Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
G PROTEIN-COUPLED RECEPTOR RESEMBLING GALAN1N RECEPTORS
CROSS-REFERENCE TO RELATED APPLICATIONS
Not applicable.
STATEMENT REGARDING FEDERALLY-SPONSORED R&D
Not applicable.
REFERENCE TO MICROFICHE APPENDIX
Not applicable.
FIELD OF THE INVENTION
This invention relates to novel human and rat DNAs encoding GPR54,
a G protein-coupled receptor (GPCR) related to the galanin receptors, the
proteins
encoded by the DNAs, and methods of identifying selective agonists and
antagonists
of the proteins encoded by the DNAs.
BACKGROUND OF THE INVENTION
G-protein coupled receptors (GPCRs) are a very large class of
membrane receptors that relay information from the exterior to the interior of
cells.
GPCRs function by interacting with a class of heterotrimeric proteins known as
G-
proteins. Most GPCRs function by a similar mechanism. Upon the binding of
agonist, a GPCR catalyzes the dissociation of guanosine diphosphate (GDP) from
the
a, subunit of G proteins. This allows for the binding of guanosine
triphosphate (GTP)
to the a subunit, resulting in the disassociation of the a subunit from the ~3
and y
subunits. The freed oc subunit then interacts with other cellular components,
and in
the process passes on the extracellular signal represented by the presence of
the
agonist. Occasionally, it is the freed ~3 and y subunits which transduce the
agonist
signal.
GPCRs possess common structural characteristics. They have seven
hydrophobic domains, each about 20-30 amino acids long, linked by sequences of
hydrophilic amino acids of varied length. These seven hydrophobic domains
intercalate into the plasma membrane, giving rise to a protein with seven
transmembrane domains, an extracellular amino terminus, and an intracellular
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WO 00/50563 PCT/US00/04416
carboxy terminus (Strader et al., 1994, Ann. Rev. Biochem. 63:101-132;
Schertler et
al., 1993, Nature 362:770-7721; Dohlman et al., 1991, Ann. Rev. Biochem.
60:653-
688).
GPCRs are expressed in a wide variety of tissue types and respond to a
S wide range of ligands, e.g., protein hormones, biogenic amines, peptides,
lipid derived
messengers, etc. Given their wide range of expression and ligands, it is not
surprising
that GPCRs are involved in many pathological states. This has led to great
interest in
developing modulators of GPCR activity that can be used pharmacologically. For
example, Table 1 of Stadel et al., 1997, Trends Pharmacol. Sci. 18:430-437,
lists 37
different marketed drugs that act upon GPCRs. Accordingly, there is a great
need to
understand GPCR function and to develop agents that can be used to modulate
GPCR
activity.
Galanin is widely distributed in the central and peripheral nervous
system. Galanin in most species is a 29 amino acid peptide with an amidated
carboxyl terminus. Human galanin is unique in that it is longer, 30 amino
acids, and
is not amidated. There is strong conservation of the galanin sequence, with
the amino
terminal fifteen residues being absolutely conserved in all species. Galanin
immunoreactivity and binding is abundant in the hypothalamus, the locus
coeruleus,
the hippocampus, and the anterior pituitary, as well as regions of the spinal
cord, the
pancreas, and the gastrointestinal tract.
Injection of galanin into the paraventricular nucleus (PVN) of the
hypothalamus produces a dose-dependent increase in feeding in satiated rats.
Although galanin can enhance carbohydrate ingestion, studies have shown that
it
profoundly increases fat intake. It has been suggested that galanin shifts
macronutrient preference from carbohydrate to fat. The same injections of
galanin
that increase feeding reduce energy expenditure and inhibit insulin secretion.
There is
enhanced galanin expression in the hypothalamus of genetically obese rats
compared
with their lean littermates. Injection of peptide galanin receptor antagonists
into the
PVN blocks the galanin-specific induction of increased fat intake. Specific
galanin
antisense oligonucleotides when injected into the PVN produce a specific
decrease in
galanin expression associated with a decrease in fat ingestion and total
caloric intake
while hardly affecting either protein or carbohydrate intake. Thus galanin
appears to
be a potential neurochemical marker related to the behavior of fat ingestion
and
galanin receptors are attractive targets for the development of drugs to treat
obesity
and other eating disorders.
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Galanin inhibits cholinergic function and impairs working memory in
rats. Lesions that destroy cholinergic neurons result in deficits in spatial
learning
tasks. While locally administered acetylcholine (ACh) reverses some of this
deficit,
galanin blocks this ACh-mediated improvement. Evidence from autopsy samples
from Alzheimer's disease-afflicted brains suggests an increased galinergic
innervation
of the nucleus basilis. Thus, if galinergic overactivity contributes to the
decline in
cognitive performance in Alzheimer's disease, galanin antagonists may be
therapeutically useful in alleviating cognitive impairment.
Other physiological processes in which galanin has been implicated
include nociception (Verge et al., 1993, Neurosci. Lett. 149:193-197) and
sexual
behavior (Benelli et al., 1994, Eur. J. Pharmacol. 260:279-282).
In the rat, administration of galanin intracerebroventriclarly,
subcutaneously, or intravenously increases plasma growth hormone. Infusion of
human galanin into healthy subjects also increases plasma growth hormone and
potently enhances the growth hormone response to growth hormone releasing
hormone (GHRH).
Galanin levels are particularly high in dorsal root ganglia. Sciatic
nerve resection dramatically up-regulates galanin peptide and mRNA levels.
Chronic
administration of galanin receptor antagonists (M35, M15) after axotomy
results in a
marked increase in self mutilation behavior in rats, generally considered to
be a
response to pain. Application of antisense oligonucleotides specific for
galanin to the
proximal end of a transected sciatic nerve suppressed the increase in galanin
peptide
levels with a parallel increase in autotomy. Galanin injected intrathecally
acts
synergistically with morphine to produce analgesia, this antinociceptive
effect of
morphine is blocked by galanin receptor antagonists. Thus, galanin agonists
may
have some utility in relieving neural pain.
The actions of galanin are mediated by at least three high affinity
galanin receptors that are coupled by pertussis toxin sensitive Gi/Go proteins
to
inhibition of adenylate cyclase activity, closure of L-type Ca++ channels, and
opening
of ATP-sensitive K+ channels (Habert-Ortoli et al., 1994, Proc. Natl. Acad.
Sci. USA
91:9780-9783; Howard et al., 1997, FEBS Lett. 405:285-290; Wang et al., 1997,
J.
Biol. Chem. 272:31949-31952; Kolakowski et al., 1998, J. Neurochem 71:2239-
2251 ). Specific binding of 125I_galanin (Kd approximately 1 nM) has been
demonstrated in areas paralleling localization of galanin immunoreactivity:
hypothalamus, ventral hippocampus, basal forebrain, spinal cord, pancreas, and
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pituitary. In most tissues, the amino terminus (GAL 1-15) is sufficient for
high
affinity receptor binding and agonist activity.
A galanin receptor cDNA was isolated by expression cloning from a
human Bowes melanoma cell line. (Habert-Ortoli, et al. 1994. Proc. Nat. Acad.
Sci"
USA 91: 9780-9783). This receptor, GALR1, is expressed in human fetal brain
and
small intestine, but little else is known of its distribution. Gal(1-16) is at
least 1,000
times more active than pGAL(3-29) as an inhibitor of 1251-porcine galanin
binding to
this receptor transiently expressed in COS cells. It remains to be determined
whether
this receptor subtype represents the hypothalamic receptor that mediates
galanin
specific feeding behavior.
Galanin receptors have been described in several international patent
publications (WO 98/03548; WO 97/46681; WO 97/26853; WO 98/29439; WO
98/29440; WO 98/29441; WO 95/22608). European Patent Application EP 711830
also describes a galanin receptor.
It would be desirable to identify additional galanin receptors so that
they can be used to further characterize this biological system and to
identify galanin
receptor subtype selective agonists and antagonists.
SUMMARY OF THE INVENTION
The present invention is directed to novel human and rat DNAs that
encode a G-protein coupled receptor, GPR54. The DNAs encoding GPR54 are
substantially free from other nucleic acids and have the nucleotide sequences
shown
as SEQ.ID.NO.:1 (human GPR54) and SEQ.ID.N0.:2 (rat GPR54). Also provided
are GPR54 proteins encoded by the novel DNA sequences. The GPR54 proteins are
substantially free from other proteins and have the amino acid sequences shown
as
SEQ.ID.N0.:3 (human GPR54) and SEQ.ID.N0.:4 (rat GPR54). Methods of
expressing GPR54 in recombinant systems and of identifying agonists and
antagonists
of GPR54 are provided.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 A-B shows the complete cDNA sequence and amino acid
sequence of human GPR54. The DNA sequence shown is SEQ.ID.NO.:1. The
amino acid sequence shown is SEQ.ID.N0.:3.
Figure 2A-B shows the complete cDNA sequence of rat GPR54
(SEQ.ID.N0.:2).
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Figure 3 shows the complete amino acid sequence of human GPR54
(SEQ.ID.N0.:3).
Figure 4 shows the complete amino acid sequence of rat GPR54
(SEQ.>D.N0.:4).
Figure SA-B shows the location of the rat GPR54 open reading frame.
The nucleotide sequence shown is (SEQ.ID.N0.:2). The amino acid sequence shown
is (SEQ.ID.N0.:4).
Figure 6 shows the results of a Northern blot of rat GPR54 mRNA in
rat brain. Each lane contained 5 ~g of poly(A)+ RNA isolated from various
tissues.
Figure 7A-D shows darkfield autoradiograms of sagittal and coronal
sections of rat brain showing the localization of GPR54 receptor mRNA. Figure
7A
shows a lateral representative section at 0.9 mm. Also shown are
representative
sections at levels relative to the bregma at -3.3 mm (Figure 7B), -3.8 mm
(Figure 7C),
and -6.3 mm (Figure 7D). Aco = cortical nucleus of the amygdala; Ahy =
anterior
hypothalamic area; Arc = hypothalamic arcuate nucleus; IC = inferior
colliculus; CA,
field of Ammon's horn; DG, dentate gyrus; DM, dorsomedial hypothalamic
nucleus;
LC, locus coeruleus; LH, lateral hypothalamic area, LHb, lateral habenular
nucleus;
MeA, medial nucleus of the amygdala; MPO, medial preoptic area; MRN,
mesencephalic reticular nucleus; PAG, periaqueductal gray; PB, parabrachial
nucleus;
PF, parafascicular thalamic nucleus; PH, posterior hypothalamic nucleus; PMV,
ventral premammillary nucleus; PO, primary olfactory cortex; RSpI,
retrosplenial
cortex; SC, superior colliculus; SHy, septohypothalamic nucleus; VTA, ventral
tegmental area; ZI, zona incerta.
Figure 8 shows an alignment of the amino acid sequence of rat GPR54
(SEQ.ID.N0.:4) with the amino acid sequence of rat GALRl (SEQ.ID.NO.:S), rat
GALR2 (SEQ.ID.N0.:6), rat GALR3 (SEQ.ID.N0.:7), and the rat opiod receptor
DOR (SEQ.117.N0.:8).
Figure 9 shows an alignment of the amino acid sequences of rat
GPR54 (SEQ.ID.N0.:4) and human GPR54 (SEQ.ID.N0.:3).
DETAILED DESCRIPTION OF THE INVENTION
For the purposes of this invention:
"Substantially free from other proteins" means at least 90%, preferably
95%, more preferably 99%, and even more preferably 99.9%, free of other
proteins.
Thus, a GPR54 protein preparation that is substantially free from other
proteins will
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contain, as a percent of its total protein, no more than 10%, preferably no
more than
5%, more preferably no more than 1 %, and even more preferably no more than
0.1 %,
of non-GPR54 proteins. Whether a given GPR54 protein preparation is
substantially
free from other proteins can be determined by such conventional techniques of
S assessing protein purity as, e.g., sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) combined with appropriate detection methods, e.g.,
silver staining or immunoblotting.
"Substantially free from other nucleic acids" means at least 90%,
preferably 95%, more preferably 99%, and even more preferably 99.9%, free of
other
nucleic acids. Thus, a GPR54 DNA preparation that is substantially free from
other
nucleic acids will contain, as a percent of its total nucleic acid, no more
than 10%,
preferably no more than 5%, more preferably no more than 1 %, and even more
preferably no more than 0.1 %, of non-GPR54 nucleic acids. Whether a given
GPR54
DNA preparation is substantially free from other nucleic acids can be
determined by
such conventional techniques of assessing nucleic acid purity as, e.g.,
agarose gel
electrophoresis combined with appropriate staining methods, e.g., ethidium
bromide
staining, or by sequencing.
"Functional equivalent" means a receptor which does not have exactly
the same amino acid sequence as naturally occurring GPR54, due to alternative
splicing, substitutions, deletions, mutations, or additions, but retains
substantially the
same biological activity as GPR54. Such functional equivalents will have
significant
amino acid sequence identity with naturally occurnng GPR54. Genes and DNA
encoding such functional equivalents can be detected by reduced stringency
hybridization with a DNA sequence encoding naturally occurnng GPR54. For the
purposes of this invention, naturally occurnng GPR54 has the amino acid shown
as
SEQ.m.N0.:3 or SEQ.>D.N0.:4. A nucleic acid encoding a functional equivalent
has
at least about 50% identity at the nucleotide sequence level to SEQ.>D.NO.:1
or
SEQ.ID.N0.:2.
A polypeptide has "substantially the same biological activity" as
GPR54 if that polypeptide has a Kd for a ligand that is no more than 5-fold
greater
than the Kd of GPR54 having SEQ.ID.N0.:3 or SEQ.>D.N0.:4 for the same ligand.
A polypeptide also has "substantially the same biological activity" as GPR54
if that
polypeptide is capable of mediating the same functional response as naturally
occurnng GPR54 when exposed to the same ligand as naturally occurring GPR54.
Examples of functional responses are: pigment aggregation in Xenopus
melanophores,
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changes in membrane currents in Xenopus oocytes, modulation of cAMP levels,
changes in calcium concentration, changes in inositol phosphate levels, and
coupling
to inwardly rectifying potassium channels. One skilled in the art would be
familiar
with a variety of methods of measuring the functional responses of other G-
protein
coupled receptors and would be able to apply those methods to GPR54 (see,
e.g.,
Lerner, 1994, Trends Neurosci. 17:142-146 [changes in pigment distribution in
melanophore cells]; Yokomizo et al., 1997, Nature 387:620-624 [changes in cAMP
or calcium concentration, chemotaxis]; Howard et al., 1996, Science 273:974-
977
[changes in membrane currents in Xenopus oocytes]; McKee et al., 1997, Mol.
Endocrinol. 11:415-423 [changes in calcium concentration measured using the
aequorin assay]; Offermanns & Simon, 1995, J. Biol. Chem. 270:15175, 15180
[changes in inositol phosphate levels]). Zlokarnik et al., 1998, Science
279:84-88 and
U.S. Patent No. 5,741,657 describe a reporter gene assay that can be adapted
to
measure GPR54 functional responses. The assay utilizes an inducible promoter-
driven (3-lactamase that cleaves a fluorescent substrate. Cleavage of the
substrate
leads to a change in fluoresence resonance energy transfer (FRET) between
different
portions of the substrate that is proportional to the magnitude of induction
of the [3-
lactamase. Thus, the level of activation of the inducible promoter determines
the
amount of FRET measured. This level of induction of the promoter is in turn
determined by the level of the substance (e.g., cAMP) the promoter is induced
by. By
choosing a promoter that is induced by a functional response that results from
the
interaction of a ligand and GPR54 (e.g., changes in cAMP levels), one can use
this
assay to measure GPR54 functional responses.
Depending upon the cells in which GPR54 is expressed, and thus the
G-proteins with which GPR54 is coupled, certain of such methods as described
above
may be appropriate for measuring the functional responses of GPR54. It is well
within the competence of one skilled in the art to select the appropriate
method of
measuring functional responses for a given experimental system.
A "conservative amino acid substitution" refers to the replacement of
one amino acid residue by another, chemically similar, amino acid residue.
Examples
of such conservative substitutions are: substitution of one hydrophobic
residue
(isoleucine, leucine, valine, or methionine) for another; substitution of one
polar
residue for another polar residue of the same charge (e.g., arginine for
lysine; glutamic
acid for aspartic acid).
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By "isolated GPR54 protein" or "isolated GPR54 DNA" is meant
GPR54 protein or DNA encoding GPR54 that has been isolated from a natural
source.
Use of the term "isolated" indicates that GPR54 protein or DNA has been
removed
from its normal cellular environment. Thus, an isolated GPR54 protein may be
in a
cell-free solution or placed in a different cellular environment from that in
which it
occurs naturally. The term isolated does not imply that an isolated GPR54
protein is
the only protein present, but instead means that an isolated GPR54 protein is
at least
95% free of non-amino acid material (e.g., nucleic acids, lipids,
carbohydrates)
naturally associated with the GPR54 protein. Thus, a GPR54 protein that is
expressed
in bacteria or even in eukaryotic cells which do not naturally (i.e., without
human
intervention) express it through recombinant means is an "isolated GPR54
protein."
Similarly, DNA encoding GPR54 that is present in bacteria or even in
eukaryotic cells
which do not naturally (i.e., without human intervention) contain it through
recombinant means is an "isolated DNA encoding GPR54."
The present invention pertains to the discovery of DNA encoding a
galanin receptor-like protein. Two degenerate primers (P 1 and P2, see Example
1 )
based on conserved GPCR sequences in transmembrane segment 3 (TM3) and
transmembrane segment 7 (TM7), respectively, were used to amplify an aliquot
of a
rat brain cDNA library with proof reading Pfu polymerase. The amplified DNA
was
excised and subcloned into the pBluescript vector. One of the resulting rat
clones
appeared to partially encode a galanin/opioid-like receptor. The partial cDNA
was
labeled with 32P dCTP-a and used to screen the cDNA library employed in the
degenerate PCR. Two positive plaques were purified and their inserts amplified
by
PCR using Pfu polymerase and primers flanking the cloning site of the ~,gtl 1
vector.
The PCR products were subcloned into pBluescript and sequenced. Sequence
analysis revealed that each plaque encoded a region of a putative GPCR from
TM3 to
the carboxy terminus identical to each other and the original probe. A second
round
of screening of 1 x 106 plaques freshly plated from the same library yielded
an
additional three positive plaques. PCR amplification of these positive plaques
with
~,gtl 1 flanking primers, each paired with an internal primer, revealed that
only one of
these positive plaques contained the entire open reading frame (ORF). This
plaque
was purified, the insert subcloned into pBluescript and was confirmed to
contain the
5' end of the full-length open reading frame. Finally, two specific primers
from the 5'
and 3' ends of the ORF were used to amplify with pfu polymerase the full
length rat
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cDNA 1.2 Kb clone, named GPR54. Sequence analysis revealed the cloned GPR54
ORF to be identical to the previous phage clones and the original probe.
GPR54 contained an ORF of 1,185 by encoding a protein of 395 amino
acids.
Using GPR54 in a BLAST search (Altschul, 1997, Nucleic Acids Res
25:3389-3402), the highest identity was observed with the galanin and opioid
receptor
families. Specifically, GPR54 shared an amino acid sequence identity in the TM
regions with rat galanin receptors GalRl(45%), GalR3 (45%), GalR2 (44%), and
rat
opioid receptor DOR (37%) (Figure 8). Conserved residues and consensus
sequences
of the rhodopsin superfamily of GPCRs present in GPR54 included an asparagine
in
TM1, an aspartate in TM2, prolines in TMs 4 through 7, three consensus
sequences
for N-linked glycosylation in the amino terminus, cysteines in the first and
second
extracellular loops, a PKA/PKC consensus sequence in the second intracellular
loop,
a PKC consensus sequence in the third intracellular loop, and three possible
1 S palmitoylation cysteine sites in the carboxy tail. Significantly, various
residues in the
human GalRl receptor shown to be important for high-affinity galanin binding
(corresponding to His262, His265, G1u269, and Phe280 in rat GalRl; (Kask et
al.,
1996, EMBO J. 15:236-244 (Kask); Berthold et al., 1997, Eur. J. Biochem.
249:601-
606 (Berthold)) were not conserved in GPR54. Among these however, only His262
is
conserved among the three galanin receptors. In addition, the substitution of
a
tyrosine residue found in GPR54, GalR2 and GalR3 in place of Phe280 in GalR1
was
shown to have no significant effect on galanin binding (Kask) as opposed to
previous
studies where Phe280 was replaced by alanine in GalRl (Berthold).
Both Northern blot and in situ hybridization analyses of GPR54 were
performed at high stringencies and with a DNA probe encoding GPR54 from TM3 to
TM7 and with low identities to the genes encoding galanin and related
receptors. The
tissue distribution of GPR54 was obtained by northern blot analysis using
poly(A)+
RNA isolated from various rat tissues (Figure 6). In the brain, multiple RNA
transcripts with a complex pattern were detected in the medulla pons,
midbrain,
hippocampus, cortex, frontal cortex, and striatum. The most intense band was
approximately 3.7 Kb in length, with a single, larger transcript of
approximately 12
Kb length detected in the liver and intestine only. No transcripts were
revealed in the
cerebellum or kidney tissues.
Using in situ hybridization of rat brain sections, the distribution of
GPR54 mRNA was found to be discretely localized to many areas (Figure 7). The
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highest levels of expression were seen in hypothalamic and amygdaloid nuclei.
GPR54 mRNA was highly expressed in the zona incerta, ventral tegmental area,
dentate gyrus, hypothalamic arcuate nucleus, dorsomedial hypothalamic nucleus,
primary olfactory cortex, lateral habenular nucleus, lateral hypothalamic
area, locus
coeruleus, and the cortical and medial nuclei of the amygdala. GPR54 mRNA was
also concentrated in the superior colliculus, medial preoptic area, anterior
hypothalamic area, posterior hypothalamic nucleus, periaqueductal gray,
parafascicular thalamic nucleus, parabrachial nucleus, and ventral
premammillary
nucleus. The signals detected in the septohypothalamic nucleus, inferior
colliculus,
medial nucleus of the amygdala, mesencephalic reticular nucleus and
retrosplenial
cortex were diffuse and less abundant.
GPR54's CNS expression pattern was found to resemble those of
galanin receptors. Specifically, rat GalRl mRNA expression is abundant in
several
brain regions including the hypothalamus, amygdala, hippocampus and locus
coeruleus (Parker et al., 1995, Mol. Brain Res. 34:179-189). Rat GalR2 mRNA
expression is found in the mammilary nuclei, the dentate gyrus and posterior
hypothalamic and arcuate nuclei (Kolakowski et al., 1998, J. Neurochem. 71,
2239-
2251 ). Rat GalR3 is found to be abundantly expressed in the CA regions of
Ammon's
horn and the dentate gyrus with transcripts also detected in thalamic,
hypothalamic,
mammilary and amygdaloid nuclei (Kolakowski et al., 1998, J. Neurochem. 71,
2239-
2251 ).
The identity and overlapping expression patterns of GPR54 with the
galanin receptors suggested that the encoded receptor may demonstrate binding
to
galanin. In preparation for expression and binding studies, the 1.2 kb cDNA
fragment
encoding the ORF of GPR54 was subcloned into the multiple cloning site of the
pcDNA3 expression vector and transiently transfected into COS-7 cells. No
specific
binding was observed with 125I_human galanin. In contrast, specific and high
affinity
binding was observed under similar conditions with 125I_human galanin in
membranes prepared from COS cells transfected with human GalRl, consistent
with a
previous report for GalR2 and GalR3 (Kolakowski et al., 1998, J. Neurochem.
71,
2239-2251 ).
A BLAST search with the rat GPR54 sequence revealed high identity
with a human 3.5 Mb contig located in chromosome 19p l 3.3 containing a serine
protease gene cluster (GenBank accession number AC005379). Sequence analysis
revealed a previously unrecognized 3.3 kb intron-containing human orthologue
of
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GPR54 encoding a protein 398 amino acids in length and sharing a translated
amino
acid identity of 81% (100% identity in the TM regions) with rat GPR54. The
genomic
sequence revealed four introns located in TM2 0800 bp, interrupting the
translated
FYL.ANL sequence), TM3 0800 bp, interrupting IQQ..VSV), TM4 0250 bp,
interrupting WVG..SAA) and in the third intracellular loop 0180 bp,
interrupting
ALQ..GQV).
One aspect of this invention is an isolated DNA comprising
nucleotides encoding a polypeptide having the amino acid sequence SEQ.>D.N0.:3
or
SEQ.ID.N0.:4. This isolated DNA can be substantially free from other nucleic
acids
and can be either single stranded or double stranded, i.e., paired with its
complementary sequence. Also within the present invention is isolated RNA
corresponding to this DNA.
Another aspect of this invention is the identification and cloning of a
cDNA which encodes GPR54, a G protein-coupled receptor. This cDNA is
substantially free from other nucleic acids and can be either single stranded
or double
stranded. The present invention provides a cDNA molecule substantially free
from
other nucleic acids having the nucleotide sequence shown in Figure 1 as
SEQ.ID.NO.:l or in Figure 2 as SEQ.>D.N0.:2. SEQ.ID.NO.:1 contains an open
reading frame (positions 1-1,194 of SEQ.>D.NO.:1) encoding a protein of 398
amino
acids. SEQ.>T7.N0.:2 contains an open reading frame (positions 61-1,245 of
SEQ.>D.N0.:2) encoding a protein of 395 amino acids. (see Figure SA-B).
Thus, the present invention also provides a DNA molecule
substantially free from other nucleic acids comprising the nucleotide sequence
of
positions 1-1,194 of SEQ.ID.NO.:1 as well as a DNA molecule substantially free
from other nucleic acids comprising the nucleotide sequence of positions 61-
1,245 of
SEQ.ID.N0.:2. The present invention also provides recombinant DNA molecules
comprising the nucleotide sequence of positions 1-1,194 of SEQ.ID.NO.:l or
positions 61-1,245 of SEQ.ID.N0.:2.
Based on their predicted amino acid sequences, the human and rat
GPR54 proteins most likely represent novel G-protein coupled receptors (GPCRs)
since these GPR54 proteins obtain many of the characteristic features of
GPCRs, e.g..
(a) seven transmembrane domains;
(b) three intracellular loops;
(c) three extracellular loops; and
(d) the GPCR triplet signature sequence.
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Northern blot and in situ hybridization analyses such as Figure 6 and
Figure 7 showed that GPR54 RNA is widely expressed in rat brain regions (pons,
midbrain, thalamus, hypothalamus, hippocampus, amygdala, cortex, frontal
cortex,
and striatum) as well as peripheral regions (liver and intestine).
The novel DNA sequences of the present invention encoding GPR54,
in whole or in part, can be linked with other DNA sequences, i.e., DNA
sequences to
which GPR54 is not naturally linked, to form "recombinant DNA molecules"
containing GPR54 sequences. The novel DNA sequences of the present invention
can
be inserted into vectors in order to direct recombinant expression of GPR54.
Such
vectors may be comprised of DNA or RNA; for most purposes DNA vectors are
preferred. Typical vectors include plasmids, modified viruses, bacteriophage,
cosmids, yeast artificial chromosomes, and other forms of episomal or
integrated
DNA that can encode GPR54. One skilled in the art can readily determine an
appropriate vector for a particular use.
1 S Included in the present invention are DNA sequences that hybridize to
SEQ.ID.NO.:1 or SEQ.ID.N0.:2 under stringent conditions. By way of example,
and
not limitation, a procedure using conditions of high stringency is as follows:
Prehybridization of filters containing DNA is earned out for 2 hr. to
overnight at 65°C
in buffer composed of 6X SSC, SX Denhardt's solution, and 100 pg/ml denatured
salmon sperm DNA. Filters are hybridized for 12 to 48 hrs at 65°C in
prehybridization mixture containing 100 pg/ml denatured salmon sperm DNA and 5-
20 X 106 cpm of 32P-labeled probe. Washing of filters is done at 37°C
for 1 hr in a
solution containing 2X SSC, 0.1% SDS. This is followed by a wash in O.1X SSC,
0.1% SDS at SO°C for 45 min. before autoradiography.
Other procedures using conditions of high stringency would include
either a hybridization step earned out in SXSSC, SX Denhardt's solution, 50%
formamide at 42°C for 12 to 48 hours or a washing step earned out in
0.2X SSPE,
0.2% SDS at 65°C for 30 to 60 minutes.
Reagents mentioned in the foregoing procedures for carrying out high
stringency hybridization are well known in the art. Details of the composition
of
these reagents can be found in, e.g., Sambrook, Fritsch, and Maniatis, 1989,
Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor
Laboratory Press. In addition to the foregoing, other conditions of high
stringency
which may be used are well known in the art.
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The degeneracy of the genetic code is such that, for all but two amino
acids, more than a single codon encodes a particular amino acid. This allows
for the
construction of synthetic DNA that encodes the GPR54 protein where the
nucleotide
sequence of the synthetic DNA differs significantly from the nucleotide
sequence of
SEQ.>D.NO.:1 or SEQ.ID.N0.:2, but still encodes the same GPR54 protein as
SEQ.ID.NO.:l or SEQ.ID.N0.:2. Such synthetic DNAs are intended to be within
the
scope of the present invention. If it is desired to express such synthetic
DNAs in a
particular host cell or organism, the codon usage of such synthetic DNAs can
be
adjusted to reflect the codon usage of that particular host cell or organism,
thus
leading to higher levels of expression of GPR54 protein in the host.
Another aspect of the present invention includes host cells that have
been engineered to contain and/or express DNA sequences encoding GPR54. Such
recombinant host cells can be cultured under suitable conditions to produce
GPR54.
An expression vector containing DNA encoding GPR54 can be used for expression
of
GPR54 in a recombinant host cell. Recombinant host cells may be prokaryotic or
eukaryotic, including but not limited to, bacteria such as E. coli, fungal
cells such as
yeast, mammalian cells including, but not limited to, cell lines of human,
bovine,
porcine, monkey, and rodent origin, and insect cells including but not limited
to,
Drosophila and silkworm derived cell lines. Cell lines derived from mammalian
species which are suitable for recombinant expression of GPR54 and which are
commercially available, include but are not limited to, L cells L-M(TK-) (ATCC
CCL
1.3), L cells L-M (ATCC CCL 1.2), HEK 293 (ATCC CRL 1573), Raji (ATCC CCL
86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651),
CHO-Kl (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658),
HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5
(ATCC CCL 171 ), Xenopus melanophores, and Xenopus oocytes.
Human embryonic kidney (HEK 293) cells and Chinese hamster ovary
(CHO) cells are particularly suitable for expression of the GPR54 protein
because
these cells express a large number of G-proteins. Thus, it is likely that at
least one of
these G-proteins will be able to functionally couple the signal generated by
interaction
of GPR54 and its ligands, thus transmitting this signal to downstream
effectors,
eventually resulting in a measurable change in some assayable component, e.g.,
cAMP level, expression of a reporter gene, hydrolysis of inositol lipids, or
intracellular Ca2+ levels.
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Other cells that are particularly suitable for expression of the GPR54
protein are immortalized melanophore pigment cells from Xenopus laevis. Such
melanophore pigment cells can be used for functional assays using recombinant
expression of GPR54 in a manner similar to the use of such melanophore pigment
cells for the functional assay of other recombinant GPCRs (Graminski et al.,
1993, J.
Biol. Chem. 268:5957-5964; Lerner, 1994, Trends Neurosci. 17:142-146; Potenza
&
Lerner, 1992, Pigment Cell Res. 5:372-378).
A variety of mammalian expression vectors can be used to express
recombinant GPR54 in mammalian and other cells. Commercially available
mammalian expression vectors which are suitable include, but are not limited
to,
pCR2.l (Invitrogen), pMClneo (Stratagene), pSGS (Stratagene), pcDNAI and
pcDNAIamp, pcDNA3, pcDNA3.l, pCR3.1 (Invitrogen), EBO-pSV2-neo (ATCC
37593), pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224),
pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), and pSV2-dhfr (ATCC 37146).
For expression in non-mammalian cells, various suitable expression vectors are
known in the art. The choice of vector will depend upon cell type used, level
of
expression desired, and the like. Following expression in recombinant cells,
GPR54
can be purified to a level that is substantially free from other proteins by
conventional
techniques, e.g., salt fractionation, ion exchange chromatography, size
exclusion
chromatography, hydroxylapatite adsorption chromatography, hydrophobic
interaction
chromatography, and preparative gel electrophoresis.
The present invention includes GPR54 protein substantially free from
other proteins. The amino acid sequence of the full-length human GPR54 protein
is
shown in Figure 3 as SEQ.ID.N0.:3. The amino acid sequence of the full-length
rat
GPR54 protein is shown in Figure 4 as SEQ.ID.N0.:4. Thus, the present
invention
includes GPR54 proteins substantially free from other proteins having the
amino acid
sequence of SEQ.ID.N0.:3 or SEQ.ID.N0.:4.
As with many receptor proteins, it is possible to modify many of the
amino acids of GPR54, particularly those which are not found in the ligand
binding
domain, and still retain substantially the same biological activity as the
original
receptor. Thus, the present invention includes modified GPR54 polypeptides
which
have amino acid deletions, additions, or substitutions but that still retain
substantially
the same biological activity as naturally occurring GPR54. It is generally
accepted
that single amino acid substitutions do not usually alter the biological
activity of a
protein (see, e.g., Molecular Biolo~y of the Gene, Watson et al., 1987, Fourth
Ed.,
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The Benjamin/Cummings Publishing Co., Inc., page 226; and Cunningham & Wells,
1989, Science 244:1081-1085). Accordingly, the present invention includes
polypeptides where one amino acid substitution has been made in SEQ.>D.N0.:3
or
SEQ.>D.N0.:4 wherein the polypeptides still retain substantially the same
biological
S activity as naturally occurring GPR54. The present invention also includes
polypeptides where two or more amino acid substitutions have been made in
SEQ.>D.N0.:3 or SEQ.>D.N0.:4 wherein the polypeptides still retain
substantially the
same biological activity as naturally occurring GPR54. In particular, the
present
invention includes embodiments where the above-described substitutions are
conservative substitutions. In particular, the present invention includes
embodiments
where the above-described substitutions do not occur in the ligand-binding
domain of
GPR54.
When deciding which amino acid residues of GPR54 may be
substituted to produce polypeptides that are functional equivalents of GPR54,
one
skilled in the art would be guided by a comparison of the amino acid sequence
of
GPR54 with the amino acid sequences of related proteins, e.g., the human,
mouse, or
rat GALRl, GALR2, or GALR3 receptors, as well as the rat opiod receptor DOR
(see,
e.g., Figure 8). Such a comparison would allow one skilled in the art to
minimize the
number of amino acid substitutions made in regions that are highly conserved
between GPR54 and the related proteins. Accordingly, the present invention
includes
polypeptides where two or more amino acid substitutions have been made in
SEQ.ID.N0.:3 or SEQ.~.N0.:4 where the polypeptides still retain substantially
the
same biological activity as naturally occurnng GPR54 and where the
substitutions are
conservative and do not occur in positions where GPR54 and any of the human,
mouse, or rat GALRl, GALR2, or GALR3 receptors share the same amino acid, or
do
not occur in positions where GPR54 and the rat opiod DOR receptor share the
same
amino acid (see Figure 8). In particular embodiments, the substitutions do not
occur
in positions where GPR54 and any of the rat GALR1, GALR2, or GALR3 receptors
share the same amino acid (see Figure 8).
One skilled in the art would also recognize that polypeptides that are
functional equivalents of GPR54 and have changes from the GPR54 amino acid
sequence that are small deletions or insertions of amino acids could also be
produced
by following the same guidelines, i.e., minimizing the differences in amino
acid
sequence between GPR54 and related proteins. Small deletions or insertions are
generally in the range of about 1 to 5 amino acids. The effect of such small
deletions
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or insertions on the biological activity of the modified GPR54 polypeptide can
easily
be assayed by producing the polypeptide synthetically or by making the
required
changes in DNA encoding GPR54 and then expressing the DNA recombinantly and
assaying the protein produced synthetically or by such recombinant expression.
Assays that could be used include simple binding assays to determine if the
modified
GPR54 polypeptide is capable of binding the same ligands, with approximately
the
same affinity, as naturally occurring GPR54 protein. Alternatively, one can
use
functional assays such as assays such as those described herein.
The present invention also includes C-terminal truncated forms of
GPR54, particularly those which encompass the extracellular portion of the
receptor,
but lack the intracellular signaling portion of the receptor. Such truncated
receptors
are useful in various binding assays described herein, for crystallization
studies, and
for structure-activity-relationship studies.
The present invention also includes chimeric GPR54 proteins.
Chimeric GPR54 proteins consist of a contiguous polypeptide sequence of GPR54
fused in frame to a polypeptide sequence of a non-GPR54 protein. For example,
the
N-terminal domain and seven transmembrane spanning domains of GPR54 fused at
the C-terminus in frame to a G protein would be a chimeric GPR54 protein.
The present invention also includes GPR54 proteins that are in the
form of multimeric structures, e.g., dimers. Such multimers of other G-protein
coupled receptors are known (Hebert et al., 1996, J. Biol. Chem. 271, 16384-
16392;
Ng et al., 1996, Biochem. Biophys. Res. Comm. 227, 200-204; Romano et al.,
1996,
J. Biol. Chem. 271, 28612-28616). The dimers may be homodimers containing two
GPR54 proteins or the dimers may be heterodimers containing GPR54 and another
protein.
The present invention also includes isolated forms of GPR54 proteins.
The present invention includes methods of identifying compounds that
specifically bind to GPR54 protein, as well as compounds identified by such
methods.
The specificity of binding of compounds having affinity for GPR54 is shown by
measuring the affinity of the compounds for recombinant cells expressing the
cloned
receptor or for membranes from such cells. Expression of the cloned receptor
and
screening for compounds that bind to GPR54, or that inhibit the binding of a
known
ligand of GPR54 to such cells, or membranes prepared from such cells, provides
an
effective method for the rapid selection of compounds with high affinity for
GPR54.
Such ligands or compounds can be radiolabeled, but can also be nonisotopic
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compounds that can be used to displace bound radiolabeled ligands or that can
be
used as activators or inhibitors in functional assays. Compounds identified by
the
above method are likely to be agonists or antagonists of GPR54 and may be
peptides,
proteins, or non-proteinaceous organic molecules. Such compounds are likely to
be
pharmacologically useful modulators of GPR54 activity.
Therefore, the present invention includes assays by which GPR54
agonists and antagonists may be identified. Methods for identifying agonists
and
antagonists of other receptors are well known in the art and can be adapted to
identify
agonists and antagonists of GPR54. Accordingly, the present invention includes
a
method for determining whether a substance is a potential agonist or
antagonist of
GPR54 that comprises:
(a) transfecting cells with an expression vector encoding GPR54;
(b) allowing the transfected cells to grow for a time sufficient to
allow GPR54 to be expressed;
(c) exposing the cells to a labeled ligand of GPR54 in the presence
and in the absence of the substance;
(d) measuring the binding of the labeled ligand to GPR54;
where if the amount of binding of the labeled ligand is less in the
presence of the substance than in the absence of the substance, then the
substance is a
potential agonist or antagonist of GPR54.
The conditions under which step (c) of the method is practiced are
conditions that are typically used in the art for the study of protein-ligand
interactions:
e.g., physiological pH; salt conditions such as those represented by such
commonly
used buffers as PBS or in tissue culture media; a temperature of about
4°C to about
55°C.
The present invention also includes a method for determining whether
a substance is capable of binding to GPR54, i.e., whether the substance is a
potential
agonist or an antagonist of GPR54, where the method comprises:
(a) providing test cells by transfecting cells with an expression
vector that directs the expression of GPR54 in the cells;
(b) exposing the test cells to the substance;
(c) measuring the amount of binding of the substance to GPR54 in
the test cells;
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(d) comparing the amount of binding of the substance to GPR54 in
the test cells with the amount of binding of the substance to control cells
that have not
been transfected with GPR54;
wherein if the amount of binding of the substance is greater in the test
cells as compared to the control cells, the substance is capable of binding to
GPR54.
Determining whether the substance is an agonist or antagonist can then be
accomplished by the use of functional assays such as, e.g., the assay
involving the use
of promiscuous G-proteins described below.
The conditions under which step (b) of the method is practiced are
conditions that are typically used in the art for the study of protein-ligand
interactions:
e.g., physiological pH; salt conditions such as those represented by such
commonly
used buffers as PBS or in tissue culture media; a temperature of about
4°C to about
55°C.
In a particular embodiment of the above-described methods, the cells
are eukaryotic cells. In another embodiment, the cells are mammalian cells. In
other
embodiments, the cells are L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC
CCL 1.2), HEK 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL
70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL
61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2),
C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) or MRC-5 (ATCC CCL 171).
The assays described above can be carried out with cells that have been
transiently or stably transfected with GPR54. Transfection is meant to include
any
method known in the art for introducing GPR54 into the test cells. For
example,
transfection includes calcium phosphate or calcium chloride mediated
transfection,
lipofection, infection with a retroviral construct containing GPR54, and
electroporation.
Where binding of the substance or ligand to GPR54 is measured, such
binding can be measured by employing a labeled substance or ligand. The
substance
or ligand can be labeled in any convenient manner known to the art, e.g.,
radioactively, fluorescently, enzymatically.
In particular embodiments of the above-described methods, GPR54 has
an amino acid sequence of SEQ.ID.N0.:3 or SEQ.m.N0.:4.
The above-described methods can be modified in that, rather than
exposing the test cells to the substance, membranes can be prepared from the
test cells
and those membranes can be exposed to the substance. Such a modification
utilizing
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membranes rather than cells is well known in the art and is described in,
e.g., Hess et
al., 1992, Biochem. Biophys. Res. Comm. 184:260-268.
Accordingly, the present invention provides a method for determining
whether a substance is capable of binding to GPR54 comprising:
(a) providing test cells by transfecting cells with an expression
vector that directs the expression of GPR54 in the cells;
(b) preparing membranes containing GPR54 from the test cells and
exposing the membranes to a ligand of GPR54 under conditions such that the
ligand
binds to the GPR54 in the membranes;
(c) subsequently or concurrently to step (b), exposing the
membranes from the test cells to a substance;
(d) measuring the amount of binding of the ligand to the GPR54 in
the membranes in the presence and the absence of the substance;
(e) comparing the amount of binding of the ligand to GPR54 in the
membranes in the presence and the absence of the substance where a decrease in
the
amount of binding of the ligand to GPR54 in the membranes in the presence of
the
substance indicates that the substance is capable of binding to GPR54.
In particular embodiments, GPR54 has an amino acid sequence of
SEQ.ID.N0.:3 or SEQ.>D.N0.:4.
The present invention provides a method for determining whether a
substance is capable of binding to GPR54 comprising:
(a) providing test cells by transfecting cells with an expression
vector that directs the expression of GPR54 in the cells;
(b) preparing membranes containing GPR54 from the test cells and
exposing the membranes from the test cells to the substance;
(c) measuring the amount of binding of the substance to the
GPR54 in the membranes from the test cells;
(d) comparing the amount of binding of the substance to GPR54 in
the membranes from the test cells with the amount of binding of the substance
to
membranes from control cells that have not been transfected with GPR54;
where if the amount of binding of the substance to GPR54 in the
membranes from the test cells is greater than the amount of binding of the
substance
to the membranes from the control cells, then the substance is capable of
binding to
GPR54.
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In particular embodiments, GPR54 has an amino acid sequence of
SEQ.ID.N0.:3 or SEQ.LD.N0.:4.
As a further modification of the above-described methods, RNA
encoding GPR54 can be prepared, e.g., by in vitro transcription using a
plasmid
containing GPR54 under the control of a bacteriophage T7 promoter, and the RNA
can be microinjected into Xenopus oocytes in order to cause the expression of
GPR54
in the oocytes. Substances are then tested for binding to the GPR54 expressed
in the
oocytes. Alternatively, rather than detecting binding, the effect of the
substances on
the electrophysiological properties of the oocytes can be determined.
The present invention includes assays by which GPR54 agonists and
antagonists may be identified by their ability to stimulate or antagonize a
functional
response mediated by GPR54. One skilled in the art would be familiar with a
variety
of methods of measuring the functional responses of G-protein coupled
receptors (see,
e.g., Lerner, 1994, Trends Neurosci. 17:142-146 [changes in pigment
distribution in
melanophore cells]; Yokomizo et al., 1997, Nature 387:620-624 [changes in cAMP
or calcium concentration; chemotaxis]; Howard et al., 1996, Science 273:974-
977
[changes in membrane currents in Xenopus oocytes]; McKee et al., 1997, Mol.
Endocrinol. 11:415-423 [changes in calcium concentration measured using the
aequorin assay]; Offermanns & Simon, 1995, J. Biol. Chem. 270:15175, 15180
[changes in inositol phosphate levels]).
Accordingly, the present invention provides a method of identifying
agonists and antagonists of GPR54 comprising:
(a) providing test cells by transfecting cells with an expression
vector that directs the expression of GPR54 in the cells;
(b) exposing the test cells to a substance that is suspected of being
an agonist or an antagonist of GPR54;
(c) measuring the amount of a functional response of the test cells
that have been exposed to the substance;
(d) comparing the amount of the functional response exhibited by
the test cells with the amount of the functional response exhibited by control
cells;
wherein if the amount of the functional response exhibited by the test
cells differs from the amount of the functional response exhibited by the
control cells,
the substance is an agonist or antagonist of GPR54;
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where the control cells are cells that have not been transfected with
GPR54 but have been exposed to the substance or are test cells that have not
been
exposed to the substance.
In particular embodiments, GPR54 has an amino acid sequence of
SEQ.)D.N0.:3 or SEQ.>D.N0.:4.
In particular embodiments, the functional response is selected from the
group consisting of changes in pigment distribution in melanophore cells;
changes in
cAMP or calcium concentration; changes in membrane currents in Xenopus
oocytes;
and changes in inositol phosphate levels.
GPR54 belongs to the class of proteins known as G-protein coupled
receptors (GPCRs). GPCRs transmit signals across cell membranes upon the
binding
of ligand. The ligand-bound GPCR interacts with a heterotrimeric G-protein,
causing
the Ga subunit of the G-protein to disassociate from the G(3 and Cry subunits.
The Ga
subunit can then go on to activate a variety of second messenger systems.
Generally, a particular GPCR is only coupled to a particular type of G-
protein. Thus, to observe a functional response from the GPCR, it is necessary
to
ensure that the proper G-protein is present in the system containing the GPCR.
It has
been found, however, that there are certain G-proteins that are "promiscuous."
These
promiscuous G-proteins will couple to, and thus transduce a functional signal
from,
virtually any GPCR. See Offermanns & Simon, 1995, J. Biol. Chem. 270:1 S 175,
15180 (Offermanns). Offermanns described a system in which cells are
transfected
with expression vectors that result in the expression of one of a large number
of
GPCRs as well as the expression of one of the promiscuous G-proteins Gals or
Gal6. Upon the addition of an agonist of the GPCR to the transfected cells,
the
GPCR was activated and was able, via Gals or Gal6, to activate the (3 isoform
of
phospholipase C, leading to an increase in inositol phosphate levels in the
cells.
Therefore, by making use of these promiscuous G-proteins as in
Offermanns, it is possible to set up functional assays for GPR54, even in the
absence
of knowledge of the G-protein with which GPR54 is coupled in vivo. One
possibility
is to create a fusion or chimeric protein composed of the extracellular and
membrane
spanning portion of GPR54 fused to a promiscuous G-protein. Such a fusion
protein
would be expected to transduce a signal following binding of ligand to the
GPR54
portion of the fusion protein. Accordingly, the present invention provides a
method of
identifying antagonists of GPR54 comprising:
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(a) providing cells that expresses a chimeric GPR54 protein fused
at its C-terminus to a promiscuous G-protein;
(b) exposing the cells to an agonist of GPR54;
(c) subsequently or concurrently to step (b), exposing the cells to a
S substance that is a suspected antagonist of GPR54;
(d) measuring the level of inositol phosphates in the cells;
where a decrease in the level of inositol phosphates in the cells in the
presence of the substance as compared to the level of inositol phosphates in
the cells
in the absence of the substance indicates that the substance is an antagonist
of GPR54.
Another possibility for utilizing promiscuous G-proteins in connection
with GPR54 includes a method of identifying agonists of GPR54 comprising:
(a) providing cells that expresses both GPR54 and a promiscuous
G-protein;
(b) exposing the cells to a substance that is a suspected agonist of
GPR54;
(c) measuring the level of inositol phosphates in the cells;
where an increase in the level of inositol phosphates in the cells as
compared to the level of inositol phosphates in the cells in the absence of
the
suspected agonist indicates that the substance is an agonist of GPR54.
Levels of inositol phosphates can be measured by monitoring calcium
mobilization. Intracellular calcium mobilization is typically assayed in whole
cells
under a microscope using fluorescent dyes or in cell suspensions via
luminescence
using the aequorin assay.
In a particular embodiment of the above-described method, the cells
are eukaryotic cells. In another embodiment, the cells are mammalian cells. In
other
embodiments, the cells are L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC
CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70),
COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61),
3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I
(ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171 ), Xenopus
oocytes, or Xenopus melanophores.
In a particular embodiment of the above-described method, the cells
are transfected with expression vectors that direct the expression of GPR54
and the
promiscuous G-protein in the cells.
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The conditions under which step (b) of the method is practiced are
conditions that are typically used in the art for the study of protein-ligand
interactions:
e.g., physiological pH; salt conditions such as those represented by such
commonly
used buffers as PBS or in tissue culture media; a temperature of about
4°C to about
55°C.
In a particular embodiment of the above-described method, the
promiscuous G-protein is selected from the group consisting of Gals or Gal6.
Expression vectors containing Gals or Gal6 are known in the art. See, e.g.,
Offermanns; Buhl et al., 1993, FEBS Lett. 323:132-134; Amatruda et al., 1993,
J.
Biol. Chem. 268:10139-10144.
The above-described assay can be modified to form a method to
identify antagonists of GPR54. Such a method is also part of the present
invention
and comprises:
(a) providing cells that expresses both GPR54 and a promiscuous
G-protein;
(b) exposing the cells to a substance that is an agonist of GPR54;
(c) subsequently or concurrently to step (b), exposing the cells to a
substance that is a suspected antagonist of GPR54;
(d) measuring the level of inositol phosphates in the cells;
where a decrease in the level of inositol phosphates in the cells in the
presence of the suspected antagonist as compared to the level of inositol
phosphates in
the cells in the absence of the suspected antagonist indicates that the
substance is an
antagonist of GPR54.
In a particular embodiment of the above-described method, the cells
are eukaryotic cells. In another embodiment, the cells are mammalian cells. In
other
embodiments, the cells are L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC
CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70),
COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-Kl (ATCC CCL 61),
3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I
(ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171 ), Xenopus
oocytes, or Xenopus melanophores.
The conditions under which steps (b) and (c) of the method are
practiced are conditions that are typically used in the art for the study of
protein-ligand
interactions: e.g., physiological pH; salt conditions such as those
represented by such
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commonly used buffers as PBS or in tissue culture media; a temperature of
about 4°C
to about 55°C.
In a particular embodiment of the above-described method, the cells
are transfected with expression vectors that direct the expression of GPR54
and the
promiscuous G-protein in the cells.
In a particular embodiment of the above-described method, the
promiscuous G-protein is selected from the group consisting of Gals or Ga.l6.
In particular embodiments of the above-described methods, GPR54 has
an amino acid sequence of SEQ.~:N0.:3 or SEQ.m.N0.:4.
While the above-described methods are explicitly directed to testing
whether "a" substance is an agonist or antagonist of GPR54, it will be clear
to one
skilled in the art that such methods can be adapted to test collections of
substances,
e.g., combinatorial libraries, to determine whether any members of such
collections
are activators or inhibitors of GPR54. Accordingly, the use of collections of
substances, or individual members of such collections, as the substance in the
above-
described methods is within the scope of the present invention.
Agonists and antagonists of GPR54 that are identified by the above-
described methods are expected to have utility in the treatment of diseases
that
involve the inappropriate expression of GPR54. In particular, given the
resemblance
between GPR54 and the galanin receptors, it is expected that agonists and
antagonists
of GPR54 will have pharmacological activity and be useful in a manner similar
to that
in which agonists and antagonists of the galanin receptors are useful.
Therefore,
agonists and antagonists of GPR54 are expected to be useful in the treatment
of:
eating disorders and obesity; Alzheimer's disease and other disorders
affecting
memory; pain; sexual disorders; and growth hormone imbalances.
The present invention includes pharmaceutical compositions
comprising agonists and antagonists of GPR54. The agonists and antagonists are
generally combined with pharmaceutically acceptable Garners to form
pharmaceutical
compositions. Examples of such carriers and methods of formulation of
pharmaceutical compositions containing agonists and antagonists and Garners
can be
found in Remington's Pharmaceutical Sciences. To form a pharmaceutically
acceptable composition suitable for effective administration, such
compositions will
contain a therapeutically effective amount of the agonists and antagonists.
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Therapeutic or prophylactic compositions are administered to an
individual in amounts sufficient to treat or prevent conditions where GPR54
activity is
abnormal. The effective amount can vary according to a variety of factors such
as the
individual's condition, weight, gender, and age. Other factors include the
mode of
administration. The appropriate amount can be determined by a skilled
physician.
Compositions can be used alone at appropriate dosages. Alternatively,
co-administration or sequential administration of other agents can be
desirable.
The compositions can be administered in a wide variety of therapeutic
dosage forms in conventional vehicles for administration. For example, the
compositions can be administered in such oral dosage forms as tablets,
capsules (each
including timed release and sustained release formulations), pills, powders,
granules,
elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by
injection.
Likewise, they can also be administered in intravenous (both bolus and
infusion),
intraperitoneal, subcutaneous, topical with or without occlusion, or
intramuscular
form, all using forms well known to those of ordinary skill in the
pharmaceutical arts.
Advantageously, compositions can be administered in a single daily
dose, or the total daily dosage can be administered in divided doses of two,
three or
four times daily. Furthermore, compositions can be administered in intranasal
form
via topical use of suitable intranasal vehicles, or via transdermal routes,
using those
forms of transdermal skin patches well known to those of ordinary skill in
that art. To
be administered in the form of a transdermal delivery system, the dosage
administration will, of course, be continuous rather than intermittent
throughout the
dosage regimen.
The dosage regimen utilizing the compositions is selected in
accordance with a variety of factors including type, species, age, weight, sex
and
medical condition of the patient; the severity of the condition to be treated;
the route
of administration; the renal, hepatic and cardiovascular function of the
patient; and the
particular composition thereof employed. A physician of ordinary skill can
readily
determine and prescribe the effective amount of the composition required to
prevent,
counter or arrest the progress of the condition. Optimal precision in
achieving
concentrations of composition within the range that yields efficacy without
toxicity
requires a regimen based on the kinetics of the composition's availability to
target
sites. This involves a consideration of the distribution, equilibrium, and
elimination
of a composition.
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The present invention also includes methods of expressing GPR54 in
recombinant systems and then utilizing the recombinantly expressed GPR54
receptor
protein for counter-screening. When screening compounds in order to identify
potential pharmaceuticals that specifically interact with a target receptor,
it is
necessary to ensure that the compounds identified are as specific as possible
for the
target receptor. To do this, it is necessary to screen the compounds against
as wide an
array as possible of receptors that are similar to the target receptor. Thus,
in order to
find compounds that are potential pharmaceuticals that interact with receptor
A, it is
necessary not only to ensure that the compounds interact with receptor A (the
"plus
target") and produce the desired pharmacological effect through receptor A, it
is also
necessary to determine that the compounds do not interact with receptors B, C,
D, etc.
(the "minus targets"). In general, as part of a screening program, it is
important to
have as many minus targets as possible (see Hodgson, 1992, Bio/Technology
10:973-
980, at 980). Therefore, GPR54 proteins and DNA encoding GPR54 proteins have
utility in counter-screens. That is, they can be used as "minus targets" in
counter-
screens in connection with screening projects designed to identify compounds
that
specifically interact with other G-protein coupled receptors.
The DNA of the present invention, or hybridization probes based upon
the DNA, can be used in chromosomal mapping studies in order to identify the
precise
chromosomal location of the GPR54 gene or of genes encoding proteins related
to
GPR54. While the present inventors have determined that the human GPR54 gene
is
located at chromosome 19p 13.3, it may be desirable to perform mapping studies
to
even more precisely locate the human GPR54 gene. Such mapping studies can be
carned out using well-known genetic and/or chromosomal mapping techniques such
as, e.g., linkage analysis with respect to known chromosomal markers or in
situ
hybridization. See, e.g., Verma et al., 1988, Human Chromosomes: A Manual of
Basic Technigues, Pergamon Press, New York, NY. After identifying the precise
chromosomal location of the GPR54 gene or genes encoding proteins related to
GPR54, this information can be compared with the locations of known disease-
causing genes contained in genetic map data (such as the data found in the
genome
issue of Science (1994, 265:1981-2144). In this way, one can correlate the
chromosomal location of the GPR54 gene or of genes encoding proteins related
to
GPR54 with the locations of known disease-causing genes and thus help to limit
the
region of DNA containing such disease-causing genes. This will simplify the
process
of cloning such disease-causing genes. Also, once linkage between the precise
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chromosomal location of the GPR54 gene or of genes encoding proteins related
to
GPR54 and the locations of a known disease-causing gene is established, that
linkage
can be used diagnostically to identify restriction fragment length
polymorphisms
(RFLPs) in the vicinity of the GPR54 gene or of genes encoding proteins
related to
GPR54. Such RFLPs will be associated with the disease-causing gene and thus
can
be used to identify individuals carrying the disease-causing gene.
For such chromosomal mapping studies as described herein, it may be
advantageous to use, in addition to the DNA of the present invention, the
reverse
complement of the DNA of the present invention or RNA corresponding to the DNA
of the present invention.
Nucleotide sequences that are complementary to the GPR54 sequences
disclosed herein can be synthesized for use in antisense therapy. Such
antisense
molecules can be DNA, stable derivatives of DNA such as phosphorothioates or
methyl phosphonates, RNA, stable derivatives of RNA such as 2'-O-alkyl RNA, or
other forms of GPR54 antisense molecules. GPR54 antisense molecules can be
introduced into cells by a variety of methods, e.g., microinjection, liposome
encapsulation, or by expression from vectors harboring the antisense sequence.
GPR54 antisense therapy is expected to be particularly useful in the treatment
of
conditions where it is beneficial to reduce GPR54 activity.
The present invention also includes antibodies to the GPR54 protein.
Such antibodies may be polyclonal antibodies or monoclonal antibodies and are
useful
in treating disorders that involve the inappropriate expression or activity of
the
GPR54 protein. The antibodies of the present invention are raised against the
entire
GPR54 protein or against suitable antigenic fragments of the protein that are
coupled
to suitable Garners, e.g., serum albumin or keyhole limpet hemocyanin, by
methods
well known in the art. Methods of identifying suitable antigenic fragments of
a
protein are known in the art. See, e.g., Hopp & Woods, 1981, Proc. Natl. Acad.
Sci.
USA 78:3824-3828; and Jameson & Wolf, 1988, CABIOS (Computer Applications in
the Biosciences) 4:181-186.
For the production of polyclonal antibodies, GPR54 protein or an
antigenic fragment, coupled to a suitable carrier, is injected on a periodic
basis into an
appropriate non-human host animal such as, e.g., rabbits, sheep, goats, rats,
mice.
The animals are bled periodically and sera obtained are tested for the
presence of
antibodies to the injected antigen. The injections can be intramuscular,
intraperitoneal, subcutaneous, and the like, and can be accompanied with
adjuvant.
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For the production of monoclonal antibodies, GPR54 protein or an
antigenic fragment, coupled to a suitable Garner, is injected into an
appropriate non-
human host animal as above for the production of polyclonal antibodies. In the
case
of monoclonal antibodies, the animal is generally a mouse. The animal's spleen
cells
S are then immortalized, often by fusion with a myeloma cell, as described in
Kohler &
Milstein, 1975, Nature 256:495-497. For a fuller description of the production
of
monoclonal antibodies, see Antibodies: A Laboratory Manual, Harlow & Lane,
eds.,
Cold Spring Harbor Laboratory Press, 1988.
Gene therapy may be used to introduce GPR54 polypeptides into the
cells of target organs. Nucleotides encoding GPR54 polypeptides can be ligated
into
viral vectors which mediate transfer of the nucleotides by infection of
recipient cells.
Suitable viral vectors include retrovirus, adenovirus, adeno-associated virus,
herpes
virus, vaccinia virus, and polio virus based vectors. Alternatively,
nucleotides
encoding GPR54 polypeptides can be transferred into cells for gene therapy by
non-
viral techniques including receptor-mediated targeted transfer using ligand-
nucleotide
conjugates, lipofection, membrane fusion, or direct microinjection. These
procedures
and variations thereof are suitable for ex vivo as well as in vivo gene
therapy. Gene
therapy with GPR54 polypeptides will be particularly useful for the treatment
of
diseases where it is beneficial to elevate GPR54 activity.
A cDNA fragment encoding full-length GPR54 can be isolated from an
appropriate human cDNA library by using the polymerise chain reaction (PCR)
employing suitable primer pairs. Such primer pairs can be selected based upon
the
cDNA sequence for GPR54 shown in Figure 1 as SEQ.>D.NO.: l . Suitable primer
pairs would be, e.g.:
5'-ATG CAC ACC GTG GCT ACG TCC-3' (SEQ.>D.NO.:11 ) and
5'-TCA GAG AGG GGC GTT GTC CTC-3' (SEQ.>D.N0.:12).
The above primers may contain restriction sites in their S' ends to
facilitate cloning of the amplified cDNA into suitable vectors, e.g.,
pcDNA3.1. The
above primers are meant to be illustrative. One skilled in the art would
recognize that
a variety of other suitable primers can be designed.
PCR reactions can be carried out with a variety of thermostable
enzymes including but not limited to AmpliTaq, AmpliTaq Gold, or Vent
polymerise.
For AmpliTaq, reactions can be carried out in 10 mM Tris-Cl, pH 8.3, 2.0 mM
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MgCl2, 200 ~M for each dNTP, 50 mM KCI, 0.2 uM for each primer, 10 ng of DNA
template, 0.05 units/~l of AmpliTaq. The reactions are heated at 95°C
for 3 minutes
and then cycled 35 times using the cycling parameters of 95°C, 20
seconds, 62°C, 20
seconds, 72°C, 3 minutes. In addition to these conditions, a variety of
suitable PCR
protocols can be found in PCR Primer, A Laboratory Manual, edited by C.W.
Dieffenbach and G.S. Dveksler, 1995, Cold Spring Harbor Laboratory Press; or
PCR
Protocols: A Guide to Methods and Applications, Michael et al., eds., 1990,
Academic Press .
A suitable cDNA library from which a clone encoding GPR54 can be
isolated would be a human cDNA library made from RNA from brain tissue. Such
libraries can be prepared by methods well-known in the art. Alternatively,
several
commercially available libraries would be suitable, e.g., cDNA libraries such
as
human fetal brain, catalog #937227 from Stratagene, Inc., La Jolla, CA, USA,
and
human brain hypothalamus, catalog #HL1172a, from Clontech Laboratories, Inc.,
Palo Alto, CA, USA. The primary clones of such libraries can be subdivided
into
pools with each pool containing approximately 20,000 clones and each pool can
be
amplified separately.
By this method, a cDNA fragment encoding an open reading frame of
398 amino acids (SEQ.ID.N0.:3) can be obtained. This cDNA fragment can be
cloned into a suitable cloning vector or expression vector. For example, the
fragment
can be cloned into the mammalian expression vector pcDNA3.1 (Invitrogen, San
Diego, Ca). GPR54 protein can then be produced by transfernng an expression
vector
encoding GPR54 into suitable host cells and growing the host cells under
appropriate
conditions. GPR54 protein can then be isolated by methods well known in the
art.
As an alternative to the above-described PCR method, a cDNA clone
encoding GPR54 can be isolated from a cDNA library using as a probe
oligonucleotides specific for GPR54 and methods well known in the art for
screening
cDNA libraries with oligonucleotide probes. Such methods are described in,
e.g.,
Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual; Cold Spring
Harbor Laboratory, Cold Spring Harbor, New York; Glover, D.M. (ed.), 1985, DNA
Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K., Vol. I, II.
Oligonucleotides that are specific for GPR54 and that can be used to screen
cDNA
libraries can be readily designed based upon the cDNA sequence of GPR54 shown
in
Figure 1 as SEQ.ID.NO.:1 and can be synthesized by methods well-known in the
art.
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Genomic clones containing the GPR54 gene can be obtained from
commercially available human PAC or BAC libraries, e.g., from Research
Genetics,
Huntsville, AL. Alternatively, one may prepare genomic libraries, for example
in Pl
artificial chromosome vectors, from which genomic clones containing the GPR54
can
be isolated, using probes based upon the GPR54 nucleotide sequences disclosed
herein. Methods of preparing such libraries are known in the art (Ioannou et
a1.,1994,
Nature Genet. 6:84-89).
The following non-limiting examples are presented to better illustrate
the invention.
EXAMPLE 1
PCR amplification and cDNA library screening
A rat brain S' Stretch cDNA library (Clontech) was amplified by the
polymerise chain reaction (PCR) using proof reading Pfu polymerise
(Stratagene)
and degenerate oligonucleotides based upon sequences encoding GPCR conserved
transmembrane (TM) region 3
P1: 5'-CTGACCGGCATGABDETFGADCGHTA-3' (SEQ.ID.N0.:9)
and transmembrane (TM) region 7
P2: 5'-GAAGGCGTAGAFBAIJGGKTT) -3' (SEQ.ID.NO.:10)
whereB=Core,D=CorT,E=AorGorT,F=CorGorT,H=AorC,I=Aor
CorGorT,J=AorCorG,K=Aorta.
PCR conditions were as follows: denaturation at 94°C for 30 sec,
annealing at 55, 48, 45, 42, or 40°C for 40 sec, and extension at
72°C for 30 sec, for
30 cycles, followed by a 7 min extension at 72°C. The PCR products were
extracted
with phenol/chloroform, precipitated with ethanol and electrophoresed on a low
melting point agarose gel. PCR product bands in the expected size range were
excised from the gel, ligated into the EcoRV site of pBluescript SK(-)
(Stratagene)
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and sequenced. One insert appeared to encode a novel GPCR and was labeled with
~32p~ dCTP-a, (NEN) by nick translation (Amersham) and used to screen the same
library amplified above as previously described (Marchese et al., 1994,
Genomics
23:609-618). Positive phage clones were plaque purified and their inserts
amplified
by PCR using Pfu polymerise and primers flanking the Igtl 1 EcoRI cloning
site. The
PCR products were blunt-end ligated into the EcoRV site of pBluescript SK(-)
(Stratagene) and sequenced on both strands.
EXAMPLE 2
Northern blot anal
Rat mRNAs from several rat tissues were extracted as described
previously (Marchese et al., 1994, Genomics 23:609-618). Briefly, total RNA
was
extracted by the method of Chomczynski & Sacchi, 1987, Anal. Biochem. 162:156-
159 and poly (A)+ RNA isolated using oligo(dT) cellulose spin columns
(Pharmacia,
Uppsala, Sweden). RNA was denatured and size fractionated on a 1 %
formaldehyde
agarose gel, transferred onto nylon membrane and immobilized by UV
irradiation.
The blots were hybridized with a 32P-labeled DNA fragment encoding GPR54,
washed with 2X SSPE and 0.1% SDS at 50°C for 20 min and again with O.1X
SSPE
and 0.1% SDS at 50°C for 2 h and exposed to X-ray film at -70°C
in the presence of
an intensifying screen.
EXAMPLE 3
In situ hybridization analysis
~ 35S-labeled DNA fragment encoding GPR54 was used as a probe
for in situ hybridization. Preparation of rat brain sections and in situ
hybridization
procedures were done as previously described (O'Dowd et al., 1996, FEBS Lett
394:325-329).
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EXAMPLE 4
Expression of GPR54 cDNA in COS-7 mammalian cells
The African Green Monkey SV40 transformed kidney cell line (COS-7
cells), obtained from the American Type Culture Collection, was grown in
Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated
fetal
calf serum (Sigma), 50 units/ml penicillin, 50 ~g/ml streptomycin (Flow
Laboratories,
McLean, VA), and 2 mM glutamine (Flow Laboratories) at 37°C under an
atmosphere
of 6% C02. 5 X 106 cells per 175- cm2 culture flask were seeded in 20 ml of
media
and transiently transfected at 80% confluence with either 2.75, 5.5, or 11.65
~g of
pcDNA3-GPR54 or pcIneo-hGALRl plasmids and 70 ~l of LipofectAMINE reagent
(Life Technologies, Inc.), following recommendations of the manufacturer. Two
days
after transfection, cells were harvested following dissociation in enzyme-free
dissociation solution (Specialty Media, Lavallette, NJ).
EXAMPLE 5
Membrane preparation and radioli~and binding assays
Membranes were prepared from transfected cells by disruption by
pressurized nitrogen cavitation in ice-cold membrane buffer (10 mM Tris, pH
7.4, 10
mM phenylmethylsulfonylfluoride, 10 mM phosphoramidon). After a low speed
(1100 x g for 10 min. at 4°C) and a high speed centrifugation (38,700 x
g for 15 min.
at 4°C), membranes were resuspended in buffer and their protein
concentration
determined (Bio-Rad assay kit). Binding of 125I_human galanin (specific
activity of
2200 Ci/mmol, DuPont NEN) was measured in membranes using a buffer of 25 mM
Tris, pH 7.4, 0.3% BSA, 2 mM MgCl2, 4 mg/ml phosphoramidon, and 10 mM
leupeptin in a total volume of 250 ml. 200 pM of 125I_human galanin was used.
Reactions were initiated by the addition of membranes and the incubation was
allowed to proceed at room temperature for 2 hours. Non-specific binding was
defined as the amount of radioactivity remaining bound in the presence of 10
mM
unlabeled human galanin. Incubations were terminated by rapid filtration
through
GF/C filters which had been presoaked with 0.1 % polyethylamine using a TOMTEC
(Orange, CT) cell harvester.
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The present invention is not to be limited in scope by the specific
embodiments described herein. Indeed, various modifications of the invention
in
addition to those described herein will become apparent to those skilled in
the art
from the foregoing description. Such modifications are intended to fall within
the
scope of the appended claims.
Various publications are cited herein, the disclosures of which are
incorporated by reference in their entireties.
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