
human immunodeficiency virus, maloney virus, cytomegalovirus
immediate early promoter, Epstein Barr virus, rous sarcoma virus,
human actin, human myosin, human hemoglobin, human muscle creatine,
and human metalothionein.
[0111] Additional regulatory sequences can also be included in
preferred vectors. Preferred examples of suitable regulatory
sequences are represented by the Shine-Dalgarno of the replicase
gene of the phage MS-2 and of the gene cII of bacteriophage lambda.
The Shine-Dalgarno sequence may be directly followed by DNA
encoding DmGPCR and result in the expression of the mature DmGPCR
protein.
[0112] Moreover, suitable expression vectors can include an
appropriate marker that allows the screening of the transformed
host cells. The transformation of the selected host is carried out
using any one of the various techniques well known to the expert in
the art and described in Sambrook et al., supra.
[0113] An origin of replication can also be provided either by
construction of the vector to include an exogenous origin or may be
provided by the host cell chromosomal replication mechanism. If the
vector is integrated into the host cell chromosome, the latter may
be sufficient. Alternatively, rather than using vectors which
contain viral origins of replication, one skilled in the art can
transform mammalian cells by the method of co-transformation with a
selectable marker and DmGPCR DNA. An example of a suitable marker
is dihydrofolate reductase (DHFR) or thymidine kinase (see, U.S.
Pat. No. 4,399,216).
[0114] Nucleotide sequences encoding GPCR may be recombined with
vector DNA in accordance with conventional techniques, including
blunt-ended or staggered-ended termini for ligation, restriction
enzyme digestion to provide appropriate termini, filling in of
cohesive ends as appropriate, alkaline phosphatase treatment to
avoid undesirable joining, and ligation with appropriate ligases.
Techniques for such manipulation are disclosed by Sambrook et al.,
supra and are well known in the art. Methods for construction of
mammalian expression vectors are disclosed in, for example, Okayama
et al., Mol. Cell. Biol., 1983, 3, 280, Cosman et al., Mol.
Immunol., 1986, 23, 935, Cosman et al., Nature, 1984, 312, 768,
EP-A-0367566, and WO 91/18982, each of which is incorporated herein
by reference in its entirety.
[0115] Another aspect of the present invention is directed to
transformed host cells having an expression vector comprising any
of the nucleic acid molecules described above. Expression of the
nucleotide sequence occurs when the expression vector is introduced
into an appropriate host cell. Suitable host cells for expression
of the polypeptides of the invention include, but are not limited
to, prokaryotes, yeast, and eukaryotes. If a prokaryotic expression
vector is employed, then the appropriate host cell would be any
prokaryotic cell capable of expressing the cloned sequences.
Suitable prokaryotic cells include, but are not limited to,
bacteria of the genera Escherichia, Bacillus, Salmonella,
Pseudomonas, Streptomyces, and Staphylococcus.
[0116] If a eukaryotic expression vector is employed, then the
appropriate host cell would be any eukaryotic cell capable of
expressing the cloned sequence. Preferably, eukaryotic cells are
cells of higher eukaryotes. Suitable eukaryotic cells include, but
are not limited to, non-human mammalian tissue culture cells and
human tissue culture cells. Preferred host cells include, but are
not limited to, insect cells, HeLa cells, Chinese hamster ovary
cells (CHO cells), African green monkey kidney cells (COS cells),
human 293 cells, and murine 3T3 fibroblasts. Propagation of such
cells in cell culture has become a routine procedure (see, Tissue
Culture, Academic Press, Kruse and Patterson, eds. (1973), which is
incorporated herein by reference in its entirety).
[0117] In addition, a yeast host may be employed as a host cell.
Preferred yeast cells include, but are not limited to, the genera
Saccharomyces, Pichia, and Kluveromyces. Preferred yeast hosts are
S. cerevisiae and P. pastoris. Preferred yeast vectors can contain
an origin of replication sequence from a 2T yeast plasmid, an
autonomously replication sequence (ARS), a promoter region,
sequences for polyadenylation, sequences for transcription
termination, and a selectable marker gene. Shuttle vectors for
replication in both yeast and E. coli are also included herein.
[0118] Alternatively, insect cells may be used as host cells. In a
preferred embodiment, the polypeptides of the invention are
expressed using a baculovirus expression system (see, Luckow et
al., Bio/Technology, 1988, 6, 47, Baculovirus Expression Vectors: A
Laboratory Manual, O'Rielly et al. (Eds.), W.H. Freeman and
Company, New York, 1992, and U.S. Pat. No. 4,879,236, each of which
is incorporated herein by reference in its entirety). In addition,
the MAXBAC.TM. complete baculovirus expression system (Invitrogen)
can, for example, be used for production in insect cells.
[0119] Also comprehended by the present invention are antibodies
(e.g., monoclonal and polyclonal antibodies, single chain
antibodies, chimeric antibodies, bifunctional/bispecific
antibodies, humanized antibodies, human antibodies, and
complementary determining region (CDR)-grafted antibodies,
including compounds which include CDR sequences which specifically
recognize a polypeptide of the invention) specific for DmGPCR or
fragments thereof. Preferred antibodies of the invention are human
antibodies which are produced and identified according to methods
described in WO93/11236, published Jun. 20, 1993, which is
incorporated herein by reference in its entirety. Antibody
fragments, including Fab, Fab', F(ab').sub.2, and F.sub.v, are also
provided by the invention. The term "specific for," when used to
describe antibodies of the invention, indicates that the variable
regions of the antibodies of the invention recognize and bind
DmGPCR polypeptides exclusively (i.e., are able to distinguish
DmGPCR polypeptides from other known GPCR polypeptides by virtue of
measurable differences in binding affinity, despite the possible
existence of localized sequence identity, homology, or similarity
between DmGPCR and such polypeptides). It will be understood that
specific antibodies may also interact with other proteins (for
example, S. aureus protein A or other antibodies in ELISA
techniques) through interactions with sequences outside the
variable region of the antibodies, and, in particular, in the
constant region of the molecule. Screening assays to determine
binding specificity of an antibody of the invention are well known
and routinely practiced in the art. For a comprehensive discussion
of such assays, see Harlow et al. (Eds.), Antibodies A Laboratory
Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y.
(1988), Chapter 6. Antibodies that recognize and bind fragments of
the DmGPCR polypeptides of the invention are also contemplated,
provided that the antibodies are specific for DmGPCR polypeptides.
Antibodies of the invention can be produced using any method well
known and routinely practiced in the art.
[0120] Non-human antibodies may be humanized by any of the methods
known in the art. In one method, the non-human CDRs are inserted
into a human antibody or consensus antibody framework sequence.
Further changes can then be introduced into the antibody framework
to modulate affinity or immunogenicity.
[0121] Antibodies of the invention are useful for, e.g.,
therapeutic purposes (by modulating activity of DmGPCR), diagnostic
purposes to detect or quantitate DmGPCR, and purification of
DmGPCR. Kits comprising an antibody of the invention for any of the
purposes described herein are also comprehended. In general, a kit
of the invention also includes a control antigen for which the
antibody is immunospecific.
[0122] Another aspect of the present invention is directed to
methods of inducing an immune response in a mammal against a
polypeptide of the invention by administering to the mammal an
amount of the polypeptide sufficient to induce an immune response.
The amount will be dependent on the animal species, size of the
animal, and the like but can be determined by those skilled in the
art.
[0123] Another aspect of the present invention is directed to
compositions, including pharmaceutical compositions, comprising any
of the nucleic acid molecules or recombinant expression vectors
described above and an acceptable carrier or diluent. Preferably,
the carrier or diluent is pharmaceutically acceptable. Suitable
carriers are described in the most recent edition of Remington's
Pharmaceutical Sciences, A. Osol, a standard reference text in this
field, which is incorporated herein by reference in its entirety.
Preferred examples of such carriers or diluents include, but are
not limited to, water, saline, Ringer's solution, dextrose
solution, and 5% human serum albumin. Liposomes and nonaqueous
vehicles such as fixed oils may also be used. The formulations are
sterilized by commonly used techniques.
[0124] With the knowledge of the nucleotide sequence information
disclosed in the present invention, one skilled in the art can
identify and obtain nucleotide sequences which encode FaRP-binding
GPCRs from different sources (i.e., different tissues or different
organisms) through a variety of means well known to the skilled
artisan and as disclosed by, for example, Sambrook et al.,
"Molecular cloning: a laboratory manual", Second Edition, Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), which is
incorporated herein by reference in its entirety.
[0125] For example, DNA that encodes GPCR may be obtained by
screening of mRNA, cDNA, or genomic DNA with oligonucleotide probes
generated from the DmGPCR gene sequence information provided
herein. Probes may be labeled with a detectable group, such as a
fluorescent group, a radioactive atom or a chemiluminescent group
in accordance with procedures known to the skilled artisan and used
in conventional hybridization assays, as described by, for example,
Sambrook et al.
[0126] Specific binding molecules, including natural ligands and
synthetic compounds, can be identified or developed using isolated
or recombinant DmGPCR products, DmGPCR variants, or preferably,
cells expressing such products. Binding partners are useful for
purifying DmGPCR products and detection or quantification of DmGPCR
products in fluid and tissue samples using known immunological
procedures. Binding molecules are also manifestly useful in
modulating (i.e., blocking, inhibiting or stimulating) biological
activities of DmGPCR, especially those activities involved in
signal transduction.
[0127] The DNA and amino acid sequence information provided by the
present invention also makes possible identification of binding
partner compounds with which a DmGPCR polypeptide or polynucleotide
will interact. Methods to identify binding partner compounds
include solution assays, in vitro assays wherein DmGPCR
polypeptides are immobilized, and cell-based assays. Identification
of binding partner compounds of DmGPCR polypeptides provides
candidates for therapeutic or prophylactic intervention in
pathologies associated with DmGPCR normal and aberrant biological
activity.
[0128] The invention includes several assay systems for identifying
DmGPCR binding partners. In solution assays, methods of the
invention comprise the steps of (a) contacting a DmGPCR polypeptide
with one or more candidate binding partner compounds and (b)
identifying the compounds that bind to the DmGPCR polypeptide.
Identification of the compounds that bind the DmGPCR polypeptide
can be achieved by isolating the DmGPCR polypeptide/binding partner
complex, and separating the binding partner compound from the
DmGPCR polypeptide. An additional step of characterizing the
physical, biological, and/or biochemical properties of the binding
partner compound is also comprehended in another embodiment of the
invention. In one aspect, the DmGPCR polypeptide/binding partner
complex is isolated using an antibody immunospecific for either the
DmGPCR polypeptide or the candidate binding partner compound.
[0129] In still other embodiments, either the DmGPCR polypeptide or
the candidate binding partner compound comprises a label or tag
that facilitates its isolation, and methods of the invention to
identify binding partner compounds include a step of isolating the
DmGPCR polypeptide/binding partner complex through interaction with
the label or tag. An exemplary tag of this type is a poly-histidine
sequence, generally around six histidine residues, that permits
isolation of a compound so labeled using nickel chelation. Other
labels and tags, such as the FLAG.RTM. tag (Eastman Kodak,
Rochester, N.Y.), well known and routinely used in the art, are
embraced by the invention.
[0130] In one variation of an in vitro assay, the invention
provides a method comprising the steps of (a) contacting an
immobilized DmGPCR polypeptide with a candidate binding partner
compound and (b) detecting binding of the candidate compound to the
DmGPCR polypeptide. In an alternative embodiment, the candidate
binding partner compound is immobilized and binding of DmGPCR is
detected. Immobilization is accomplished using any of the methods
well known in the art, including covalent bonding to a support, a
bead, or a chromatographic resin, as well as non-covalent, high
affinity interactions such as antibody binding, or use of
streptavidin/biotin binding wherein the immobilized compound
includes a biotin moiety. Detection of binding can be accomplished
(i) using a radioactive label on the compound that is not
immobilized, (ii) using of a fluorescent label on the
non-immobilized compound, (iii) using an antibody immunospecific
for the non-immobilized compound, (iv) using a label on the
non-immobilized compound that excites a fluorescent support to
which the immobilized compound is attached, as well as other
techniques well known and routinely practiced in the art.
[0131] The invention also provides cell-based assays to identify
binding partner compounds of a DmGPCR polypeptide. In one
embodiment, the invention provides a method comprising the steps of
contacting a DmGPCR polypeptide expressed on the surface of a cell
with a candidate binding partner compound and detecting binding of
the candidate binding partner compound to the DmGPCR polypeptide.
In a preferred embodiment, the detection comprises detecting a
calcium flux or other physiological event in the cell caused by the
binding of the molecule.
[0132] Agents that modulate (i.e., increase, decrease, or block)
DmGPCR activity or expression may be identified by incubating a
putative modulator with a cell containing a DmGPCR polypeptide or
polynucleotide and determining the effect of the putative modulator
on DmGPCR activity or expression. The selectivity of a compound
that modulates the activity of DmGPCR can be evaluated by comparing
its effects on DmGPCR to its effect on other GPCR compounds.
Selective modulators may include, for example, antibodies and other
proteins, peptides, or organic molecules which specifically bind to
a DmGPCR polypeptide or a DmGPCR-encoding nucleic acid. Modulators
of DmGPCR activity will be therapeutically useful in treatment of
diseases and physiological conditions in which normal or aberrant
DmGPCR activity is involved. DmGPCR polynucleotides, polypeptides,
and modulators may be used in the treatment of such diseases and
conditions as infections, such as viral infections caused by HIV-1
or HIV-2; pain; cancers; Parkinson's disease; hypotension;
hypertension; and psychotic and neurological disorders, including
anxiety, schizophrenia, manic depression, delirium, dementia,
severe mental retardation and dyskinesias, such as Huntington's
disease or Tourette's Syndrome, among others. DmGPCR
polynucleotides and polypeptides, as well as DmGPCR modulators, may
also be used in diagnostic assays for such diseases or
conditions.
[0133] Methods of the invention to identify modulators include
variations on any of the methods described above to identify
binding partner compounds, the variations including techniques
wherein a binding partner compound has been identified and the
binding assay is carried out in the presence and absence of a
candidate modulator. A modulator is identified in those instances
where binding between the DmGPCR polypeptide and the binding
partner compound changes in the presence of the candidate modulator
compared to binding in the absence of the candidate modulator
compound. A modulator that increases binding between the DmGPCR
polypeptide and the binding partner compound is described as an
enhancer or activator, and a modulator that decreases binding
between the DmGPCR polypeptide and the binding partner compound is
described as an inhibitor.
[0134] The invention also comprehends high-throughput screening
(HTS) assays to identify compounds that interact with or inhibit
biological activity (i.e., affect enzymatic activity, binding
activity, etc.) of a DmGPCR polypeptide. HTS assays permit
screening of large numbers of compounds in an efficient manner.
Cell-based HTS systems are contemplated to investigate DmGPCR
receptor-ligand interaction. HTS assays are designed to identify
"hits" or "lead compounds" having the desired property, from which
modifications can be designed to improve the desired property.
Chemical modification of the "hit" or "lead compound" is often
based on an identifiable structure/activity relationship between
the "hit" and the DmGPCR polypeptide.
[0135] Another aspect of the present invention is directed to
methods of identifying compounds that bind to either DmGPCR or
nucleic acid molecules encoding DmGPCR, comprising contacting
DmGPCR, or a nucleic acid molecule encoding the same, with a
compound, and determining whether the compound binds DmGPCR, or a
nucleic acid molecule encoding the same. Binding can be determined
by binding assays which are well known to the skilled artisan,
including, but not limited to, gel-shift assays, Western blots,
radiolabeled competition assay, phage-based expression cloning,
co-fractionation by chromatography, co-precipitation, cross
linking, interaction trap/two-hybrid analysis, southwestern
analysis, ELISA, and the like, which are described in, for example,
Current Protocols in Molecular Biology, 1999, John Wiley &
Sons, NY, which is incorporated herein by reference in its
entirety. The compounds to be screened include (which may include
compounds which are suspected to bind DmGPCR, or a nucleic acid
molecule encoding the same), but are not limited to, extracellular,
intracellular, biologic or chemical origin.
[0136] The methods of the invention also embrace neuropeptides that
are attached to a label, such as a radiolabel (e.g., .sup.125I,
.sup.35S, .sup.32P, .sup.33P, .sup.3H), a fluorescence label, a
chemiluminescent label, an enzymic label and an immunogenic label.
Modulators falling within the scope of the invention include, but
are not limited to, non-peptide molecules such as non-peptide
mimetics, non-peptide allosteric effectors, and peptides. The
DmGPCR polypeptide or polynucleotide employed in such a test may
either be free in solution, attached to a solid support, borne on a
cell surface or located intracellularly or associated with a
portion of a cell. One skilled in the art can, for example, measure
the formation of complexes between DmGPCR and the compound being
tested. Alternatively, one skilled in the art can examine the
diminution in complex formation between DmGPCR and its substrate
caused by the compound being tested.
[0137] Another aspect of the present invention is directed to
methods of identifying compounds which modulate (i.e., increase or
decrease) activity of DmGPCR comprising contacting DmGPCR with a
compound, and determining whether the compound modifies activity of
DmGPCR. The activity in the presence of the test compared is
measured to the activity in the absence of the test compound. Where
the activity of the sample containing the test compound is higher
than the activity in the sample lacking the test compound, the
compound will have increased activity. Similarly, where the
activity of the sample containing the test compound is lower than
the activity in the sample lacking the test compound, the compound
will have inhibited activity.
[0138] The present invention is particularly useful for screening
compounds by using DmGPCR in any of a variety of drug screening
techniques. The compounds to be screened include (which may include
compounds which are suspected to modulate DmGPCR activity), but are
not limited to, extracellular, intracellular, biologic or chemical
origin. The DmGPCR polypeptide employed in such a test may be in
any form, preferably, free in solution, attached to a solid
support, borne on a cell surface or located intracellularly. One
skilled in the art can, for example, measure the formation of
complexes between DmGPCR and the compound being tested.
Alternatively, one skilled in the art can examine the diminution in
complex formation between DmGPCR and its substrate caused by the
compound being tested.
[0139] The activity of DmGPCR polypeptides of the invention can be
determined by, for example, examining the ability to bind or be
activated by chemically synthesized peptide ligands. Alternatively,
the activity of the DmGPCRs can be assayed by examining their
ability to bind calcium ions, hormones, chemokines, neuropeptides,
neurotransmitters, nucleotides, lipids, odorants, and photons.
Alternatively, the activity of the GPCRs can be determined by
examining the activity of effector molecules including, but not
limited to, adenylate cyclase, phospholipases and ion channels.
Thus, modulators of GPCR activity may alter a GPCR receptor
function, such as a binding property of a receptor or an activity
such as G protein-mediated signal transduction or membrane
localization. In various embodiments of the method, the assay may
take the form of an ion flux assay, a yeast growth assay, a
non-hydrolyzable GTP assay such as a [.sup.35S]-GTP S assay, a cAMP
assay, an inositol triphosphate assay, a diacylglycerol assay, an
Aequorin assay, a Luciferase assay, a FLIPR assay for intracellular
Ca.sup.2+ concentration, a mitogenesis assay, a MAP Kinase activity
assay, an arachidonic acid release assay (e.g., using
[.sup.3H]-arachidonic acid), and an assay for extracellular
acidification rates, as well as other binding or function-based
assays of DmGPCR activity that are generally known in the art. In
several of these embodiments, the invention comprehends the
inclusion of any of the G proteins known in the art, such as
G.sub.16, G.sub.15, or chimeric G.sub.qd5, G.sub.qs5, G.sub.qo5,
G.sub.q25, and the like. DmGPCR activity can be determined by
methodologies that are used to assay for FaRP activity, which is
well known to those skilled in the art. Biological activities of
DmGPCR receptors according to the invention include, but are not
limited to, the binding of a natural or an unnatural ligand, as
well as any one of the functional activities of GPCRs known in the
art. Non-limiting examples of GPCR activities include transmembrane
signaling of various forms, which may involve G protein association
and/or the exertion of an influence over G protein binding of
various guanidylate nucleotides; another exemplary activity of
GPCRs is the binding of accessory proteins or polypeptides that
differ from known G proteins.
[0140] The modulators of the invention exhibit a variety of
chemical structures, which can be generally grouped into
non-peptide mimetics of natural GPCR receptor ligands, peptide and
non-peptide allosteric effectors of GPCR receptors, and peptides
that may function as activators or inhibitors (competitive,
uncompetitive and non-competitive) (e.g., antibody products) of
GPCR receptors. The invention does not restrict the sources for
suitable modulators, which may be obtained from natural sources
such as plant, animal or mineral extracts, or non-natural sources
such as small molecule libraries, including the products of
combinatorial chemical approaches to library construction, and
peptide libraries. Examples of peptide modulators of GPCR receptors
exhibit the following primary structures: GLGPRPLRFamide,
GNSFLRFamide, GGPQGPLRFamide, GPSGPLRFamide, PDVDHVFLRFamide, and
pyro-EDVDHVFLRFamide.
[0141] Other assays can be used to examine enzymatic activity
including, but not limited to, photometric, radiometric, HPLC,
electrochemical, and the like, which are described in, for example,
Enzyme Assays: A Practical Approach, eds. R. Eisenthal and M. J.
Danson, 1992, Oxford University Press, which is incorporated herein
by reference in its entirety.
[0142] The use of cDNAs encoding GPCRs in drug discovery programs
is well-known; assays capable of testing thousands of unknown
compounds per day in high-throughput screens (HTSs) are thoroughly
documented. The literature is replete with examples of the use of
radiolabelled ligands in HTS binding assays for drug discovery (see
Williams, Medicinal Research Reviews, 1991, 11, 147-184; Sweetnam,
et al., J Natural Products, 1993, 56, 441-455 for review).
Recombinant receptors are preferred for binding assay HTS because
they allow for better specificity (higher relative purity), provide
the ability to generate large amounts of receptor material, and can
be used in a broad variety of formats (see Hodgson, Bio/Technology,
1992, 10, 973-980; each of which is incorporated herein by
reference in its entirety).
[0143] A variety of heterologous systems is available for
functional expression of recombinant receptors that are well known
to those skilled in the art. Such systems include bacteria
(Strosberg, et al., Trends in Pharmacological Sciences, 1992, 13,
95-98), yeast (Pausch, Trends in Biotechnology, 1997, 15, 487-494),
several kinds of insect cells (Vanden Broeck, Int. Rev. Cytology,
1996, 164, 189-268), amphibian cells (Jayawickreme et al., Current
Opinion in Biotechnology, 1997, 8, 629-634) and several mammalian
cell lines (CHO, HEK293, COS, etc.; see Gerhardt, et al., Eur. J.
Pharmacology, 1997, 334, 1-23). These examples do not preclude the
use of other possible cell expression systems, including cell lines
obtained from nematodes (PCT application WO 98/37177).
[0144] In preferred embodiments of the invention, methods of
screening for compounds which modulate GPCR activity comprise
contacting test compounds with DmGPCR and assaying for the presence
of a complex between the compound and DmGPCR. In such assays, the
ligand is typically labeled. After suitable incubation, free ligand
is separated from that present in bound form, and the amount of
free or uncomplexed label is a measure of the ability of the
particular compound to bind to DmGPCR.
[0145] In another embodiment of the invention, high throughput
screening for compounds having suitable binding affinity to DmGPCR
is employed. Briefly, large numbers of different small peptide test
compounds are synthesised on a solid substrate. The peptide test
compounds are contacted with DmGPCR and washed. Bound DmGPCR is
then detected by methods well known in the art. Purified
polypeptides of the invention can also be coated directly onto
plates for use in the aforementioned drug screening techniques. In
addition, non-neutralizing antibodies can be used to capture the
protein and immobilize it on the solid support.
[0146] Generally, an expressed DmGPCR can be used for HTS binding
assays in conjunction with its defined ligand, in this case the
corresponding neuropeptide that activates it. The identified
peptide is labeled with a suitable radioisotope, including, but not
limited to, .sup.125I, .sup.3H, .sup.35S or .sup.32P, by methods
that are well known to those skilled in the art. Alternatively, the
peptides may be labeled by well-known methods with a suitable
fluorescent derivative (Baindur, et al., Drug Dev. Res., 1994, 33,
373-398; Rogers, Drug Discovery Today, 1997, 2, 156-160).
Radioactive ligand specifically bound to the receptor in membrane
preparations made from the cell line expressing the recombinant
protein can be detected in HTS assays in one of several standard
ways, including filtration of the receptor-ligand complex to
separate bound ligand from unbound ligand (Williams, Med. Res.
Rev., 1991, 11, 147-184; Sweetnam, et al., J. Natural Products,
1993, 56, 441-455). Alternative methods include a scintillation
proximity assay (SPA) or a FlashPlate format in which such
separation is unnecessary (Nakayama, Cur. Opinion Drug Disc. Dev.,
1998, 1, 85-91 Bosse, et al., J. Biomolecular Screening, 1998, 3,
285-292.). Binding of fluorescent ligands can be detected in
various ways, including fluorescence energy transfer (FRET), direct
spectrophotofluorometric analysis of bound ligand, or fluorescence
polarization (Rogers, Drug Discovery Today, 1997, 2, 156-160; Hill,
Cur. Opinion Drug Disc. Dev., 1998, 1, 92-97).
[0147] It is well known that activation of heterologous receptors
expressed in recombinant systems results in a variety of biological
responses, which are mediated by G proteins expressed in the host
cells. Occupation of a GPCR by an agonist results in exchange of
bound GDP for GTP at a binding site on the G.sub..alpha. subunit;
one can use a radioactive, non-hydrolyzable derivative of GTP,
GTP.gamma.[.sup.35S], to measure binding of an agonist to the
receptor (Sim et al., Neuroreport, 1996, 7, 729-733). One can also
use this binding to measure the ability of antagonists to bind to
the receptor by decreasing binding of GTP.gamma.[.sup.35S] in the
presence of a known agonist. One could therefore construct a HTS
based on GTP.gamma.[.sup.35S] binding, though this is not the
preferred method.
[0148] The G proteins required for functional expression of
heterologous GPCRs can be native constituents of the host cell or
can be introduced through well-known recombinant technology. The G
proteins can be intact or chimeric. Often, a nearly universally
competent G protein (e.g., G.sub..alpha.16) is used to couple any
given receptor to a detectable response pathway. G protein
activation results in the stimulation or inhibition of other native
proteins, events that can be linked to a measurable response.
[0149] Examples of such biological responses include, but are not
limited to, the following: the ability to survive in the absence of
a limiting nutrient in specifically engineered yeast cells (Pausch,
Trends in Biotechnology, 1997, 15, 487-494); changes in
intracellular Ca.sup.2+ concentration as measured by fluorescent
dyes (Murphy, et al., Cur. Opinion Drug Disc. Dev., 1998, 1,
192-199). Fluorescence changes can also be used to monitor
ligand-induced changes in membrane potential or intracellular pH;
an automated system suitable for HTS has been described for these
purposes (Schroeder, et al., J. Biomolecular Screening, 1996, 1,
75-80). Melanophores prepared from Xenopus laevis show a
ligand-dependent change in pigment organization in response to
heterologous GPCR activation; this response is adaptable to HTS
formats (Jayawickreme, et al., Cur. Opinion Biotechnology, 1997, 8,
629-634). Assays are also available for the measurement of common
second messengers, including cAMP, phosphoinositides and
arachidonic acid, but these are not generally preferred for
HTS.
[0150] Preferred methods of HTS employing these receptors include
permanently transfected CHO cells, in which agonists and
antagonists can be identified by the ability to specifically alter
the binding of GTP.gamma.[.sup.35S] in membranes prepared from
these cells. In another embodiment of the invention, permanently
transfected CHO cells could be used for the preparation of
membranes which contain significant amounts of the recombinant
receptor proteins; these membrane preparations would then be used
in receptor binding assays, employing the radiolabelled ligand
specific for the particular receptor. Alternatively, a functional
assay, such as fluorescent monitoring of ligand-induced changes in
internal Ca.sup.2+ concentration or membrane potential in
permanently transfected CHO cells containing each of these
receptors individually or in combination would be preferred for
HTS. Equally preferred would be an alternative type of mammalian
cell, such as HEK293 or COS cells, in similar formats. More
preferred would be permanently transfected insect cell lines, such
as Drosophila S2 cells. Even more preferred would be recombinant
yeast cells expressing the Drosophila melanogaster receptors in HTS
formats well known to those skilled in the art (e.g., Pausch,
Trends in Biotechnology, 1997, 15, 487-494).
[0151] The invention contemplates a multitude of assays to screen
and identify inhibitors of ligand binding to DmGPCR receptors. In
one example, the DmGPCR receptor is immobilized and interaction
with a binding partner is assessed in the presence and absence of a
candidate modulator such as an inhibitor compound. In another
example, interaction between the DmGPCR receptor and its binding
partner is assessed in a solution assay, both in the presence and
absence of a candidate inhibitor compound. In either assay, an
inhibitor is identified as a compound that decreases binding
between the DmGPCR receptor and its binding partner. Another
contemplated assay involves a variation of the di-hybrid assay
wherein an inhibitor of protein/protein interactions is identified
by detection of a positive signal in a transformed or transfected
host cell, as described in PCT publication number WO 95/20652,
published Aug. 3, 1995.
[0152] Candidate modulators contemplated by the invention include
compounds selected from libraries of either potential activators or
potential inhibitors. There are a number of different libraries
used for the identification of small molecule modulators,
including: (1) chemical libraries, (2) natural product libraries,
and (3) combinatorial libraries comprised of random peptides,
oligonucleotides or organic molecules. Chemical libraries consist
of random chemical structures, some of which are analogs of known
compounds or analogs of compounds that have been identified as
"hits" or "leads" in other drug discovery screens, some of which
are derived from natural products, and some of which arise from
non-directed synthetic organic chemistry. Natural product libraries
are collections of microorganisms, animals, plants, or marine
organisms which are used to create mixtures for screening by: (1)
fermentation and extraction of broths from soil, plant or marine
microorganisms or (2) extraction of plants or marine organisms.
Natural product libraries include polyketides, non-ribosomal
peptides, and variants (non-naturally occurring) thereof. For a
review, see Science 282:63-68 (1998). Combinatorial libraries are
composed of large numbers of peptides, oligonucleotides, or organic
compounds as a mixture. These libraries are relatively easy to
prepare by traditional automated synthesis methods, PCR, cloning,
or proprietary synthetic methods. Of particular interest are
non-peptide combinatorial libraries. Still other libraries of
interest include peptide, protein, peptidomimetic, multiparallel
synthetic collection, recombinatorial, and polypeptide libraries.
For a review of combinatorial chemistry and libraries created
therefrom, see Myers, Curr. Opin. Biotechnol. 8:701-707 (1997).
Identification of modulators through use of the various libraries
described herein permits modification of the candidate "hit" (or
"lead") to optimize the capacity of the "hit" to modulate
activity.
[0153] Still other candidate inhibitors contemplated by the
invention can be designed and include soluble forms of binding
partners, as well as such binding partners as chimeric, or fusion,
proteins. A "binding partner" as used herein broadly encompasses
non-peptide modulators, as well as such peptide modulators as
neuropeptides other than natural ligands, antibodies, antibody
fragments, and modified compounds comprising antibody domains that
are immunospecific for the expression product of the identified
DmGPCR gene.
[0154] Other assays may be used to identify specific neuropeptide
ligands of a DmGPCR receptor, including assays that identify
ligands of the target protein through measuring direct binding of
test ligands to the target protein, as well as assays that identify
ligands of target proteins through affinity ultrafiltration with
ion spray mass spectroscopy/HPLC methods or other physical and
analytical methods. Alternatively, such binding interactions are
evaluated indirectly using the yeast two-hybrid system described in
Fields et al., Nature, 340:245-246 (1989), and Fields et al.,
Trends in Genetics, 10:286-292 (1994), both of which are
incorporated herein by reference. The two-hybrid system is a
genetic assay for detecting interactions between two proteins or
polypeptides. It can be used to identify proteins that bind to a
known protein of interest, or to delineate domains or residues
critical for an interaction. Variations on this methodology have
been developed to clone genes that encode DNA binding proteins, to
identify peptides that bind to a protein, and to screen for drugs.
The two-hybrid system exploits the ability of a pair of interacting
proteins to bring a transcription activation domain into close
proximity with a DNA binding domain that binds to an upstream
activation sequence (UAS) of a reporter gene, and is generally
performed in yeast. The assay requires the construction of two
hybrid genes encoding (1) a DNA-binding domain that is fused to a
first protein and (2) an activation domain fused to a second
protein. The DNA-binding domain targets the first hybrid protein to
the UAS of the reporter gene; however, because most proteins lack
an activation domain, this DNA-binding hybrid protein does not
activate transcription of the reporter gene. The second hybrid
protein, which contains the activation domain, cannot by itself
activate expression of the reporter gene because it does not bind
the UAS. However, when both hybrid proteins are present, the
noncovalent interaction of the first and second proteins tethers
the activation domain to the UAS, activating transcription of the
reporter gene. For example, when the first protein is a GPCR gene
product, or fragment thereof, that is known to interact with
another protein or nucleic acid, this assay can be used to detect
agents that interfere with the binding interaction. Expression of
the reporter gene is monitored as different test agents are added
to the system. The presence of an inhibitory agent results in lack
of a reporter signal.
[0155] When the function of the DmGPCR gene product is unknown and
no ligands are known to bind the gene product, the yeast two-hybrid
assay can also be used to identify proteins that bind to the gene
product. In an assay to identify proteins that bind to a DmGPCR
receptor, or fragment thereof, a fusion polynucleotide encoding
both a DmGPCR receptor (or fragment) and a UAS binding domain
(i.e., a first protein) may be used. In addition, a large number of
hybrid genes each encoding a different second protein fused to an
activation domain are produced and screened in the assay.
Typically, the second protein is encoded by one or more members of
a total cDNA or genomic DNA fusion library, with each second
protein coding region being fused to the activation domain. This
system is applicable to a wide variety of proteins, and it is not
even necessary to know the identity or function of the second
binding protein. The system is highly sensitive and can detect
interactions not revealed by other methods; even transient
interactions may trigger transcription to produce a stable mRNA
that can be repeatedly translated to yield the reporter
protein.
[0156] Other assays may be used to search for agents that bind to
the target protein. One such screening method to identify direct
binding of test ligands to a target protein is described in U.S.
Pat. No. 5,585,277, incorporated herein by reference. This method
relies on the principle that proteins generally exist as a mixture
of folded and unfolded states, and continually alternate between
the two states. When a test ligand binds to the folded form of a
target protein (i.e., when the test ligand is a ligand of the
target protein), the target protein molecule bound by the ligand
remains in its folded state. Thus, the folded target protein is
present to a greater extent in the presence of a test ligand which
binds the target protein, than in the absence of a ligand. Binding
of the ligand to the target protein can be determined by any method
which distinguishes between the folded and unfolded states of the
target protein. The function of the target protein need not be
known in order for this assay to be performed. Virtually any agent
can be assessed by this method as a test ligand, including, but not
limited to, metals, polypeptides, proteins, lipids,
polysaccharides, polynucleotides and small organic molecules.
[0157] Another method for identifying ligands of a target protein
is described in Wieboldt et al., Anal. Chem., 69:1683-1691 (1997),
incorporated herein by reference. This technique screens
combinatorial libraries of 20-30 agents at a time in solution phase
for binding to the target protein. Agents that bind to the target
protein are separated from other library components by simple
membrane washing. The specifically selected molecules that are
retained on the filter are subsequently liberated from the target
protein and analyzed by HPLC and pneumatically assisted
electrospray (ion spray) ionization mass spectroscopy. This
procedure selects library components with the greatest affinity for
the target protein, and is particularly useful for small molecule
libraries.
[0158] Other embodiments of the invention comprise using
competitive screening assays in which neutralizing antibodies
capable of binding a polypeptide of the invention specifically
compete with a test compound for binding to the polypeptide. In
this manner, the antibodies can be used to detect the presence of
any peptide that shares one or more antigenic determinants with
DmGPCR. Radiolabeled competitive binding studies are described in
A. H. Lin et al. Antimicrobial Agents and Chemotherapy, 1997, vol.
41, no. 10. pp. 2127-2131, the disclosure of which is incorporated
herein by reference in its entirety.
[0159] In other embodiments of the invention, the polypeptides of
the invention are employed as a research tool for identification,
characterization and purification of interacting, regulatory
proteins. Appropriate labels are incorporated into the polypeptides
of the invention by various methods known in the art and the
polypeptides are used to capture interacting molecules. For
example, molecules are incubated with the labeled polypeptides,
washed to removed unbound polypeptides, and the polypeptide complex
is quantified. Data obtained using different concentrations of
polypeptide are used to calculate values for the number, affinity,
and association of polypeptide with the protein complex.
[0160] Labeled polypeptides are also useful as reagents for the
purification of molecules with which the polypeptide interacts
including, but not limited to, inhibitors. In one embodiment of
affinity purification, a polypeptide is covalently coupled to a
chromatography column. Cells and their membranes are extracted, and
various cellular subcomponents are passed over the column.
Molecules bind to the column by virtue of their affinity to the
polypeptide. The polypeptide-complex is recovered from the column,
dissociated and the recovered molecule is subjected to protein
sequencing. This amino acid sequence is then used to identify the
captured molecule or to design degenerate oligonucleotides for
cloning the corresponding gene from an appropriate cDNA
library.
[0161] Alternatively, compounds may be identified which exhibit
similar properties to the ligand for the DmGPCR of the invention,
but which are smaller and exhibit a longer half time than the
endogenous ligand in a human or animal body. When an organic
compound is designed, a molecule according to the invention is used
as a "lead" compound. The design of mimetics to known
pharmaceutically active compounds is a well known approach in the
development of pharmaceuticals based on such "lead" compounds.
Mimetic design, synthesis and testing are generally used to avoid
randomly screening a large number of molecules for a target
property. Furthermore, structural data deriving from the analysis
of the deduced amino acid sequences encoded by the DNAs of the
present invention are useful to design new drugs, more specific and
therefore with a higher pharmacological potency.
[0162] Comparison of the protein sequence of the present invention
with the sequences present in all the available databases showed a
significant homology with the transmembrane portion of G protein
coupled receptors. Accordingly, computer modelling can be used to
develop a putative tertiary structure of the proteins of the
invention based on the available information of the transmembrane
domain of other proteins. Thus, novel ligands based on the
predicted structure of DmGPCR can be designed.
[0163] In a particular embodiment, the novel molecules identified
by the screening methods according to the invention are low
molecular weight organic molecules, in which case a composition or
pharmaceutical composition can be prepared thereof for oral intake,
such as in tablets. The compositions, or pharmaceutical
compositions, comprising the nucleic acid molecules, vectors,
polypeptides, antibodies and compounds identified by the screening
methods described herein, can be prepared for any route of
administration including, but not limited to, oral, intravenous,
cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal.
The nature of the carrier or other ingredients will depend on the
specific route of administration and particular embodiment of the
invention to be administered. Examples of techniques and protocols
that are useful in this context are, inter alia, found in
Remington's Pharmaceutical Sciences, 16.sup.th edition, Osol, A
(ed.), 1980, which is incorporated herein by reference in its
entirety.
[0164] The dosage of these low molecular weight compounds will
depend on the disease state or condition to be treated and other
clinical factors such as weight and condition of the human or
animal and the route of administration of the compound. For
treating human or animals, between approximately 0.5 mg/kg of body
weight to 500 mg/kg of body weight of the compound can be
administered. Therapy is typically administered at lower dosages
and is continued until the desired therapeutic outcome is
observed.
[0165] Another aspect of the present invention is the use of the
DmGPCR nucleotide sequences disclosed herein for identifying
homologs of the DmGPCR, in other animals, including but not limited
to humans and other mammals, and invertebrates. Any of the
nucleotide sequences disclosed herein, or any portion thereof, can
be used, for example, as probes to screen databases or nucleic acid
libraries, such as, for example, genomic or cDNA libraries, to
identify homologs, using screening procedures well known to those
skilled in the art. Accordingly, homologs having at least 50%, more
preferably at least 60%, more preferably at least 70%, more
preferably at least 80%, more preferably at least 90%, more
preferably at least 95%, and most preferably at least 100% homology
with DmGPCR sequences can be identified.
[0166] The present compounds and methods, including nucleic acid
molecules, polypeptides, antibodies, compounds identified by the
screening methods described herein, have a variety of
pharmaceutical applications and may be used, for example, to treat
or prevent unregulated cellular growth, such as cancer cell and
tumour growth. In a particular embodiment, the present molecules
are used in gene therapy. For a review of gene therapy procedures,
see e.g. Anderson, Science, 1992, 256, 808-813, which is
incorporated herein by reference in its entirety.
[0167] The present invention also encompasses a method of agonizing
(stimulating) or antagonizing a DmGPCR natural binding partner
associated activity in a mammal comprising administering to said
mammal an agonist or antagonist to one of the above disclosed
polypeptides in an amount sufficient to effect said agonism or
antagonism. One embodiment of the present invention, then, is a
method of treating diseases in a mammal with an agonist or
antagonist of the protein of the present invention comprises
administering the agonist or antagonist to a mammal in an amount
sufficient to agonize or antagonize DmGPCR-associated
functions.
[0168] In an effort to discover novel treatments for diseases,
biomedical researchers and chemists have designed, synthesized, and
tested molecules that inhibit the function of protein polypeptides.
Some small organic molecules form a class of compounds that
modulate the function of protein polypeptides. Examples of
molecules that have been reported to inhibit the function of
protein kinases include, but are not limited to, bis monocyclic,
bicyclic or heterocyclic aryl compounds (PCT WO 92/20642, published
Nov. 26, 1992 by Maguire et al.), vinylene-azaindole derivatives
(PCT WO 94/14808, published Jul. 7, 1994 by Ballinari et al.),
1-cyclopropyl-4-pyridyl-quinolones (U.S. Pat. No. 5,330,992),
styryl compounds (U.S. Pat. No. 5,217,999), styryl-substituted
pyridyl compounds (U.S. Pat. No. 5,302,606), certain quinazoline
derivatives (EP Application No. 0 566 266 A1), seleoindoles and
selenides (PCT WO 94/03427, published Feb. 17, 1994 by Denny et
al.), tricyclic polyhydroxylic compounds (PCT WO 92/21660,
published Dec. 10, 1992 by Dow), and benzylphosphonic acid
compounds (PCT WO 91/15495, published Oct. 17, 1991 by Dow et al),
all of which are incorporated by reference herein, including any
drawings.
[0169] Compounds that can traverse cell membranes and are resistant
to acid hydrolysis are potentially advantageous as therapeutics as
they can become highly bioavailable after being administered orally
to patients. However, many of these protein inhibitors only weakly
inhibit function. In addition, many inhibit a variety of protein
kinases and will therefore cause multiple side-effects as
therapeutics for diseases.
[0170] Some indolinone compounds, however, form classes of acid
resistant and membrane permeable organic molecules. WO 96/22976
(published Aug. 1, 1996 by Ballinari et al.) describes hydrosoluble
indolinone compounds that harbor tetralin, naphthalene, quinoline,
and indole substituents fused to the oxindole ring. These bicyclic
substituents are in turn substituted with polar groups including
hydroxylated alkyl, phosphate, and ether substituents. U.S. patent
application Ser. No. 08/702,232, filed Aug. 23, 1996, entitled
"Indolinone Combinatorial Libraries and Related Products and
Methods for the Treatment of Disease" by Tang et al. (Lyon &
Lyon Docket No. 221/187) and 08/485,323, filed Jun. 7, 1995,
entitled "Benzylidene-Z-Indoline Compounds for the Treatment of
Disease" by Tang et al. (Lyon & Lyon Docket No. 223/298) and
International Patent Publication WO 96/22976, published Aug. 1,
1996 by Ballinari et al., all of which are incorporated herein by
reference in their entirety, including any drawings, describe
indolinone chemical libraries of indolinone compounds harboring
other bicyclic moieties as well as monocyclic moieties fused to the
oxindole ring application Ser. No. 08/702,232, filed Aug. 23, 1996,
entitled "Indolinone Combinatorial Libraries and Related Products
and Methods for the Treatment of Disease" by Tang et al. (Lyon
& Lyon Docket No. 221/187), 08/485,323, filed Jun. 7, 1995,
entitled "Benzylidene-Z-Indoline Compounds for the Treatment of
Disease" by Tang et al. (Lyon & Lyon Docket No. 223/298), and
WO 96/22976, published Aug. 1, 1996 by Ballinari et al. teach
methods of indolinone synthesis, methods of testing the biological
activity of indolinone compounds in cells, and inhibition patterns
of indolinone derivatives, both of which are incorporated by
reference herein, including any drawings.
[0171] Other examples of substances capable of modulating kinase
activity include, but are not limited to, tyrphostins,
quinazolines, quinoxolines, and quinolines. The quinazolines,
tyrphostins, quinolines, and quinoxolines referred to above include
well known compounds such as those described in the literature. For
example, representative publications describing quinazolines
include Barker et al., EPO Publication No. 0 520 722 A1; Jones et
al., U.S. Pat. No. 4,447,608; Kabbe et al., U.S. Pat. No.
4,757,072; Kaul and Vougioukas, U.S. Pat. No. 5,316,553; Kreighbaum
and Corner, U.S. Pat. No. 4,343,940; Pegg and Wardleworth, EPO
Publication No. 0 562 734 A1; Barker et al., Proc. of Am. Assoc.
for Cancer Research 32:327 (1991); Bertino, J. R., Cancer Research
3:293-304 (1979); Bertino, J. R., Cancer Research 9(2 part
1):293-304 (1979); Curtin et al., Br. J. Cancer 53:361-368 (1986);
Fernandes et al., Cancer Research 43:1117-1123 (1983); Ferris et
al. J. Org. Chem. 44(2):173-178; Fry et al., Science 265:1093-1095
(1994); Jackman et al., Cancer Research 51:5579-5586 (1981); Jones
et al. J. Med. Chem. 29(6):1114-1118; Lee and Skibo, Biochemistry
26(23):7355-7362 (1987); Lemus et al., J. Org. Chem. 54:3511-3518
(1989); Ley and Seng, Synthesis 1975:415-522 (1975); Maxwell et
al., Magnetic Resonance in Medicine 17:189-196 (1991); Mini et al.,
Cancer Research 45:325-330 (1985); Phillips and Castle, J.
Heterocyclic Chem. 17(19):1489-1596 (1980); Reece et al., Cancer
Research 47(11):2996-2999 (1977); Sculier et al., Cancer Immunol.
and Immunother. 23:A65 (1986); Sikora et al., Cancer Letters
23:289-295 (1984); and Sikora et al., Analytical Biochem.
172:344-355 (1988), all of which are incorporated herein by
reference in their entirety, including any drawings.
[0172] Quinoxaline is described in Kaul and Vougioukas, U.S. Pat.
No. 5,316,553, incorporated herein by reference in its entirety,
including any drawings.
[0173] Quinolines are described in Dolle et al., J. Med. Chem.
37:2627-2629 (1994); MaGuire, J. Med. Chem. 37:2129-2131 (1994);
Burke et al., J. Med. Chem. 36:425-432 (1993); and Burke et al.
BioOrganic Med. Chem. Letters 2:1771-1774 (1992), all of which are
incorporated by reference in their entirety, including any
drawings.
[0174] Tyrphostins are described in Allen et al., Clin. Exp.
Immunol. 91:141-156 (1993); Anafi et al., Blood 82:12:3524-3529
(1993); Baker et al., J. Cell Sci. 102:543-555 (1992); Bilder et
al., Amer. Physiol. Soc. pp. 6363-6143:C721-C730 (1991); Brunton et
al., Proceedings of Amer. Assoc. Cancer Rsch. 33:558 (1992);
Bryckaert et al., Experimental Cell Research 199:255-261 (1992);
Dong et al., J. Leukocyte Biology 53:53-60 (1993); Dong et al., J.
Immunol. 151(5):2717-2724 (1993); Gazit et al., J. Med. Chem.
32:2344-2352 (1989); Gazit et al., "J. Med. Chem. 36:3556-3564
(1993); Kaur et al., Anti-Cancer Drugs 5:213-222 (1994); Kaur et
al., King et al., Biochem. J. 275:413-418 (1991); Kuo et al.,
Cancer Letters 74:197-202 (1993); Levitzki, A., The FASEB J.
6:3275-3282 (1992); Lyall et al., J. Biol. Chem. 264:14503-14509
(1989); Peterson et al., The Prostate 22:335-345 (1993); Pillemer
et al.; Int. J. Cancer 50:80-85 (1992); Posner et al., Molecular
Pharmacology 45:673-683 (1993); Rendu et al., Biol. Pharmacology
44(5):881-888 (1992); Sauro and Thomas, Life Sciences 53:371-376
(1993); Sauro and Thomas, J. Pharm. and Experimental Therapeutics
267(3): 119-1125 (1993); Wolbring et al., J. Biol. Chem.
269(36):22470-22472 (1994); and Yoneda et al., Cancer Research
51:4430-4435 (1991); all of which are incorporated herein by
reference in their entirety, including any drawings.
[0175] Other compounds that could be used as modulators include
oxindolinones such as those described in U.S. patent application
Ser. No. 08/702,232 filed Aug. 23, 1996, incorporated herein by
reference in its entirety, including any drawings.
[0176] Methods of determining the dosages of compounds to be
administered to a patient and modes of administering compounds to
an organism are disclosed in U.S. application Ser. No. 08/702,282,
filed Aug. 23, 1996 and International patent publication number WO
96/22976, published Aug. 1, 1996, both of which are incorporated
herein by reference in their entirety, including any drawings,
figures or tables. Those skilled in the art will appreciate that
such descriptions are applicable to the present invention and can
be easily adapted to it.
[0177] The proper dosage depends on various factors such as the
type of disease being treated, the particular composition being
used and the size and physiological condition of the patient.
Therapeutically effective doses for the compounds described herein
can be estimated initially from cell culture and animal models. For
example, a dose can be formulated in animal models to achieve a
circulating concentration range that initially takes into account
the IC50 as determined in cell culture assays. The animal model
data can be used to more accurately determine useful doses in
humans.
[0178] Plasma half-life and biodistribution of the drug and
metabolites in the plasma, tumors and major organs can also be
determined to facilitate the selection of drugs most appropriate to
inhibit a disorder. Such measurements can be carried out. For
example, HPLC analysis can be performed on the plasma of animals
treated with the drug and the location of radiolabeled compounds
can be deter-mined using detection methods such as X-ray, CAT scan
and MRI. Compounds that show potent inhibitory activity in the
screening assays, but have poor pharmacokinetic characteristics,
can be optimized by altering the chemical structure and retesting.
In this regard, compounds displaying good pharmaco-kinetic
characteristics can be used as a model.
[0179] Toxicity studies can also be carried out by measuring the
blood cell composition. For example, toxicity studies can be
carried out in a suitable animal model as follows: 1) the compound
is administered to mice (an untreated control mouse should also be
used); 2) blood samples are periodically obtained via the tail vein
from one mouse in each treatment group; and 3) the samples are
analyzed for red and white blood cell counts, blood cell
composition and the percent of lymphocytes versus polymorphonuclear
cells. A comparison of results for each dosing regime with the
controls indicates if toxicity is present.
[0180] At the termination of each toxicity study, further studies
can be carried out by sacrificing the animals (preferably, in
accordance with the American Veterinary Medical Association
guidelines Report of the American Veterinary Medical Assoc. Panel
on Euthanasia, Journal of American Veterinary Medical Assoc.,
202:229-249, 1993). Representative animals from each treatment
group can then be examined by gross necropsy for immediate evidence
of metastasis, unusual illness or toxicity. Gross abnormalities in
tissue are noted and tissues are examined histologically. Compounds
causing a reduction in body weight or blood components are less
preferred, as are compounds having an adverse effect on major
organs. In general, the greater the adverse effect the less
preferred the compound.
[0181] For the treatment of cancers the expected daily dose of a
hydrophobic pharmaceutical agent is between 1 to 500 mg/day,
preferably 1 to 250 mg/day, and most preferably 1 to 50 mg/day.
Drugs can be delivered less frequently provided plasma levels of
the active moiety are sufficient to maintain therapeutic
effectiveness. Plasma levels should reflect the potency of the
drug. Generally, the more potent the compound the lower the plasma
levels necessary to achieve efficacy.
[0182] DmGPCR mRNA transcripts may be found in many tissues,
including peripheral blood lymphocytes, spleen, bone marrow,
salivary gland, heart, thyroid gland, adrenal gland, pancreas,
liver, colon, lung, prostate, small intestine, muscle, stomach,
placenta and fetal liver. The sequences provided in SEQ ID NO: 1,
3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23 may be used to find the
full-length clone of an DmGPCR receptor. The clone of the receptor
will, as detailed above, enable screening for the endogenous
neurotransmitter/hormone which activates the receptor and for
compounds with potential utility in treating disorders including
cardiovascular disorders, reperfusion restenosis, coronary
thrombosis, clotting disorders, unregulated cell growth disorders
such as cancer, glaucoma, obesity, metabolic disorders,
inflammatory disorders, and CNS disorders.
[0183] For example, DmGPCR may be useful in the treatment of
respiratory ailments such as asthma, where T cells are implicated
by the disease. Contraction of airway smooth muscle is stimulated
by thrombin. Cicala et al (1999) Br J Pharmacol 126:478-484.
Additionally, in bronchiolitis obliterans, it has been noted that
activation of thrombin receptors may be deleterious. Hauck et al.
(1999) Am J Physiol 277:L22-L29. Furthermore, mast cells have also
been shown to have thrombin receptors. Cirino et al (1996) J Exp
Med 183:821-827. DmGPCR may also be useful in remodelling of airway
structures in chronic pulmonary inflammation via stimulation of
fibroblast procollagen synthesis. See, e.g., Chambers et al. (1998)
Biochem J 333:121-127; Trejo et al. (1996) J Biol Chem
271:21536-21541.
[0184] In another example, increased release of sCD40L and
expression of CD40L by T cells after activation of thrombin
receptors suggests that DmGPCR may be useful in the treatment of
unstable angina due to the role of T cells and inflammation. See
Aukrust et al. (1999) Circulation 100:614-620.
[0185] A further example is the treatment of inflammatory diseases,
such as psoriasis, inflammatory bowel disease, multiple sclerosis,
rheumatoid arthritis, and thyroiditis. Due to the tissue expression
profile of DmGPCR, inhibition of thrombin receptors may be
beneficial for these diseases. See, e.g., Morris et al. (1996) Ann
Rheum Dis 55:841-843. In addition to T cells, NK cells and
monocytes are also critical cell types which contribute to the
pathogenesis of these diseases. See, e.g., Naldini & Carney
(1996) Cell Immunol 172:3542; Hoffman & Cooper (1995) Blood
Cells Mol Dis 21:156-167; Colotta et al. (1994) Am J Pathol
144:975-985.
[0186] Expression of DmGPCR in bone marrow & spleen suggests
that it may play a role in the proliferation of hematopoietic
progenitor cells. See DiCuccio et al. (1996) Exp Hematol
24:914-918.
[0187] As another example, DmGPCR may be useful in the treatment of
acute and/or traumatic brain injury. Astrocytes have been
demonstrated to express thrombin receptors. Activation of thrombin
receptors may be involved in astrogliosis following brain injury.
Therefore, inhibition of receptor activity may be beneficial for
limiting neuroinflammation. Scar formation mediated by astrocytes
may also be limited by inhibiting thrombin receptors. See, e.g,
Pindon et al. (1998) Eur J Biochem 255:766-774; Ubl & Reiser.
(1997) Glia 21:361-369; Grabham & Cunningham (1995) J Neurochem
64:583-591.
[0188] DmGPCR receptor activation may mediate neuronal and
astrocyte apoptosis and prevention of neurite outgrowth. Inhibition
would be beneficial in both chronic and acute brain injury. See,
e.g., Donovan et al. (1997) J Neurosci 17:5316-5326; Turgeon et al.
(1998) J Neurosci 18:6882-6891; Smith-Swintosky et al. (1997) J
Neurochem 69:1890-1896; Gill et al. (1998) Brain Res 797:321-327;
Suidan et al. (1996) Semin Thromb Hemost 22:125-133.
[0189] The following Table 4 contains the sequences of the
polynucleotides and polypeptides of the invention.
TABLE-US-00004 TABLE 4 The following DNA sequence for DmGPCR1
<SEQ ID NO. 1> was identified in D. melanogaster.
ATGGCCAACTTAAGCTGGCTGAGCACCATCACCACCACCTCCTCCTCCAT
CAGCACCAGCCAGCTGCCATTGGTCAGCACAACCAACTGGAGCCTAACGT
CGCCGGGAACTACTAGCGCTATCTTGGCGGATGTGGCTGCATCGGATGAG
GATAGGAGCGGCGGGATCATTCACAACCAGTTCGTGCAAATCTTCTTCTA
CGTCCTGTACGCCACGGTCTTTGTCCTGGGTGTCTTCGGAAATGTCCTGG
TTTGCTACGTAGTTCTGAGGAATCGGGCCATGCAGACTGTGACCAATATA
TTCATCACGAATCTGGCCCTGTCGGACATATTGCTCTGCGTCCTGGCGGT
GCCATTTACTCCGCTTTACACGTTCATGGGTCGCTGGGCCTTCGGCAGGA
GTCTGTGCCATCTGGTGTCCTTTGCCCAGGGATGCAGCATCTACATATCC
ACGCTGACCCTCACCTCGATTGCCATCGATCGGTACTTCGTTATCATATA
CCCCTTCCATCCGCGCATGAAGCTCTCCACCTGCATCGGGATCATAGTGA
GCATCTGGGTGATAGCCCTGCTGGCCACCGTTCCCTACGGCATGTACATG
AAGATGACCAACGAGCTGGTGAACGGAACGCAGACAGGCAACGAGACCCT
GGTGGAGGCCACTCTAATGCTAAACGGAAGCTTTGTGGCCCAGGGATCAG
GATTCATCGAGGCGCCGGACTCTACCTCGGCCACCCAGGCCTATATGCAG
GTGATGACCGCCGGATCAACGGGACCGGAGATGCCCTATGTGCGGGTGTA
CTGCGAGGAGAACTGGCCATCGGAGCAGTACCGGAAGGTGTTCGGTGCCA
TCACAACCACTCTGCAGTTTGTGCTGCCCTTCTTCATCATCTCGATTTGC
TACGTGTGGATATCGGTGAAGCTAAACCAGCGGGCCAGGGCCAAGCCGGG
ATCGAAATCCTCGAGACGGGAGGAGGCGGATCGGGATCGCAAGAAGCGCA
CCAACCGCATGCTCATCGCCATGGTGGCGGTATTCGGACTCAGCTGGCTG
CCCATCAATGTGGTCAACATATTCGATGACTTCGATGACAAGTCCAACGA
GTGGCGCTTCTACATCCTATTCTTCTTTGTGGCCCACTCTATTGCCATGA
GCTCCACCTGCTACAATCCCTTCCTGTACGCCTGGCTGAACGAGAACTTC
CGCAAGGAGTTCAGCACGTGCTGCCCTGCTTTAATCCCTCGAACAACAAC
ATCATCAACATCACCAGGGGCTATAATCGGAGTGATCGGAACACCTGTGG
TCCGCGACTGCATCATGGCAAGGGGGATGGTGGCATGGGCGGTGGCAGTC
TGGACGCCGACGACCAGGACGAGAACGGCATCACCCAGGAGACCTGTCTG
CCCAAGGAGAAGCTGCTGATTATCCCCAGGGAGCCGACTTACGGCAATGG
CACGGGTGCCGTGTCGCCAATCCTTAGCGGGCGCGGCATTAACGCCGCCC
TGGTGCACGGTGGCGACCATCAGATGCACCAGCTGCAGCCGTCACACCAT
CAACAGGTGGAGCTGACGAGGCGAATCCGCCGGCGGACAGACGAGACGGA
CGGGGATTACCTGGACTCCGGCGACGAGCAGACCGTGGAGGTGCGCTTCA
GCGAGACGCCGTTCGTCAGCACGGATAATACCACCGGGATCAGCATTCTG
GAGACGAGTACGAGTCACTGCCAGGACTCGGATGTGATGGTCGAGCTGGG
CGAGGCAATCGGCGCCGGTGGTGGGGCAGAGCTGGGGAGGCGAATCAACT GA The following
amino acid sequence <SEQ ID NO. 2> is the amino acid sequence
for the protein encoded by the DNA sequence of SEQ ID NO. 1:
MANLSWLSTITTTSSSISTSQLPLVSTTNWSLTSPGTTSAILADVAASDE
DRSGGIIHNQFVQIFFYVLYATVFVLGVFGNVLVCYVVLRNRANQTVTNI
FITNLALSDILLCVLAVPFTPLYTFMGRWAFGRSLCHLVSFAQGCSIYIS
TLTLTSIAIDRYFVIIYPFHPRMKLSTCIGIIVSIWVIALLATVPYGMYM
KMTNELVNGTQTGNETLVEATLMLNGSFVAQGSGFIEAPDSTSATQAYMQ
VMTAGSTGPEMPYVRVYCEENWPSEQYRKVFGAITTTLQFVLPFFIISIC
YVWISVKLNQRARAKPGSKSSRREEADRDRKKRTNRNLIANVAVFGLSWL
PINVVNIFDDFDDKSNEWRFYILFFFVAHSIAMSSTCYNPFLYAWLNENF
RKEFKHVLPCFNPSNNNIINITRGYNRSDRNTCGPRLHHGKGDGGMGGGS
LDADDQDENGITQETCLPKEKLLIIPREPTYGNGTGAVSPILSGRGINAA
LVHGGDHQMHQLQPSHHQQVELTRRIRRRTDETDGDYLDSGDEQTVEVRF
SETPFVSTDNTTGISILETSTSHCQDSDVMVELGEAIGAGGGAELGRRIN The following
DNA sequence for DmGPCR2a <SEQ ID NO. 3> was identified in D.
melanogaster: ATGAATCAGACGGAGCCCGCCCAGCTGGCAGATGGGGAGCATCTGAGTGG
ATACGCCAGCAGCAGCAACAGCGTGCGCTATCTGGACGACCGGCATCCGC
TGGACTACCTTGACCTGGGCACGGTGCACGCCCTCAACACCACTGCCATC
AACACCTCGGATCTGAATGAGACTGGGAGCAGGCCGCTGGACCCGGTGCT
TATCGATAGGTTCCTGAGCAACAGGGCGGTGGACAGCCCCTGGTACCACA
TGCTCATCAGCATGTACGGCGTGCTAATCGTCTTCGGCGCCCTAGGCAAC
ACCCTGGTTGTTATAGCCGTCATCCGGAAGCCCATCATGCGCACTGCTCG
CAATCTGTTCATCCTCAACCTGGCCATATCGGACCTACTTTTATGCCTAG
TCACCATGCCGCTGACCTTGATGGAGATCCTGTCCAAGTACTGGCCCTAC
GGCTCCTGCTCCATCCTGTGCAAAACGATTGCCATGCTGCAGGCACTTTG
TATTTTCGTGTCGACAATATCCATAACGGCCATTGCCTTCGACAGATATC
AGGTGATCGTGTACCCCACGCGGGACAGCCTGCAGTTCGTGGGCGCGGTG
ACGATCCTGGCGGGGATCTGGGCACTGGCACTGCTGCTGGCCTCGCCGCT
GTTCGTCTACAAGGAGCTGATCAACACAGACACGCCGGCACTCCTGCAGC
AGATCGGCCTGCAGGACACGATCCCGTACTGCATTGAGGACTGGCCAAGT
CGCAACGGGCGCTTCTACTACTCGATCTTCTCGCTGTGCGTACAATACCT
GGTGCCCATCCTGATCGTCTCGGTGGCATACTTCGGGATCTACAACAAGC
TGAAGAGCCGCATCACCGTGGTGGCTGTGCAGGCGTCCTCCGCTCAGCGG
AAGGTGGAGCGGGGGCGGCGGATGAAGCGCACCAACTGCCTACTGATCAG
CATCGCCATCATCTTTGGCGTTTCTTGGCTGCCGCTGAACTTTTTCAACC
TGTACGCGGACATGGAGCGCTCGCCGGTCACTCAGAGCATGCTAGTCCGC
TACGCCATCTGCCACATGATCGGCATGAGCTCCGCCTGCTCCAACCCGTT
GCTCTACGGCTGGCTCAACGACAACTTCCGTAAAGAATTTCAAGAACTGC
TCTGCCGTTGCTCAGACACTAATGTTGCTCTTAACGGTCACACGACAGGC
TGCAACGTCCAGGCGGCGGCGCGCAAGCGTCGCAAGTTGGGCGCCGAACT
CTCCAAAGGCGAACTCAAGCTGCTGGGGCCAGGCGGCGCCCAGAGCGGTA
CCGCCGGCGGGGAAGGCGGTCTGGCGGCCACCGACTTCATGACCGGCCAC
CACGAGGGCGGACTGCGCAGCGCCATAACCGAGTCGGTGGCCCTCACGGA
CCACAACCCCGTGCCCTCGGAGGTCACCAAGCTGATGCCGCGGTA The following amino
acid sequence <SEQ ED NO.4> is the amino acid sequence for
the protein encoded by the DNA sequence of SEQ ID NO. 3:
MENTTMLANISLNATRNEENITSFFTDEEWLAINGTLPWIVGFFFGVIAI
TGFFGNLLVILVVVFNNNMRSTTNLMIVNLAAADLMFVILCIPFTATDYM
VYYWPYGRFWCRSVQYLIVVTAFASIYTLVLMSIDRFLAVVHPIRSRMMR
TENITLIAIVTLWIVVLVVSVPVAFTHDVVVDYDAKKNITYGMCTFTTND
FLGPRTYQVTFFISSYLLPLMIISGLYMRMIMRLWRQGTGVRNSKESQRG
RKRVTRLVVVVVIAFASLWLPVQLILLLKSLDVIETNTLTKLVIQVTAQT
LAYSSSCINPLLYAFLSENFRKAFYKAVNCSSRYQNYTSDLPPPRKTSCA RTSTTGL The
following DNA sequence for DmGPCR2b <SEQ ID NO. 5> was
identified in D. melanogaster:
ATGAATCAGACGGAGCCCGCCCAGCTGGCAGATGGGGAGCATCTGAGTGG
ATACGCCAGCAGCAGCAACAGCGTGCGCTATCTGGACGACCGGCATCCGC
TGGACTACCTTGACCTGGGCACGGTGCACGCCCTCAACACCACTGCCATC
AACACCTCGGATCTGAATGAGACTGGGAGCAGGCCGCTGGACCCGGTGCT
TATCGATAGGTTCCTGAGCAACAGGGCGGTGGACAGCCCCTGGTACCACA
TGCTCATCAGCATGTACGGCGTGCTAATCGTCTTCGGCGCCCTAGGCAAC
ACCCTGGTTGTTATAGCCGTCATCCGGAAGCCCATCATGCGCACTGCTCG
CAATCTGTTCATCCTCAACCTGGCCATATCGGACCTACTTTTATGCCTAG
TCACCATGCCGCTGACCTTGATGGAGATCCTGTCCAAGTACTGGCCCTAC
GGCTCCTGCTCCATCCTGTGCAAAACGATTGCCATGCTGCAGGCACTTTG
TATTTTCGTGTCGACAATATCCATAACGGCCATTGCCTTCGACAGATATC
AGGTGATCGTGTACCCCACGCGGGACAGCCTGCAGTTCGTGGGCGCGGTG
ACGATCCTGGCGGGGATCTGGGCACTGGCACTGCTGCTGGCCTCGCCGCT
GTTCGTCTACAAGGAGCTGATCAACACAGACACGCCGGCACTCCTGCAGC
AGATCGGCCTGCAGGACACGATCCCGTACTGCATTGAGGACTGGCCAAGT
CGCAACGGGCGCTTCTACTACTCGATCTTCTCGCTGTGCGTACAATACCT
GGTGCCCATCCTGATCGTCTCGGTGGCATACTTCGGGATCTACAACAAGC
TGAAGAGCCGCATCACCGTGGTGGCTGTGCAGGCGTCCTCCGCTCAGCGG
AAGGTGGAGCGGGGGCGGCGGATGAAGCGCACCAACTGCCTACTGATCAG
CATCGCCATCATCTTTGGCGTTTCTTGGCTGCCGCTGAACTTTTTCAACC
TGTACGCGGACATGGAGCGCTCGCCGGTCACTCAGAGCATGCTAGTCCGC
TACGCCATCTGCCACATGATCGGCATGAGCTCCGCCTGCTCCAACCCGTT
GCTCTACGGCTGGCTCAACGACAACTTCCGCTGCAACGTCCAGGCGGCGG
CGCGCAAGCGTCGCAAGTTGGGCGCCGAACTCTCCAAAGGCGAACTCAAG
CTGCTGGGGCCAGGCGGCGCCCAGAGCGGTACCGCCGGCGGGGAAGGCGG
TCTGGCGGCCACCGACTTCATGACCGGCCACCACGAGGGCGGACTGCGCA
GCGCCATAACCGAGTCGGTGGCCCTCACGGACCACAACCCCGTGCCCTCG
GAGGTCACCAAGCTGATGCCGCGGTA The following amino acid sequence
<SEQ ID NO. 6> is the amino acid sequence for the protein
encoded by the DNA sequence of SEQ ID NO. 5:
MNQTEPAQLADGEHLSGYASSSNSVRYLDDRHPLDYLDLGTVHALNTTAI
NTSDLNETGSRPLDPVLIDRFLSNRAVDSPWYHMLISMYGVLIVFGALGN
TLVVIAVIRKPIMRTARNLFILNLAISDLLLCLVTMPLTLMEILSKYWPY
GSCSILCKTIAMLQALCIFVSTISITAIAFDRYQVIVYPTRDSLQFVGAV
TILAGIWALALLLASPLFVYKELINTDTPALLQQIGLQDTIPYCIEDWPS
RNGRFYYSIFSLCVQYLVPILIVSVAYFGIYNKLKSRITVVAVQASSAQR
KVERGRRMKRTNCLLISIAIIFGVSWLPLNFFNLYADMERSPVTQSMLVR
YAICHMIGMSSACSNPLLYGWLNDNFRCNVQAAARKRRKLGAELSKGELK
LLGPGGAQSGTAGGEGGLAATDFMTGHHEGGLRSAITESVALTDHNPVPS EVTKLMPR The
following DNA sequence for DmGPCR4 <SEQ ID NO. 7> was
identified in D. melanogaster:
ATGGAGAACACCACAATGCTGGCTAATATTAGCCTAAATGCAACCAGAAA
TGAGGAGAATATCACCTCATTCTTCACCGACGAAGAGTGGCTGGCCATCA
ATGGCACTTTGCCGTGGATAGTGGGATTCTTCTTCGGCGTCATCGCCATC
ACGGGATTCTTCGGCAACCTGCTGGTCATCCTGGTGGTGGTCTTCAACAA
CAACATGCGCTCCACCACCAACCTGATGATTGTCAATCTGGCTGCCGCTG
ATCTGATGTTCGTAATCCTCTGCATTCCCTTCACGGCCACCGATTACATG
GTGTACTACTGGCCATATGGAAGGTTCTGGTGCCGCAGTGTCCAGTACCT
GATTGTGGTGACCGCCTTCGCCTCCATCTACACGCTGGTGCTAATGTCCA
TCGATCGGTTCCTGGCGGTGGTTCATCCCATTCGCTCGCGGATGATGAGG
ACGGAGAACATTACCCTGATTGCCATCGTGACTCTGTGGATCGTGGTGCT
GGTCGTTTCGGTGCCAGTGGCCTTCACCCACGACGTGGTGGTGGACTACG
ATGCAAAGAAGAACATCACCTACGGCATGTGCACCTTCACGACGAACGAC
TTCCTTGGTCCGCGCACCTACCAGGTCACCTTCTTCATCAGCTCCTACCT
GCTGCCCCTGATGATCATCAGCGGTCTCTACATGCGCATGATCATGCGGC
TCTGGCGCCAGGGAACCGGCGTCCGCATGTCCAAGGAGTCGCAGCGCGGT
CGCAAGCGGGTCACCCGACTCGTCGTCGTGGTGGTCATCGCCTTCGCCTC
GCTCTGGCTGCCTGTCCAGCTCATCCTGCTGCTCAAGTCACTGGATGTCA
TCGAGACGAACACCCTCACCAAGCTAGTCATCCAGGTCACCGCCCAGACT
CTGGCCTACAGCAGCTCGTGTATCAATCCGCTGCTCTACGCCTTCCTCTC
CGAGAATTTCCGGAAGGCCTTCTATAAGGCCGTTAACTGCTCCTCTCGAT
ACCAGAACTACACATCTGATTTGCCGCCGCCGCGCAAGACGTCCTGTGCC
AGGACCTCCACCACTGGACTCTA The following amino acid sequence <SEQ
ID NO. 8> is the amino acid sequence for the protein encoded by
the DNA sequence of SEQ ID NO. 7:
MENTTMLANISLNATRNEENITSFFTDEEWLAINGTLPWIVGFFFGVIAI
TGFFGNLLVILVVVFNNNMRSTTNLMIVNLAAADLMFVILCIPFTATDYM
VYYWPYGRFWCRSVQYLIVVTAFASIYTLVLMSIDRFLAVVHPIRSRMMR
TENITLIAIVTLWIVVLVVSVPVAFTHDVVVDYDAKKNITYGMCTFTTND
FLGPRTYQVTFFISSYLLPLMIISGLYMRMIMRLWRQGTGVRMSKESQRG
RKRVTRLVVVVVIAFASLWLPVQLILLLKSLDVIETNTLTKLVIQVTAQT
LAYSSSCINPLLYAFLSENFRKAFYKAVNCSSRYQNYTSDLPPPRKTSCA RTSTTGL The
following DNA sequence for DmGPCR5a <SEQ ID NO. 9> was
identified in D. melanogaster
ATGGAGAATCGCAGTGACTTCGAGGCGGATGACTACGGCGACATCAGTTG
GAGCAATTGGAGCAACTGGAGCACCCCCGCCGGCGTCCTTTTCTCGGCCA
TGAGCAGCGTGCTCTCGGCCAGCAACCATACGCCCCTGCCGGACTTTGGC
CAGGAGCTCGCCCTATCCACCAGCTCCTTCAATCACAGCCAGACCCTATC
CACCGACCAGCCCGCCGTCGGGGACGTGGAAGACGCGGCCGAGGATGCGG
CGGCGTCCATGGAGACGGGCTCGTTTGCATTTGTGGTCCCGTGGTGGCGT
CAGGTGCTCTGGAGCATCCTCTTCGGCGGCATGGTCATTGTGGCGACGGG
CGGTAACCTGATTGTTGTCTGGATCGTGATGACGACCAAGCGGATGCGGA
CGGTAACCAACTATTTCATAGTGAATCTCTCCATCGCGGACGCCATGGTG
TCCAGCCTAAACGTCACCTTCAACTACTACTATATGCTGGATAGCGACTG
GCCCTTCGGCGAGTTCTACTGCAAGTTGTCCCAGTTCATCGCGATGCTAA
GCATCTGCGCCTCAGTGTTCACCCTAATGGCCATCTCCATCGACAGATAC
GTGGCCATCATCCGGCCACTGCAGCCGCGGATGAGCAAGCGGTGCAACCT
GGCCATCGCGGCGGTCATCTGGCTGGCCTCCACGCTCATCTCCTGCCCCA
TGATGATCATCTACCGCACGGAGGAGGTGCCGGTCCGCGGGCTCAGCAAC
CGCACGGTCTGCTACCCGGAGTGGCCCGATGGGCCCACCAATCACTCCAC
GATGGAGTCCCTCTACAACATCCTCATCATCATYCTAACCTACTTCCTGC
CCATCGTCTCCATGACGGTCACCTACTCGCGCGTGGGCATCGAGCTCTGG
GGATCCAAGACCATCGGCGAGTGCACGCCCCGCCAGGTGGARAAYGTGCG
GAGTAAGCGAAGGGTGGTGAAGATGATGATTGTGGTCGTCCTGATATTCG
CCATCTGCTGGCTGCCGTTCCACAGCTACTTCATAATCACATCCTGCTAC
CCGGCCATCACGGAGGCGCCCTTCATCCAGGAACTCTACCTGGCCATCTA
CTGGCTGGCCATGAGCAACTCCATGTACAATCCCATTATATACTGCTGGA
TGAATTCGCGCTTTCGCTATGGTTTCAAGATGGTCTTCCGCTGGTGCCTG
TTTGTGCGCGTGGGCACTGAACCCTTTAGTCGGCGGGAGAACCTGACATC
CCGGTACTCCTGCTCCGGTTCCCCGGATCACAATCGCATCAAGCGCAATG
ATACCCAGAAATCGATACTTTATACCTGTCCCAGCTCACCCAAGTCGCAT
CGAATTTCGCACAGCGGAACAGGTCGCAGTGCGACGCTGCGGAACAGTCT
GCCGGCGGAGTCACTGTCGTCCGGCGGATCTGGTGGTGGAGGGCACAGGA
AACGGTTGTCCTACCAGCAGGAAATGCAGCAGCGTTGGTCAGGACCCAAT
AGTGCCACCGCAGTGACCAATTCCAGCAGTACGGCCAACACCACCCAACT GCTCTCCTG The
following amino acid sequence <SEQ ID NO. 10> is the amino
acid sequence for the protein encoded by the DNA sequence of SEQ ID
NO. 9: MENRSDFEADDYGDISWSNWSNWSTPAGVLFSAMSSVLSASNHTPLPDFG
QELALSTSSFNHSQTLSTDQPAVGDVEDAAEDAAASMETGSFAFVVPWWR
QVLWSILFGGMVIVATGGNLIVVWIVMTTKRMRTVTNYFIVNLSIADAMV
SSLNVTFNYYYMLDSDWPFGEFYCKLSQFIAMLSICASVFTLMAISIDRY
VAIIRPLQPRMSKRCNLAIAAVIWLASTLISCPMMIIYRTEEVPVRGLSN
RTVCYPEWPDGPTNHSTMESLYNILIIILTYFLPIVSMTVTYSRVGIELW
GSKTIGECTPRQVENVRSKRRVVKMMIVVVLIFAICWLPFHSYFIETSCY
PAITEAPFIQELYLAIYWLAMSNSMYNPIIYCWMNSRFRYGFKMVFRWCL
FVRVGTEPFSRRENLTSRYSCSGSPDHNRIKRNDTQKSILYTCPSSPKSH
RISHSGTGRSATLRNSLPAESLSSGGSGGGGHRKRLSYQQEMQQRWSGPN
SATAVTNSSSTANTTQLLS The following DNA sequence for DmGPCR5b <SEQ
ID NO. 11> was identified in D. melanogaster.
ATGGAGAATCGCAGTGACTTCGAGGCGGATGACTACGGCGACATCAGTTG
GAGCAATTGGAGCAATTGGAGCAACTGGAGCACCCCCGCCGGCGTCCTTT
TCTCGGCCATGAGCAGCGTGCTCTCGGCCAGCAACCATACGCCTCTGCCG
GACTTTGGCCAGGAGCTCGCCCTATCCACCAGCTCCTTCAATCACAGCCA
GACCCTATCCACCGACCTGCCCGCCGTCGGGGACGTGGAAGACGCGGCCG
AGGATGCGGCGGCGTCCATGGAGACGGGCTCGTTTGCATTTGTGGTCCCG
TGGTGGCGTCAGGTGCTCTGGAGCATCCTCTTCGGCGGCATGGTCATTGT
GGCGACGGGCGGTAACCTGATTGTTGTCTGGATCGTGATGACGACCAAGC
GGATGCGGACGGTAACCAACTATTTCATAGTAAATCTCTCCATCGCGGAC
GCCATGGTGTCCAGCCTGAACGTCACCTTCAACTACTACTACATGCTGGA
TAGCGACTGGCCCTTCGGCGAGTTCTACTGCAAGTTGTCCCAGTTCATCG
CGATGCTAAGCATCTGCGCCTCAGTGTTCACCCTAATGGCCATCTCCATC
GACAGATACGTGGCCATCATCCGGCCACTGCAGCCGCGGATGAGCAAGCG
GTGCAACCTGGCCATCGCGGCGGTCATCTGGCTGGCCTCCACGCTCATCT
CCTGCCCCATGATGATCATCTACCGCACGGAGGAGGTGCCGGTCCGCGGG
CTCAGCAACCGCACGGTCTGCTACCCGGAGTGGCCCGATGGGCCCACCAA
TCACTCCACGATGGAGTCCCTCTACAACATCCTCATCATCATTCTAACCT
ACTTCCTGCCCATCGTCTCCATGACGGTCACCTACTCGCGCGTGGGCATC
GAGCTCTGGGGATCCAAGACCATCGGCGAGTGCACGCCCCGCCAGGTGGA
GAATGTGCGGAGTAAGCGAAGGGTGGTGAAGATGATGATTGTGGTCGTCC
TGATATTCGCCATCTGCTGGCTGCCGTTCCACAGCTACTTCATAATCACA
TCCTGCTACCCGGCCATCACGGAGGCGCCCTTCATCCAGGAACTTTACCT
GGCCATCTACTGGCTGGCCATGAGCAACTCCATGTACAATCCCATTATAT
ACTGCTGGATGAATTCGCGCTTTCGCTATGGTTTCAAGATGGTCTTCCGC
TGGTGCCTGTTTGTGCGCGTGGGCACTGAACCCTTTAGTCGGCGGGAGAA
CCTGACATCCCGGTACTCCTGCTCCGGTTCCCCGGATCACAATCGCATCA
AGCGCAATGATACCCAGAAATCGATACTTTATACCTGTCCCAGCTCACCC
AAGTCGCATCGAATTTCGCACAGCGGAACAGGTCGCAGTGCGACGCTGAG
GAACAGTCTGCCGGCGGAGTCATTGTCGTCCGGTGGATCTGGAGGTGGAG
GACACAGGAAACGGTTGTCCTACCAGCAGGAAATGCAGCAGCGGTGGTCA
GGACCCAATAGTGCCACCGCAGTGACCAATTCCAGCAGTACGGCCAACAC
CACCCAACTGCTCTCCTG The following amino acid sequence <SEQ ID NO.
12> is the amino acid sequence for the protein encoded by the
DNA sequence of SEQ ID NO. 11:
MENRSDFEADDYGDISWSNWSNWSNWSTPAGVLFSAMSSVLSASNHTPLP
DFGQELALSTSSFNHSQTLSTDLPAVGDVEDAAEDAAASMETGSFAFVVP
WWRQVLWSILFGGMVIVATGGNLIVVWIVMTTKRMRTVTNYFIVNLSIAD
AMVSSLNVTFNYYYMLDSDWPFGEFYCKLSQFIAMLSICASVFTLMAISI
DRYVAIIRFLQPRMSKRCNLAIAAVIWLASTLISCPMMIIYRTEEVPVRG
LSNRTVCYPEWPDGPTNHSTMESLYNTLIIILTYFLPIVSMTVTYSRVGI
ELWGSKTIGECTPRQVENVRSKRRVVKMMIVVVLIFAICWLPFHSYFIIT
SCYPAITEAPFIQELYLAIYWLAMSNSMYNPIIYCWMNSRFRYGFKMVFR
WCLFVRVGTEPFSRRENLTSRYSCSGSPDHNRIKRNDTQKSILYTCPSSP
KSHRISHSGTGRSATLRNSLPAESLSSGGSGGGGHRKRLSYQQEMQQRWS
GPNSATAVTNSSSTANTTQLLS The following DNA sequence for DmGPCR6aL
<SEQ ID NO. 13> was identified in D. melanogaster
ATGGAGCACCACAATAGCCATCTGTTGCCTGGTGGCAGCGAGAAGATGTA
CTACATAGCTCACCAGCAGCCGATGCTGCGGAACGAGGATGATAACTACC
AGGAGGGGTACTTCATCAGGCCGGACCCTGCATCCTTACTTTACAATACC
ACCGCACTGCCAGCGGACGATGAAGGGTCCAACTATGGATATGGCTCCAC
CACAACGCTCAGTGGCCTCCAGTTCGAGACCTATAATATCACTGTGATGA
TGAACTTTAGCTGTGACGACTATGACCTTCTATCGGAGGACATGTGGTCT
AGTGCCTACTTTAAGATCATCGTCTACATGCTCTACATTCCCATCTTTAT
CTTCGCCCTGATCGGCAACGGAACGGTCTGCTATATCGTCTATTCCACAC
CTCGCATGCGCACGGTCACCAATTACTTTATAGCCAGCTTGGCCATCGGC
GACATCCTGATGTCCTTCTTCTGCGTTCCGTCGTCCTTCATCTCGCTGTT
CATCCTGAACTACTGGCCTTTTGGCCTGGCCCTCTGTCACTTTGTGAACT
ACTCGCAGGCGGTCTCAGTTCTGGTCAGCGCCTATACTTTGGTGGCAATT
AGCATTGACCGCTACATAGCCATTATGTGGCCATTAAAGCCACGCATCAC
AAAACGCTATGCCACCTTCATCATCGCCGGCGTTTGGTTTATTGCACTTG
CCACCGCACTTCCCATACCCATCGTCTCTGGACTCGACATCCCAATGTCG
CCGTGGCACACGAAATGCGAGAAATACATTTGCCGCGAAATGTGGCCGTC
GCGGACGCAGGAGTACTACTACACCCTGTCCCTCTTCGCGCTGCAGTTCG
TCGTGCCGCTGGGCGTGCTCATCTTCACCTACGCCCGGATCACCATTCGC
GTCTGGGCGAAACGACCGCCAGGCGAGGCGGAAACCAACCGCGACCAGCG
GATGGCACGCTCCAAACGGAAGATGGTCAAAATGATGGTGACGGTTGTGA
TTGTGTTCACCTGCTGTTGGCTGCCCTTCAATATTTTGCAGCTTTTACTG
AACGACGAGGAGTTCGCCCACTGGGATCCTCTGCCGTATGTATGGTTCGC
GTTTCACTGGCTGGCCATGTCGCACTGCTGCTACAATCCGATCATCTACT
GCTACATGAACGCCCGTTTCAGGAGCGGATTCGTCCAGCTGATGCACCGT
ATGCCCGGCCTGCGTCGCTGGTGCTGCCTGCGGAGCGTCGGTGATCGCAT
GAACGCAACTTCCGGAACGGGTCCAGCACTTCCTCTCAATCGAATGAACA
CATCCACCACCTACATCAGCGCTCGTCGAAAGCCACGAGCGACATCTTTG
CGAGCGAACCCATTATCATGCGGCGAGACGTCACCACTGCGGTA The following amino
acid sequence <SEQ ID NO. 14> is the amino acid sequence for
the protein encoded by the DNA sequence of SEQ ID NO. 13:
MEHHNSHLLPGGSEKMYYIAHQQPMLRNEDDNYQEGYFIRPDPASLLYNT
TALPADDEGSNYGYGSTTTLSGLQFETYNITVMMNFSCDDYDLLSEDMWS
SAYFKIIVYMLYTPIFIFALIGNGTVCYIVYSTPRMRTVTNYFIASLAIG
DILMSFFCVPSSFISLFILNYWPFGLALCHFVNYSQAVSVLVSAYTLVAI
SIDRYIAIMWPLKPRITKRYATFIIAGVWFIALATALPIPIVSGLDIPMS
PWHTKCEKYICREMWPSRTQEYYYTLSLFALQFVVPLGVLIFTYARITIR
VWAKRPPGEAETNRDQRNARSKRKMVKMMLTVVIVFTCCWLPFNILQLLL
NDEEFAHWDPLPYVWFAFHWLAMSHCCYNPIIYCYMNARFRSGFVQLMHR
MPGLRRWCCLRSVGDRMNATSGTGPALPLNRMNTSTTYISARRKPRATSL RANPLSCGETSPLR
The following DNA sequence for DmGPCR6bL <SEQ ID NO. 15> was
identified in D. melanogaster:
ATGGAGCACCACAATAGCCATCTGTTGCCTGGTGGCAGCGAGAAGATGTA
CTACATAGCTCACCAGCAGCCGATGCTGCGGAACGAGGATGATAACTACC
AGGAGGGGTACTTCATCAGGCCGGACCCTGCATCCTTACTTTACAATACC
ACCGCACTGCCAGCGGACGATGAAGGGTCCAACTATGGATATGGCTCCAC
CACAACGCTCAGTGGCCTCCAGTTCGAGACCTATAATATCACTGTGATGA
TGAACTTTAGCTGTGACGACTATGACCTTCTATCGGAGGACATGTGGTCT
AGTGCCTACTTTAAGATCATCGTCTACATGCTCTACATTCCCATCTTTAT
CTTCGCCCTGATCGGCAACGGAACGGTCTGCTATATCGTCTATTCCACAC
CTCGCATGCGCACGGTCACCAATTACTTTATAGCCAGCTTGGCCATCGGC
GACATCCTGATGTCCTTCTTCTGCGTTCCGTCGTCCTTCATCTCGCTGTT
CATCCTGAACTACTGGCCTTTTGGCCTGGCCCTCTGTCACTTTGTGAACT
ACTCGCAGGCGGTCTCAGTTCTGGTCAGCGCCTATACTTTGGTGGCAATT
AGCATTGACCGCTACATAGCCATTATGTGGCCATTAAAGCCACGCATCAC
AAAACGCTATGCCACCTTCATCATCGCCGGCGTTTGGTTTATTGCACTTG
CCACCGCACTTCCCATACCCATCGTCTCTGGACTCGACATCCCAATGTCG
CCGTGGCACACGAAATGCGAGAAATACATTTGCCGCGAAATGTGGCCGTC
GCGGACGCAGGAGTACTACTACACCCTGTCCCTCTTCGCGCTGCAGTTCG
TCGTGCCGCTGGGCGTGCTCATCTTCACCTACGCCCGGATCACCATTCGC
GTCTGGGCGAAACGACCGCCAGGCGAGGCGGAAACCAACCGCGACCAGCG
GATGGCACGCTCCAAACGGAAGATGGTCAAAATGATGCTGACGGTTGTGA
TTGTGTTCACCTGCTGTTGGCTGCCCTTCAATATTTTGCAGCTTTTACTG
AACGACGAGGAGTTCGCCCACTGGGATCCTCTGCCGTATGTGTGGTTCGC
GTTTCACTGGCTGGCCATGTCGCACTGCTGCTACAATCCGATCATCTACT
GCTACATGAACGCCCGTTTCAGGAGCGGATTCGTCCAGCTGATGCACCGT
ATGCCCGGCCTGCGTCGCTGGTGCTGCCTGCGGAGCGTCGGTGATCGCAT
GAACGCAACTTCCGGTGAGATGACTACGAAGTACCATCGCCATGTCGGCG
ATGCCCTATTCCGGAAACCCAAAATATGCATTAGGAACGGGTCCAGCACT
TCCTCTCAATCGAATGAACACATCCACCACCTACATCAGCGCTCGTCGAA
AGCCACGAGCGACATCTTTGCGAGCGAACCCATTATCATGCGGCGAGACG
TCACCACTGCGGTAGCTGTCATATCAAAAAATAAAACTGATTCACCGGTG
CGCCGATCGGGAAGCTCAGGTGGAACAGAAGCAAACATAAGAAGCACCGA GTTTTG The
following amino acid sequence <SEQ ID NO. 16> is the amino
acid sequence for the protein encoded by the DNA sequence of SEQ ID
NO. 15: MEHHNSHLLPGGSEKMYYIAHQQPMLRNEDDNYQEGYFIRPDPASLLYNT
TALPADDEGSNYGYGSTTTLSGLQFETYNITVMMNFSCDDYDLLSEDMWS
SAYFKIIVYMLYIPIFIFALIGNGTVCYIVYSTPRMRTVTNYFIASLAIG
DILMSFFCVPSSFISLFILNYWPFGLALCHFVNYSQAVSVLVSAYTLVAI
SIDRYIAIMWPLKPRITKRYATFIIAGVWFIALATALPIPIVSGLDIPMS
PWHTKCEKYICREMWPSRTQEYYYTLSLFALQFVVPLGVLIFTYARITIR
VWAKRPPGEAETNRDQRMARSKRKMVKMMLTVVIVFTCCWLPFNILQLLL
NDEEFARWDPLPYVWFAFHWLAMSHCCYNPITYCYMNARFRSGFVQLMHR
HPGLRRWCCLRSVGDRMNATSGEMTTKYHRHVGDALFRKPKICIRNGSST
SSQSNEHIHHLHQRSSKATSDIFASEPIIMRRDVTTAVAVISKNKTDSPV
RRSGSSGGTEANIRSTEF The following DNA sequence for DmGPCR7 <SEQ
ID NO. 17> was identified in D. melanogaster:
ATGGCAATGGACTTAATCGAGCAGGAGTCCCGCCTGGAATTCCTGCCCGG
AGCCGAGGAGGAAGCAGAATTTGAGCGTCTATACGCGGCTCCCGCTGAGA
TTGTGGCCCTGTTGTCCATTTTCTATGGGGGAATCAGTATCGTGGCCGTC
ATTGGCAACACTTTGGTCATCTGGGTGGTGGCCACGACCAGGCAAATGCG
GACCGTGACAAATATGTATATCGCTAATTTGGCTTTTGCCGATGTGATTA
TTGGCCTCTTCTGCATACCATTTCAGTTCCAGGCTGCCCTGCTGCAGAGT
TGGAACCTGCCGTGGTTCATGTGCAGCTTCTGCCCCTTCGTCCAGGCCCT
GAGTGTAAATGTCTCGGTATTCACGCTGACCGCCATTGCAATCGATCGGC
ATAGGGCCATCATTAATCCACTTAGGGCACGTCCCACCAAGTTCGTATCG
AAGTTCATAATTGGTGGAATTTGGATGCTGGCCCTGCTATTTGCGGTGCC
CTTTGCCATTGCCTTTCGTGTGGAGGAGTTGACCGAAAGATTTCGCGAGA
ACAATGAGACCTACAATGTGACGCGGCCATTCTGCATGAACAAGAACCTA
TCCGATGATCAATTGCAATCCTTTCGCTACACCCTGGTTTTTGTGCAGTA
TCTGGTTCCATTCTGTGTCATCAGCTTTGTCTACATCCAGATGGCGGTAC
GATTGTGGGGCACACGTGCTCCTGGTAACGCACAGGATTCACGGGACATA
ACGCTGTTGAAAAACAAGAAGAAGGTCATCAAAATGCTGATTATCGTGGT
CATTATCTTTGGACTCTGCTGGCTGCCACTGCAGCTCTATAATATTCTGT
ATGTCACGATACCGGAAATCAACGACTACCACTTCATTAGCATCGTCTGG
TTTTGCTGCGATTGGCTGGCCATGAGCAATAGCTGCTACAATCCCTTTAT
TTATGGCATCTACAATGAAAAATTTAAGCGGGAATTCAACAAGCGATTTG
CGGCCTGTTTCTGCAAGTTCAAGACGAGCATGGACGCCCACGAAAGGACC
TTTTCGATGCACACCCGCGCCAGCTCCATAAGGTCAACCTACGCCAACTC
CTCGATGCGAATCCGGAGTAATCTCTTTGGTCCGGCGCGTGGTGGTGTCA
ACAATGGGAAGCCGGGCTTGCATATGCCGCGGGTGCATGGATCCGGTGCT
AACAGCGGCATTTACAACGGAAGTAGTGGGCAGAACAACAATGTCAATGG
CCAACATCATCAGCATCAAAGCGTGGTTACCTTTGCGGCCACTCCGGGTG
TTTCGGCACCAGGTGTTGGCGTTGCAATGCCGCCGTGGCGGCGAAACAAC
TTCAAACCTCTGCATCCGAACGTAATCGAATGCGAGGACGACGTGGCACT
CATGGAGCTGCCATCAACCACGCCCCCCAGCGAGGAGTTGGCATCCGGGG
CCGGAGTCCAGTTGGCCCTGCTAAGCAGGGAGAGCTCCAGCTGCATTTGC
GAACAGGAATTTGGCAGCCAAACCGAATGCGATGGCACCTGCATACTCAG
CGAGGTGTCGCGAGTCCACCTGCCCGGCTCGCAGGCGAAGGACAAGGATG
CGGGCAAGTCCTTGTGGCAACCACTTTA The following amino acid sequence
<SEQ ID NO. 18> is the amino acid sequence for the protein
encoded by the DNA sequence of SEQ ID NO. 17:
MAMDLIEQESRLEFLPGAEEEAEFERLYAAPAEIVALLSIFYGGISIVAV
IGNTLVIWVVATTRQMRTVTNMYIANLAFADVIIGLFCIPFQFQAALLQS
WNLPWFMCSFCPFVQALSVNVSVFTLTAIAIDRHRAIINPLRARPTKFVS
KFIIGGIWMLALLFAVEFAIAFRVEELTERFRENNETYNVTRPFCMNKNL
SDDQLQSFRYTLVFVQYLVPFCVISFVYIQMAVRLWGTRAPGNAQDSRDI
TLLKNKKKVIKMLIIVVIIFGLCWLPLQLYNILYVTIPEINDYHFISIVW
FCCDWLAMSNSCYNPFIYGIYNEKFKREFNKRFAACFCKFKTSMDAHERT
FSMHTRASSIRSTYANSSMRIRSNLFGPARGGVNNGKPGLHMPRVHGSGA
NSGIYNGSSGQNNNVNGQHHQHQSVVTFAATPGVSAPGVGVAMPPWRRNN
FKPLHPNVIECEDDVALMELPSTTPPSEELASGAGVQLALLSRESSSCIC
EQEFGSQTECDGTCILSEVSRVHLPGSQAKDKDAGKSLWQPL The following DNA
sequence for DmGPCR8 <SEQ ID NO. 19> was identified in D.
melanogaster: ATGTTTACGTGGCTGATGATGGATGTCCTCCAGTTTGTGAAAGGGGAAAT
GACAGCCGATTCAGAGGCAAATGCCACAAATTGGTATAACACGAACGAGA
GCTTATATACCACGGAACTGAACCATAGATGGATTAGTGGTAGTTCCACA
ATTCAGCCAGAGGAGTCCCTTTATGGCACTGATTTGCCCACCTATCAACA
TTGCATAGCCACGCGGAATTCCTTTGCTGACTTGTTCACTGTGGTGCTCT
ACGGATTTGTGTGCATTATCGGATTATTTGGCAACACCCTGGTGATCTAC
GTGGTGTTGCGCTTTTCCAAAATGCAAACGGTCACGAATATATATATCCT
GAATCTGGCGGTGGCAGACGAGTGCTTCCTGATTGGAATACCCTTTCTGC
TGTACACAATGCGAATTTGCAGCTGGCGATTCGGGGAGTTTATGTGCA.about.A
GCCTACATGGTGAGCACATCCATCACCTCCTTCACCTCGTCGATTTTTCT
GCTCATCATGTCCGCGGATCGATATATAGCGGTATGCCACCCGATTTCCT
CGCCACGATATCGAACTCTGCATATTGCCAAAGTGGTCTCAGCGATTGCC
TGGTCAACTTCAGCGGTCCTCATGCTGCCCGTGATCCTTTATGCCAGCAC
TGTGGAGCAGGAGGATGGCATCAATTACTCGTGCAACATAATGTGGCCAG
ATGCGTACAAGAAGCATTCGGGCACCACCTTCATACTGTACACATTTTTC
CTAGGATTCGCCACACCGCTGTGCTTTATCCTGAGTTTCTACTACTTGGT
TATAAGGAAACTGCGATCGGTGGGTCCCAAACCAGGAACGAAGTCCAAGG
AGAAGAGGCGGGCTCACAGGAAGGTCACTCGACTGGTACTGACGGTGATA
AGTGTATACATTCTATGTTGGCTCCCTCACTGGATTTCTCAGGTGGCCCT
GATTCACTCGAATCCCGCGCAAAGGGACCTCTCCCGACTGGAAATACTCA
TTTTCCTACTTCTGGGGGCACTGGTTTACTCGAATTCGGCGGTGAATCCC
ATACTTTATGCCTTCCTAAGTGAGAACTTCCGGAAGAGCTTCTTCAAGGC
CTTTACCTGTATGAATAAGCAGGATATCAACGCTCAACTCCAGCTGGAGC
CCAGTGTTTTCACCAAACAGGGCAGTAAAAAGAGGGGTGGCTCCAAGCGC
CTGTTGACCAGCAATCCGCAGATTCCTCCACTGCTGCCACTGAATGCGGG
TAACAACAATTCATCGACCACCACATCCTCGACCACGACAGCGGAAAAGA
CCGGAACCACGGGGACACAGAAATCATGCAATTCCAATGGCAAAGTGACA
GCTCCGCCGGAGAATTTGATTATATGTTTGAGCGAGCAGCAGGAGGCATT
TTGCACCACCGCGAGAAGAGGATCGGGCGCAGTGCAGCAGACAGATTTGT A The following
amino acid sequence <SEQ ID NO. 20> is the amino acid
sequence for the protein encoded by the DNA sequence of SEQ ID NO.
19: MFTWLMMDVLQFVKGEMTADSEANATNWYNTNESLYTTELNHRWISGSST
IQPEESLYGTDLPTYQHCIATRNSFADLFTVVLYGFVCIIGLFGNTLVIY
VVLRFSKMQTVTNIYILNLAVADECFLIGIPFLLYTMRICSWRFGEFMCK
AYMVSTSITSFTSSIFLLIMSADRYIAVCHPISSPRYRTLHIAKVVSAIA
WSTSAVLMLPVILYASTVEQEDGINYSCNIMWPDAYKKHSGTTFILYTFF
LGFATPLCFILSFYYLVIRKLRSVGPKPGTKSKEKRRAHRKVTRLVLTVI
SVYILCWLPHWISQVALIHSNPAQRDLSRLEILIFLLLGALVYSNSAVNP
ILYAFLSENFRKSFFKAFTCMNKQDINAQLQLEPSVFTKQGSKKRGGSKR
LLTSNPQIPPLLPLNAGNNNSSTTTSSTTTAEKTGTTGTQKSCNSNGKVT
APPENLIICLSEQQEAFCTTARRGSGAVQQTDL The following DNA sequence for
DmGPCR9 <SEQ ID NO. 21> was identified in D. melanogaster
ATGTTCAACTACGAGGAGGGGGATGCCGACCAGGCGGCCATGGCTGCAGC
GGCTGCCTATAGGGCACTGCTCGACTACTATGCCAATGCGCCAAGTGCGG
CGGGTCACATAGTGTCGCTCAACGTGGCACCCTACAATGGAACTGGAAAC
GGAGGCACTGTCTCCTTGGCGGGCAATGCGACAAGCAGCTATGGCGATGA
TGATAGGGATGGCTATATGGACACCGAGCCCAGTGACCTGGTCACCGAAC
TGGCCTTCTCCCTGGGCACCAGTTCAAGTCCAAGTCCCAGTTCCACACCC
GCTTCCAGCTCCAGTACTTCCACTGGCATGGCCGTCTGGCTGATACCCAG
CTATAGCATGATTCTGCTGTTCGCCGTGCTGGGCAACCTGCTGGTCATCT
CGACGCTGGTGCAGAATCGCCGGATGCGTACCATAACCAACGTGTTCCTG
CTCAACCTGGCCATATCGGACATGCTGCTGGGCGTGCTCTGCATGCCCGT
CACCCTGGTGGGCACCCTGCTGCGAAACTTCATCTTTGGCGAGTTCCTCT
GCAAGCTCTTTCAGTTCTCGCAAGCCGCCTCCGTGGCCGTTTCGTCCTGG
ACCTTGGTGGCCATATCCTGTGAGCGCTACTACGCGATATGCCATCCACT
GCGCTCGCGATCCTGGCAGACAATCAGTCACGCCTACAAGATCATCGGCT
TCATCTGGCTGGGCGGCATCCTCTGCATGACGCCCATAGCGGTCTTTAGT
CAATTGATACCCACCAGTCGACCGGGCTACTGCAAGTGCCGTGAGTTTTG
GCCCGACCAGGGATACGAGCTCTTCTACAACATCCTGCTGGACTTCCTGC
TGCTCGTCCTGCCGCTTCTCGTCCTCTGCGTGGCCTACATCCTCATCACG
CGTACCCTGTACGTAGGCATGGCCAAGGACAGCGGACGCATCCTGCAGCA
ATCGCTGCCTGTTTCCGCTACAACGGCCGGCGGAAGCGCACCGAATCCGG
GCACCAGCAGCAGTAGTAACTGCATCCTGGTCCTGACCGCCACCGCAGTC
TATAATGAAAATAGTAACAATAATAATGGAAATTCAGAGGGATCCGCAGG
CGGAGGATCAACCAATATGGCAACGACCACCTTGACAACGAGACCAACGG
CTCCAACTGTGATCACCACCACCACGACGACCACGGTGACGCTGGCCAAG
ACCTCCTCGCCCAGCATTCGCGTCCACGATGCGGCACTTCGCAGGTCCAA
CGAGGCCAAGACCCTGGAGAGCAAGAAGCGTGTGGTCAAGATGCTGTTCG
TCCTGGTGCTGGAGTTTTTCATCTGCTGGACTCCGCTGTACGTGATCAAC
ACGATGGTCATGCTGATCGGACCGGTGGTGTACGAGTATGTCGACTACAC
GGCCATCAGTTTCCTCCAGCTGCTGGCCTACTCATCCAGCTGCTGCAATC
CGATCACCTACTGCTTCATGAACGCCAGCTTCCGGCGCGCCTTTGTCGAC
ACCTTCAAGGGTCTGCCCTGGCGTCGTGGAGCAGGTGCCAGCGGAGGCGT
CGGTGGTGCTGCTGGTGGAGGACTCTCCGCCAGCCAGGCGGGCGCAGGCC
CGGGCGCCTATGCGAGTGCCAACACCAACATTAGTCTCAATCCCGGCCTA
GCCATGGGTATGGGCACCTGGCGGAGTCGCTCACGCCACGAGTTTCTCAA
TGCGGTGGTGACCACCAATAGTGCCGCCGCCGCCGTCAACAGTCCTCAGC TCTA The
following amino acid sequence <SEQ ID NO. 22> is the amino
acid sequence for the protein encoded by the DNA sequence of SEQ ID
NO. 21: MFNYEEGDADQAAMAAAAAYRALLDYYANAPSAAGHIVSLNVAPYNGTGN
GGTVSLAGNATSSYGDDDRDGYMDTEPSDLVTELAFSLGTSSSPSPSSTP
ASSSSTSTGMPVWLIPSYSMILLFAVLGNLLVISTLVQNRRMRTITNVFL
LNLAISDMLLGVLCMPVTLVGTLLRNFIFGEFLCKLFQFSQAASVAVSSW
TLVAISCERYYAICHPLRSRSWQTISRAYKIIGFIWLGGILCMTPIAVFS
QLIPTSRPGYCKCREFWPDQGYELFYNILLDFLLLVLPLLVLCVAYILIT
RTLYVGMAKDSGRILQQSLPVSATTAGGSAPNPGTSSSSNCILVLTATAV
YNENSNNNNGNSEGSAGGGSTNMATTTLTTRPTAPTVITTTTTTTVTLAK
TSSPSIRVHDAALRRSNEAKTLESKKRVVKMLFVLVLEFFICWTPLYVIN
TMVMLIGPVVYEYVDYTAISFLQLLAYSSSCCNPITYCFMNASFRRAFVD
TFKGLPWRRGAGASGGVGGAAGGGLSASQAGAGPGAYASANTNISLNPGL
AMGMGTWRSRSRHEFLNAVVTTNSAAAAVNSPQL The following DNA sequence for
DmGPCR10 <SEQ ID NO. 23> was identified in D. melanogaster:
ATGTACGCCTCCTTGATGGACGTTGGCCAGACGTTGGCAGCCAGGCTGGC
GGATAGCGACGGCAACGGGGCCAATGACAGCGGACTCCTGGCAACCGGAC
AAGGTCTGGAGCAGGAGCAGGAGGGTCTGGCACTGGATATGGGCCACAAT
GCCAGCGCCGACGGCGGAATAGTACCGTATGTGCCCGTGCTGGACCGCCC
GGAGACGTACATTGTCACCGTGCTGTACACGCTCATCTTCATTGTGGGAG
TTTTGGGCAACGGCACGCTGGTCATCATCTTCTTTCGCCACCGCTCCATG
CGCAACATACCCAACACATACATTCTTTCACTGGCCCTGGCTGATCTGTT
GGTTATATTGGTGTGTGTACCTGTGGCCACGATTGTCTACACGCAGGAAA
GCTGGCCCTTTGAGCGGAACATGTGCCGCATCAGCGAGTTCTTTAAGGAC
ATATCCATCGGGGTGTCCGTGTTTACACTGACCGCCCTTTCCGGCGAGCG
GTACTGCGCCATTGTAAATCCCCTACGCAAGCTTCAGACCAAGCCGCTCA
CTGTCTTTACTGCGGTGATGATCTGGATCCTGGCCATCCTACTGGGCATG
CCTTCGGTTCTTTTCTCCGACATCAAGTCCTACCCTGTGTTCACAGCCAC
CGGTAACATGACCATTGAAGTGTGCTCCCCATTTCGCGACCCGGAGTATG
CAAAGTTCATGGTGGCGGGCAAGGCACTGGTGTACTACCTGTTGCCGCTG
TCCATCATTGGGGCGCTATACATCATGATGGCCAAGCGGCTCCATATGAG
CGCCCGCAACATGCCCGGCGAACAGCAGAGCATGCAGAGCCGCACCCAGG
CTAGGGCCCGACTCCATGTGGCGCGCATGGTGGTAGCATTCGTGGTGGTG
TTCTTCATCTGCTTCTTCCCGTACCACGTGTTTGAGCTGTGGTACCACTT
CTACCCAACGGCTGAGGAGGACTTCGATGAGTTCTGGAACGTGCTGCGCA
TCCTTCCTAAACTCGTGCGTCAACCCCGTGGCCTCTACTGCGTGTCCGGG
GTGTTTCGGCAGCACTTTAATCGCTACCTCTGCTGCATCTGCGTCAAGCG
GCAGCCGCACCTGCGGCAGCACTCAACGGCCACTGGAATGATGGACAATA
CCAGTGTGATGTCCATGCGCCGCTCCACGTACGTGGGTGGAACCGCTGGC
AATCTGCGGGCCTCGCTGCACCGGAACAGCAATCACGGAGTTGGTGGAGC
TGGAGGTGGAGTAGGAGGAGGAGTAGGGTCAGGTCGTGTGGGCAGCTTTC
ATCGGCAGGACTCGATGCCCCTGCAGCACGGAAATGCCCACGGAGGTGGT
GCGGGCGGGGGATCCTCCGGACTTGGAGCCGGCGGGCGGACGGCGGCAGT
GAGCGAAAAGAGCTTTATAAATCGTTACGAAAGTGGCGTAATGCGCTACT AA The following
amino acid sequence <SEQ ID NO. 24> is the amino acid
sequence for the protein encoded by the DNA sequence of SEQ ID NO.
23: MYASLMDVGQTLAARLADSDGNGANDSGLLATGQGLEQEQEGLALDMGHN
ASADGGIVPYVPVLDRPETYIVTVLYTLIFIVGVLGNGTLVIIFFRHRSM
RNIPNTYILSLALADLLVILVCVPVATIVYTQESWPFERNMCRISEFFKD
ISIGVSVFTLTALSGERYCAIVNPLRKLQTKPLTVFTAVMIWILAILLGM
PSVLFSDIKSYPVFTATGNMTIEVCSPFRDPEYAKFMVAGKALVYYLLPL
SIIGALYIMMAKRLHMSARNMPGEQQSMQSRTQARARLHVARMVVAFVVV
FFICFFPYHVFELWYHFYPTAEEDFDEFWNVLRILPKLVRQPRGLYCVSG
VFRQHFNRYLCCICVKRQPHLRQHSTATGMMDNTSVMSMRRSTYVGGTAG
NLRASLHRNSWHGVGGAGGGVGGGVGSGRVGSFHRQDSMPLQHGNAHGGG
AGGGSSGLGAGGRTAAVSEKSFINRYESGVMRY
[0190] In accordance with the Budapest Treaty, clones of the
present invention have been deposited at the Agricultural Research
Culture Collection (NRRL) International Depository Authority, 1815
N. University Street, Peoria, Ill. 61604, U.S.A. Accession numbers
and deposit dates are provided below in Table 5.
TABLE-US-00005 TABLE 5 Date of Clone NRRL Accession No. Deposit
DmGPCR1 <SEQ ID NO: 1> NRRL B-30347 19 Oct. 2000 DmGPCR2a
<SEQ ID NO: 3> NRRL B-30348 19 Oct. 2000 DmGPCR4 <SEQ ID
NO: 7> NRRL B-30349 19 Oct. 2000 DmGPCR5a <SEQ ID NO: 9>
NRRL B-30350 19 Oct. 2000 DmGPCR6aL <SEQ ID NO: 13> NRRL
B-30351 19 Oct. 2000 DmGPCR6bL <SEQ ID NO: 15> NRRL B-30352
19 Oct. 2000 DmGPCR7 <SEQ ID NO: 17> NRRL B-30353 19 Oct.
2000 DmGPCR8 <SEQ ID NO: 19> NRRL B-30354 19 Oct. 2000
DmGPCR9 <SEQ ID NO: 21> NRRL B-30355 19 Oct. 2000
[0191] The invention is further illustrated by way of the following
examples which are intended to elucidate the invention. These
examples are not intended, nor are they to be construed, as
limiting the scope of the invention. It will be clear that the
invention may be practiced otherwise than as particularly described
herein. Numerous modifications and variations of the present
invention are possible in view of the teachings herein and,
therefore, are within the scope of the invention.
[0192] It is intended that each of the patents, applications, and
printed publications mentioned in this patent document be hereby
incorporated by reference in their entirety.
[0193] Example 1 presented below is actual whereas the remaining
Examples are prophetic.
EXAMPLES
Example 1
Identification of DmGPCRs
[0194] A Celera genomic D. melanogaster database was converted to a
database of predicted proteins and a mRNA database using a variety
of gene finding software tools to predict the mRNAs that would be
generated (the "PnuFlyPep" database). Procedures for analyzing
genomic databases using gene finding software tools are known to
those skilled in the art.
[0195] The nucleotide sequences of several C. elegans FaRP GPCRs
were used as query sequences against the mRNA database described
above. This database was searched for regions of similarity using a
variety of tools, including FASTA and Gapped BLAST (Altschul et
al., Nuc. Acids Res., 1997, 25, 3389, which is incorporated herein
by reference in its entirety).
[0196] Briefly, the BLAST algorithm, which stands for Basic Local
Alignment Search Tool is suitable for determining sequence
similarity (Altschul et al., J. Mol. Biol., 1990, 215, 403-410,
which is incorporated herein by reference in its entirety).
Software for performing BLAST analyses is publicly available
through the National Center for Biotechnology Information
(http://www.ncbi.nlrn.nih.gov/). This algorithm involves first
identifying high scoring sequence pair (HSPs) by identifying short
words of length W in the query sequence that either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighbourhood word score threshold (Altschul et al., supra).
These initial neighbourhood word hits act as seeds for initiating
searches to find HSPs containing them. The word hits are extended
in both directions along each sequence for as far as the cumulative
alignment score can be increased. Extension for the word hits in
each direction are halted when: 1) the cumulative alignment score
falls off by the quantity X from its maximum achieved value; 2) the
cumulative score goes to zero or below, due to the accumulation of
one or more negative-scoring residue alignments; or 3) the end of
either sequence is reached. The Blast algorithm parameters W, T and
X determine the sensitivity and speed of the alignment. The Blast
program uses as defaults a word length (W) of 11, the BLOSUM62
scoring matrix (see Henikoff et al., Proc. Natl. Acad. Sci. USA,
1992, 89, 10915-10919, which is incorporated herein by reference in
its entirety) alignments (B) of 50, expectation (E) of 10, M=5,
N=4, and a comparison of both strands.
[0197] The BLAST algorithm (Karlin et al., Proc. Natl. Acad. Sci.
USA, 1993, 90, 5873-5787, which is incorporated herein by reference
in its entirety) and Gapped BLAST perform a statistical analysis of
the similarity between two sequences. One measure of similarity
provided by the BLAST algorithm is the smallest sum probability
(P(N)), which provides an indication of the probability by which a
match between two nucleotide or amino acid sequences would occur by
chance. For example, a nucleic acid is considered similar to a GPCR
gene or cDNA if the smallest sum probability in comparison of the
test nucleic acid to a GPCR nucleic acid is less than about 1,
preferably less than about 0.1, more preferably less than about
0.01, and most preferably less than about 0.001.
[0198] The mRNAs corresponding to the predicted proteins were
retrieved from the database of predicted mRNAs used to prepare the
PnuFlyPep database. These are identified as the following
nucleotide sequences: SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, and 17,
each having a statistically significant overlapping homology to the
query sequence. The nucleotide sequences SEQ ID NOs: 3, 4, 5, 6,
and 9-16 (corresponding to DmGPCRs 2a, 2b, 5a, 5b, 6a, and 6b were
obtained from PCR cloning and sequencing of another identified
sequence (not shown). Each of these sequences represent a splice
variant of a DmGPCR gene.
Example 2
Cloning of DmGPCRs
[0199] cDNA Preparation
[0200] cDNA was prepared from either adult Drosophila melanogaster
poly A.sup.+ RNA (Clontech Laboratories, Palo Alto, Calif.) or
adult Drosophila melanogaster total RNA (below). To obtain total
RNA, parent stocks of Drosophila melanogaster (Biological Supply
Company, Burlington, N.C.) were anesthetized by chilling, and 5 to
6 adults were added to a culture vessel containing 10 ml H.sub.20,
10 ml Formula 4-24 Instant Drosophila Medium and 6 to 10 grains of
active dry yeast (Biological Supply Company). A polyurethane foam
plug was placed at end of each vessel, and flies were incubated at
room temperature (RT) for 4 to 6 weeks. At maturity, the vessels
were chilled, and the anesthetized flies were poured into a 50 ml
polypropylene tube held in liquid N.sub.2. The frozen flies were
stored at -70.degree. C. until they were ground with a mortar and
pestle in the presence of liquid N.sub.2. The powdered tissue along
with some liquid N.sub.2 was decanted into 50 ml polypropylene
tubes on dry ice. Following evaporation of the liquid N.sub.2, the
powdered tissue was stored at -70.degree. C.
[0201] To prepare RNA, 300 mg of powdered tissue was placed into
polypropylene tubes on dry ice, and 5 ml of 6 M guanidine
hydrochloride in 0.1 M NaOAc, pH 5.2 was added. All solutions were
either treated with DEPC, or prepared with DEPC-treated dH.sub.2O,
all glassware was baked, or virgin plastic labware was used, to
reduce problems with RNAse contamination. Tubes were vortex-mixed
then placed on ice. The powdered tissue was homogenized by
successive passage through 20, 21, and 22 gauge needles. The tubes
were centrifuged (1000.times.g for 10 min), then 2.5 to 3 ml of
supernatant was layered on top of 8 ml 5.7 M cesium chloride in 0.1
M NaOAc contained in 14.times.95 mm Ultra-Clear centrifuge tubes
(Beckman Instruments, Inc., Palo Alto, Calif.). The samples were
centrifuged at 25000 rpm for 18 h at 18.degree. C. in an L8-70
ultracentrifuge (Beckman Instruments, Inc.). The supernatant was
decanted, and the tube was inverted and allowed to drain. The RNA
pellet was suspended in 200 .mu.l of RNAse-free dH.sub.2O (Qiagen
Inc., Valencia, Calif.), then rinsed twice with 100 .mu.l
RNAse-free dH.sub.2O (total, 400 .mu.l). The RNA was precipitated
by the addition of 44 .mu.l of 3M NaOAc, pH5.2, and 1 ml cold 100%
ethanol. Following overnight storage at -70.degree. C., the tube
was centrifuged at 14000 rpm for 1 h (Eppendorf microfuge 5402),
rinsed with 75% ethanol (prepared with DEPC-treated dH.sub.2O),
then the pellet was dissolved in RNAse-free dH.sub.2O. Absorbances
at 260 or 280 nm were determined in 10 mM Tris-HCl, pH 7.5, and
used to estimate RNA concentration and purity.
[0202] First-strand cDNA was prepared according to the procedure
supplied with the Superscript II enzyme (GIBCO BRL, Rockville,
Md.). Either 500 ng (2 .mu.l) of poly A.sup.+ RNA or 3 .mu.g (4
.mu.l) of total RNA was added to microfuge tubes containing
RNAse-free dH.sub.2O and 250 ng (2.5 .mu.l) random primers. The
tubes (12 .mu.l) were incubated at 70.degree. C. for 10 min,
chilled on ice, then 4 .mu.l of 5.times. first strand buffer, 2
.mu.l of 0.1 M DTT and 1 .mu.l of 10 mM DNTP mix were added.
Following incubation at 25.degree. C. for 10 min, then at
42.degree. C. for 2 min, 1 .mu.l (200 units) of Superscript II was
added, and incubation continued at 42.degree. C. for 50 min. The
enzyme was inactivated by incubation at 70.degree. C. for 15 min.
To remove RNA complimentary to the cDNA, 2 .mu.l (2 units) of RNAse
H (Boehringer Mannheim, Indianapolis, Ind.) was added, followed by
incubation at 37.degree. C. for 20 min. The cDNA was stored at
-20.degree. C.
[0203] PCR Reactions
[0204] Either a standard 50/100 .mu.l PCR reaction or Hot Start PCR
Reaction, using Ampliwax beads, (Perkin Elmer Cetus, Norwalk Conn.)
was used to amplify the Drosophila melanogaster G protein-coupled
receptors (DmGPCRs). Distilled H.sub.2O was used to dissolve the
primers (Genosys Biotechnologies, Inc., The Woodlands, Tex.); 5'-
and 3'-primers at 10 .mu.M concentrations, internal primers at 1
.mu.M. Each PCR reaction contained 2 to 4 units of rTth XL DNA
polymerase, 1.2 to 1.5 mM Mg(OAc).sub.2, 200 .mu.M each dNTP and
200 or 400 nM each primer. For Hot Start PCR, 32 or 36 .mu.l
`lower` cocktail (dH.sub.2O, 3.3.times.XL-buffer, dNTP and
Mg(OAc).sub.2 was added to 2 or 4 .mu.l of each primer (total
volume, 40 .mu.l). An Ampliwax bead (Perkin Elmer Cetus), was
added, tubes incubated at 75.degree. C. for 5 min, cooled at RT,
then 60 .mu.l `upper` cocktail (dH.sub.2O, 3.3.times.XL-buffer,
rTth and template) was added. PCR amplifications were performed in
a Perkin Elmer Series 9600 thermal cycler. The typical program for
the thermal cycler included: 1 min at 94.degree. C., followed by 30
cycles of amplification (0.5 min at 94.degree. C., 0.5 min at
60.degree. C., 2 min at 72.degree. C.), followed by 6 min at
60.degree. C. In order to create 3' A-overhangs on the PCR product
(`tailing`), 1 .mu.l Taq polymerase (Invitrogen, Carlsbad, Calif.)
was added at the end of the PCR amplification, and tubes incubated
at 72.degree. C. for 10 min. The reaction mixtures were analyzed on
1% agarose gel prepared in TAE buffer (5). PCR products were
typically purified using QIAquick spun columns (QIAGEN).
[0205] Ligation and Transformation
[0206] Ligation of all PCR products into PCR 3.1 vector
(Invitrogen) and transformation of the ligated products into One
Shot.TM. TOP10F` competent cells (Invitrogen) were done according
to the manufacturer's directions. Transformants to be screened for
inserts were propagated in LB broth containing 50 .mu.g
ampicillin/ml. Colonies with inserts were identified either by a
boiling-lysis plasmid mini-prep procedure (5) or by a `colony PCR`
procedure that directly amplified the plasmid DNA from the
transformed bacteria (6).
[0207] DNA Sequencing
[0208] DNA for sequencing was prepared using Qiagen anion-exchange
plasmid kits (QIAGEN-tip 20) to isolate the DNA from 5 ml LB
cultures grown at 37.degree. C. overnight as per the manufacturer's
directions. Four primers (T7, M13 reverse, `sense` and `antisense`,
see Table 1) were typically used for sequencing each DNA.
Dye-terminator sequencing chemistry was used, either the BigDye
Terminator reagents (Applied Biosystems, Foster City, Calif.) or
DYEnamic.TM. ET terminator kit (Amersham Pharmacia Biotech, Inc.,
Piscataway, N.J.). Manufacturer's recommendations were followed for
preparation of the sequencing reactions. Primers and unincorporated
nucleotides were removed using Centri-Sep spun columns (Princeton
Separations, Adelphia, N.J.). Sequencing reactions were analyzed on
a Applied Biosystems 377 automated DNA sequencer. DNA sequences
were assembled and analyzed using Sequencher (Gene Codes, Ann
Arbor, Mich.), the GCG group of sequence analysis programs
(Wisconsin Package Version 10.1, Genetics Computer Group (GCG),
Madison, Wis.), and functions available through the Vector NTI 5.5
suite of programs (Informax, Bethesda, Md.).
[0209] The results of cloning and sequencing of the DmGPCRs of the
present invention are as follows:
[0210] DmGPCR1
[0211] PCR primers designed to the cDNA corresponding to
PnuFlyPep34651 were used to successfully amplify a PCR product from
a cDNA preparation prepared from Drosophila polyA.sup.+ mRNA. The
resulting product was cloned and sequenced. The experimentally
obtained sequence was identical to the predicted sequence. An
intact clone was obtained and designated `DmGPCR1.`
[0212] DmGPCR2
[0213] Initial attempts to amplify a PCR product using primers
designed to the cDNA corresponding to PnuFlyPep67585 were
unsuccessful. Alignment of the predicted sequence to the existing
C. elegans receptors, and to other neuropeptide receptors, showed
that the 5' end of the predicted sequence was unusually long, and
suggested that there may have been an error in gene prediction on
that side. Using the genomic sequence as a guide, a variety of
alternative 5' PCR primers were designed and tested. One of these
primer combinations, using cDNA prepared from total RNA, was
successful in giving a product of the right size. Sequencing of
clones derived from the PCR reaction showed that the amplified
product contained the anticipated 5' and 3' ends, and was identical
to the predicted sequence with the exception that the predicted
sequence was missing a small stretch of 6 amino acids. Comparison
of the clones also revealed that two splicing isoforms were
present, one similar to the predicted sequence (designated
`DmGPCR2a`), and the other missing a stretch of 23 amino acids
located just past TM VII into the intracellular C-terminus of the
molecule (designated DmGPCR2b`).
[0214] DmGPCR3
[0215] A gene corresponding to the DmGPCR3 predicted protein had
already been reported in the literature. This gene (GenBank
accession M77168) was described as NKD, "a developmentally
regulated tachykinin receptor". Monnier D, et al., Journal of
Biological Chemistry 1992; 267(2):1298-302. Comparison of the
M77168 and PnuFlyPep68505 sequences showed that the predicted
sequences were significantly different from the cDNA. The cDNA had
a longer 5' end, was missing an exon encoding 51 amino acids, and
was significantly shorter on the 3' end. PCR primers were designed
to the published sequence, and a PCR product was obtained using
cDNA prepared from total RNA. This product was identical in
structure to the reported NKD sequence.
[0216] DmGPCR4
[0217] The cDNA corresponding to PnuFlyPep 67393 was used to design
PCR primers for the amplification of DmGPCR4. Using a cDNA library
prepared from total Drosophila mRNA, a PCR product was obtained and
cloned. Comparison of the clones with the sequence predicted by
PnuFlyPep revealed that the sequences were identical with the
exception that one exon predicted by HMMGene was not present in any
of the cloned PCR products. DmGPCR4 has been recently cloned by
Lenz et al., Biochem. Biophys. Res. Comm., 273:571-577 (2000), and
was classified as a second putative allatostatin receptor.
[0218] DmGPCR5
[0219] DmGPCR5 (PnuFlyPep67522) had already been cloned and
described in the literature as a `Drosophila receptor for
tachykinin-related peptides` (M77168). Li X J, et al., EMBO Journal
1991; 10(11):3221-9. At first appearance, the predicted cDNA
corresponding to the PnuFlyPep protein was identical to the
published sequence. PCR primers were used to successfully amplify a
PCR product of the appropriate size from a cDNA mixture prepared
from Drosophila melanogaster poly A.sup.+ mRNA. Sequencing of the
cloned PCR products revealed that, although the overall splicing
pattern was the same, two sequencing errors were present in the
published sequence. These errors resulted in a frameshift mutation
followed by a compensatory frameshift mutation, resulting in a
difference of 13 amino acids between the experimentally determined
and reported sequences, starting at amino acid position 46. This
cloned gene was designated `DmGPCR5a`
[0220] Additionally, a splicing isoform was found for DmGPCR5. This
variant encoded an extra three amino acids in the N-terminal
extracellular domain. This variant was designated `DmGPCR5b`.
[0221] DmGPCR6
[0222] The GPCR corresponding to PnuFlyPep15731 had already been
described in the literature as a `Neuropeptide Y` receptor (M81490.
Li X J, et al., Journal of Biological Chemistry 1992; 267(1):9-12.
The PnuFlyPep-predicted sequence was different from M81490 at both
ends of the molecule. PnuFlyPep15731 contained an extra 15 amino
acids on the N-terminus as compared to M81490. The 3' end of
PnuFlyPep 15731 was also different from M81490, being truncated and
not containing conserved TM VI and TM VII residues.
[0223] The initial PCR primers were designed using the sequence of
M81490. Using these primers, and a template derived from total
mRNA, a PCR product was obtained. Examination of the cloned PCR
product revealed that it used an identical processing pattern to
M81490. This clone was designated `DmGPCR6a`.
[0224] During the cloning of DmGPCR6a an additional splicing
isoform was discovered. This isoform was generated by use of an
alternative splice acceptor site to generate an alternative 3' end
of the molecule using much of the same sequence as the `6a` form
but in a different reading frame. Additionally, the open reading
frame for this clone extended past the original 3' PCR primer.
Examination of the genomic sequence on the 3' end revealed a number
of likely candidate exons. PCR primers corresponding to a number of
these possible exons were tested until one was found that would
amplify a PCR product. This product was designated `6b`.
Examination of the genomic sequence also predicted that the
initiator ATG predicted by PnuFlyPep15731 was in-frame with the
M81490 initiation codon containing an extra 15 amino acids, and
that it was likely that the PnuFlyPep15731 start codon was the
authentic start codon. A new 5' PCR primer was designed that
incorporated the PnuFlyPep 15731 start codon and was used in
conjunction with the two 3' PCR primers to amplify and clone
`DmGPCR6aL` and `DmGPCR6bL` (`long`).
[0225] DmGPCR7
[0226] Initial attempts to amplify the DmGPCR7 gene product were
unsuccessful. Alignment of the predicted sequence (PnuFlyPep67863)
with other GPCRs suggested that the error was probably in the
prediction of the 3' end of the molecule. The predicted sequence
had a 3' end that was far longer than that of most other GPCRs.
Examination of the genomic sequence suggested that the likely error
was in the prediction of a splicing event that removed an in-frame
stop codon that would have resulted in a molecule of the
appropriate size. A 3' PCR primer was designed within that intron.
Additionally, a new 5' PCR primer was designed to utilize an
in-frame ATG just upstream of the predicted start codon. PCR
amplification of cDNA derived from total mRNA resulted in a product
of the expected size.
[0227] DmGPCR8
[0228] DmGPCR8 was successfully amplified using PCR primers
designed to the PnuFlyPep predicted sequence. cDNA derived from
poly A.sup.+ RNA was used as template for the PCR reaction. All six
of the sequenced clones were identical in structure to the
PnuFlyPep-predicted sequence. A polymorphism was noted at position
#68 (DNA sequence), with half of the clones having a "C" at this
position, and half an "A." This change does result in an amino acid
change, Asp or Glu, respectively. The Celera sequence noted an "A,"
so an "A" clone (Glu) was arbitrarily chosen for further study. No
"A" clones were obtained in the correct orientation, thus a
subcloning step, utilizing Pme I to remove the insert from the
original pCR3.1 clone and a Pme 1-digested pCR3.1 vector, was used
to reverse the orientation.
[0229] DmGPCR9
[0230] DmGPCR9 was cloned using PCR primers designed to the
PnuFlyPep predicted sequence and a cDNA template prep prepared from
poly A.sup.+ RNA. The genomic structure was correctly predicted in
PnuFlyPep.
[0231] DmGPCR10
[0232] Initial attempts to generate a PCR product with primers
designed for DmGPCR10 (PnuFlyPep70325) were unsuccessful.
Examination of the predicted cDNA showed that the predicted
sequence was unusual in that it did not contain the highly
conserved "WXP" motif in TM VI, nor the "NPXXF" motif in TM VII,
though several other conserved residues were present. Examination
of genomic sequences up to 80 kb downstream of the last exon did
not reveal any other potential exons. Attempts to obtain an intact
clone for DmGPCR10 were not undertaken.
[0233] DmGPCR11 (Allatostatin-Like Peptide Receptor)
[0234] PCR primers for the `allatostatin-like peptide receptor were
designed using the published sequence. Birgul N, et al., EMBO
Journal 1999; 18(21):5892-900. A PCR product was obtained using
cDNA derived from a total mRNA prep, and was cloned and sequenced.
The final cDNA coded for a protein identical to that described in
publication.
Example 3
Northern Blot Analysis
[0235] Northern blots may be performed to examine the expression of
mRNA. The sense orientation oligonucleotide and the
antisense-orientation oligonucleotide, described above, are used as
primers to amplify a portion of the GPCR cDNA sequence of a
nucleotide sequence selected from the group consisting of SEQ ID
NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21.
[0236] Multiple human tissue northern blot from Clontech (Human II
#7767-1) are hybridized with the probe. Pre-hybridization is
carried out at 42 C for 4 hours in 5.times.SSC, 1.times.Denhardt's
reagent, 0.1% SDS, 50% formamide, 250 mg/ml salmon sperm DNA.
Hybridization is performed overnight at 42.degree. C. in the same
mixture with the addition of about 1.5.times.106 cpm/ml of labeled
probe.
[0237] The probe is labeled with .alpha.-32P-dCTP by Rediprime DNA
labelling system (Amersham Pharmacia), purified on Nick Column
(Amersham Pharmacia) and added to the hybridization solution. The
filters are washed several times at 42 C in 0.2.times.SSC, 0.1%
SDS. Filters are exposed to Kodak XAR film (Eastman Kodak Company,
Rochester, N.Y., USA) with intensifying screen at -80 C.
Example 4
Recombinant Expression of DmGPCR in Eukaryotic Cells
[0238] A. Expression of DmGPCR in Mammalian Cells
[0239] To produce DmGPCR protein, a DmGPCR-encoding polynucleotide
is expressed in a suitable host cell using a suitable expression
vector and standard genetic engineering techniques. For example,
the DmGPCR-encoding sequence described in Example 1 is subcloned
into the commercial expression vector pzeoSV2 (Invitrogen, San
Diego, Calif.) and transfected into Chinese Hamster Ovary (CHO)
cells using the transfection reagent FuGENE 6 (Boehringer-Mannheim)
and the transfection protocol provided in the product insert. Other
eukaryotic cell lines, including human embryonic kidney (HEK 293),
CHO cells, and COS cells, are suitable as well. Cells stably
expressing DmGPCR are selected by growth in the presence of 100
.mu.g/ml zeocin (Stratagene, LaJolla, Calif.). Optionally, DmGPCR
may be purified from the cells using standard chromatographic
techniques. To facilitate purification, antisera is raised against
one or more synthetic peptide sequences that correspond to portions
of the DmGPCR amino acid sequence, and the antisera is used to
affinity purify DmGPCR. The DmGPCR also may be expressed in-frame
with a tag sequence (e.g., polyhistidine, hemagluttinin, FLAG) to
facilitate purification. Moreover, it will be appreciated that many
of the uses for DmGPCR polypeptides, such as assays described
below, do not require purification of DmGPCR from the host
cell.
[0240] B. Expression of DmGPCR in 293 Cells
[0241] For expression of DmGPCR in mammalian CHO cells, a plasmid
bearing the relevant DmGPCR coding sequence is prepared, using
vector pSecTag2A (Invitrogen). Vector pSecTag2A contains the murine
IgK chain leader sequence for secretion, the c-myc epitope for
detection of the recombinant protein with the anti-myc antibody, a
C-terminal polyhistidine for purification with nickel chelate
chromatography, and a Zeocin resistant gene for selection of stable
transfectants. The forward primer for amplification of this GPCR
cDNA is determined by routine procedures and preferably contains a
5' extension of nucleotides to introduce the HindIII cloning site
and nucleotides matching the GPCR sequence. The reverse primer is
also determined by routine procedures and preferably contains a 5'
extension of nucleotides to introduce an XhoI restriction site for
cloning and nucleotides corresponding to the reverse complement of
the DmGPCR sequence. The PCR conditions are 55 C as the annealing
temperature. The PCR product is gel purified and cloned into the
HindIII-XhoI sites of the vector.
[0242] The DNA is purified using Qiagen chromatography columns and
transfected into 293 cells using DOTAP transfection media
(Boehringer Mannheim, Indianapolis, Ind.). Transiently transfected
cells are tested for expression after 24 hours of transfection,
using western blots probed with antiHis and anti-DmGPCR peptide
antibodies. Permanently transfected cells are selected with Zeocin
and propagated. Production of the recombinant protein is detected
from both cells and media by western blots probed with anti-His,
anti-Myc or anti-GPCR peptide antibodies.
[0243] C. Expression of DmGPCR in COS cells
[0244] For expression of the DmGPCR in COS7 cells, a polynucleotide
molecule having a nucleotide sequence selected from the group
consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or
23 can be cloned into vector p3-CI. This vector is a pUC18-derived
plasmid that contains the HCMV (human cytomegalovirus)
promoter-intron located upstream from the bGH (bovine growth
hormone) polyadenylation sequence and a multiple cloning site. In
addition, the plasmid contains the dhrf (dihydrofolate reductase)
gene which provides selection in the presence of the drug
methotrexane (MTX) for selection of stable transformants.
[0245] The forward primer is determined by routine procedures and
preferably contains a 5' extension which introduces an XbaI
restriction site for cloning, followed by nucleotides which
correspond to a nucleotide sequence selected from the group
consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or
23. The reverse primer is also determined by routine procedures and
preferably contains 5'-extension of nucleotides which introduces a
SalI cloning site followed by nucleotides which correspond to the
reverse complement of a nucleotide sequence selected from the group
consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or
23.
[0246] The PCR consists of an initial denaturation step of 5 min at
95 C, 30 cycles of 30 sec denaturation at 95 C, 30 sec annealing at
58 C and 30 sec extension at 72 C, followed by 5 min extension at
72 C. The PCR product is gel purified and ligated into the XbaI and
SalI sites of vector p3-CI. This construct is transformed into E.
coli cells for amplification and DNA purification. The DNA is
purified with Qiagen chromatography columns and transfected into
COS 7 cells using Lipofectamine reagent from BRL, following the
manufacturer's protocols. Forty eight and 72 hours after
transfection, the media and the cells are tested for recombinant
protein expression.
[0247] DmGPCR expressed from a COS cell culture can be purified by
concentrating the cell-growth media to about 10 mg of protein/ml,
and purifying the protein by, for example, chromatography. Purified
DmGPCR is concentrated to 0.5 mg/ml in an Amicon concentrator
fitted with a YM-10 membrane and stored at -80 C.
[0248] D. Expression of DmGPCR in Insect Cells
[0249] For expression of DmGPCR in a baculovirus system, a
polynucleotide molecule having a nucleotide sequence selected from
the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, or 23 can be amplified by PCR. The forward primer is
determined by routine procedures and preferably contains a 5'
extension which adds the NdeI cloning site, followed by followed by
nucleotides which correspond to a nucleotide sequence selected from
the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, or 23. The reverse primer is also determined by routine
procedures and preferably contains a 5' extension which introduces
the KpnI cloning site, followed by followed by nucleotides which
correspond to the reverse complement of a nucleotide sequence
selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9,
11, 13, 15, 17, 19, 21, or 23.
[0250] The PCR product is gel purified, digested with NdeI and
KpnI, and cloned into the corresponding sites of vector pAcHTL-A
(Pharmingen, San Diego, Calif.). The pAcHTL expression vector
contains the strong polyhedrin promoter of the Autographa
californica nuclear polyhedrosis virus (AcMNPV), and a 6.times.His
tag upstream from the multiple cloning site. A protein kinase site
for phosphorylation and a thrombin site for excision of the
recombinant protein precede the multiple cloning site is also
present. Of course, many other baculovirus vectors could be used in
place of pAcHTL-A, such as pAc373, pVL941 and pAcIM1. Other
suitable vectors for the expression of GPCR polypeptides can be
used, provided that the vector construct includes appropriately
located signals for transcription, translation, and trafficking,
such as an in-frame AUG and a signal peptide, as required. Such
vectors are described in Luckow et al., Virology 170:31-39, among
others.
[0251] The virus is grown and isolated using standard baculovirus
expression methods, such as those described in Summers et al. (A
Manual of Methods for Baculovirus Vectors and Insect Cell Culture
Procedures, Texas Agricultural Experimental Station Bulletin No.
1555 (1987)).
[0252] In a preferred embodiment, pAcHLT-A containing DmGPCR gene
is introduced into baculovirus using the "BaculoGold" transfection
kit (Pharmingen, San Diego, Calif.) using methods established by
the manufacturer. Individual virus isolates are analyzed for
protein production by radiolabeling infected cells with
.sup.35S-methionine at 24 hours post infection. Infected cells are
harvested at 48 hours post infection, and the labeled proteins are
visualized by SDS-PAGE. Viruses exhibiting high expression levels
can be isolated and used for scaled up expression.
[0253] For expression of a DmGPCR polypeptide in a Sf9 cells, a
polynucleotide molecule having a nucleotide sequence selected from
the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, or 23, can be amplified by PCR using the primers and
methods described above for baculovirus expression. The DmGPCR cDNA
is cloned into vector pAcHLT-A (Pharmingen) for expression in Sf9
insect. The insert is cloned into the NdeI and KpnI sites, after
elimination of an internal NdeI site (using the same primers
described above for expression in baculovirus). DNA is purified
with Qiagen chromatography columns and expressed in Sf9 cells.
Preliminary Western blot experiments from non purified plaques are
tested for the presence of the recombinant protein of the expected
size which reacted with the GPCR-specific antibody. These results
are confirmed after further purification and expression
optimization in HiG5 cells.
Example 5
Interaction Trap/Two-Hybrid System
[0254] In order to assay for DmGPCR-interacting proteins, the
interaction trap/two-hybrid library screening method can be used.
This assay was first described in Fields, et al., Nature, 1989,
340, 245, which is incorporated herein by reference in its
entirety. A protocol is published in Current Protocols in Molecular
Biology 1999, John Wiley & Sons, NY and Ausubel, F. M. et al.
1992, Short protocols in molecular biology, fourth edition, Greene
and Wiley-interscience, NY, which is incorporated herein by
reference in its entirety. Kits are available from Clontech, Palo
Alto, Calif. (Matchmaker Two-Hybrid System 3).
[0255] A fusion of the nucleotide sequences encoding all or partial
DmGPCR and the yeast transcription factor GAL4 DNA-binding domain
(DNA-BD) is constructed in an appropriate plasmid (ie. pGBKT7)
using standard subcloning techniques. Similarly, a GAL4 active
domain (AD) fusion library is constructed in a second plasmid (ie.
pGADT7) from cDNA of potential GPCR-binding proteins (for protocols
on forming cDNA libraries, see Sambrook et al. 1989, Molecular
cloning: a laboratory manual, second edition, Cold Spring Harbor
Press, Cold Spring Harbor, N.Y.), which is incorporated herein by
reference in its entirety. The DNA-BD/GPCR fusion construct is
verified by sequencing, and tested for autonomous reporter gene
activation and cell toxicity, both of which would prevent a
successful two-hybrid analysis. Similar controls are performed with
the AD/library fusion construct to ensure expression in host cells
and lack of transcriptional activity. Yeast cells are transformed
(ca. 105 transformants/mg DNA) with both the GPCR and library
fusion plasmids according to standard procedure (Ausubel, et al.,
1992, Short protocols in molecular biology, fourth edition, Greene
and Wiley-interscience, NY, which is incorporated herein by
reference in its entirety). In vivo binding of DNA-BD/GPCR with
AD/library proteins results in transcription of specific yeast
plasmid reporter genes (i.e., lacZ, HIS3, ADE2, LEU2). Yeast cells
are plated on nutrient-deficient media to screen for expression of
reporter genes. Colonies are dually assayed for
.beta.-galactosidase activity upon growth in Xgal
(5-bromo-4-chloro-3-indolyl-.beta.-D-galactoside) supplemented
media (filter assay for .beta.-galactosidase activity is described
in Breeden, et al., Cold Spring Harb. Symp. Quant. Biol., 1985, 50,
643, which is incorporated herein by reference in its entirety).
Positive AD-library plasmids are rescued from transformants and
reintroduced into the original yeast strain as well as other
strains containing unrelated DNA-BD fusion proteins to confirm
specific DmGPCR/library protein interactions. Insert DNA is
sequenced to verify the presence of an open reading frame fused to
GAL4 AD and to determine the identity of the DmGPCR-binding
protein.
Example 6
Mobility Shift DNA-Binding Assay Using Gel Electrophoresis
[0256] A gel electrophoresis mobility shift assay can rapidly
detect specific protein-DNA interactions. Protocols are widely
available in such manuals as Sambrook et al. 1989, Molecular
cloning: a laboratory manual, second edition, Cold Spring Harbor
Press, Cold Spring Harbor, N.Y. and Ausubel, F. M. et al., 1992,
Short Protocols in Molecular Biology, fourth edition, Greene and
Wiley-interscience, NY, each of which is incorporated herein by
reference in its entirety.
[0257] Probe DNA (<300 bp) is obtained from synthetic
oligonucleotides, restriction endonuclease fragments, or PCR
fragments and end-labeled with 32P. An aliquot of purified DmGPCR
(ca. 15 .mu.g) or crude DmGPCR extract (ca. 15 ng) is incubated at
constant temperature (in the range 22-37 C) for at least 30 minutes
in 10-15 .mu.l of buffer (ie. TAE or TBE, pH 8.0-8.5) containing
radiolabeled probe DNA, nonspecific carrier DNA (ca. 1 .mu.g), BSA
(300 .mu.g/ml), and 10% (v/v) glycerol. The reaction mixture is
then loaded onto a polyacrylamide gel and run at 30-35 mA until
good separation of free probe DNA from protein-DNA complexes
occurs. The gel is then dried and bands corresponding to free DNA
and protein-DNA complexes are detected by autoradiography.
Example 7
Antibodies to DmGPCR
[0258] Standard techniques are employed to generate polyclonal or
monoclonal antibodies to the DmGPCR receptor, and to generate
useful antigen-binding fragments thereof or variants thereof,
including "humanized" variants. Such protocols can be found, for
example, in Sambrook et al. (1989) and Harlow et al. (Eds.),
Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold
Spring Harbor, N.Y. (1988). In one embodiment, recombinant DmGPCR
polypeptides (or cells or cell membranes containing such
polypeptides) are used as antigen to generate the antibodies. In
another embodiment, one or more peptides having amino acid
sequences corresponding to an immunogenic portion of DmGPCR (e.g.
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more
amino acids) are used as antigen. Peptides corresponding to
extracellular portions of DmGPCR, especially hydrophilic
extracellular portions, are preferred. The antigen may be mixed
with an adjuvant or linked to a hapten to increase antibody
production.
[0259] A. Polyclonal or Monoclonal Antibodies
[0260] As one exemplary protocol, recombinant DmGPCR or a synthetic
fragment thereof is used to immunize a mouse for generation of
monoclonal antibodies (or larger mammal, such as a rabbit, for
polyclonal antibodies). To increase antigenicity, peptides are
conjugated to Keyhole Lympet Hemocyanin (Pierce), according to the
manufacturer's recommendations. For an initial injection, the
antigen is emulsified with Freund's Complete Adjuvant and injected
subcutaneously. At intervals of two to three weeks, additional
aliquots of DmGPCR antigen are emulsified with Freund's Incomplete
Adjuvant and injected subcutaneously. Prior to the final booster
injection, a serum sample is taken from the immunized mice and
assayed by western blot to confirm the presence of antibodies that
immunoreact with DmGPCR. Serum from the immunized animals may be
used as a polyclonal antisera or used to isolate polyclonal
antibodies that recognize DmGPCR. Alternatively, the mice are
sacrificed and their spleen removed for generation of monoclonal
antibodies.
[0261] To generate monoclonal antibodies, the spleens are placed in
10 ml serum-free RPMI 1640, and single cell suspensions are formed
by grinding the spleens in serum-free RPMI 1640, supplemented with
2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin,
and 100 .mu.g/ml streptomycin (RPMI) (Gibco, Canada). The cell
suspensions are filtered and washed by centrifugation and
resuspended in serum-free RPMI. Thymocytes taken from three naive
Balb/c mice are prepared in a similar manner and used as a Feeder
Layer. NS-1 myeloma cells, kept in log phase in RPMI with 10% fetal
bovine serum (FBS) (Hyclone Laboratories, Inc., Logan, Utah) for
three days prior to fusion, are centrifuged and washed as well.
[0262] To produce hybridoma fusions, spleen cells from the
immunized mice are combined with NS-1 cells and centrifuged, and
the supernatant is aspirated. The cell pellet is dislodged by
tapping the tube, and 2 ml of 37.degree. C. PEG 1500 (50% in 75 mM
HEPES, pH 8.0) (Boehringer-Mannheim) is stirred into the pellet,
followed by the addition of serum-free RPMI. Thereafter, the cells
are centrifuged, resuspended in RPMI containing 15% FBS, 100 .mu.M
sodium hypoxanthine, 0.4 .mu.M aminopterin, 16 .mu.M thymidine
(HAT) (Gibco), 25 units/ml IL-6 (Boehringer-Mannheim) and
1.5.times.10.sup.6 thymocytes/ml, and plated into 10 Corning
flat-bottom 96-well tissue culture plates (Corning, Corning
N.Y.).
[0263] On days 2, 4, and 6 after the fusion, 100 .mu.l of medium is
removed from the wells of the fusion plates and replaced with fresh
medium. On day 8, the fusions are screened by ELISA, testing for
the presence of mouse IgG that binds to DmGPCR. Selected fusion
wells are further cloned by dilution until monoclonal cultures
producing anti-DmGPCR antibodies are obtained.
[0264] B. Humanization of Anti-DmGPCR Monoclonal Antibodies
[0265] The expression pattern of DmGPCR as reported herein and the
proven track record of GPCRs as targets for therapeutic
intervention suggest therapeutic indications for DmGPCR inhibitors
(antagonists). DmGPCR-neutralizing antibodies comprise one class of
therapeutics useful as DmGPCR antagonists. Following are protocols
to improve the utility of anti-DmGPCR monoclonal antibodies as
therapeutics in humans by "humanizing" the monoclonal antibodies to
improve their serum half-life and render them less immunogenic in
human hosts (i.e., to prevent human antibody response to non-human
anti-DmGPCR antibodies).
[0266] The principles of humanization have been described in the
literature and are facilitated by the modular arrangement of
antibody proteins. To minimize the possibility of binding
complement, a humanized antibody of the IgG4 isotype is
preferred.
[0267] For example, a level of humanization is achieved by
generating chimeric antibodies comprising the variable domains of
non-human antibody proteins of interest with the constant domains
of human antibody molecules. (See, e.g., Morrison et al., Adv.
Immunol., 44:65-92 (1989)). The variable domains of
DmGPCR-neutralizing anti-DmGPCR antibodies are cloned from the
genomic DNA of a B-cell hybridoma or from cDNA generated from mRNA
isolated from the hybridoma of interest. The V region gene
fragments are linked to exons encoding human antibody constant
domains, and the resultant construct is expressed in suitable
mammalian host cells (e.g., myeloma or CHO cells).
[0268] To achieve an even greater level of humanization, only those
portions of the variable region gene fragments that encode
antigen-binding complementarity determining regions ("CDR") of the
non-human monoclonal antibody genes are cloned into human antibody
sequences. (See, e.g., Jones et al., Nature 321:522-525 (1986);
Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,
Science 239:1534-36 (1988); and Tempest et al., Bio/Technology
9:266-71 (1991)). If necessary, the .beta.-sheet framework of the
human antibody surrounding the CDR3 regions also is modified to
more closely mirror the three dimensional structure of the
antigen-binding domain of the original monoclonal antibody. (See
Kettleborough et al., Protein Engin., 4:773-783 (1991); and Foote
et al., J. Mol. Biol., 224:487-499 (1992)).
[0269] In an alternative approach, the surface of a non-human
monoclonal antibody of interest is humanized by altering selected
surface residues of the non-human antibody, e.g., by site-directed
mutagenesis, while retaining all of the interior and contacting
residues of the non-human antibody. See Padlan, Molecular Immunol.,
28(4/5):489-98 (1991).
[0270] The foregoing approaches are employed using
DmGPCR-neutralizing anti-DmGPCR monoclonal antibodies and the
hybridomas that produce them to generate humanized
DmGPCR-neutralizing antibodies useful as therapeutics to treat or
palliate conditions wherein DmGPCR expression or ligand-mediated
DmGPCR signaling is detrimental.
Example 8
Assays to Identify Modulators of DmGPCR Activity
[0271] Set forth below are several nonlimiting assays for
identifying modulators (agonists and antagonists) of DmGPCR
activity. Among the modulators that can be identified by these
assays are natural ligand compounds of the receptor; synthetic
analogs and derivatives of natural ligands; antibodies, antibody
fragments, and/or antibody-like compounds derived from natural
antibodies or from antibody-like combinatorial libraries; and/or
synthetic compounds identified by high-throughput screening of
libraries; and the like. All modulators that bind DmGPCR are useful
for identifying DmGPCR in tissue samples (e.g., for diagnostic
purposes, pathological purposes, and the like). Agonist and
antagonist modulators are useful for up-regulating and
down-regulating DmGPCR activity, respectively, to treat disease
states characterized by abnormal levels of DmGPCR activity. The
assays may be performed using single putative modulators, and/or
may be performed using a known agonist in combination with
candidate antagonists (or visa versa).
[0272] A. cAMP Assays
[0273] In one type of assay, levels of cyclic adenosine
monophosphate (cAMP) are measured in DmGPCR-transfected cells that
have been exposed to candidate modulator compounds. Protocols for
cAMP assays have been described in the literature. (See, e.g.,
Sutherland et al., Circulation 37: 279 (1968); Frandsen et al.,
Life Sciences 18: 529-541 (1976); Dooley et al., Journal of
Pharmacology and Experimental Therapeutics 283 (2): 735-41 (1997);
and George et al., Journal of Biomolecular Screening 2 (4): 235-40
(1997)). An exemplary protocol for such an assay, using an Adenylyl
Cyclase Activation FlashPlate.RTM. Assay from NEN.TM. Life Science
Products, is set forth below.
[0274] Briefly, the DmGPCR coding sequence (e.g., a cDNA or
intronless genomic DNA) is subcloned into a commercial expression
vector, such as pzeoSV2 (Invitrogen), and transiently transfected
into Chinese Hamster Ovary (CHO) cells using known methods, such as
the transfection protocol provided by Boehringer-Mannheim when
supplying the FuGENE 6 transfection reagent. Transfected CHO cells
are seeded into 96-well microplates from the FlashPlate.RTM. assay
kit, which are coated with solid scintillant to which antisera to
cAMP has been bound. For a control, some wells are seeded with wild
type (untransfected) CHO cells. Other wells in the plate receive
various amounts of a cAMP standard solution for use in creating a
standard curve.
[0275] One or more test compounds (i.e., candidate modulators) are
added to the cells in each well, with water and/or compound-free
medium/diluent serving as a control or controls. After treatment,
cAMP is allowed to accumulate in the cells for exactly 15 minutes
at room temperature. The assay is terminated by the addition of
lysis buffer containing [.sup.125I]-labeled cAMP, and the plate is
counted using a Packard Topcount.TM. 96-well microplate
scintillation counter. Unlabeled cAMP from the lysed cells (or from
standards) and fixed amounts of [.sup.125I]-cAMP compete for
antibody bound to the plate. A standard curve is constructed, and
cAMP values for the unknowns are obtained by interpolation. Changes
in intracellular cAMP levels of cells in response to exposure to a
test compound are indicative of DmGPCR modulating activity.
Modulators that act as agonists of receptors which couple to the
G.sub.s subtype of G proteins will stimulate production of cAMP,
leading to a measurable 3-10 fold increase in cAMP levels. Agonists
of receptors which couple to the G.sub.i/o subtype of G proteins
will inhibit forskolin-stimulated cAMP production, leading to a
measurable decrease in cAMP levels of 50-100%. Modulators that act
as inverse agonists will reverse these effects at receptors that
are either constitutively active or activated by known
agonists.
[0276] B. Aequorin Assays
[0277] In another assay, cells (e.g., CHO cells) are transiently
co-transfected with both a DmGPCR expression construct and a
construct that encodes the photoprotein apoaquorin. In the presence
of the cofactor coelenterazine, apoaquorin will emit a measurable
luminescence that is proportional to the amount of intracellular
(cytoplasmic) free calcium. (See generally, Cobbold, et al.
"Aequorin measurements of cytoplasmic free calcium," In: McCormack
J. G. and Cobbold P. H., eds., Cellular Calcium: A Practical
Approach. Oxford: IRL Press (1991); Stables et al., Analytical
Biochemistry 252: 115-26 (1997); and Haugland, Handbook of
Fluorescent Probes and Research Chemicals. Sixth edition. Eugene
Oreg.: Molecular Probes (1996).)
[0278] In one exemplary assay, DmGPCR is subcloned into the
commercial expression vector pzeoSV2 (Invitrogen) and transiently
co-transfected along with a construct that encodes the photoprotein
apoaquorin (Molecular Probes, Eugene, Oreg.) into CHO cells using
the transfection reagent FuGENE 6 (Boehringer-Mannheim) and the
transfection protocol provided in the product insert.
[0279] The cells are cultured for 24 hours at 37.degree. C. in MEM
(Gibco/BRL, Gaithersburg, Md.) supplemented with 10% fetal bovine
serum, 2 mM glutamine, 10 U/ml penicillin and 10 .mu.g/ml
streptomycin, at which time the medium is changed to serum-free MEM
containing 5 .mu.M coelenterazine (Molecular Probes, Eugene,
Oreg.). Culturing is then continued for two additional hours at
37.degree. C. Subsequently, cells are detached from the plate using
VERSEN (Gibco/BRL), washed, and resuspended at 200,000 cells/ml in
serum-free MEM.
[0280] Dilutions of candidate DmGPCR modulator compounds are
prepared in serum-free MEM and dispensed into wells of an opaque
96-well assay plate at 50 .mu.l/well. Plates are then loaded onto
an MLX microtiter plate luminometer (Dynex Technologies, Inc.,
Chantilly, Va.). The instrument is programmed to dispense 50 .mu.l
cell suspensions into each well, one well at a time, and
immediately read luminescence for 15 seconds. Dose-response curves
for the candidate modulators are constructed using the area under
the curve for each light signal peak. Data are analyzed with
SlideWrite, using the equation for a one-site ligand, and EC.sub.50
values are obtained. Changes in luminescence caused by the
compounds are considered indicative of modulatory activity.
Modulators that act as agonists at receptors which couple to the
G.sub.q subtype of G proteins give an increase in luminescence of
up to 100 fold. Modulators that act as inverse agonists will
reverse this effect at receptors that are either constitutively
active or activated by known agonists.
[0281] C. Luciferase Reporter Gene Assay
[0282] The photoprotein luciferase provides another useful tool for
assaying for modulators of DmGPCR activity. Cells (e.g., CHO cells
or COS 7 cells) are transiently co-transfected with both a DmGPCR
expression construct (e.g., DmGPCR in pzeoSV2) and a reporter
construct which includes a gene for the luciferase protein
downstream from a transcription factor binding site, such as the
cAMP-response element (CRE), AP-1, or NF-kappa B. Agonist binding
to receptors coupled to the G.sub.s subtype of G proteins leads to
increases in cAMP, thereby activating the CRE transcription factor
and resulting in expression of the luciferase gene. Agonist binding
to receptors coupled to the G.sub.q subtype of G protein leads to
production of diacylglycerol that activates protein kinase C, which
activates the AP-1 or NF-kappa B transcription factors, in turn
resulting in expression of the luciferase gene. Expression levels
of luciferase reflect the activation status of the signaling
events. (See generally, George et al., Journal of Biomolecular
Screening 2(4): 235-240 (1997); and Stratowa et al., Current
Opinion in Biotechnology 6: 574-581 (1995)). Luciferase activity
may be quantitatively measured using, e.g., luciferase assay
reagents that are commercially available from Promega (Madison,
Wis.).
[0283] In one exemplary assay, CHO cells are plated in 24-well
culture dishes at a density of 100,000 cells/well one day prior to
transfection and cultured at 37.degree. C. in MEM (Gibco/BRL)
supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml
penicillin and 10 .mu.g/ml streptomycin. Cells are transiently
co-transfected with both a DmGPCR expression construct and a
reporter construct containing the luciferase gene. The reporter
plasmids CRE-luciferase, AP-1-luciferase and NF-kappaB-luciferase
may be purchased from Stratagene (LaJolla, Calif.). Transfections
are performed using the FuGENE 6 transfection reagent
(Boehringer-Mannheim) according to the supplier's instructions.
Cells transfected with the reporter construct alone are used as a
control. Twenty-four hours after transfection, cells are washed
once with PBS pre-warmed to 37 C. Serum-free MEM is then added to
the cells either alone (control) or with one or more candidate
modulators and the cells are incubated at 37.degree. C. for five
hours. Thereafter, cells are washed once with ice-cold PBS and
lysed by the addition of 100 .mu.l of lysis buffer per well from
the luciferase assay kit supplied by Promega After incubation for
15 minutes at room temperature, 15 .mu.l of the lysate is mixed
with 50 .mu.l of substrate solution (Promega) in an opaque-white,
96-well plate, and the luminescence is read immediately on a
Wallace model 1450 MicroBeta scintillation and luminescence counter
(Wallace Instruments, Gaithersburg, Md.).
[0284] Differences in luminescence in the presence versus the
absence of a candidate modulator compound are indicative of
modulatory activity. Receptors that are either constitutively
active or activated by agonists typically give a 3-20-fold
stimulation of luminescence compared to cells transfected with the
reporter gene alone. Modulators that act as inverse agonists will
reverse this effect.
[0285] D. Intracellular Calcium Measurement Using FLIPR
[0286] Changes in intracellular calcium levels are another
recognized indicator of G protein-coupled receptor activity, and
such assays can be employed to screen for modulators of DmGPCR
activity. For example, CHO cells stably transfected with a DmGPCR
expression vector are plated at a density of 4.times.10.sup.4
cells/well in Packard black-walled, 96-well plates specially
designed to discriminate fluorescence signals emanating from the
various wells on the plate. The cells are incubated for 60 minutes
at 37.degree. C. in modified Dulbecco's PBS (D-PBS) containing 36
mg/L pyruvate and 1 g/L glucose with the addition of 1% fetal
bovine serum and one of four calcium indicator dyes (Fluo-3.TM. AM,
Fluo-4.TM. AM, Calcium Green.TM.-1 AM, or Oregon Green.TM. 488
BAPTA-1 AM), each at a concentration of 4 .mu.M. Plates are washed
once with modified D-PBS without 1% fetal bovine serum and
incubated for 10 minutes at 37.degree. C. to remove residual dye
from the cellular membrane. In addition, a series of washes with
modified D-PBS without 1% fetal bovine serum is performed
immediately prior to activation of the calcium response.
[0287] A calcium response is initiated by the addition of one or
more candidate receptor agonist compounds, calcium ionophore A23187
(10 .mu.M; positive control), or ATP (4 .mu.M; positive control).
Fluorescence is measured by Molecular Device's FLIPR with an argon
laser (excitation at 488 nm). (See, e.g., Kuntzweiler et al., Drug
Development Research, 44(1):14-20 (1998)). The F-stop for the
detector camera was set at 2.5 and the length of exposure was 0.4
milliseconds. Basal fluorescence of cells was measured for 20
seconds prior to addition of candidate agonist, ATP, or A23187, and
the basal fluorescence level was subtracted from the response
signal. The calcium signal is measured for approximately 200
seconds, taking readings every two seconds. Calcium ionophore
A23187 and ATP increase the calcium signal 200% above baseline
levels. In general, activated GPCRs increase the calcium signal
approximately 10-15% above baseline signal.
[0288] E. Mitogenesis Assay
[0289] In a mitogenesis assay, the ability of candidate modulators
to induce or inhibit DmGPCR-mediated cell division is determined.
(See, e.g., Lajiness et al., Journal of Pharmacology and
Experimental Therapeutics 267(3):1573-1581 (1993)). For example,
CHO cells stably expressing DmGPCR are seeded into 96-well plates
at a density of 5000 cells/well and grown at 37.degree. C. in MEM
with 10% fetal calf serum for 48 hours, at which time the cells are
rinsed twice with serum-free MEM. After rinsing, 80 .mu.l of fresh
MEM, or MEM containing a known mitogen, is added along with 20
.mu.l MEM containing varying concentrations of one or more
candidate modulators or test compounds diluted in serum-free
medium. As controls, some wells on each plate receive serum-free
medium alone, and some receive medium containing 10% fetal bovine
serum. Untransfected cells or cells transfected with vector alone
also may serve as controls.
[0290] After culture for 16-18 hours, 1 .mu.Ci of
[.sup.3H]-thymidine (2 Ci/mmol) is added to the wells and cells are
incubated for an additional 2 hours at 37.degree. C. The cells are
trypsinized and collected on filter mats with a cell harvester
(Tomtec); the filters are then counted in a Betaplate counter. The
incorporation of [.sup.3H]-thymidine in serum-free test wells is
compared to the results achieved in cells stimulated with serum
(positive control). Use of multiple concentrations of test
compounds permits creation and analysis of dose-response curves
using the non-linear, least squares fit equation:
A=B.times.[C/(D+C)]+G where A is the percent of serum stimulation;
B is the maximal effect minus baseline; C is the EC.sub.50; D is
the concentration of the compound; and G is the maximal effect.
Parameters B, C and G are determined by Simplex optimization.
[0291] Agonists that bind to the receptor are expected to increase
[.sup.3H]-thymidine incorporation into cells, showing up to 80% of
the response to serum. Antagonists that bind to the receptor will
inhibit the stimulation seen with a known agonist by up to
100%.
[0292] F. [.sup.35S]GTP.gamma.S Binding Assay
[0293] Because G protein-coupled receptors signal through
intracellular G proteins whose activity involves GTP binding and
hydrolysis to yield bound GDP, measurement of binding of the
non-hydrolyzable GTP analog [.sup.35S]GTP.gamma.S in the presence
and absence of candidate modulators provides another assay for
modulator activity. (See, e.g., Kowal et al., Neuropharmacology
37:179-187 (1998).)
[0294] In one exemplary assay, cells stably transfected with a
DmGPCR expression vector are grown in 10 cm tissue culture dishes
to subconfluence, rinsed once with 5 ml of ice-cold
Ca.sup.2+/Mg.sup.2+-free phosphate-buffered saline, and scraped
into 5 ml of the same buffer. Cells are pelleted by centrifugation
(500.times.g, 5 minutes), resuspended in TEE buffer (25 mM Tris, pH
7.5, 5 mM EDTA, 5 mM EGTA), and frozen in liquid nitrogen. After
thawing, the cells are homogenized using a Dounce homogenizer (one
ml TEE per plate of cells), and centrifuged at 1,000.times.g for 5
minutes to remove nuclei and unbroken cells.
[0295] The homogenate supernatant is centrifuged at 20,000.times.g
for 20 minutes to isolate the membrane fraction, and the membrane
pellet is washed once with TEE and resuspended in binding buffer
(20 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM MgCl.sub.2, 1 mM EDTA).
The resuspended membranes can be frozen in liquid nitrogen and
stored at -70.degree. C. until use.
[0296] Aliquots of cell membranes prepared as described above and
stored at -70.degree. C. are thawed, homogenized, and diluted into
buffer containing 20 mM HEPES, 10 mM MgCl.sub.2, 1 mM EDTA, 120 mM
NaCl, 10 .mu.M GDP, and 0.2 mM ascorbate, at a concentration of
10-50 .mu.g/ml. In a final volume of 90 .mu.l, homogenates are
incubated with varying concentrations of candidate modulator
compounds or 100 .mu.M GTP for 30 minutes at 30.degree. C. and then
placed on ice. To each sample, 10 .mu.l guanosine
5'-O-(3[.sup.35S]thio) triphosphate (NEN, 1200 Ci/mmol;
[.sup.35S]-GTP.gamma.S), was added to a final concentration of
100-200 .mu.M. Samples are incubated at 30.degree. C. for an
additional 30 minutes, 1 ml of 10 mM HEPES, pH 7.4, 10 mM
MgCl.sub.2, at 4.degree. C. is added and the reaction is stopped by
filtration.
[0297] Samples are filtered over Whatman GF/B filters and the
filters are washed with 20 ml ice-cold 10 mM HEPES, pH 7.4, 10 mM
MgCl.sub.2. Filters are counted by liquid scintillation
spectroscopy. Nonspecific binding of [.sup.35S]-GTP.gamma.S is
measured in the presence of 100 .mu.M GTP and subtracted from the
total. Compounds are selected that modulate the amount of
[.sup.35S]-GTP.gamma.S binding in the cells, compared to
untransfected control cells. Activation of receptors by agonists
gives up to a five-fold increase in [.sup.35S]GTP.gamma.S binding.
This response is blocked by antagonists.
[0298] G. MAP Kinase Activity Assay
[0299] Evaluation of MAP kinase activity in cells expressing a GPCR
provides another assay to identify modulators of GPCR activity.
(See, e.g., Lajiness et al., Journal of Pharmacology and
Experimental Therapeutics 267(3):1573-1581 (1993) and Boulton et
al., Cell 65:663-675 (1991).)
[0300] In one embodiment, CHO cells stably transfected with DmGPCR
are seeded into 6-well plates at a density of 70,000 cells/well 48
hours prior to the assay. During this 48-hour period, the cells are
cultured at 37.degree. C. in MEM medium supplemented with 10% fetal
bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 .mu.g/ml
streptomycin. The cells are serum-starved for 1-2 hours prior to
the addition of stimulants.
[0301] For the assay, the cells are treated with medium alone or
medium containing either a candidate agonist or 200 nM Phorbol
ester-myristoyl acetate (i.e., PMA, a positive control), and the
cells are incubated at 37.degree. C. for varying times. To stop the
reaction, the plates are placed on ice, the medium is aspirated,
and the cells are rinsed with 1 ml of ice-cold PBS containing 1 mM
EDTA. Thereafter, 200 .mu.l of cell lysis buffer (12.5 mM MOPS, pH
7.3, 12.5 mM glycerophosphate, 7.5 mM MgCl.sub.2, 0.5 mM EGTA, 0.5
mM sodium vanadate, 1 mM benzamidine, 1 mM dithiothreitol, 10
.mu.g/ml leupeptin, 10 .mu.g/ml aprotinin, 2 .mu.g/ml pepstatin A,
and 1 .mu.M okadaic acid) is added to the cells. The cells are
scraped from the plates and homogenized by 10 passages through a
233/4 G needle, and the cytosol fraction is prepared by
centrifugation at 20,000.times.g for 15 minutes.
[0302] Aliquots (5-10 .mu.l containing 1-5 .mu.g protein) of
cytosol are mixed with 1 mM MAPK Substrate Peptide (APRTPGGRR (SEQ
ID NO:), Upstate Biotechnology, Inc., N.Y.) and 50 .mu.M
[.gamma.-.sup.32P]ATP (NEN, 3000 Ci/mmol), diluted to a final
specific activity of .about.2000 cpm/pmol, in a total volume of 25
.mu.l. The samples are incubated for 5 minutes at 30.degree. C.,
and reactions are stopped by spotting 20 .mu.l on 2 cm.sup.2
squares of Whatman P81 phosphocellulose paper. The filter squares
are washed in 4 changes of 1% H.sub.3PO.sub.4, and the squares are
subjected to liquid scintillation spectroscopy to quantitate bound
label. Equivalent cytosolic extracts are incubated without MAPK
substrate peptide, and the bound label from these samples are
subtracted from the matched samples with the substrate peptide. The
cytosolic extract from each well is used as a separate point.
Protein concentrations are determined by a dye binding protein
assay (Bio-Rad Laboratories). Agonist activation of the receptor is
expected to result in up to a five-fold increase in MAPK enzyme
activity. This increase is blocked by antagonists.
[0303] H. [.sup.3H]Arachidonic Acid Release
[0304] The activation of GPCRs also has been observed to potentiate
arachidonic acid release in cells, providing yet another useful
assay for modulators of GPCR activity. (See, e.g., Kanterman et
al., Molecular Pharmacology 39:364-369 (1991).) For example, CHO
cells that are stably transfected with a DmGPCR expression vector
are plated in 24-well plates at a density of 15,000 cells/well and
grown in MEM medium supplemented with 10% fetal bovine serum, 2 mM
glutamine, 10 U/ml penicillin and 10 .mu.g/ml streptomycin for 48
hours at 37.degree. C. before use. Cells of each well are labeled
by incubation with [.sup.3H]-arachidonic acid (Amersham Corp., 210
Ci/mmol) at 0.5 .mu.Ci/ml in 1 ml MEM supplemented with 10 mM
HEPES, pH 7.5, and 0.5% fatty-acid-free bovine serum albumin for 2
hours at 37.degree. C. The cells are then washed twice with 1 ml of
the same buffer.
[0305] Candidate modulator compounds are added in 1 ml of the same
buffer, either alone or with 10 .mu.M ATP and the cells are
incubated at 37.degree. C. for 30 minutes. Buffer alone and
mock-transfected cells are used as controls. Samples (0.5 ml) from
each well are counted by liquid scintillation spectroscopy.
Agonists which activate the receptor will lead to potentiation of
the ATP-stimulated release of [.sup.3H]-arachidonic acid. This
potentiation is blocked by antagonists.
[0306] I. Extracellular Acidification Rate
[0307] In yet another assay, the effects of candidate modulators of
DmGPCR activity are assayed by monitoring extracellular changes in
pH induced by the test compounds. (See, e.g., Dunlop et al.,
Journal of Pharmacological and Toxicological Methods 40(1):47-55
(1998).) In one embodiment, CHO cells transfected with a DmGPCR
expression vector are seeded into 12 mm capsule cups (Molecular
Devices Corp.) at 4.times.10.sup.5 cells/cup in MEM supplemented
with 10% fetal bovine serum, 2 mM L-glutamine, 10 U/ml penicillin,
and 10 .mu.g/ml streptomycin. The cells are incubated in this
medium at 37.degree. C. in 5% CO.sub.2 for 24 hours.
[0308] Extracellular acidification rates are measured using a
Cytosensor microphysiometer (Molecular Devices Corp.). The capsule
cups are loaded into the sensor chambers of the microphysiometer
and the chambers are perfused with running buffer (bicarbonate-free
MEM supplemented with 4 mM L-glutamine, 10 units/ml penicillin, 10
.mu.g/ml streptomycin, 26 mM NaCl) at a flow rate of 100
.mu.l/minute. Candidate agonists or other agents are diluted into
the running buffer and perfused through a second fluid path. During
each 60-second pump cycle, the pump is run for 38 seconds and is
off for the remaining 22 seconds. The pH of the running buffer in
the sensor chamber is recorded during the cycle from 43-58 seconds,
and the pump is re-started at 60 seconds to start the next cycle.
The rate of acidification of the running buffer during the
recording time is calculated by the Cytosoft program. Changes in
the rate of acidification are calculated by subtracting the
baseline value (the average of 4 rate measurements immediately
before addition of a modulator candidate) from the highest rate
measurement obtained after addition of a modulator candidate. The
selected instrument detects 61 mV/pH unit. Modulators that act as
agonists of the receptor result in an increase in the rate of
extracellular acidification compared to the rate in the absence of
agonist. This response is blocked by modulators which act as
antagonists of the receptor.
Example 9
Matching DmGPCRs with Peptide Ligands
[0309] Cell Cultures and Transfections
[0310] Wild type Chinese hamster ovary (CHO-K1) cells (from the
American Type Culture Collection, Rockville, Md.) were cultured at
37.degree. C. in a humidified atmosphere of 5% CO.sub.2 in air in
DMEM media supplemented with 10% heat-inactivated FBS, 10 .mu.g/ml
gentamicin, 0.1 mM nonessential amino acids to give complete DMEM
media Cells were transfected with orphan GPCR DNAs in the pCR3.1
vector, using LipofectAMINE PLUS.TM., essentially according to the
manufacturer's instructions. Briefly, CHO cells were plated on 10
cm sterile tissue culture dishes (Corning Glass Works, Corning,
N.Y.) and they were about 50-60% confluent the day of transfection.
In a plastic tube, PLUS (20 .mu.l/plate) was added to cDNA plasmid
(5 .mu.g/plate) which was earlier diluted into 0.75 ml OptiMEM,
mixed and incubated at room temp for 15 min. Separately,
LipofectAMINE (30 .mu.l/plate) was mixed with 0.75 ml OptiMEM and
added to the pre-complexed DNA/PLUS mixture and incubated at room
temp. for 15 minutes. Medium on the cells was replaced with
serum-free transfection medium (plain DMEM, 5 ml/plate), and the
DNA-PLUS-LipofectAMINE complex was added (1.5 ml per plate) and
mixed gently into the medium followed by a 3 hr incubation at
37.degree. C./5% CO.sub.2. Then the medium was supplemented with
the complete DMEM medium containing 20% FBS (6.5 ml ml/plate) and
the incubation continued at 37.degree. C./5% CO.sub.2 for 24 to 48
hrs. A plasmid for Green Fluorescent Protein (GFP, 4 .mu.g/plate)
was used for transient GFP expression in CHO cells to estimate the
transfection yields under the same conditions also used for
GPCRs.
[0311] Membrane Preparation
[0312] The transfected cells were washed once with ice-cold
Dulbecco's phosphate buffered saline (PBS), 5 ml per 10 cm plate,
and scraped into 5 ml of the same buffer. Cell suspensions from
multiple plates were combined and centrifuged at 500.times.g for 10
min at 4.degree. C. The cell pellet was reconstituted in ice-cold
TEE (25 mM TRIS, 5 mM EGTA, 5 mM EDTA). Convenient aliquots were
snap-frozen in liquid nitrogen and stored at -70.degree. C. After
thawing, the cells were homogenized and centrifuged at 4.degree.
C., 500.times.g for 5 minutes to pellet nuclei and unbroken cells.
The supernatant was centrifuged at 47,000.times.g for 30 minutes at
4.degree. C. The membrane pellet was washed once with TEE,
resuspended in 20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM MgCl.sub.2,
1 mM EDTA (assay buffer), aliquoted and frozen in liquid nitrogen.
Membrane aliquots were stored at -70.degree. C. Membrane protein
concentration was determined using the BCA Protein Assay Reagent
from Pierce (Rockford, Ill.) and BSA as standard.
[0313] [.sup.35S]GTP.gamma.S Binding Assay
[0314] Aliquots of cell membranes were thawed, homogenized, and
diluted into buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 10
mM MgCl.sub.2, 1 mM EDTA (assay buffer). Initially, reaction
mixtures were prepared in 96-well polypropylene plates (Nunc). In
each well, peptide aqueous solution (20 .mu.l, 10.times.), or water
controls (20 .mu.l), 18.2 .mu.M GDP in assay buffer (0.11 ml, 10
.mu.M final), and membranes suspended in assay buffer (50 .mu.l, 10
.mu.g membrane protein) were mixed and placed on ice. The
ligand-GDP-membrane mixtures were incubated for 20 min. at room
temperature on a shaking platform and then placed on ice. To each
sample, 20 .mu.l guanosine-5'-O-(3-[.sup.35S]thio)-triphosphate
([.sup.35S]GTP.gamma.S) (600-1,200 Ci/mmol from New England
Nuclear, Boston, Mass.) was added to .about.40,000 cpm/0.2 ml, or a
final concentration of 0.1 nM. Plates with the incubation mixtures
(0.2 ml/well total) were incubated at room temperature for 45
minutes. Reaction mixture aliquots, 0.175 ml each, were then
transferred into wash buffer pretreated (100 .mu.l/well) 96-well FB
MultiScreen filter plates (Millipore) and vacuum filtered using a
MultiScreen Vacuum manifold (Millipore). Then the membranes were
washed 3 times with 0.25 ml ice-cold wash buffer/well (10 mM HEPES,
10 mM MgCl.sub.2, pH 7.4) and vacuum filtered. After the last wash,
Supermix Opti-phase scintillation fluid (25 .mu.l/well, Wallac) was
added and the plates were sealed and counted in a Trilux 1450
Microbeta counter (Wallac) for 1 minute/well. As positive controls,
membranes from CHO cells stably expressing a dopamine type 2
(rD.sub.2) receptor, were treated with 1 mM dopamine in 0.025%
ascorbic acid (100 .mu.M dopamine final) or vehicle (0.0025%
ascorbic acid final). Non-specific binding was measured in the
presence of 100 .mu.M cold GTP.gamma.S and was subtracted from the
total. Each treatment was carried out in triplicates.
[0315] Data Analysis
[0316] Ligand-induced stimulation of [.sup.35S]GTP.gamma.S binding
was expressed as fold increase over the basal activity with no
ligand added. Each treatment was run either in triplicate, or, on
occasion in duplicate and the binding (cpm) was calculated as means
+/-standard deviations. Dose-response curves for the
receptor/ligand systems were analyzed using a non-linear least
square SAS model, y=B.sub.max X/(K.sub.d+X). Other dose-response
curves were analyzed using Prism (GraphPad Software, Inc. San
Diego, Calif.) and the following equation
y=Bottom+(Top-Bottom)/(1+10.sup.LogEC50-X).
[0317] Results
[0318] Originally, we have chosen the GTP.gamma.S assay as a
functional assay because agonist-driven stimulation of GTP.gamma.S
assay reflects early events in the GPCR activation cascade,
regardless of further activation pathways of various down-stream
signaling events. This appears especially useful for the assessment
of possible activation of orphan GPCRs with unknown functions and
unknown signaling pathways. The GTP.gamma.S assay was carried out
with membranes prepared from CHO cells transiently transfected with
DNA encoding Drosophila GPCRs using a 96-well MultiScreen G/FB
filter plates and a MultiScreen vacuum manifold (Millipore) for
filtration. Since the GTP.gamma.S assay is known to poorly
recognize GPCRs coupled to the Gq class of G-proteins, a Ca.sup.+2
mobilization assay based on a FLIPR readout was used as well to
evaluate Gq coupled orphan GPCRs in CHO cells transiently
transfected with DNA encoding Drosophila GPCRs.
[0319] Using GTP.gamma.S assay, DmGPCR1 (PnuFlyPep34651) was found
to be best activated by two peptides, DPKQDFMRF-NH2 <SEQ ID
NO:26> and PDNFF-NH2 <SEQ ID NO:27> (EC.sub.50's range 370
nM to 500 nM). As reported by Nambu et al. (Neuron 1, 55-61, 1988),
these two peptides are encoded on the same precursor gene together
with nine other FaRPs. Additional FaRPs and other neuropeptides
which also stimulated GTP.gamma.S binding, although less
effectively (EC.sub.50's in the range of 5 to 10 .mu.M), included
the following peptides: TDVDHVFLRF-NH2 <SEQ ID NO:25>,
TPAEDFMRF-NH2 <SEQ ID NO:28>, SLKQDFMHF-NH2 <SEQ ID
NO:29>, SVKQDFMHF-NH2 <SEQ ID NO:30>, AAMDRY-NH2 <SEQ
ID NO:31>, and SVQDNFMHF-NH2 <SEQ ID NO:32>. In addition,
the FLIPR assay identified a Colorado potato beetle peptide,
ARGPQLRLRF-NH2 <SEQ ID NO:33>, matched to DmGPCR1 receptor
with an EC.sub.50 of 100-200 mM.
[0320] As shown by the GTP.gamma.S responses, DmGPCR4 (PnuFlyPep
67393) was activated by a Drosophila melanogaster allatostatin,
drostatin-3 (SRPYSFGL-NH2 <SEQ ID NO:161>) with an EC.sub.50
in the low nanomolar range, as well as by various Diplotera
punctata (cockroach) allatostatins, namely: GDGRLYAFGL-NH2 <SEQ
ID NO:34>, DRLYSFGL-NH2 <SEQ ID NO:35>, APSGAQRLYGFGL-NH2
<SEQ ID NO:36>, and GGSLYSFGL-NH2 <SEQ ID NO:37>
(EC.sub.50's in the range of ca. 20-280 nM). The same peptides
elicited a very strong calcium signal when tested at 10 .mu.M by
FLIPR. DmGPCR4 has been recently cloned by Lenz et al. (supra) and
classified as a second putative allatostatin receptor (DARII).
However, no pharmacological data on receptor activation have been
reported to date. To our knowledge this is the very first
experimental evidence that various allatostatins do activate this
receptor.
[0321] DmGPCR6a (M811490) was reported as a PYY receptor by Li et
al. (J Biol Chem 267, 9-12, 1992). Using the GTP.gamma.S assay, the
peptides listed in Table 6, tested at 5 .mu.M, stimulated
GTP.gamma.S binding (1.7 to 4 fold increase above the basal) to
membranes from CHO cells transfected with a DNA encoding DmGPCRa.
It is noteworthy that, in addition to a battery of insect and C.
elegans peptides that activated this receptor, also human NPFF
(FLFQPQRF-NH2 <SEQ ID NO:59>) was found to be a ligand for
DmGPCR6 (4-fold increase in GTP.gamma.S binding by 5 .mu.M
NPFF).
[0322] Dmgpcr6aL and Dmgpcr6bL are two splice variants of DmGPCR6a
(M811490). The latter was reported as a PYY receptor by Li et al.
(J Biol Chem 267, 9-12, 1992). We name both DmGPCR6aL and
DmGPCR6bL, RF-amide receptors since they recognize only peptides
that have an Arg-Phe-NH2 (RFa) sequence at the C-terminus. The
peptides that these GPCRs did not "see" have different than RFa
sequences at the C-end (e.g. SFa, QFa, YFa, RLa, DWa, RPa, HFa,
LQa, SNa etc.). In the calcium mobilization assay (FLIPR),
Dmgpcr6aL and Dmgpcr6bL showed very strong Ca.sup.+2 responses to a
battery of FaRPs tested at 10 .mu.M. The sequences shown below in
Table 6 represent all the identified active FaRPs belonging to
various species including Drosophila, C. elegans, A. suum,
Mollusca, P. redivivus, Trematoda, lobster, human, and leech: The
only exception from the C-end "RFamide rule" was the peptide
pGluDRDYRPLQF-NH2 <SEQ ID NO:120>, whose C-terminus ends with
an Gln-Phe-NH2 (QFa) sequence. Interestingly, both Dmgpcr6a.sub.L
and Dmgpcr6b.sub.L also recognized NPFF (FLFQPQRF-NH2 <SEQ ID
NO:152>), a mammalian peptide with the RFamide sequence at the
C-terminus. (Note in the results above that p-Glu or pQ refers to
pyroglutamic acid.)
[0323] DmGPCR9 has been matched with FDDY(SO.sub.3H)GHLRF-NH2
<SEQ ID NO: 157>, based on its very strong signal in the
calcium mobilization assay (EC.sub.50 in the low nanomolar range).
The fact that no GTP.gamma.S responses to this peptide were
detected with membranes prepared from CHO cells transfected with a
DNA encoding Dmgpcr9, indicates that Dmgpcr9 is most likely coupled
to Gq signaling pathways. FDDY(SO.sub.3H)GHLRF-NH2 <SEQ ID NO:
157> represents a Met7.fwdarw.Leu7 analog of the naturally
occurring drosulfakinin-1 (DSK-1), FDDY(SO.sub.3H)GHMRF-NH2 <SEQ
ID NO: 159>. Therefore we assign the DmGPCR9 receptor as a
sulfakinin receptor. This match is very specific since even
FDDYGHLRF-NH2 <SEQ ID NO:158>, which is an unsulfated
counterpart of FDDY(SO.sub.3H)GHLRF-NH2 <SEQ ID NO:157>,
showed only a very weak calcium signal when tested at 10 .mu.M and
none of the other 117 tested FaRPs and related peptides showed any
activity either in FLIPR or in the GTP.gamma.S assay at the DmGPCR9
receptor.
[0324] A table matching the ligands with their associated receptors
is shown below in Table 6.
TABLE-US-00006 TABLE 6 GPCR SEQ ID NO Peptide Matching Sequence
dmgpcr1 SEQ ID NO: 25 TDVDHVFLRF-NH.sub.2 SEQ ID NO: 26
DPKQDFMRF-NH.sub.2 SEQ ID NO: 27 PDNFMRF-NH.sub.2 SEQ ID NO: 28
TPAEDFMRF-NH.sub.2 SEQ ID NO: 29 SLKQDFMHF-NH.sub.2 SEQ ID NO: 30
SVKQDFMHF-NH.sub.2 SEQ ID NO: 31 AAMDRY-NH.sub.2 SEQ ID NO: 32
SVQDNFMHF-NH.sub.2 SEQ ID NO: 33 ARGPQLRLRF-NH.sub.2 dmgpcr4 SEQ ID
NO: 34 GDGRLYAFGL-NH.sub.2 SEQ ID NO: 35 DRLYSFGL-NH.sub.2 SEQ ID
NO: 36 APSGAQRLYGFGL-NH.sub.2 SEQ ID NO: 37 GGSLYSFGL-NH.sub.2
dmgpcr6 SEQ ID NO: 38 FIRF-NH.sub.2 (6a) SEQ ID NO: 39
KNEFIRF-NH.sub.2 SEQ ID NO: 40 FMRF-NH.sub.2 SEQ ID NO: 41
KSAFMRF-NH.sub.2 SEQ ID NO: 42 KPNFLRF-NH.sub.2 SEQ ID NO: 43
FLRF-NH.sub.2 SEQ ID NO: 44 YLRF-NH.sub.2 SEQ ID NO: 45
KPNFLRY-NH.sub.2 SEQ ID NO: 46 TNRNFLRF-NH.sub.2 SEQ ID NO: 47
RNKFEFIRF-NH.sub.2 SEQ ID NO: 48 AGPRFIRF-NH.sub.2 SEQ ID NO: 49
GLGPRPLRF-NH.sub.2 SEQ ID NO: 50 IL-Nle-RF-NH.sub.2 SEQ ID NO: 51
AGAKFIRF-NH.sub.2 SEQ ID NO: 52 APKPKFIRF-NH.sub.2 SEQ ID NO: 53
KSAFVLRF-NH.sub.2 SEQ ID NO: 54 TKFQDFLRF-NH.sub.2 SEQ ID NO: 55
SAEPFGTMRF-NH.sub.2 SEQ ID NO: 56 ASEDALFGTMRF-NH.sub.2 SEQ ID NO:
57 SADDSAPFGTMRF-NH.sub.2 SEQ ID NO: 58 EDGNAPFGTMRF-NH.sub.2 SEQ
ID NQ: 59 FLFQPQRF-NH.sub.2 dmgpcr6 SEQ ID NO: 60
SADPNFLRF-NH.sub.2 6aL and SEQ ID NO: 61 SQPNFLRF-NH.sub.2 6bL SEQ
ID NO: 62 ASGDPNFLRF-NH.sub.2 SEQ ID NO: 63 SDPNFLRF-NH.sub.2 SEQ
ID NO: 64 AAADPNFLRF-NH.sub.2 SEQ ID NO: 65 PNFLRF-NH.sub.2 SEQ ID
NO: 66 KPNFLRF-NH.sub.2 SEQ ID NO: 67 AGSDPNFLRF-NH.sub.2 SEQ ID
NO: 68 KPNFLRY-NH.sub.2 SEQ ID NO: 69 SPREPIRF-NH.sub.2 SEQ ID NO:
70 LRGEPIRF-NH.sub.2 SEQ ID NO: 71 SPLGTMRF-NH.sub.2 SEQ ID NO: 72
EAEEPLGTMRF-NH.sub.2 SEQ ID NO: 73 ASEDALFGTMRF-NH.sub.2 SEQ ID NO:
74 EDGNAPFGTMRF-NH.sub.2 SEQ ID NO: 75 SAEPFGTMRF-NH.sub.2 SEQ ID
NO: 76 SADDSAPFGTMRF-NH.sub.2 SEQ ID NO: 77 KPTFIRF-NH.sub.2 SEQ ID
NO: 78 ASPSFIRF-NH.sub.2 SEQ ID NO: 79 GAKFIRF-NH.sub.2 SEQ ID NO:
80 AGAKFIRF-NH.sub.2 SEQ ID NO: 81 APKPKFIRF-NH.sub.2 SEQ ID NO: 82
KSAYMRF-NH.sub.2 SEQ ID NO: 83 SPMQRSSMVRF-NH.sub.2 SEQ ID NO: 84
SPMERSAMVRF-NH.sub.2 SEQ ID NO: 85 SPMDRSKMVRF-NH.sub.2 SEQ ID NO:
86 KNEFIRF-NH.sub.2 SEQ ID NO: 87 KPSFVRF-NH.sub.2 SEQ ID NO: 88
pQPKARSGYIRF-NH.sub.2 SEQ ID NO: 89 AMRNALVRF-NH.sub.2 SEQ ID NO:
90 ASGQMRNALVRF-NH.sub.2 SEQ ID NO: 91 NGAPQPFVRF-NH.sub.2 SEQ ID
NO: 92 RNKFEFIRF-NH.sub.2 SEQ ID NO: 93 SDRPTRAMDSPLIRF-NH.sub.2
SEQ ID NO: 94 AADGAPLIRF-NH.sub.2 SEQ ID NO: 95 APEASPFIRF-NH.sub.2
SEQ ID NO: 96 ASPSAPLIRF-NH.sub.2 SEQ ID NO: 97 SPSAVPLIRF-NH.sub.2
SEQ ID NO: 98 ASSAPLIRF-NH.sub.2 SEQ ID NO: 99 KHEYLRF-NH.sub.2 SEQ
ID NO: 100 SLLDYRF-NH.sub.2 SEQ ID NO: 101 EIVFHQISPIFFRF-NH.sub.2
SEQ ID NO: 102 GGPQGPLRF-NH.sub.2 SEQ ID NO: 103 GPSGPLRF-NH.sub.2
SEQ ID NO: 104 AQTFVRF-NH.sub.2 SEQ ID NO: 105 GQTFVRF-NH.sub.2 SEQ
ID NO: 106 KSAFVRF-NH.sub.2 SEQ ID NO: 107 KSQYIRF-NH.sub.2 SEQ ID
NO: 108 DVPGVLRF-NH.sub.2 SEQ ID NO: 109 KSVPGVLRF-NH.sub.2 SEQ ID
NO: 110 SEVPGVLRF-NH.sub.2 SEQ ID NO: 111 SVPGVLRF-NH.sub.2 SEQ ID
NO: 112 DFDGAMPGVLRF-NH.sub.2 SEQ ID NO: 113 EIPGVLRF-NH.sub.2 SEQ
ID NO: 114 WANQVRF-NH.sub.2 SEQ ID NO: 115 ASWASSVRF-NH.sub.2 SEQ
ID NO: 116 AMMRF-NH.sub.2 SEQ ID NO: 117 GLGPRPLRF-NH.sub.2 SEQ ID
NO: 118 SPSAKWMRF-NH.sub.2 SEQ ID NO: 119 TKFQDFLRF-NH.sub.2 SEQ ID
NO: 120 pQDRDYRPLQF-NH.sub.2 SEQ ID NO: 121 FIRF-NH.sub.2 SEQ ID
NO: 122 AVPGVLRF-NH.sub.2 SEQ ID NO: 123 GDVPGVLRF-NH.sub.2 SEQ ID
NO: 124 SDIGISEPNFLRF-NH.sub.2 SEQ ID NO: 125 SGKPTFIRF-NH.sub.2
SEQ ID NO: 126 AEGLSSPLIRF-NH.sub.2 SEQ ID NO: 127
FDRDFMRF-NH.sub.2 SEQ ID NO: 128 AGPRFIRF-NH.sub.2 SEQ ID NO: 129
GMPGVLRF-NH.sub.2 SEQ ID NO: 130 IL-Nle-RF-NH.sub.2 SEQ ID NO: 131
LQPNFLRF-NH.sub.2 SEQ ID NO: 132 KPNFIRF-NH.sub.2 SEQ ID NO: 133
FMRF-NH.sub.2 SEQ ID NO: 134 FLRF-NH.sub.2 SEQ ID NO: 135
YIRF-NH.sub.2 SEQ ID NO: 136 GNSFLRF-NH.sub.2 SEQ ID NO: 137
DPSFLRF-NH.sub.2 SEQ ID NO: 138 pQDFMRF-NH.sub.2 SEQ ID NO: 139
KPNQDFMRF-NH.sub.2 SEQ ID NO: 140 TDVDHVFLRF-NH.sub.2 SEQ ID NO:
141 AAMDRY-NH.sub.2 SEQ ID NO: 142 SPKQDFMRF-NH.sub.2 SEQ ID NO:
143 PDNFMRF-NH.sub.2 SEQ ID NO: 144 DPKQDFMRF-NH.sub.2 SEQ ID NO:
145 TPAEDFMRF-NH.sub.2 SEQ ID NO: 146 SDNFMRF-NH.sub.2 SEQ ID NO:
147 YLRF-NH.sub.2 SEQ ID NO: 148 SDRNFLRF-NH.sub.2 SEQ ID NO: 149
TNRNFLRF-NH.sub.2 SEQ ID NO: 150 PDVDHVFLRF-NH.sub.2 SEQ ID NO: 151
pQDVDHVFLRF-NH.sub.2 SEQ ID NO: 152 FLFQPQRF-NH.sub.2 SEQ ID NO:
153 ARGPQLRLRF-NH.sub.2 SEQ ID NO: 154
FDDY(SO.sub.3H)GHLRF-NH.sub.2 SEQ ID NO: 155 FDDYGHLRF-NH.sub.2 SEQ
ID NO: 156 MDSNFIRF-NH.sub.2 dmgpcr9 SEQ ID NO: 157
FDDY(SO.sub.3H)GHLRF-NH.sub.2
Example 10
Competition Assay
[0325] Preparation of Mono-Iodinated Peptide
[0326] The peptide, is iodinated via a typical chloramine T
procedure. Added to a 2 ml glass vial are 10 .mu.l of a 1 mM water
solution of peptide, 10 .mu.l of 0.1M (pH 7.99) sodium phosphate
buffer, 1.0 mCi [.sup.125I] sodium iodide and 5 .mu.l of a 2 mg/ml
chloramine T solution (in the phosphate buffer). The mixture is
vortexed for 60 seconds and the reaction stopped by the addition of
25 .mu.l of a 5 mg/ml solution of sodium metabisulfite in phosphate
buffer. The mixture then undergoes HPLC by injecting it onto a
Vydac C18 (0.45.times.15 cm) column and subjecting it to gradient
separation. The gradient used is 70% A and 30% B at time zero to
20% A and 80% B at time 25 minutes (A=0.1 M NH.sub.4 acetate in
water. B=0.1 M NH.sub.4 acetate in water 40%: CH.sub.3CN 60%,
v:v.). Flow rate is 1.0 ml/minute. Samples are collected into 0.25
ml capture buffer (0.1M sodium phosphate buffer with 0.5% bovine
serum albumin, 0.1% Triton X100 and 0.05% Tween 20) at 30 second
intervals from t=8 to t=20 minutes. Monoiodo peptide typically
elutes at t=11 minutes and the yield is approximately 100 .mu.Ci in
0.75 ml.
[0327] Binding Assay
[0328] 96-well plates used are Millipore Multiscreen.RTM.
filtration plates (FB opaque 1.0 .mu.M glass fiber type B, cat.
#MAFBNOB50). A Millipore Multiscreen.RTM. solvent resistant
manifold (cat. #MAVMO960R) is used in conjunction with the plates
to filter the assay at termination. Each replicate is one well and
has a volume of 100 ul containing 5 ug protein (preparation
described above). Each test group contains two replicates. For each
test compound, one group is run with [.sup.125I]peptide only (for
total binding) and one with 1 .mu.M (or as designated)
concentration of the test compound and [.sup.125I]peptide (for
non-specific binding). The order of adding reagents for each
replicate is: assay buffer (20 mM HEPES, 10 mM MgCl.sub.2, 1%
bovine serum albumin, pH 7.4) test compound (made up in assay
buffer), [.sup.125I]peptide (in assay buffer) and membrane
suspension (in assay buffer). The addition of the membrane
suspension initiates the binding reaction which is run for 30
minutes at room temperature (22.degree. C.). Following the 30
minute incubation each plate is place on the filtration manifold
and vacuum is applied, pulling the liquid through the filter
(discarded) and catching the protein on the filters in each well.
For washing, the vacuum is released and 200 .mu.l assay buffer is
added to each well followed by reapplication of the vacuum. This
washing is repeated twice more (total of 3.times. washes for each
replicate). Following washing, the plastic covering on the
underside of each plate is removed and the plate placed in a bottom
sealed Microbeta.RTM. scintillation counting cassette (cat
#1450-105). 25 .mu.l of scintillant is added to each well and the
plate is placed on a rotary shaker at 80 rpm for one hour and then
allowed to sit overnight. The following day the plate is counted in
a Microbeta.RTM. scintillation counter. The mean non-specific
binding is subtracted from the mean total binding to yield specific
binding for both the standard (peptideamide) and the unknowns.
[0329] Some of the preferred embodiments of the invention described
above are outlined below and include, but are not limited to, the
following embodiments. As those skilled in the art will appreciate,
numerous changes and modifications may be made to the preferred
embodiments of the invention without departing from the spirit of
the invention. It is intended that all such variations fall within
the scope of the invention.
[0330] The entire disclosure of each publication cited herein is
hereby incorporated by reference.
Sequence CWU 1
1
16811803DNAD. melanogaster 1atggccaact taagctggct gagcaccatc
accaccacct cctcctccat cagcaccagc 60cagctgccat tggtcagcac aaccaactgg
agcctaacgt cgccgggaac tactagcgct 120atcttggcgg atgtggctgc
atcggatgag gataggagcg gcgggatcat tcacaaccag 180ttcgtgcaaa
tcttcttcta cgtcctgtac gccacggtct ttgtcctggg tgtcttcgga
240aatgtcctgg tttgctacgt agttctgagg aatcgggcca tgcagactgt
gaccaatata 300ttcatcacga atctggccct gtcggacata ttgctctgcg
tcctggcggt gccatttact 360ccgctttaca cgttcatggg tcgctgggcc
ttcggcagga gtctgtgcca tctggtgtcc 420tttgcccagg gatgcagcat
ctacatatcc acgctgaccc tcacctcgat tgccatcgat 480cggtacttcg
ttatcatata ccccttccat ccgcgcatga agctctccac ctgcatcggg
540atcatagtga gcatctgggt gatagccctg ctggccaccg ttccctacgg
catgtacatg 600aagatgacca acgagctggt gaacggaacg cagacaggca
acgagaccct ggtggaggcc 660actctaatgc taaacggaag ctttgtggcc
cagggatcag gattcatcga ggcgccggac 720tctacctcgg ccacccaggc
ctatatgcag gtgatgaccg ccggatcaac gggaccggag 780atgccctatg
tgcgggtgta ctgcgaggag aactggccat cggagcagta ccggaaggtg
840ttcggtgcca tcacaaccac tctgcagttt gtgctgccct tcttcatcat
ctcgatttgc 900tacgtgtgga tatcggtgaa gctaaaccag cgggccaggg
ccaagccggg atcgaaatcc 960tcgagacggg aggaggcgga tcgggatcgc
aagaagcgca ccaaccgcat gctcatcgcc 1020atggtggcgg tattcggact
cagctggctg cccatcaatg tggtcaacat attcgatgac 1080ttcgatgaca
agtccaacga gtggcgcttc tacatcctat tcttctttgt ggcccactct
1140attgccatga gctccacctg ctacaatccc ttcctgtacg cctggctgaa
cgagaacttc 1200cgcaaggagt tcaagcacgt gctgccctgc tttaatccct
cgaacaacaa catcatcaac 1260atcaccaggg gctataatcg gagtgatcgg
aacacctgtg gtccgcgact gcatcatggc 1320aagggggatg gtggcatggg
cggtggcagt ctggacgccg acgaccagga cgagaacggc 1380atcacccagg
agacctgtct gcccaaggag aagctgctga ttatccccag ggagccgact
1440tacggcaatg gcacgggtgc cgtgtcgcca atccttagcg ggcgcggcat
taacgccgcc 1500ctggtgcacg gtggcgacca tcagatgcac cagctgcagc
cgtcacacca tcaacaggtg 1560gagctgacga ggcgaatccg ccggcggaca
gacgagacgg acggggatta cctggactcc 1620ggcgacgagc agaccgtgga
ggtgcgcttc agcgagacgc cgttcgtcag cacggataat 1680accaccggga
tcagcattct ggagacgagt acgagtcact gccaggactc ggatgtgatg
1740gtcgagctgg gcgaggcaat cggcgccggt ggtggggcag agctggggag
gcgaatcaac 1800tga 18032600PRTD. melanogaster 2Met Ala Asn Leu Ser
Trp Leu Ser Thr Ile Thr Thr Thr Ser Ser Ser1 5 10 15Ile Ser Thr Ser
Gln Leu Pro Leu Val Ser Thr Thr Asn Trp Ser Leu 20 25 30Thr Ser Pro
Gly Thr Thr Ser Ala Ile Leu Ala Asp Val Ala Ala Ser 35 40 45Asp Glu
Asp Arg Ser Gly Gly Ile Ile His Asn Gln Phe Val Gln Ile 50 55 60Phe
Phe Tyr Val Leu Tyr Ala Thr Val Phe Val Leu Gly Val Phe Gly65 70 75
80Asn Val Leu Val Cys Tyr Val Val Leu Arg Asn Arg Ala Met Gln Thr
85 90 95Val Thr Asn Ile Phe Ile Thr Asn Leu Ala Leu Ser Asp Ile Leu
Leu 100 105 110Cys Val Leu Ala Val Pro Phe Thr Pro Leu Tyr Thr Phe
Met Gly Arg 115 120 125Trp Ala Phe Gly Arg Ser Leu Cys His Leu Val
Ser Phe Ala Gln Gly 130 135 140Cys Ser Ile Tyr Ile Ser Thr Leu Thr
Leu Thr Ser Ile Ala Ile Asp145 150 155 160Arg Tyr Phe Val Ile Ile
Tyr Pro Phe His Pro Arg Met Lys Leu Ser 165 170 175Thr Cys Ile Gly
Ile Ile Val Ser Ile Trp Val Ile Ala Leu Leu Ala 180 185 190Thr Val
Pro Tyr Gly Met Tyr Met Lys Met Thr Asn Glu Leu Val Asn 195 200
205Gly Thr Gln Thr Gly Asn Glu Thr Leu Val Glu Ala Thr Leu Met Leu
210 215 220Asn Gly Ser Phe Val Ala Gln Gly Ser Gly Phe Ile Glu Ala
Pro Asp225 230 235 240Ser Thr Ser Ala Thr Gln Ala Tyr Met Gln Val
Met Thr Ala Gly Ser 245 250 255Thr Gly Pro Glu Met Pro Tyr Val Arg
Val Tyr Cys Glu Glu Asn Trp 260 265 270Pro Ser Glu Gln Tyr Arg Lys
Val Phe Gly Ala Ile Thr Thr Thr Leu 275 280 285Gln Phe Val Leu Pro
Phe Phe Ile Ile Ser Ile Cys Tyr Val Trp Ile 290 295 300Ser Val Lys
Leu Asn Gln Arg Ala Arg Ala Lys Pro Gly Ser Lys Ser305 310 315
320Ser Arg Arg Glu Glu Ala Asp Arg Asp Arg Lys Lys Arg Thr Asn Arg
325 330 335Met Leu Ile Ala Met Val Ala Val Phe Gly Leu Ser Trp Leu
Pro Ile 340 345 350Asn Val Val Asn Ile Phe Asp Asp Phe Asp Asp Lys
Ser Asn Glu Trp 355 360 365Arg Phe Tyr Ile Leu Phe Phe Phe Val Ala
His Ser Ile Ala Met Ser 370 375 380Ser Thr Cys Tyr Asn Pro Phe Leu
Tyr Ala Trp Leu Asn Glu Asn Phe385 390 395 400Arg Lys Glu Phe Lys
His Val Leu Pro Cys Phe Asn Pro Ser Asn Asn 405 410 415Asn Ile Ile
Asn Ile Thr Arg Gly Tyr Asn Arg Ser Asp Arg Asn Thr 420 425 430Cys
Gly Pro Arg Leu His His Gly Lys Gly Asp Gly Gly Met Gly Gly 435 440
445Gly Ser Leu Asp Ala Asp Asp Gln Asp Glu Asn Gly Ile Thr Gln Glu
450 455 460Thr Cys Leu Pro Lys Glu Lys Leu Leu Ile Ile Pro Arg Glu
Pro Thr465 470 475 480Tyr Gly Asn Gly Thr Gly Ala Val Ser Pro Ile
Leu Ser Gly Arg Gly 485 490 495Ile Asn Ala Ala Leu Val His Gly Gly
Asp His Gln Met His Gln Leu 500 505 510Gln Pro Ser His His Gln Gln
Val Glu Leu Thr Arg Arg Ile Arg Arg 515 520 525Arg Thr Asp Glu Thr
Asp Gly Asp Tyr Leu Asp Ser Gly Asp Glu Gln 530 535 540Thr Val Glu
Val Arg Phe Ser Glu Thr Pro Phe Val Ser Thr Asp Asn545 550 555
560Thr Thr Gly Ile Ser Ile Leu Glu Thr Ser Thr Ser His Cys Gln Asp
565 570 575Ser Asp Val Met Val Glu Leu Gly Glu Ala Ile Gly Ala Gly
Gly Gly 580 585 590Ala Glu Leu Gly Arg Arg Ile Asn 595
60031445DNAD. melanogaster 3atgaatcaga cggagcccgc ccagctggca
gatggggagc atctgagtgg atacgccagc 60agcagcaaca gcgtgcgcta tctggacgac
cggcatccgc tggactacct tgacctgggc 120acggtgcacg ccctcaacac
cactgccatc aacacctcgg atctgaatga gactgggagc 180aggccgctgg
acccggtgct tatcgatagg ttcctgagca acagggcggt ggacagcccc
240tggtaccaca tgctcatcag catgtacggc gtgctaatcg tcttcggcgc
cctaggcaac 300accctggttg ttatagccgt catccggaag cccatcatgc
gcactgctcg caatctgttc 360atcctcaacc tggccatatc ggacctactt
ttatgcctag tcaccatgcc gctgaccttg 420atggagatcc tgtccaagta
ctggccctac ggctcctgct ccatcctgtg caaaacgatt 480gccatgctgc
aggcactttg tattttcgtg tcgacaatat ccataacggc cattgccttc
540gacagatatc aggtgatcgt gtaccccacg cgggacagcc tgcagttcgt
gggcgcggtg 600acgatcctgg cggggatctg ggcactggca ctgctgctgg
cctcgccgct gttcgtctac 660aaggagctga tcaacacaga cacgccggca
ctcctgcagc agatcggcct gcaggacacg 720atcccgtact gcattgagga
ctggccaagt cgcaacgggc gcttctacta ctcgatcttc 780tcgctgtgcg
tacaatacct ggtgcccatc ctgatcgtct cggtggcata cttcgggatc
840tacaacaagc tgaagagccg catcaccgtg gtggctgtgc aggcgtcctc
cgctcagcgg 900aaggtggagc gggggcggcg gatgaagcgc accaactgcc
tactgatcag catcgccatc 960atctttggcg tttcttggct gccgctgaac
tttttcaacc tgtacgcgga catggagcgc 1020tcgccggtca ctcagagcat
gctagtccgc tacgccatct gccacatgat cggcatgagc 1080tccgcctgct
ccaacccgtt gctctacggc tggctcaacg acaacttccg taaagaattt
1140caagaactgc tctgccgttg ctcagacact aatgttgctc ttaacggtca
cacgacaggc 1200tgcaacgtcc aggcggcggc gcgcaagcgt cgcaagttgg
gcgccgaact ctccaaaggc 1260gaactcaagc tgctggggcc aggcggcgcc
cagagcggta ccgccggcgg ggaaggcggt 1320ctggcggcca ccgacttcat
gaccggccac cacgagggcg gactgcgcag cgccataacc 1380gagtcggtgg
ccctcacgga ccacaacccc gtgccctcgg aggtcaccaa gctgatgccg 1440cggta
14454357PRTD. melanogaster 4Met Glu Asn Thr Thr Met Leu Ala Asn Ile
Ser Leu Asn Ala Thr Arg1 5 10 15Asn Glu Glu Asn Ile Thr Ser Phe Phe
Thr Asp Glu Glu Trp Leu Ala 20 25 30Ile Asn Gly Thr Leu Pro Trp Ile
Val Gly Phe Phe Phe Gly Val Ile 35 40 45Ala Ile Thr Gly Phe Phe Gly
Asn Leu Leu Val Ile Leu Val Val Val 50 55 60Phe Asn Asn Asn Met Arg
Ser Thr Thr Asn Leu Met Ile Val Asn Leu65 70 75 80Ala Ala Ala Asp
Leu Met Phe Val Ile Leu Cys Ile Pro Phe Thr Ala 85 90 95Thr Asp Tyr
Met Val Tyr Tyr Trp Pro Tyr Gly Arg Phe Trp Cys Arg 100 105 110Ser
Val Gln Tyr Leu Ile Val Val Thr Ala Phe Ala Ser Ile Tyr Thr 115 120
125Leu Val Leu Met Ser Ile Asp Arg Phe Leu Ala Val Val His Pro Ile
130 135 140Arg Ser Arg Met Met Arg Thr Glu Asn Ile Thr Leu Ile Ala
Ile Val145 150 155 160Thr Leu Trp Ile Val Val Leu Val Val Ser Val
Pro Val Ala Phe Thr 165 170 175His Asp Val Val Val Asp Tyr Asp Ala
Lys Lys Asn Ile Thr Tyr Gly 180 185 190Met Cys Thr Phe Thr Thr Asn
Asp Phe Leu Gly Pro Arg Thr Tyr Gln 195 200 205Val Thr Phe Phe Ile
Ser Ser Tyr Leu Leu Pro Leu Met Ile Ile Ser 210 215 220Gly Leu Tyr
Met Arg Met Ile Met Arg Leu Trp Arg Gln Gly Thr Gly225 230 235
240Val Arg Met Ser Lys Glu Ser Gln Arg Gly Arg Lys Arg Val Thr Arg
245 250 255Leu Val Val Val Val Val Ile Ala Phe Ala Ser Leu Trp Leu
Pro Val 260 265 270Gln Leu Ile Leu Leu Leu Lys Ser Leu Asp Val Ile
Glu Thr Asn Thr 275 280 285Leu Thr Lys Leu Val Ile Gln Val Thr Ala
Gln Thr Leu Ala Tyr Ser 290 295 300Ser Ser Cys Ile Asn Pro Leu Leu
Tyr Ala Phe Leu Ser Glu Asn Phe305 310 315 320Arg Lys Ala Phe Tyr
Lys Ala Val Asn Cys Ser Ser Arg Tyr Gln Asn 325 330 335Tyr Thr Ser
Asp Leu Pro Pro Pro Arg Lys Thr Ser Cys Ala Arg Thr 340 345 350Ser
Thr Thr Gly Leu 35551376DNAD. melanogaster 5atgaatcaga cggagcccgc
ccagctggca gatggggagc atctgagtgg atacgccagc 60agcagcaaca gcgtgcgcta
tctggacgac cggcatccgc tggactacct tgacctgggc 120acggtgcacg
ccctcaacac cactgccatc aacacctcgg atctgaatga gactgggagc
180aggccgctgg acccggtgct tatcgatagg ttcctgagca acagggcggt
ggacagcccc 240tggtaccaca tgctcatcag catgtacggc gtgctaatcg
tcttcggcgc cctaggcaac 300accctggttg ttatagccgt catccggaag
cccatcatgc gcactgctcg caatctgttc 360atcctcaacc tggccatatc
ggacctactt ttatgcctag tcaccatgcc gctgaccttg 420atggagatcc
tgtccaagta ctggccctac ggctcctgct ccatcctgtg caaaacgatt
480gccatgctgc aggcactttg tattttcgtg tcgacaatat ccataacggc
cattgccttc 540gacagatatc aggtgatcgt gtaccccacg cgggacagcc
tgcagttcgt gggcgcggtg 600acgatcctgg cggggatctg ggcactggca
ctgctgctgg cctcgccgct gttcgtctac 660aaggagctga tcaacacaga
cacgccggca ctcctgcagc agatcggcct gcaggacacg 720atcccgtact
gcattgagga ctggccaagt cgcaacgggc gcttctacta ctcgatcttc
780tcgctgtgcg tacaatacct ggtgcccatc ctgatcgtct cggtggcata
cttcgggatc 840tacaacaagc tgaagagccg catcaccgtg gtggctgtgc
aggcgtcctc cgctcagcgg 900aaggtggagc gggggcggcg gatgaagcgc
accaactgcc tactgatcag catcgccatc 960atctttggcg tttcttggct
gccgctgaac tttttcaacc tgtacgcgga catggagcgc 1020tcgccggtca
ctcagagcat gctagtccgc tacgccatct gccacatgat cggcatgagc
1080tccgcctgct ccaacccgtt gctctacggc tggctcaacg acaacttccg
ctgcaacgtc 1140caggcggcgg cgcgcaagcg tcgcaagttg ggcgccgaac
tctccaaagg cgaactcaag 1200ctgctggggc caggcggcgc ccagagcggt
accgccggcg gggaaggcgg tctggcggcc 1260accgacttca tgaccggcca
ccacgagggc ggactgcgca gcgccataac cgagtcggtg 1320gccctcacgg
accacaaccc cgtgccctcg gaggtcacca agctgatgcc gcggta 13766458PRTD.
melanogaster 6Met Asn Gln Thr Glu Pro Ala Gln Leu Ala Asp Gly Glu
His Leu Ser1 5 10 15Gly Tyr Ala Ser Ser Ser Asn Ser Val Arg Tyr Leu
Asp Asp Arg His 20 25 30Pro Leu Asp Tyr Leu Asp Leu Gly Thr Val His
Ala Leu Asn Thr Thr 35 40 45Ala Ile Asn Thr Ser Asp Leu Asn Glu Thr
Gly Ser Arg Pro Leu Asp 50 55 60Pro Val Leu Ile Asp Arg Phe Leu Ser
Asn Arg Ala Val Asp Ser Pro65 70 75 80Trp Tyr His Met Leu Ile Ser
Met Tyr Gly Val Leu Ile Val Phe Gly 85 90 95Ala Leu Gly Asn Thr Leu
Val Val Ile Ala Val Ile Arg Lys Pro Ile 100 105 110Met Arg Thr Ala
Arg Asn Leu Phe Ile Leu Asn Leu Ala Ile Ser Asp 115 120 125Leu Leu
Leu Cys Leu Val Thr Met Pro Leu Thr Leu Met Glu Ile Leu 130 135
140Ser Lys Tyr Trp Pro Tyr Gly Ser Cys Ser Ile Leu Cys Lys Thr
Ile145 150 155 160Ala Met Leu Gln Ala Leu Cys Ile Phe Val Ser Thr
Ile Ser Ile Thr 165 170 175Ala Ile Ala Phe Asp Arg Tyr Gln Val Ile
Val Tyr Pro Thr Arg Asp 180 185 190Ser Leu Gln Phe Val Gly Ala Val
Thr Ile Leu Ala Gly Ile Trp Ala 195 200 205Leu Ala Leu Leu Leu Ala
Ser Pro Leu Phe Val Tyr Lys Glu Leu Ile 210 215 220Asn Thr Asp Thr
Pro Ala Leu Leu Gln Gln Ile Gly Leu Gln Asp Thr225 230 235 240Ile
Pro Tyr Cys Ile Glu Asp Trp Pro Ser Arg Asn Gly Arg Phe Tyr 245 250
255Tyr Ser Ile Phe Ser Leu Cys Val Gln Tyr Leu Val Pro Ile Leu Ile
260 265 270Val Ser Val Ala Tyr Phe Gly Ile Tyr Asn Lys Leu Lys Ser
Arg Ile 275 280 285Thr Val Val Ala Val Gln Ala Ser Ser Ala Gln Arg
Lys Val Glu Arg 290 295 300Gly Arg Arg Met Lys Arg Thr Asn Cys Leu
Leu Ile Ser Ile Ala Ile305 310 315 320Ile Phe Gly Val Ser Trp Leu
Pro Leu Asn Phe Phe Asn Leu Tyr Ala 325 330 335Asp Met Glu Arg Ser
Pro Val Thr Gln Ser Met Leu Val Arg Tyr Ala 340 345 350Ile Cys His
Met Ile Gly Met Ser Ser Ala Cys Ser Asn Pro Leu Leu 355 360 365Tyr
Gly Trp Leu Asn Asp Asn Phe Arg Cys Asn Val Gln Ala Ala Ala 370 375
380Arg Lys Arg Arg Lys Leu Gly Ala Glu Leu Ser Lys Gly Glu Leu
Lys385 390 395 400Leu Leu Gly Pro Gly Gly Ala Gln Ser Gly Thr Ala
Gly Gly Glu Gly 405 410 415Gly Leu Ala Ala Thr Asp Phe Met Thr Gly
His His Glu Gly Gly Leu 420 425 430Arg Ser Ala Ile Thr Glu Ser Val
Ala Leu Thr Asp His Asn Pro Val 435 440 445Pro Ser Glu Val Thr Lys
Leu Met Pro Arg 450 45571073DNAD. melanogaster 7atggagaaca
ccacaatgct ggctaatatt agcctaaatg caaccagaaa tgaggagaat 60atcacctcat
tcttcaccga cgaagagtgg ctggccatca atggcacttt gccgtggata
120gtgggattct tcttcggcgt catcgccatc acgggattct tcggcaacct
gctggtcatc 180ctggtggtgg tcttcaacaa caacatgcgc tccaccacca
acctgatgat tgtcaatctg 240gctgccgctg atctgatgtt cgtaatcctc
tgcattccct tcacggccac cgattacatg 300gtgtactact ggccatatgg
aaggttctgg tgccgcagtg tccagtacct gattgtggtg 360accgccttcg
cctccatcta cacgctggtg ctaatgtcca tcgatcggtt cctggcggtg
420gttcatccca ttcgctcgcg gatgatgagg acggagaaca ttaccctgat
tgccatcgtg 480actctgtgga tcgtggtgct ggtcgtttcg gtgccagtgg
ccttcaccca cgacgtggtg 540gtggactacg atgcaaagaa gaacatcacc
tacggcatgt gcaccttcac gacgaacgac 600ttccttggtc cgcgcaccta
ccaggtcacc ttcttcatca gctcctacct gctgcccctg 660atgatcatca
gcggtctcta catgcgcatg atcatgcggc tctggcgcca gggaaccggc
720gtccgcatgt ccaaggagtc gcagcgcggt cgcaagcggg tcacccgact
cgtcgtcgtg 780gtggtcatcg ccttcgcctc gctctggctg cctgtccagc
tcatcctgct gctcaagtca 840ctggatgtca tcgagacgaa caccctcacc
aagctagtca tccaggtcac cgcccagact 900ctggcctaca gcagctcgtg
tatcaatccg ctgctctacg ccttcctctc cgagaatttc 960cggaaggcct
tctataaggc cgttaactgc tcctctcgat accagaacta cacatctgat
1020ttgccgccgc cgcgcaagac gtcctgtgcc aggacctcca ccactggact cta
10738357PRTD. melanogaster 8Met Glu Asn Thr Thr Met Leu Ala Asn Ile
Ser Leu Asn Ala Thr Arg1 5 10 15Asn Glu Glu Asn Ile Thr Ser Phe Phe
Thr Asp Glu Glu Trp Leu Ala 20 25 30Ile Asn Gly Thr Leu Pro Trp Ile
Val Gly Phe Phe Phe Gly Val Ile 35 40 45Ala Ile Thr Gly Phe Phe Gly
Asn Leu Leu Val Ile Leu Val Val Val 50 55 60Phe Asn Asn Asn Met Arg
Ser Thr Thr Asn Leu Met Ile Val Asn Leu65 70 75
80Ala Ala Ala Asp Leu Met Phe Val Ile Leu Cys Ile Pro Phe Thr Ala
85 90 95Thr Asp Tyr Met Val Tyr Tyr Trp Pro Tyr Gly Arg Phe Trp Cys
Arg 100 105 110Ser Val Gln Tyr Leu Ile Val Val Thr Ala Phe Ala Ser
Ile Tyr Thr 115 120 125Leu Val Leu Met Ser Ile Asp Arg Phe Leu Ala
Val Val His Pro Ile 130 135 140Arg Ser Arg Met Met Arg Thr Glu Asn
Ile Thr Leu Ile Ala Ile Val145 150 155 160Thr Leu Trp Ile Val Val
Leu Val Val Ser Val Pro Val Ala Phe Thr 165 170 175His Asp Val Val
Val Asp Tyr Asp Ala Lys Lys Asn Ile Thr Tyr Gly 180 185 190Met Cys
Thr Phe Thr Thr Asn Asp Phe Leu Gly Pro Arg Thr Tyr Gln 195 200
205Val Thr Phe Phe Ile Ser Ser Tyr Leu Leu Pro Leu Met Ile Ile Ser
210 215 220Gly Leu Tyr Met Arg Met Ile Met Arg Leu Trp Arg Gln Gly
Thr Gly225 230 235 240Val Arg Met Ser Lys Glu Ser Gln Arg Gly Arg
Lys Arg Val Thr Arg 245 250 255Leu Val Val Val Val Val Ile Ala Phe
Ala Ser Leu Trp Leu Pro Val 260 265 270Gln Leu Ile Leu Leu Leu Lys
Ser Leu Asp Val Ile Glu Thr Asn Thr 275 280 285Leu Thr Lys Leu Val
Ile Gln Val Thr Ala Gln Thr Leu Ala Tyr Ser 290 295 300Ser Ser Cys
Ile Asn Pro Leu Leu Tyr Ala Phe Leu Ser Glu Asn Phe305 310 315
320Arg Lys Ala Phe Tyr Lys Ala Val Asn Cys Ser Ser Arg Tyr Gln Asn
325 330 335Tyr Thr Ser Asp Leu Pro Pro Pro Arg Lys Thr Ser Cys Ala
Arg Thr 340 345 350Ser Thr Thr Gly Leu 35591559DNAD. melanogaster
9atggagaatc gcagtgactt cgaggcggat gactacggcg acatcagttg gagcaattgg
60agcaactgga gcacccccgc cggcgtcctt ttctcggcca tgagcagcgt gctctcggcc
120agcaaccata cgcccctgcc ggactttggc caggagctcg ccctatccac
cagctccttc 180aatcacagcc agaccctatc caccgaccag cccgccgtcg
gggacgtgga agacgcggcc 240gaggatgcgg cggcgtccat ggagacgggc
tcgtttgcat ttgtggtccc gtggtggcgt 300caggtgctct ggagcatcct
cttcggcggc atggtcattg tggcgacggg cggtaacctg 360attgttgtct
ggatcgtgat gacgaccaag cggatgcgga cggtaaccaa ctatttcata
420gtgaatctct ccatcgcgga cgccatggtg tccagcctaa acgtcacctt
caactactac 480tatatgctgg atagcgactg gcccttcggc gagttctact
gcaagttgtc ccagttcatc 540gcgatgctaa gcatctgcgc ctcagtgttc
accctaatgg ccatctccat cgacagatac 600gtggccatca tccggccact
gcagccgcgg atgagcaagc ggtgcaacct ggccatcgcg 660gcggtcatct
ggctggcctc cacgctcatc tcctgcccca tgatgatcat ctaccgcacg
720gaggaggtgc cggtccgcgg gctcagcaac cgcacggtct gctacccgga
gtggcccgat 780gggcccacca atcactccac gatggagtcc ctctacaaca
tcctcatcat catyctaacc 840tacttcctgc ccatcgtctc catgacggtc
acctactcgc gcgtgggcat cgagctctgg 900ggatccaaga ccatcggcga
gtgcacgccc cgccaggtgg araaygtgcg gagtaagcga 960agggtggtga
agatgatgat tgtggtcgtc ctgatattcg ccatctgctg gctgccgttc
1020cacagctact tcataatcac atcctgctac ccggccatca cggaggcgcc
cttcatccag 1080gaactctacc tggccatcta ctggctggcc atgagcaact
ccatgtacaa tcccattata 1140tactgctgga tgaattcgcg ctttcgctat
ggtttcaaga tggtcttccg ctggtgcctg 1200tttgtgcgcg tgggcactga
accctttagt cggcgggaga acctgacatc ccggtactcc 1260tgctccggtt
ccccggatca caatcgcatc aagcgcaatg atacccagaa atcgatactt
1320tatacctgtc ccagctcacc caagtcgcat cgaatttcgc acagcggaac
aggtcgcagt 1380gcgacgctgc ggaacagtct gccggcggag tcactgtcgt
ccggcggatc tggtggtgga 1440gggcacagga aacggttgtc ctaccagcag
gaaatgcagc agcgttggtc aggacccaat 1500agtgccaccg cagtgaccaa
ttccagcagt acggccaaca ccacccaact gctctcctg 155910519PRTD.
melanogaster 10Met Glu Asn Arg Ser Asp Phe Glu Ala Asp Asp Tyr Gly
Asp Ile Ser1 5 10 15Trp Ser Asn Trp Ser Asn Trp Ser Thr Pro Ala Gly
Val Leu Phe Ser 20 25 30Ala Met Ser Ser Val Leu Ser Ala Ser Asn His
Thr Pro Leu Pro Asp 35 40 45Phe Gly Gln Glu Leu Ala Leu Ser Thr Ser
Ser Phe Asn His Ser Gln 50 55 60Thr Leu Ser Thr Asp Gln Pro Ala Val
Gly Asp Val Glu Asp Ala Ala65 70 75 80Glu Asp Ala Ala Ala Ser Met
Glu Thr Gly Ser Phe Ala Phe Val Val 85 90 95Pro Trp Trp Arg Gln Val
Leu Trp Ser Ile Leu Phe Gly Gly Met Val 100 105 110Ile Val Ala Thr
Gly Gly Asn Leu Ile Val Val Trp Ile Val Met Thr 115 120 125Thr Lys
Arg Met Arg Thr Val Thr Asn Tyr Phe Ile Val Asn Leu Ser 130 135
140Ile Ala Asp Ala Met Val Ser Ser Leu Asn Val Thr Phe Asn Tyr
Tyr145 150 155 160Tyr Met Leu Asp Ser Asp Trp Pro Phe Gly Glu Phe
Tyr Cys Lys Leu 165 170 175Ser Gln Phe Ile Ala Met Leu Ser Ile Cys
Ala Ser Val Phe Thr Leu 180 185 190Met Ala Ile Ser Ile Asp Arg Tyr
Val Ala Ile Ile Arg Pro Leu Gln 195 200 205Pro Arg Met Ser Lys Arg
Cys Asn Leu Ala Ile Ala Ala Val Ile Trp 210 215 220Leu Ala Ser Thr
Leu Ile Ser Cys Pro Met Met Ile Ile Tyr Arg Thr225 230 235 240Glu
Glu Val Pro Val Arg Gly Leu Ser Asn Arg Thr Val Cys Tyr Pro 245 250
255Glu Trp Pro Asp Gly Pro Thr Asn His Ser Thr Met Glu Ser Leu Tyr
260 265 270Asn Ile Leu Ile Ile Ile Leu Thr Tyr Phe Leu Pro Ile Val
Ser Met 275 280 285Thr Val Thr Tyr Ser Arg Val Gly Ile Glu Leu Trp
Gly Ser Lys Thr 290 295 300Ile Gly Glu Cys Thr Pro Arg Gln Val Glu
Asn Val Arg Ser Lys Arg305 310 315 320Arg Val Val Lys Met Met Ile
Val Val Val Leu Ile Phe Ala Ile Cys 325 330 335Trp Leu Pro Phe His
Ser Tyr Phe Ile Ile Thr Ser Cys Tyr Pro Ala 340 345 350Ile Thr Glu
Ala Pro Phe Ile Gln Glu Leu Tyr Leu Ala Ile Tyr Trp 355 360 365Leu
Ala Met Ser Asn Ser Met Tyr Asn Pro Ile Ile Tyr Cys Trp Met 370 375
380Asn Ser Arg Phe Arg Tyr Gly Phe Lys Met Val Phe Arg Trp Cys
Leu385 390 395 400Phe Val Arg Val Gly Thr Glu Pro Phe Ser Arg Arg
Glu Asn Leu Thr 405 410 415Ser Arg Tyr Ser Cys Ser Gly Ser Pro Asp
His Asn Arg Ile Lys Arg 420 425 430Asn Asp Thr Gln Lys Ser Ile Leu
Tyr Thr Cys Pro Ser Ser Pro Lys 435 440 445Ser His Arg Ile Ser His
Ser Gly Thr Gly Arg Ser Ala Thr Leu Arg 450 455 460Asn Ser Leu Pro
Ala Glu Ser Leu Ser Ser Gly Gly Ser Gly Gly Gly465 470 475 480Gly
His Arg Lys Arg Leu Ser Tyr Gln Gln Glu Met Gln Gln Arg Trp 485 490
495Ser Gly Pro Asn Ser Ala Thr Ala Val Thr Asn Ser Ser Ser Thr Ala
500 505 510Asn Thr Thr Gln Leu Leu Ser 515111568DNAD. melanogaster
11atggagaatc gcagtgactt cgaggcggat gactacggcg acatcagttg gagcaattgg
60agcaattgga gcaactggag cacccccgcc ggcgtccttt tctcggccat gagcagcgtg
120ctctcggcca gcaaccatac gcctctgccg gactttggcc aggagctcgc
cctatccacc 180agctccttca atcacagcca gaccctatcc accgacctgc
ccgccgtcgg ggacgtggaa 240gacgcggccg aggatgcggc ggcgtccatg
gagacgggct cgtttgcatt tgtggtcccg 300tggtggcgtc aggtgctctg
gagcatcctc ttcggcggca tggtcattgt ggcgacgggc 360ggtaacctga
ttgttgtctg gatcgtgatg acgaccaagc ggatgcggac ggtaaccaac
420tatttcatag taaatctctc catcgcggac gccatggtgt ccagcctgaa
cgtcaccttc 480aactactact acatgctgga tagcgactgg cccttcggcg
agttctactg caagttgtcc 540cagttcatcg cgatgctaag catctgcgcc
tcagtgttca ccctaatggc catctccatc 600gacagatacg tggccatcat
ccggccactg cagccgcgga tgagcaagcg gtgcaacctg 660gccatcgcgg
cggtcatctg gctggcctcc acgctcatct cctgccccat gatgatcatc
720taccgcacgg aggaggtgcc ggtccgcggg ctcagcaacc gcacggtctg
ctacccggag 780tggcccgatg ggcccaccaa tcactccacg atggagtccc
tctacaacat cctcatcatc 840attctaacct acttcctgcc catcgtctcc
atgacggtca cctactcgcg cgtgggcatc 900gagctctggg gatccaagac
catcggcgag tgcacgcccc gccaggtgga gaatgtgcgg 960agtaagcgaa
gggtggtgaa gatgatgatt gtggtcgtcc tgatattcgc catctgctgg
1020ctgccgttcc acagctactt cataatcaca tcctgctacc cggccatcac
ggaggcgccc 1080ttcatccagg aactttacct ggccatctac tggctggcca
tgagcaactc catgtacaat 1140cccattatat actgctggat gaattcgcgc
tttcgctatg gtttcaagat ggtcttccgc 1200tggtgcctgt ttgtgcgcgt
gggcactgaa ccctttagtc ggcgggagaa cctgacatcc 1260cggtactcct
gctccggttc cccggatcac aatcgcatca agcgcaatga tacccagaaa
1320tcgatacttt atacctgtcc cagctcaccc aagtcgcatc gaatttcgca
cagcggaaca 1380ggtcgcagtg cgacgctgag gaacagtctg ccggcggagt
cattgtcgtc cggtggatct 1440ggaggtggag gacacaggaa acggttgtcc
taccagcagg aaatgcagca gcggtggtca 1500ggacccaata gtgccaccgc
agtgaccaat tccagcagta cggccaacac cacccaactg 1560ctctcctg
156812522PRTD. melanogaster 12Met Glu Asn Arg Ser Asp Phe Glu Ala
Asp Asp Tyr Gly Asp Ile Ser1 5 10 15Trp Ser Asn Trp Ser Asn Trp Ser
Asn Trp Ser Thr Pro Ala Gly Val 20 25 30Leu Phe Ser Ala Met Ser Ser
Val Leu Ser Ala Ser Asn His Thr Pro 35 40 45Leu Pro Asp Phe Gly Gln
Glu Leu Ala Leu Ser Thr Ser Ser Phe Asn 50 55 60His Ser Gln Thr Leu
Ser Thr Asp Leu Pro Ala Val Gly Asp Val Glu65 70 75 80Asp Ala Ala
Glu Asp Ala Ala Ala Ser Met Glu Thr Gly Ser Phe Ala 85 90 95Phe Val
Val Pro Trp Trp Arg Gln Val Leu Trp Ser Ile Leu Phe Gly 100 105
110Gly Met Val Ile Val Ala Thr Gly Gly Asn Leu Ile Val Val Trp Ile
115 120 125Val Met Thr Thr Lys Arg Met Arg Thr Val Thr Asn Tyr Phe
Ile Val 130 135 140Asn Leu Ser Ile Ala Asp Ala Met Val Ser Ser Leu
Asn Val Thr Phe145 150 155 160Asn Tyr Tyr Tyr Met Leu Asp Ser Asp
Trp Pro Phe Gly Glu Phe Tyr 165 170 175Cys Lys Leu Ser Gln Phe Ile
Ala Met Leu Ser Ile Cys Ala Ser Val 180 185 190Phe Thr Leu Met Ala
Ile Ser Ile Asp Arg Tyr Val Ala Ile Ile Arg 195 200 205Pro Leu Gln
Pro Arg Met Ser Lys Arg Cys Asn Leu Ala Ile Ala Ala 210 215 220Val
Ile Trp Leu Ala Ser Thr Leu Ile Ser Cys Pro Met Met Ile Ile225 230
235 240Tyr Arg Thr Glu Glu Val Pro Val Arg Gly Leu Ser Asn Arg Thr
Val 245 250 255Cys Tyr Pro Glu Trp Pro Asp Gly Pro Thr Asn His Ser
Thr Met Glu 260 265 270Ser Leu Tyr Asn Ile Leu Ile Ile Ile Leu Thr
Tyr Phe Leu Pro Ile 275 280 285Val Ser Met Thr Val Thr Tyr Ser Arg
Val Gly Ile Glu Leu Trp Gly 290 295 300Ser Lys Thr Ile Gly Glu Cys
Thr Pro Arg Gln Val Glu Asn Val Arg305 310 315 320Ser Lys Arg Arg
Val Val Lys Met Met Ile Val Val Val Leu Ile Phe 325 330 335Ala Ile
Cys Trp Leu Pro Phe His Ser Tyr Phe Ile Ile Thr Ser Cys 340 345
350Tyr Pro Ala Ile Thr Glu Ala Pro Phe Ile Gln Glu Leu Tyr Leu Ala
355 360 365Ile Tyr Trp Leu Ala Met Ser Asn Ser Met Tyr Asn Pro Ile
Ile Tyr 370 375 380Cys Trp Met Asn Ser Arg Phe Arg Tyr Gly Phe Lys
Met Val Phe Arg385 390 395 400Trp Cys Leu Phe Val Arg Val Gly Thr
Glu Pro Phe Ser Arg Arg Glu 405 410 415Asn Leu Thr Ser Arg Tyr Ser
Cys Ser Gly Ser Pro Asp His Asn Arg 420 425 430Ile Lys Arg Asn Asp
Thr Gln Lys Ser Ile Leu Tyr Thr Cys Pro Ser 435 440 445Ser Pro Lys
Ser His Arg Ile Ser His Ser Gly Thr Gly Arg Ser Ala 450 455 460Thr
Leu Arg Asn Ser Leu Pro Ala Glu Ser Leu Ser Ser Gly Gly Ser465 470
475 480Gly Gly Gly Gly His Arg Lys Arg Leu Ser Tyr Gln Gln Glu Met
Gln 485 490 495Gln Arg Trp Ser Gly Pro Asn Ser Ala Thr Ala Val Thr
Asn Ser Ser 500 505 510Ser Thr Ala Asn Thr Thr Gln Leu Leu Ser 515
520131394DNAD. melanogaster 13atggagcacc acaatagcca tctgttgcct
ggtggcagcg agaagatgta ctacatagct 60caccagcagc cgatgctgcg gaacgaggat
gataactacc aggaggggta cttcatcagg 120ccggaccctg catccttact
ttacaatacc accgcactgc cagcggacga tgaagggtcc 180aactatggat
atggctccac cacaacgctc agtggcctcc agttcgagac ctataatatc
240actgtgatga tgaactttag ctgtgacgac tatgaccttc tatcggagga
catgtggtct 300agtgcctact ttaagatcat cgtctacatg ctctacattc
ccatctttat cttcgccctg 360atcggcaacg gaacggtctg ctatatcgtc
tattccacac ctcgcatgcg cacggtcacc 420aattacttta tagccagctt
ggccatcggc gacatcctga tgtccttctt ctgcgttccg 480tcgtccttca
tctcgctgtt catcctgaac tactggcctt ttggcctggc cctctgtcac
540tttgtgaact actcgcaggc ggtctcagtt ctggtcagcg cctatacttt
ggtggcaatt 600agcattgacc gctacatagc cattatgtgg ccattaaagc
cacgcatcac aaaacgctat 660gccaccttca tcatcgccgg cgtttggttt
attgcacttg ccaccgcact tcccataccc 720atcgtctctg gactcgacat
cccaatgtcg ccgtggcaca cgaaatgcga gaaatacatt 780tgccgcgaaa
tgtggccgtc gcggacgcag gagtactact acaccctgtc cctcttcgcg
840ctgcagttcg tcgtgccgct gggcgtgctc atcttcacct acgcccggat
caccattcgc 900gtctgggcga aacgaccgcc aggcgaggcg gaaaccaacc
gcgaccagcg gatggcacgc 960tccaaacgga agatggtcaa aatgatgctg
acggttgtga ttgtgttcac ctgctgttgg 1020ctgcccttca atattttgca
gcttttactg aacgacgagg agttcgccca ctgggatcct 1080ctgccgtatg
tatggttcgc gtttcactgg ctggccatgt cgcactgctg ctacaatccg
1140atcatctact gctacatgaa cgcccgtttc aggagcggat tcgtccagct
gatgcaccgt 1200atgcccggcc tgcgtcgctg gtgctgcctg cggagcgtcg
gtgatcgcat gaacgcaact 1260tccggaacgg gtccagcact tcctctcaat
cgaatgaaca catccaccac ctacatcagc 1320gctcgtcgaa agccacgagc
gacatctttg cgagcgaacc cattatcatg cggcgagacg 1380tcaccactgc ggta
139414464PRTD. melanogaster 14Met Glu His His Asn Ser His Leu Leu
Pro Gly Gly Ser Glu Lys Met1 5 10 15Tyr Tyr Ile Ala His Gln Gln Pro
Met Leu Arg Asn Glu Asp Asp Asn 20 25 30Tyr Gln Glu Gly Tyr Phe Ile
Arg Pro Asp Pro Ala Ser Leu Leu Tyr 35 40 45Asn Thr Thr Ala Leu Pro
Ala Asp Asp Glu Gly Ser Asn Tyr Gly Tyr 50 55 60Gly Ser Thr Thr Thr
Leu Ser Gly Leu Gln Phe Glu Thr Tyr Asn Ile65 70 75 80Thr Val Met
Met Asn Phe Ser Cys Asp Asp Tyr Asp Leu Leu Ser Glu 85 90 95Asp Met
Trp Ser Ser Ala Tyr Phe Lys Ile Ile Val Tyr Met Leu Tyr 100 105
110Ile Pro Ile Phe Ile Phe Ala Leu Ile Gly Asn Gly Thr Val Cys Tyr
115 120 125Ile Val Tyr Ser Thr Pro Arg Met Arg Thr Val Thr Asn Tyr
Phe Ile 130 135 140Ala Ser Leu Ala Ile Gly Asp Ile Leu Met Ser Phe
Phe Cys Val Pro145 150 155 160Ser Ser Phe Ile Ser Leu Phe Ile Leu
Asn Tyr Trp Pro Phe Gly Leu 165 170 175Ala Leu Cys His Phe Val Asn
Tyr Ser Gln Ala Val Ser Val Leu Val 180 185 190Ser Ala Tyr Thr Leu
Val Ala Ile Ser Ile Asp Arg Tyr Ile Ala Ile 195 200 205Met Trp Pro
Leu Lys Pro Arg Ile Thr Lys Arg Tyr Ala Thr Phe Ile 210 215 220Ile
Ala Gly Val Trp Phe Ile Ala Leu Ala Thr Ala Leu Pro Ile Pro225 230
235 240Ile Val Ser Gly Leu Asp Ile Pro Met Ser Pro Trp His Thr Lys
Cys 245 250 255Glu Lys Tyr Ile Cys Arg Glu Met Trp Pro Ser Arg Thr
Gln Glu Tyr 260 265 270Tyr Tyr Thr Leu Ser Leu Phe Ala Leu Gln Phe
Val Val Pro Leu Gly 275 280 285Val Leu Ile Phe Thr Tyr Ala Arg Ile
Thr Ile Arg Val Trp Ala Lys 290 295 300Arg Pro Pro Gly Glu Ala Glu
Thr Asn Arg Asp Gln Arg Met Ala Arg305 310 315 320Ser Lys Arg Lys
Met Val Lys Met Met Leu Thr Val Val Ile Val Phe 325 330 335Thr Cys
Cys Trp Leu Pro Phe Asn Ile Leu Gln Leu Leu Leu Asn Asp 340 345
350Glu Glu Phe Ala His Trp Asp Pro Leu Pro Tyr Val Trp Phe Ala Phe
355 360 365His Trp Leu Ala Met Ser His Cys Cys Tyr Asn Pro Ile Ile
Tyr Cys 370
375 380Tyr Met Asn Ala Arg Phe Arg Ser Gly Phe Val Gln Leu Met His
Arg385 390 395 400Met Pro Gly Leu Arg Arg Trp Cys Cys Leu Arg Ser
Val Gly Asp Arg 405 410 415Met Asn Ala Thr Ser Gly Thr Gly Pro Ala
Leu Pro Leu Asn Arg Met 420 425 430Asn Thr Ser Thr Thr Tyr Ile Ser
Ala Arg Arg Lys Pro Arg Ala Thr 435 440 445Ser Leu Arg Ala Asn Pro
Leu Ser Cys Gly Glu Thr Ser Pro Leu Arg 450 455 460151556DNAD.
melanogaster 15atggagcacc acaatagcca tctgttgcct ggtggcagcg
agaagatgta ctacatagct 60caccagcagc cgatgctgcg gaacgaggat gataactacc
aggaggggta cttcatcagg 120ccggaccctg catccttact ttacaatacc
accgcactgc cagcggacga tgaagggtcc 180aactatggat atggctccac
cacaacgctc agtggcctcc agttcgagac ctataatatc 240actgtgatga
tgaactttag ctgtgacgac tatgaccttc tatcggagga catgtggtct
300agtgcctact ttaagatcat cgtctacatg ctctacattc ccatctttat
cttcgccctg 360atcggcaacg gaacggtctg ctatatcgtc tattccacac
ctcgcatgcg cacggtcacc 420aattacttta tagccagctt ggccatcggc
gacatcctga tgtccttctt ctgcgttccg 480tcgtccttca tctcgctgtt
catcctgaac tactggcctt ttggcctggc cctctgtcac 540tttgtgaact
actcgcaggc ggtctcagtt ctggtcagcg cctatacttt ggtggcaatt
600agcattgacc gctacatagc cattatgtgg ccattaaagc cacgcatcac
aaaacgctat 660gccaccttca tcatcgccgg cgtttggttt attgcacttg
ccaccgcact tcccataccc 720atcgtctctg gactcgacat cccaatgtcg
ccgtggcaca cgaaatgcga gaaatacatt 780tgccgcgaaa tgtggccgtc
gcggacgcag gagtactact acaccctgtc cctcttcgcg 840ctgcagttcg
tcgtgccgct gggcgtgctc atcttcacct acgcccggat caccattcgc
900gtctgggcga aacgaccgcc aggcgaggcg gaaaccaacc gcgaccagcg
gatggcacgc 960tccaaacgga agatggtcaa aatgatgctg acggttgtga
ttgtgttcac ctgctgttgg 1020ctgcccttca atattttgca gcttttactg
aacgacgagg agttcgccca ctgggatcct 1080ctgccgtatg tgtggttcgc
gtttcactgg ctggccatgt cgcactgctg ctacaatccg 1140atcatctact
gctacatgaa cgcccgtttc aggagcggat tcgtccagct gatgcaccgt
1200atgcccggcc tgcgtcgctg gtgctgcctg cggagcgtcg gtgatcgcat
gaacgcaact 1260tccggtgaga tgactacgaa gtaccatcgc catgtcggcg
atgccctatt ccggaaaccc 1320aaaatatgca ttaggaacgg gtccagcact
tcctctcaat cgaatgaaca catccaccac 1380ctacatcagc gctcgtcgaa
agccacgagc gacatctttg cgagcgaacc cattatcatg 1440cggcgagacg
tcaccactgc ggtagctgtc atatcaaaaa ataaaactga ttcaccggtg
1500cgccgatcgg gaagctcagg tggaacagaa gcaaacataa gaagcaccga gttttg
155616518PRTD. melanogaster 16Met Glu His His Asn Ser His Leu Leu
Pro Gly Gly Ser Glu Lys Met1 5 10 15Tyr Tyr Ile Ala His Gln Gln Pro
Met Leu Arg Asn Glu Asp Asp Asn 20 25 30Tyr Gln Glu Gly Tyr Phe Ile
Arg Pro Asp Pro Ala Ser Leu Leu Tyr 35 40 45Asn Thr Thr Ala Leu Pro
Ala Asp Asp Glu Gly Ser Asn Tyr Gly Tyr 50 55 60Gly Ser Thr Thr Thr
Leu Ser Gly Leu Gln Phe Glu Thr Tyr Asn Ile65 70 75 80Thr Val Met
Met Asn Phe Ser Cys Asp Asp Tyr Asp Leu Leu Ser Glu 85 90 95Asp Met
Trp Ser Ser Ala Tyr Phe Lys Ile Ile Val Tyr Met Leu Tyr 100 105
110Ile Pro Ile Phe Ile Phe Ala Leu Ile Gly Asn Gly Thr Val Cys Tyr
115 120 125Ile Val Tyr Ser Thr Pro Arg Met Arg Thr Val Thr Asn Tyr
Phe Ile 130 135 140Ala Ser Leu Ala Ile Gly Asp Ile Leu Met Ser Phe
Phe Cys Val Pro145 150 155 160Ser Ser Phe Ile Ser Leu Phe Ile Leu
Asn Tyr Trp Pro Phe Gly Leu 165 170 175Ala Leu Cys His Phe Val Asn
Tyr Ser Gln Ala Val Ser Val Leu Val 180 185 190Ser Ala Tyr Thr Leu
Val Ala Ile Ser Ile Asp Arg Tyr Ile Ala Ile 195 200 205Met Trp Pro
Leu Lys Pro Arg Ile Thr Lys Arg Tyr Ala Thr Phe Ile 210 215 220Ile
Ala Gly Val Trp Phe Ile Ala Leu Ala Thr Ala Leu Pro Ile Pro225 230
235 240Ile Val Ser Gly Leu Asp Ile Pro Met Ser Pro Trp His Thr Lys
Cys 245 250 255Glu Lys Tyr Ile Cys Arg Glu Met Trp Pro Ser Arg Thr
Gln Glu Tyr 260 265 270Tyr Tyr Thr Leu Ser Leu Phe Ala Leu Gln Phe
Val Val Pro Leu Gly 275 280 285Val Leu Ile Phe Thr Tyr Ala Arg Ile
Thr Ile Arg Val Trp Ala Lys 290 295 300Arg Pro Pro Gly Glu Ala Glu
Thr Asn Arg Asp Gln Arg Met Ala Arg305 310 315 320Ser Lys Arg Lys
Met Val Lys Met Met Leu Thr Val Val Ile Val Phe 325 330 335Thr Cys
Cys Trp Leu Pro Phe Asn Ile Leu Gln Leu Leu Leu Asn Asp 340 345
350Glu Glu Phe Ala His Trp Asp Pro Leu Pro Tyr Val Trp Phe Ala Phe
355 360 365His Trp Leu Ala Met Ser His Cys Cys Tyr Asn Pro Ile Ile
Tyr Cys 370 375 380Tyr Met Asn Ala Arg Phe Arg Ser Gly Phe Val Gln
Leu Met His Arg385 390 395 400Met Pro Gly Leu Arg Arg Trp Cys Cys
Leu Arg Ser Val Gly Asp Arg 405 410 415Met Asn Ala Thr Ser Gly Glu
Met Thr Thr Lys Tyr His Arg His Val 420 425 430Gly Asp Ala Leu Phe
Arg Lys Pro Lys Ile Cys Ile Arg Asn Gly Ser 435 440 445Ser Thr Ser
Ser Gln Ser Asn Glu His Ile His His Leu His Gln Arg 450 455 460Ser
Ser Lys Ala Thr Ser Asp Ile Phe Ala Ser Glu Pro Ile Ile Met465 470
475 480Arg Arg Asp Val Thr Thr Ala Val Ala Val Ile Ser Lys Asn Lys
Thr 485 490 495Asp Ser Pro Val Arg Arg Ser Gly Ser Ser Gly Gly Thr
Glu Ala Asn 500 505 510Ile Arg Ser Thr Glu Phe 515171628DNAD.
melanogaster 17atggcaatgg acttaatcga gcaggagtcc cgcctggaat
tcctgcccgg agccgaggag 60gaagcagaat ttgagcgtct atacgcggct cccgctgaga
ttgtggccct gttgtccatt 120ttctatgggg gaatcagtat cgtggccgtc
attggcaaca ctttggtcat ctgggtggtg 180gccacgacca ggcaaatgcg
gaccgtgaca aatatgtata tcgctaattt ggcttttgcc 240gatgtgatta
ttggcctctt ctgcatacca tttcagttcc aggctgccct gctgcagagt
300tggaacctgc cgtggttcat gtgcagcttc tgccccttcg tccaggccct
gagtgtaaat 360gtctcggtat tcacgctgac cgccattgca atcgatcggc
atagggccat cattaatcca 420cttagggcac gtcccaccaa gttcgtatcg
aagttcataa ttggtggaat ttggatgctg 480gccctgctat ttgcggtgcc
ctttgccatt gcctttcgtg tggaggagtt gaccgaaaga 540tttcgcgaga
acaatgagac ctacaatgtg acgcggccat tctgcatgaa caagaaccta
600tccgatgatc aattgcaatc ctttcgctac accctggttt ttgtgcagta
tctggttcca 660ttctgtgtca tcagctttgt ctacatccag atggcggtac
gattgtgggg cacacgtgct 720cctggtaacg cacaggattc acgggacata
acgctgttga aaaacaagaa gaaggtcatc 780aaaatgctga ttatcgtggt
cattatcttt ggactctgct ggctgccact gcagctctat 840aatattctgt
atgtcacgat accggaaatc aacgactacc acttcattag catcgtctgg
900ttttgctgcg attggctggc catgagcaat agctgctaca atccctttat
ttatggcatc 960tacaatgaaa aatttaagcg ggaattcaac aagcgatttg
cggcctgttt ctgcaagttc 1020aagacgagca tggacgccca cgaaaggacc
ttttcgatgc acacccgcgc cagctccata 1080aggtcaacct acgccaactc
ctcgatgcga atccggagta atctctttgg tccggcgcgt 1140ggtggtgtca
acaatgggaa gccgggcttg catatgccgc gggtgcatgg atccggtgct
1200aacagcggca tttacaacgg aagtagtggg cagaacaaca atgtcaatgg
ccaacatcat 1260cagcatcaaa gcgtggttac ctttgcggcc actccgggtg
tttcggcacc aggtgttggc 1320gttgcaatgc cgccgtggcg gcgaaacaac
ttcaaacctc tgcatccgaa cgtaatcgaa 1380tgcgaggacg acgtggcact
catggagctg ccatcaacca cgccccccag cgaggagttg 1440gcatccgggg
ccggagtcca gttggccctg ctaagcaggg agagctccag ctgcatttgc
1500gaacaggaat ttggcagcca aaccgaatgc gatggcacct gcatactcag
cgaggtgtcg 1560cgagtccacc tgcccggctc gcaggcgaag gacaaggatg
cgggcaagtc cttgtggcaa 1620ccacttta 162818542PRTD. melanogaster
18Met Ala Met Asp Leu Ile Glu Gln Glu Ser Arg Leu Glu Phe Leu Pro1
5 10 15Gly Ala Glu Glu Glu Ala Glu Phe Glu Arg Leu Tyr Ala Ala Pro
Ala 20 25 30Glu Ile Val Ala Leu Leu Ser Ile Phe Tyr Gly Gly Ile Ser
Ile Val 35 40 45Ala Val Ile Gly Asn Thr Leu Val Ile Trp Val Val Ala
Thr Thr Arg 50 55 60Gln Met Arg Thr Val Thr Asn Met Tyr Ile Ala Asn
Leu Ala Phe Ala65 70 75 80Asp Val Ile Ile Gly Leu Phe Cys Ile Pro
Phe Gln Phe Gln Ala Ala 85 90 95Leu Leu Gln Ser Trp Asn Leu Pro Trp
Phe Met Cys Ser Phe Cys Pro 100 105 110Phe Val Gln Ala Leu Ser Val
Asn Val Ser Val Phe Thr Leu Thr Ala 115 120 125Ile Ala Ile Asp Arg
His Arg Ala Ile Ile Asn Pro Leu Arg Ala Arg 130 135 140Pro Thr Lys
Phe Val Ser Lys Phe Ile Ile Gly Gly Ile Trp Met Leu145 150 155
160Ala Leu Leu Phe Ala Val Pro Phe Ala Ile Ala Phe Arg Val Glu Glu
165 170 175Leu Thr Glu Arg Phe Arg Glu Asn Asn Glu Thr Tyr Asn Val
Thr Arg 180 185 190Pro Phe Cys Met Asn Lys Asn Leu Ser Asp Asp Gln
Leu Gln Ser Phe 195 200 205Arg Tyr Thr Leu Val Phe Val Gln Tyr Leu
Val Pro Phe Cys Val Ile 210 215 220Ser Phe Val Tyr Ile Gln Met Ala
Val Arg Leu Trp Gly Thr Arg Ala225 230 235 240Pro Gly Asn Ala Gln
Asp Ser Arg Asp Ile Thr Leu Leu Lys Asn Lys 245 250 255Lys Lys Val
Ile Lys Met Leu Ile Ile Val Val Ile Ile Phe Gly Leu 260 265 270Cys
Trp Leu Pro Leu Gln Leu Tyr Asn Ile Leu Tyr Val Thr Ile Pro 275 280
285Glu Ile Asn Asp Tyr His Phe Ile Ser Ile Val Trp Phe Cys Cys Asp
290 295 300Trp Leu Ala Met Ser Asn Ser Cys Tyr Asn Pro Phe Ile Tyr
Gly Ile305 310 315 320Tyr Asn Glu Lys Phe Lys Arg Glu Phe Asn Lys
Arg Phe Ala Ala Cys 325 330 335Phe Cys Lys Phe Lys Thr Ser Met Asp
Ala His Glu Arg Thr Phe Ser 340 345 350Met His Thr Arg Ala Ser Ser
Ile Arg Ser Thr Tyr Ala Asn Ser Ser 355 360 365Met Arg Ile Arg Ser
Asn Leu Phe Gly Pro Ala Arg Gly Gly Val Asn 370 375 380Asn Gly Lys
Pro Gly Leu His Met Pro Arg Val His Gly Ser Gly Ala385 390 395
400Asn Ser Gly Ile Tyr Asn Gly Ser Ser Gly Gln Asn Asn Asn Val Asn
405 410 415Gly Gln His His Gln His Gln Ser Val Val Thr Phe Ala Ala
Thr Pro 420 425 430Gly Val Ser Ala Pro Gly Val Gly Val Ala Met Pro
Pro Trp Arg Arg 435 440 445Asn Asn Phe Lys Pro Leu His Pro Asn Val
Ile Glu Cys Glu Asp Asp 450 455 460Val Ala Leu Met Glu Leu Pro Ser
Thr Thr Pro Pro Ser Glu Glu Leu465 470 475 480Ala Ser Gly Ala Gly
Val Gln Leu Ala Leu Leu Ser Arg Glu Ser Ser 485 490 495Ser Cys Ile
Cys Glu Gln Glu Phe Gly Ser Gln Thr Glu Cys Asp Gly 500 505 510Thr
Cys Ile Leu Ser Glu Val Ser Arg Val His Leu Pro Gly Ser Gln 515 520
525Ala Lys Asp Lys Asp Ala Gly Lys Ser Leu Trp Gln Pro Leu 530 535
540191451DNAD. melanogaster 19atgtttacgt ggctgatgat ggatgtcctc
cagtttgtga aaggggaaat gacagccgat 60tcagaggcaa atgccacaaa ttggtataac
acgaacgaga gcttatatac cacggaactg 120aaccatagat ggattagtgg
tagttccaca attcagccag aggagtccct ttatggcact 180gatttgccca
cctatcaaca ttgcatagcc acgcggaatt cctttgctga cttgttcact
240gtggtgctct acggatttgt gtgcattatc ggattatttg gcaacaccct
ggtgatctac 300gtggtgttgc gcttttccaa aatgcaaacg gtcacgaata
tatatatcct gaatctggcg 360gtggcagacg agtgcttcct gattggaata
ccctttctgc tgtacacaat gcgaatttgc 420agctggcgat tcggggagtt
tatgtgcaaa gcctacatgg tgagcacatc catcacctcc 480ttcacctcgt
cgatttttct gctcatcatg tccgcggatc gatatatagc ggtatgccac
540ccgatttcct cgccacgata tcgaactctg catattgcca aagtggtctc
agcgattgcc 600tggtcaactt cagcggtcct catgctgccc gtgatccttt
atgccagcac tgtggagcag 660gaggatggca tcaattactc gtgcaacata
atgtggccag atgcgtacaa gaagcattcg 720ggcaccacct tcatactgta
cacatttttc ctaggattcg ccacaccgct gtgctttatc 780ctgagtttct
actacttggt tataaggaaa ctgcgatcgg tgggtcccaa accaggaacg
840aagtccaagg agaagaggcg ggctcacagg aaggtcactc gactggtact
gacggtgata 900agtgtataca ttctatgttg gctccctcac tggatttctc
aggtggccct gattcactcg 960aatcccgcgc aaagggacct ctcccgactg
gaaatactca ttttcctact tctgggggca 1020ctggtttact cgaattcggc
ggtgaatccc atactttatg ccttcctaag tgagaacttc 1080cggaagagct
tcttcaaggc ctttacctgt atgaataagc aggatatcaa cgctcaactc
1140cagctggagc ccagtgtttt caccaaacag ggcagtaaaa agaggggtgg
ctccaagcgc 1200ctgttgacca gcaatccgca gattcctcca ctgctgccac
tgaatgcggg taacaacaat 1260tcatcgacca ccacatcctc gaccacgaca
gcggaaaaga ccggaaccac ggggacacag 1320aaatcatgca attccaatgg
caaagtgaca gctccgccgg agaatttgat tatatgtttg 1380agcgagcagc
aggaggcatt ttgcaccacc gcgagaagag gatcgggcgc agtgcagcag
1440acagatttgt a 145120483PRTD. melanogaster 20Met Phe Thr Trp Leu
Met Met Asp Val Leu Gln Phe Val Lys Gly Glu1 5 10 15Met Thr Ala Asp
Ser Glu Ala Asn Ala Thr Asn Trp Tyr Asn Thr Asn 20 25 30Glu Ser Leu
Tyr Thr Thr Glu Leu Asn His Arg Trp Ile Ser Gly Ser 35 40 45Ser Thr
Ile Gln Pro Glu Glu Ser Leu Tyr Gly Thr Asp Leu Pro Thr 50 55 60Tyr
Gln His Cys Ile Ala Thr Arg Asn Ser Phe Ala Asp Leu Phe Thr65 70 75
80Val Val Leu Tyr Gly Phe Val Cys Ile Ile Gly Leu Phe Gly Asn Thr
85 90 95Leu Val Ile Tyr Val Val Leu Arg Phe Ser Lys Met Gln Thr Val
Thr 100 105 110Asn Ile Tyr Ile Leu Asn Leu Ala Val Ala Asp Glu Cys
Phe Leu Ile 115 120 125Gly Ile Pro Phe Leu Leu Tyr Thr Met Arg Ile
Cys Ser Trp Arg Phe 130 135 140Gly Glu Phe Met Cys Lys Ala Tyr Met
Val Ser Thr Ser Ile Thr Ser145 150 155 160Phe Thr Ser Ser Ile Phe
Leu Leu Ile Met Ser Ala Asp Arg Tyr Ile 165 170 175Ala Val Cys His
Pro Ile Ser Ser Pro Arg Tyr Arg Thr Leu His Ile 180 185 190Ala Lys
Val Val Ser Ala Ile Ala Trp Ser Thr Ser Ala Val Leu Met 195 200
205Leu Pro Val Ile Leu Tyr Ala Ser Thr Val Glu Gln Glu Asp Gly Ile
210 215 220Asn Tyr Ser Cys Asn Ile Met Trp Pro Asp Ala Tyr Lys Lys
His Ser225 230 235 240Gly Thr Thr Phe Ile Leu Tyr Thr Phe Phe Leu
Gly Phe Ala Thr Pro 245 250 255Leu Cys Phe Ile Leu Ser Phe Tyr Tyr
Leu Val Ile Arg Lys Leu Arg 260 265 270Ser Val Gly Pro Lys Pro Gly
Thr Lys Ser Lys Glu Lys Arg Arg Ala 275 280 285His Arg Lys Val Thr
Arg Leu Val Leu Thr Val Ile Ser Val Tyr Ile 290 295 300Leu Cys Trp
Leu Pro His Trp Ile Ser Gln Val Ala Leu Ile His Ser305 310 315
320Asn Pro Ala Gln Arg Asp Leu Ser Arg Leu Glu Ile Leu Ile Phe Leu
325 330 335Leu Leu Gly Ala Leu Val Tyr Ser Asn Ser Ala Val Asn Pro
Ile Leu 340 345 350Tyr Ala Phe Leu Ser Glu Asn Phe Arg Lys Ser Phe
Phe Lys Ala Phe 355 360 365Thr Cys Met Asn Lys Gln Asp Ile Asn Ala
Gln Leu Gln Leu Glu Pro 370 375 380Ser Val Phe Thr Lys Gln Gly Ser
Lys Lys Arg Gly Gly Ser Lys Arg385 390 395 400Leu Leu Thr Ser Asn
Pro Gln Ile Pro Pro Leu Leu Pro Leu Asn Ala 405 410 415Gly Asn Asn
Asn Ser Ser Thr Thr Thr Ser Ser Thr Thr Thr Ala Glu 420 425 430Lys
Thr Gly Thr Thr Gly Thr Gln Lys Ser Cys Asn Ser Asn Gly Lys 435 440
445Val Thr Ala Pro Pro Glu Asn Leu Ile Ile Cys Leu Ser Glu Gln Gln
450 455 460Glu Ala Phe Cys Thr Thr Ala Arg Arg Gly Ser Gly Ala Val
Gln Gln465 470 475 480Thr Asp Leu211754DNAD. melanogaster
21atgttcaact acgaggaggg ggatgccgac caggcggcca tggctgcagc ggctgcctat
60agggcactgc tcgactacta tgccaatgcg ccaagtgcgg cgggtcacat agtgtcgctc
120aacgtggcac cctacaatgg aactggaaac ggaggcactg tctccttggc
gggcaatgcg 180acaagcagct atggcgatga tgatagggat ggctatatgg
acaccgagcc cagtgacctg 240gtcaccgaac tggccttctc
cctgggcacc agttcaagtc caagtcccag ttccacaccc 300gcttccagct
ccagtacttc cactggcatg cccgtctggc tgatacccag ctatagcatg
360attctgctgt tcgccgtgct gggcaacctg ctggtcatct cgacgctggt
gcagaatcgc 420cggatgcgta ccataaccaa cgtgttcctg ctcaacctgg
ccatatcgga catgctgctg 480ggcgtgctct gcatgcccgt caccctggtg
ggcaccctgc tgcgaaactt catctttggc 540gagttcctct gcaagctctt
tcagttctcg caagccgcct ccgtggccgt ttcgtcctgg 600accttggtgg
ccatatcctg tgagcgctac tacgcgatat gccatccact gcgctcgcga
660tcctggcaga caatcagtca cgcctacaag atcatcggct tcatctggct
gggcggcatc 720ctctgcatga cgcccatagc ggtctttagt caattgatac
ccaccagtcg accgggctac 780tgcaagtgcc gtgagttttg gcccgaccag
ggatacgagc tcttctacaa catcctgctg 840gacttcctgc tgctcgtcct
gccgcttctc gtcctctgcg tggcctacat cctcatcacg 900cgtaccctgt
acgtaggcat ggccaaggac agcggacgca tcctgcagca atcgctgcct
960gtttccgcta caacggccgg cggaagcgca ccgaatccgg gcaccagcag
cagtagtaac 1020tgcatcctgg tcctgaccgc caccgcagtc tataatgaaa
atagtaacaa taataatgga 1080aattcagagg gatccgcagg cggaggatca
accaatatgg caacgaccac cttgacaacg 1140agaccaacgg ctccaactgt
gatcaccacc accacgacga ccacggtgac gctggccaag 1200acctcctcgc
ccagcattcg cgtccacgat gcggcacttc gcaggtccaa cgaggccaag
1260accctggaga gcaagaagcg tgtggtcaag atgctgttcg tcctggtgct
ggagtttttc 1320atctgctgga ctccgctgta cgtgatcaac acgatggtca
tgctgatcgg accggtggtg 1380tacgagtatg tcgactacac ggccatcagt
ttcctccagc tgctggccta ctcatccagc 1440tgctgcaatc cgatcaccta
ctgcttcatg aacgccagct tccggcgcgc ctttgtcgac 1500accttcaagg
gtctgccctg gcgtcgtgga gcaggtgcca gcggaggcgt cggtggtgct
1560gctggtggag gactctccgc cagccaggcg ggcgcaggcc cgggcgccta
tgcgagtgcc 1620aacaccaaca ttagtctcaa tcccggccta gccatgggta
tgggcacctg gcggagtcgc 1680tcacgccacg agtttctcaa tgcggtggtg
accaccaata gtgccgccgc cgccgtcaac 1740agtcctcagc tcta 175422584PRTD.
melanogaster 22Met Phe Asn Tyr Glu Glu Gly Asp Ala Asp Gln Ala Ala
Met Ala Ala1 5 10 15Ala Ala Ala Tyr Arg Ala Leu Leu Asp Tyr Tyr Ala
Asn Ala Pro Ser 20 25 30Ala Ala Gly His Ile Val Ser Leu Asn Val Ala
Pro Tyr Asn Gly Thr 35 40 45Gly Asn Gly Gly Thr Val Ser Leu Ala Gly
Asn Ala Thr Ser Ser Tyr 50 55 60Gly Asp Asp Asp Arg Asp Gly Tyr Met
Asp Thr Glu Pro Ser Asp Leu65 70 75 80Val Thr Glu Leu Ala Phe Ser
Leu Gly Thr Ser Ser Ser Pro Ser Pro 85 90 95Ser Ser Thr Pro Ala Ser
Ser Ser Ser Thr Ser Thr Gly Met Pro Val 100 105 110Trp Leu Ile Pro
Ser Tyr Ser Met Ile Leu Leu Phe Ala Val Leu Gly 115 120 125Asn Leu
Leu Val Ile Ser Thr Leu Val Gln Asn Arg Arg Met Arg Thr 130 135
140Ile Thr Asn Val Phe Leu Leu Asn Leu Ala Ile Ser Asp Met Leu
Leu145 150 155 160Gly Val Leu Cys Met Pro Val Thr Leu Val Gly Thr
Leu Leu Arg Asn 165 170 175Phe Ile Phe Gly Glu Phe Leu Cys Lys Leu
Phe Gln Phe Ser Gln Ala 180 185 190Ala Ser Val Ala Val Ser Ser Trp
Thr Leu Val Ala Ile Ser Cys Glu 195 200 205Arg Tyr Tyr Ala Ile Cys
His Pro Leu Arg Ser Arg Ser Trp Gln Thr 210 215 220Ile Ser His Ala
Tyr Lys Ile Ile Gly Phe Ile Trp Leu Gly Gly Ile225 230 235 240Leu
Cys Met Thr Pro Ile Ala Val Phe Ser Gln Leu Ile Pro Thr Ser 245 250
255Arg Pro Gly Tyr Cys Lys Cys Arg Glu Phe Trp Pro Asp Gln Gly Tyr
260 265 270Glu Leu Phe Tyr Asn Ile Leu Leu Asp Phe Leu Leu Leu Val
Leu Pro 275 280 285Leu Leu Val Leu Cys Val Ala Tyr Ile Leu Ile Thr
Arg Thr Leu Tyr 290 295 300Val Gly Met Ala Lys Asp Ser Gly Arg Ile
Leu Gln Gln Ser Leu Pro305 310 315 320Val Ser Ala Thr Thr Ala Gly
Gly Ser Ala Pro Asn Pro Gly Thr Ser 325 330 335Ser Ser Ser Asn Cys
Ile Leu Val Leu Thr Ala Thr Ala Val Tyr Asn 340 345 350Glu Asn Ser
Asn Asn Asn Asn Gly Asn Ser Glu Gly Ser Ala Gly Gly 355 360 365Gly
Ser Thr Asn Met Ala Thr Thr Thr Leu Thr Thr Arg Pro Thr Ala 370 375
380Pro Thr Val Ile Thr Thr Thr Thr Thr Thr Thr Val Thr Leu Ala
Lys385 390 395 400Thr Ser Ser Pro Ser Ile Arg Val His Asp Ala Ala
Leu Arg Arg Ser 405 410 415Asn Glu Ala Lys Thr Leu Glu Ser Lys Lys
Arg Val Val Lys Met Leu 420 425 430Phe Val Leu Val Leu Glu Phe Phe
Ile Cys Trp Thr Pro Leu Tyr Val 435 440 445Ile Asn Thr Met Val Met
Leu Ile Gly Pro Val Val Tyr Glu Tyr Val 450 455 460Asp Tyr Thr Ala
Ile Ser Phe Leu Gln Leu Leu Ala Tyr Ser Ser Ser465 470 475 480Cys
Cys Asn Pro Ile Thr Tyr Cys Phe Met Asn Ala Ser Phe Arg Arg 485 490
495Ala Phe Val Asp Thr Phe Lys Gly Leu Pro Trp Arg Arg Gly Ala Gly
500 505 510Ala Ser Gly Gly Val Gly Gly Ala Ala Gly Gly Gly Leu Ser
Ala Ser 515 520 525Gln Ala Gly Ala Gly Pro Gly Ala Tyr Ala Ser Ala
Asn Thr Asn Ile 530 535 540Ser Leu Asn Pro Gly Leu Ala Met Gly Met
Gly Thr Trp Arg Ser Arg545 550 555 560Ser Arg His Glu Phe Leu Asn
Ala Val Val Thr Thr Asn Ser Ala Ala 565 570 575Ala Ala Val Asn Ser
Pro Gln Leu 580231452DNAD. melanogaster 23atgtacgcct ccttgatgga
cgttggccag acgttggcag ccaggctggc ggatagcgac 60ggcaacgggg ccaatgacag
cggactcctg gcaaccggac aaggtctgga gcaggagcag 120gagggtctgg
cactggatat gggccacaat gccagcgccg acggcggaat agtaccgtat
180gtgcccgtgc tggaccgccc ggagacgtac attgtcaccg tgctgtacac
gctcatcttc 240attgtgggag ttttgggcaa cggcacgctg gtcatcatct
tctttcgcca ccgctccatg 300cgcaacatac ccaacacata cattctttca
ctggccctgg ctgatctgtt ggttatattg 360gtgtgtgtac ctgtggccac
gattgtctac acgcaggaaa gctggccctt tgagcggaac 420atgtgccgca
tcagcgagtt ctttaaggac atatccatcg gggtgtccgt gtttacactg
480accgcccttt ccggcgagcg gtactgcgcc attgtaaatc ccctacgcaa
gcttcagacc 540aagccgctca ctgtctttac tgcggtgatg atctggatcc
tggccatcct actgggcatg 600ccttcggttc ttttctccga catcaagtcc
taccctgtgt tcacagccac cggtaacatg 660accattgaag tgtgctcccc
atttcgcgac ccggagtatg caaagttcat ggtggcgggc 720aaggcactgg
tgtactacct gttgccgctg tccatcattg gggcgctata catcatgatg
780gccaagcggc tccatatgag cgcccgcaac atgcccggcg aacagcagag
catgcagagc 840cgcacccagg ctagggcccg actccatgtg gcgcgcatgg
tggtagcatt cgtggtggtg 900ttcttcatct gcttcttccc gtaccacgtg
tttgagctgt ggtaccactt ctacccaacg 960gctgaggagg acttcgatga
gttctggaac gtgctgcgca tccttcctaa actcgtgcgt 1020caaccccgtg
gcctctactg cgtgtccggg gtgtttcggc agcactttaa tcgctacctc
1080tgctgcatct gcgtcaagcg gcagccgcac ctgcggcagc actcaacggc
cactggaatg 1140atggacaata ccagtgtgat gtccatgcgc cgctccacgt
acgtgggtgg aaccgctggc 1200aatctgcggg cctcgctgca ccggaacagc
aatcacggag ttggtggagc tggaggtgga 1260gtaggaggag gagtagggtc
aggtcgtgtg ggcagctttc atcggcagga ctcgatgccc 1320ctgcagcacg
gaaatgccca cggaggtggt gcgggcgggg gatcctccgg acttggagcc
1380ggcgggcgga cggcggcagt gagcgaaaag agctttataa atcgttacga
aagtggcgta 1440atgcgctact aa 145224483PRTD. melanogaster 24Met Tyr
Ala Ser Leu Met Asp Val Gly Gln Thr Leu Ala Ala Arg Leu1 5 10 15Ala
Asp Ser Asp Gly Asn Gly Ala Asn Asp Ser Gly Leu Leu Ala Thr 20 25
30Gly Gln Gly Leu Glu Gln Glu Gln Glu Gly Leu Ala Leu Asp Met Gly
35 40 45His Asn Ala Ser Ala Asp Gly Gly Ile Val Pro Tyr Val Pro Val
Leu 50 55 60Asp Arg Pro Glu Thr Tyr Ile Val Thr Val Leu Tyr Thr Leu
Ile Phe65 70 75 80Ile Val Gly Val Leu Gly Asn Gly Thr Leu Val Ile
Ile Phe Phe Arg 85 90 95His Arg Ser Met Arg Asn Ile Pro Asn Thr Tyr
Ile Leu Ser Leu Ala 100 105 110Leu Ala Asp Leu Leu Val Ile Leu Val
Cys Val Pro Val Ala Thr Ile 115 120 125Val Tyr Thr Gln Glu Ser Trp
Pro Phe Glu Arg Asn Met Cys Arg Ile 130 135 140Ser Glu Phe Phe Lys
Asp Ile Ser Ile Gly Val Ser Val Phe Thr Leu145 150 155 160Thr Ala
Leu Ser Gly Glu Arg Tyr Cys Ala Ile Val Asn Pro Leu Arg 165 170
175Lys Leu Gln Thr Lys Pro Leu Thr Val Phe Thr Ala Val Met Ile Trp
180 185 190Ile Leu Ala Ile Leu Leu Gly Met Pro Ser Val Leu Phe Ser
Asp Ile 195 200 205Lys Ser Tyr Pro Val Phe Thr Ala Thr Gly Asn Met
Thr Ile Glu Val 210 215 220Cys Ser Pro Phe Arg Asp Pro Glu Tyr Ala
Lys Phe Met Val Ala Gly225 230 235 240Lys Ala Leu Val Tyr Tyr Leu
Leu Pro Leu Ser Ile Ile Gly Ala Leu 245 250 255Tyr Ile Met Met Ala
Lys Arg Leu His Met Ser Ala Arg Asn Met Pro 260 265 270Gly Glu Gln
Gln Ser Met Gln Ser Arg Thr Gln Ala Arg Ala Arg Leu 275 280 285His
Val Ala Arg Met Val Val Ala Phe Val Val Val Phe Phe Ile Cys 290 295
300Phe Phe Pro Tyr His Val Phe Glu Leu Trp Tyr His Phe Tyr Pro
Thr305 310 315 320Ala Glu Glu Asp Phe Asp Glu Phe Trp Asn Val Leu
Arg Ile Leu Pro 325 330 335Lys Leu Val Arg Gln Pro Arg Gly Leu Tyr
Cys Val Ser Gly Val Phe 340 345 350Arg Gln His Phe Asn Arg Tyr Leu
Cys Cys Ile Cys Val Lys Arg Gln 355 360 365Pro His Leu Arg Gln His
Ser Thr Ala Thr Gly Met Met Asp Asn Thr 370 375 380Ser Val Met Ser
Met Arg Arg Ser Thr Tyr Val Gly Gly Thr Ala Gly385 390 395 400Asn
Leu Arg Ala Ser Leu His Arg Asn Ser Asn His Gly Val Gly Gly 405 410
415Ala Gly Gly Gly Val Gly Gly Gly Val Gly Ser Gly Arg Val Gly Ser
420 425 430Phe His Arg Gln Asp Ser Met Pro Leu Gln His Gly Asn Ala
His Gly 435 440 445Gly Gly Ala Gly Gly Gly Ser Ser Gly Leu Gly Ala
Gly Gly Arg Thr 450 455 460Ala Ala Val Ser Glu Lys Ser Phe Ile Asn
Arg Tyr Glu Ser Gly Val465 470 475 480Met Arg Tyr2510PRTArtificial
SequenceNovel Sequence 25Thr Asp Val Asp His Val Phe Leu Arg Phe1 5
10269PRTArtificial SequenceNovel Sequence 26Asp Pro Lys Gln Asp Phe
Met Arg Phe1 5277PRTArtificial SequenceNovel Sequence 27Pro Asp Asn
Phe Met Arg Phe1 5289PRTArtificial SequenceNovel Sequence 28Thr Pro
Ala Glu Asp Phe Met Arg Phe1 5299PRTArtificial SequenceNovel
Sequence 29Ser Leu Lys Gln Asp Phe Met His Phe1 5309PRTArtificial
SequenceNovel Sequence 30Ser Val Lys Gln Asp Phe Met His Phe1
5316PRTArtificial SequenceNovel Sequence 31Ala Ala Met Asp Arg Tyr1
5329PRTArtificial SequenceNovel Sequence 32Ser Val Gln Asp Asn Phe
Met His Phe1 53310PRTArtificial SequenceNovel Sequence 33Ala Arg
Gly Pro Gln Leu Arg Leu Arg Phe1 5 103410PRTArtificial
SequenceNovel Sequence 34Gly Asp Gly Arg Leu Tyr Ala Phe Gly Leu1 5
10358PRTArtificial SequenceNovel Sequence 35Asp Arg Leu Tyr Ser Phe
Gly Leu1 53613PRTArtificial SequenceNovel Sequence 36Ala Pro Ser
Gly Ala Gln Arg Leu Tyr Gly Phe Gly Leu1 5 10379PRTArtificial
SequenceNovel Sequence 37Gly Gly Ser Leu Tyr Ser Phe Gly Leu1
5384PRTArtificial SequenceNovel Sequence 38Phe Ile Arg
Phe1397PRTArtificial SequenceNovel Sequence 39Lys Asn Glu Phe Ile
Arg Phe1 5404PRTArtificial SequenceNovel Sequence 40Phe Met Arg
Phe1417PRTArtificial SequenceNovel Sequence 41Lys Ser Ala Phe Met
Arg Phe1 5427PRTArtificial SequenceNovel Sequence 42Lys Pro Asn Phe
Leu Arg Phe1 5434PRTArtificial SequenceNovel Sequence 43Phe Leu Arg
Phe1444PRTArtificial SequenceNovel Sequence 44Tyr Leu Arg
Phe1457PRTArtificial SequenceNovel Sequence 45Lys Pro Asn Phe Leu
Arg Tyr1 5468PRTArtificial SequenceNovel Sequence 46Thr Asn Arg Asn
Phe Leu Arg Phe1 5479PRTArtificial SequenceNovel Sequence 47Arg Asn
Lys Phe Glu Phe Ile Arg Phe1 5488PRTArtificial SequenceNovel
Sequence 48Ala Gly Pro Arg Phe Ile Arg Phe1 5499PRTArtificial
SequenceNovel Sequence 49Gly Leu Gly Pro Arg Pro Leu Arg Phe1
5502PRTArtificial SequenceNovel Sequence 50Ile Leu1518PRTArtificial
SequenceNovel Sequence 51Ala Gly Ala Lys Ile Phe Arg Phe1
5529PRTArtificial SequenceNovel Sequence 52Ala Pro Lys Pro Lys Phe
Ile Arg Phe1 5538PRTArtificial SequenceNovel Sequence 53Lys Ser Ala
Phe Val Leu Arg Phe1 5549PRTArtificial SequenceNovel Sequence 54Thr
Lys Phe Gln Asp Phe Leu Arg Phe1 55510PRTArtificial SequenceNovel
Sequence 55Ser Ala Glu Pro Phe Gly Thr Met Arg Phe1 5
105612PRTArtificial SequenceNovel Sequence 56Ala Ser Glu Asp Ala
Leu Phe Gly Thr Met Arg Phe1 5 105713PRTArtificial SequenceNovel
Sequence 57Ser Ala Asp Asp Ser Ala Pro Phe Gly Thr Met Arg Phe1 5
105812PRTArtificial SequenceNovel Sequence 58Glu Asp Gly Asn Ala
Pro Phe Gly Thr Met Arg Phe1 5 10598PRTArtificial SequenceNovel
Sequence 59Phe Leu Phe Gln Pro Gln Arg Phe1 5609PRTArtificial
SequenceNovel Sequence 60Ser Ala Asp Pro Asn Phe Leu Arg Phe1
5618PRTArtificial SequenceNovel Sequence 61Ser Gln Pro Asn Phe Leu
Arg Phe1 56210PRTArtificial SequenceNovel Sequence 62Ala Ser Gly
Asp Pro Asn Phe Leu Arg Phe1 5 10638PRTArtificial SequenceNovel
Sequence 63Ser Asp Pro Asn Phe Leu Arg Phe1 56410PRTArtificial
SequenceNovel Sequence 64Ala Ala Ala Asp Pro Asn Phe Leu Arg Phe1 5
10656PRTArtificial SequenceNovel Sequence 65Pro Asn Phe Leu Arg
Phe1 5666PRTArtificial SequenceNovel Sequence 66Lys Pro Phe Leu Arg
Phe1 56710PRTArtificial SequenceNovel Sequence 67Ala Gly Ser Asp
Pro Asn Phe Leu Arg Phe1 5 10687PRTArtificial SequenceNovel
Sequence 68Lys Pro Asn Phe Leu Arg Tyr1 5698PRTArtificial
SequenceNovel Sequence 69Ser Pro Arg Glu Pro Ile Arg Phe1
5708PRTArtificial SequenceNovel Sequence 70Leu Arg Gly Glu Pro Ile
Arg Phe1 5718PRTArtificial SequenceNovel Sequence 71Ser Pro Leu Gly
Thr Met Arg Phe1 57211PRTArtificial SequenceNovel Sequence 72Glu
Ala Glu Glu Pro Leu Gly Thr Met Arg Phe1 5 107312PRTArtificial
SequenceNovel Sequence 73Ala Ser Glu Asp Ala Leu Phe Gly Thr Met
Arg Phe1 5 107412PRTArtificial SequenceNovel Sequence 74Glu Asp Gly
Asn Ala Pro Phe Gly Thr Met Arg Phe1 5 107510PRTArtificial
SequenceNovel Sequence 75Ser Ala Glu Pro Phe Gly Thr Met Arg Phe1 5
107613PRTArtificial SequenceNovel Sequence 76Ser Ala Asp Asp Ser
Ala Pro Phe Gly Thr Met Arg Phe1 5 10777PRTArtificial SequenceNovel
Sequence 77Lys Pro Thr Phe Ile Arg Phe1 5788PRTArtificial
SequenceNovel Sequence 78Ala Ser Pro Ser Phe Ile Arg Phe1
5797PRTArtificial SequenceNovel Sequence 79Gly Ala Lys Phe Ile Arg
Phe1 5808PRTArtificial SequenceNovel Sequence 80Ala Gly Ala Lys Phe
Ile Arg Phe1 5819PRTArtificial SequenceNovel Sequence 81Ala Pro Lys
Pro Lys Phe Ile Arg Phe1 5827PRTArtificial SequenceNovel Sequence
82Lys Ser Ala Tyr Met Arg Phe1 58311PRTArtificial SequenceNovel
Sequence 83Ser Pro Met Gln Arg Ser Ser Met Val Arg Phe1 5
108411PRTArtificial SequenceNovel Sequence 84Ser Pro Met Glu Arg
Ser Ala Met Val Arg Phe1 5 108511PRTArtificial SequenceNovel
Sequence 85Ser Pro Met Asp Arg Ser Lys Met Val Arg Phe1 5
10867PRTArtificial SequenceNovel Sequence 86Lys Asn Glu Phe Ile Arg
Phe1 5877PRTArtificial SequenceNovel Sequence 87Lys Pro Ser Phe Val
Arg Phe1 58811PRTArtificial SequenceNovel Sequence 88Gln Pro Lys
Ala Arg Ser Gly Tyr Ile Arg Phe1 5 10899PRTArtificial SequenceNovel
Sequence 89Ala Met Arg Asn Ala Leu Val Arg Phe1 59012PRTArtificial
SequenceNovel Sequence 90Ala Ser Gly Gly Met Arg Asn Ala Leu Val
Arg Phe1 5
109110PRTArtificial SequenceNovel Sequence 91Asn Gly Ala Pro Gln
Pro Phe Val Arg Phe1 5 10929PRTArtificial SequenceNovel Sequence
92Arg Asn Lys Phe Glu Phe Ile Arg Phe1 59314PRTArtificial
SequenceNovel Sequence 93Ser Asp Arg Pro Thr Arg Ala Met Asp Ser
Pro Ile Arg Phe1 5 109410PRTArtificial SequenceNovel Sequence 94Ala
Ala Asp Gly Ala Pro Leu Ile Arg Phe1 5 109510PRTArtificial
SequenceNovel Sequence 95Ala Pro Glu Ala Ser Pro Phe Ile Arg Phe1 5
109610PRTArtificial SequenceNovel Sequence 96Ala Ser Pro Ser Ala
Pro Leu Ile Arg Phe1 5 109710PRTArtificial SequenceNovel Sequence
97Ser Pro Ser Ala Val Pro Leu Ile Arg Phe1 5 10989PRTArtificial
SequenceNovel Sequence 98Ala Ser Ser Ala Pro Leu Ile Arg Phe1
5997PRTArtificial SequenceNovel Sequence 99Lys His Glu Tyr Leu Arg
Phe1 51006PRTArtificial SequenceNovel Sequence 100Ser Leu Asp Tyr
Arg Phe1 510114PRTArtificial SequenceNovel Sequence 101Glu Ile Val
Phe His Gln Ile Ser Pro Ile Phe Phe Arg Phe1 5 101029PRTArtificial
SequenceNovel Sequence 102Gly Gly Pro Gln Gly Pro Leu Arg Phe1
51038PRTArtificial SequenceNovel Sequence 103Gly Pro Ser Gly Pro
Leu Arg Phe1 51047PRTArtificial SequenceNovel Sequence 104Ala Gln
Thr Phe Val Arg Phe1 51057PRTArtificial SequenceNovel Sequence
105Gly Gln Thr Phe Val Arg Phe1 51067PRTArtificial SequenceNovel
Sequence 106Lys Ser Ala Phe Val Arg Phe1 51077PRTArtificial
SequenceNovel Sequence 107Lys Ser Gln Tyr Ile Arg Phe1
51088PRTArtificial SequenceNovel Sequence 108Asp Val Pro Gly Val
Leu Arg Phe1 51099PRTArtificial SequenceNovel Sequence 109Lys Ser
Val Pro Gly Val Leu Arg Phe1 51109PRTArtificial SequenceNovel
Sequence 110Ser Glu Val Pro Gly Val Leu Arg Phe1 51118PRTArtificial
SequenceNovel Sequence 111Ser Val Pro Gly Val Leu Arg Phe1
511212PRTArtificial SequenceNovel Sequence 112Asp Phe Asp Gly Ala
Met Pro Gly Val Leu Arg Phe1 5 101138PRTArtificial SequenceNovel
Sequence 113Glu Ile Pro Gly Val Leu Arg Phe1 51147PRTArtificial
SequenceNovel Sequence 114Trp Ala Asn Gln Val Arg Phe1
51159PRTArtificial SequenceNovel Sequence 115Ala Ser Trp Ala Ser
Ser Val Arg Phe1 51165PRTArtificial SequenceNovel Sequence 116Ala
Met Met Arg Phe1 51179PRTArtificial SequenceNovel Sequence 117Gly
Leu Gly Pro Arg Pro Leu Arg Phe1 51189PRTArtificial SequenceNovel
Sequence 118Ser Pro Ser Ala Lys Trp Met Arg Phe1 51199PRTArtificial
SequenceNovel Sequence 119Thr Lys Phe Gln Asp Phe Leu Arg Phe1
512010PRTArtificial SequenceNovel Sequence 120Glu Asp Arg Asp Tyr
Arg Pro Leu Gln Phe1 5 101214PRTArtificial SequenceNovel Sequence
121Phe Ile Arg Phe11228PRTArtificial SequenceNovel Sequence 122Ala
Val Pro Gly Val Leu Arg Phe1 51239PRTArtificial SequenceNovel
Sequence 123Gly Asp Val Pro Gly Val Leu Arg Phe1
512413PRTArtificial SequenceNovel Sequence 124Ser Asp Ile Gly Ile
Ser Glu Pro Asn Phe Leu Arg Phe1 5 101259PRTArtificial
SequenceNovel Sequence 125Ser Gly Lys Pro Thr Phe Ile Arg Phe1
512611PRTArtificial SequenceNovel Sequence 126Ala Glu Gly Leu Ser
Ser Pro Leu Ile Arg Phe1 5 101278PRTArtificial SequenceNovel
Sequence 127Phe Asp Arg Asp Phe Met Arg Phe1 51288PRTArtificial
SequenceNovel Sequence 128Ala Gly Pro Arg Phe Ile Arg Phe1
51298PRTArtificial SequenceNovel Sequence 129Gly Met Pro Gly Val
Leu Arg Phe1 51302PRTArtificial SequenceNovel Sequence 130Ile
Leu11318PRTArtificial SequenceNovel Sequence 131Leu Gln Pro Asn Phe
Leu Arg Phe1 51327PRTArtificial SequenceNovel Sequence 132Lys Pro
Asn Phe Ile Arg Phe1 51334PRTArtificial SequenceNovel Sequence
133Phe Met Arg Phe11344PRTArtificial SequenceNovel Sequence 134Phe
Leu Arg Phe11354PRTArtificial SequenceNovel Sequence 135Tyr Ile Arg
Phe11367PRTArtificial SequenceNovel Sequence 136Gly Asn Ser Phe Leu
Arg Phe1 51377PRTArtificial SequenceNovel Sequence 137Asp Pro Ser
Phe Leu Arg Phe1 51386PRTArtificial SequenceNovel Sequence 138Gln
Asp Phe Met Arg Phe1 51399PRTArtificial SequenceNovel Sequence
139Lys Pro Asn Gln Asp Phe Met Arg Phe1 514010PRTArtificial
SequenceNovel Sequence 140Thr Asp Val Asp His Val Phe Leu Arg Phe1
5 101416PRTArtificial SequenceNovel Sequence 141Ala Ala Met Asp Arg
Tyr1 51429PRTArtificial SequenceNovel Sequence 142Ser Pro Lys Gln
Asp Phe Met Arg Phe1 51437PRTArtificial SequenceNovel Sequence
143Pro Asp Asn Phe Met Arg Phe1 51449PRTArtificial SequenceNovel
Sequence 144Asp Pro Lys Gln Asp Phe Met Arg Phe1 51459PRTArtificial
SequenceNovel Sequence 145Thr Pro Ala Glu Asp Phe Met Arg Phe1
51467PRTArtificial SequenceNovel Sequence 146Ser Asp Asn Phe Met
Arg Phe1 51474PRTArtificial SequenceNovel Sequence 147Tyr Leu Arg
Phe11488PRTArtificial SequenceNovel Sequence 148Ser Asp Arg Asn Phe
Leu Arg Phe1 51498PRTArtificial SequenceNovel Sequence 149Thr Asn
Arg Asn Phe Leu Arg Phe1 515010PRTArtificial SequenceNovel Sequence
150Pro Asp Val Asp His Val Phe Leu Arg Phe1 5 1015110PRTArtificial
SequenceNovel Sequence 151Gln Asp Val Asp His Val Phe Leu Arg Phe1
5 101528PRTArtificial SequenceNovel Sequence 152Phe Leu Phe Gln Pro
Gln Arg Phe1 515310PRTArtificial SequenceNovel Sequence 153Ala Arg
Gly Pro Gln Leu Arg Leu Arg Phe1 5 101549PRTArtificial
SequenceNovel Sequence 154Phe Asp Asp Tyr Gly His Leu Arg Phe1
51559PRTArtificial SequenceNovel Sequence 155Phe Asp Asp Tyr Gly
His Leu Arg Phe1 51568PRTArtificial SequenceNovel Sequence 156Met
Asp Ser Asn Phe Ile Arg Phe1 51579PRTArtificial SequenceNovel
Sequence 157Phe Asp Asp Tyr Gly His Leu Arg Phe1 51589PRTArtificial
SequenceNovel Sequence 158Phe Asp Asp Tyr Gly His Leu Arg Phe1
51599PRTArtificial SequenceNovel Sequence 159Phe Asp Asp Tyr Gly
His Met Arg Phe1 516014PRTArtificial SequenceNovel Sequence 160Gly
Gly Asp Asp Gln Phe Asp Asp Tyr Gly His Met Arg Phe1 5
101618PRTArtificial SequenceNovel Sequence 161Ser Arg Pro Tyr Ser
Phe Gly Leu1 51627PRTArtificial SequenceNovel Sequence 162Asp Tyr
Gly His Met Arg Phe1 51639PRTArtificial SequenceNovel Sequence
163Ala Pro Arg Thr Pro Gly Gly Arg Arg1 51648PRTArtificial
SequenceNovel Sequence 164Val Glu Arg Tyr Ala Phe Gly Leu1
51658PRTArtificial SequenceNovel Sequence 165Leu Pro Val Tyr Asn
Phe Gly Leu1 516611PRTArtificial SequenceNovel Sequence 166Thr Thr
Arg Pro Gln Pro Phe Asn Phe Gly Leu1 5 1016710PRTArtificial
SequenceNovel Sequence 167Glu Asp Val Asp His Val Phe Leu Arg Phe1
5 101687PRTArtificial SequenceNovel Sequence 168Gly Asn Ser Phe Leu
Arg Phe1 5
* * * * *
References