Note: Descriptions are shown in the official language in which they were submitted.
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A RECOMBINANT CELL LINE EXPRESSING GPCRxll AS A FUNCTIONAL
RECEPTOR VALIDATED BY ANGIOPEPTIN AND USEFUL FOR SCREENING
OF AGONISTS AND ANTAGONISTS
Field of the invention
[0001] The present invention is related to a newly
identified member of the superfamily of G-protein-coupled
receptors as well as to the various uses that can be made
of said receptor.
[00027 The invention is also related to the
polynucleic acid sequence (polynucleotide) encoding said
receptor.
[0003] The invention is further related to methods
using receptor polypeptide and polynucleotide applicable to
diagnostic and treatment in receptor-mediated disorders.
[0004] The invention is further related to drug-
screening methods using the receptor polypeptide and
polynucleotide, to identify agonists and antagonists
applicable to diagnostic, prevention and/or treatment of
said various disorders.
[0005] The invention further encompasses unknown
agonists and antagonists detected and recovered based on
the receptor polypeptide and polynucleotide.
[0006] The invention is further related to
procedures for producing the receptor polypeptide and
polynucleotide according to the invention, preferably by
genetic recombinant methods.
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Background of the invention
[0007] G-protein coupled receptors (GPCRs) are
proteins responsible for transducing a signal within a
cell. GPCRs have usually seven transmembrane domains. Upon
binding of a ligand to an extra-cellular portion or
fragment of a GPCR, a signal is transduced within the cell
that results in a change in a biological or physiological
property or behaviour of the cell. GPCRs, along with G-
proteins and effectors (intracellular enzymes and channels
modulated by G-proteins), are the components of a modular
signalling system that connects the state of intra-cellular
second messengers to extra-cellular inputs.
[0008] GPCR genes and gene products are potential
causative agents of disease and these receptors seem to be
of critical importance to both the central nervous system
and peripheral physiological processes.
[0009] The GPCR protein superfamily is represented
in five families . Family I, receptors typified by
rhodopsin and the beta2-adrenergic receptor and currently
represented by over 200 unique members; Family II, the
parathyroid hormone/calcitoninjsecretin receptor family;
Family III, the metabotropic glutamate receptor family,
Family IV, the CAMP receptor family, important in the
chemotaxis and development of D. discoideum; and Family V,
the fungal mating pheromone receptor such as STE2.
[0010] G proteins represent a family of
heterotrimeric proteins composed of a, (3 and y subunits,
that bind guanine nucleotides. These proteins are usually
linked to cell surface receptors (receptors containing
seven transmembrane domains).
[0011] Following ligand binding to~ the GPCR, a
conformational change is transmitted to the G protein,
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which caused the a-subunit to exchange a bound GDP molecule
for a GTP molecule and to dissociate from the (3y-subunits.
[0012] The GTP-bound form of the a, (3 and y-subunits
typically functions as an effector-modulating moiety,
leading to the production of second messengers, such as
CAMP (e. g. by activation of adenyl cyclase), diacylglycerol
or inositol phosphates.
[0013] Greater than 20 different types of a-subunits
are known in humans. These subunits associate with a small
pool of (3 and y subunits. Examples of mammalian G proteins
include Gi, Go, Gq, Gs and Gt. G proteins are described
extensively in Lodish et al., Molecular Cell
Biology,(Scientific American Books Inc., New York, N.Y.,
1995), the contents of which are incorporated herein by
reference.
[0014] Known and unknown GPCRs constitute now major
targets for drug action and development.
[0015] Therefore, it exists a need for providing new
G protein coupled receptors which could be used for the
screening of new agonists and antagonists having
advantageous potential prophylactic and therapeutical
properties.
[0016] More than 300 GPCRs have been cloned thus far
and it is generally assumed that it exists well over 1000
such receptors. Mechanistically, approximately 50-600 of
all clinically relevant drugs act by modulating the
functions of various GPCRs (Cudermann et al., J. Mol. Med.,
Vol. 73, pages 51-63, 1995).
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Summary of the invention
[0017] The present invention is related to newly
identified member of G-protein-coupled receptor, preferably
a human receptor, as well as to the polynucleotide sequence
encoding said human receptor described hereafter (SEQ ID
NO. 1 and 2).
[00l8] The present invention is also related to
other newly identified members of G-protein-coupled
receptors, preferably human receptors, as well as to the
polynucleotide sequence encoding said other human receptor
described hereafter (SEQ ID NO. 3 to SEQ ID NO. 22).
[00l9] The present invention is also related to
nucleotidic and/or amino acid sequence homologous to the
sequences corresponding to the receptor described
hereafter.
[0020] An homologous sequence (which may exist in
other mammal species) means a sequence which presents a
high sequence identity or homology (which presents an
identity higher than 70 0, 75°s, 80 0, 85%, 90 o or 95%) with
the complete human sequence described hereafter, and
preferably characterised by a similar pharmacology,
especially a preference for binding angiopeptin and/or
somatostatin analogs.
[0021] Another aspect of the present invention is
related to a specific active portion of said sequence. Said
active portion could be a receptor which comprises a
partial deletion upon the complete nucleotide or amino acid
sequence and which still maintains the active sites)
necessary for the binding of specific ligands able to
interact with said receptor.
[0022] Homologous sequences of the sequence
according to the invention may comprise similar receptors
which exist in other animal (rat, mouse, dog, etc.) or
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specific human populations, but which are involved in the
same biochemical pathway.
[0023] Such homologous sequences may comprise
addition, deletion or substitution of one or more amino
5 acids or nucleotides, which does not substantially alter
the functional characteristics of the receptor according to
the invention.
[0024] Thus, the invention encompasses also a
receptor and corresponding nucleotide sequence having
exactly the same amino acid or nucleotide sequences as
shown in the enclosed sequence listing, as well as
molecules which differ, but which are retaining the basic
qualitative binding properties of the complete receptor
according to the invention.
[0025] The invention is preferably related to said
(human) receptor characterised by the complete nucleotide
and amino acid sequences described hereafter, to unknown
(and not previously described in the state of the art)
agonist, reverse agonist and antagonist compounds or
inhibitors of said receptor. Preferably, said inhibitors
are antisens RNAs, rybo~ymes or antibodies (or specific
hypervariable (FAB, FAB'2, ...) portions thereof) that bind
specifically to said receptor or its encoding nucleotide
sequence (i.e. that have at least a 10 fold greater
affinity for said receptors than any other naturally
occurring antibody). Said specific antibodies are
preferably obtained by a process involving the injection of
a pharmaceutically acceptable preparation of suoh amino
acid sequence into a animal capable of producing antibodies
directed against said receptor.
[0026] For instance, a monoclonal antibody directed
to the receptor according to the invention is obtained by
injecting of an expression plasmid comprising the DNA
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encoding said receptor into a mouse and than fusing mouse
spleen cells with myeloma cells.
[0027] The present invention is also related to the
polynucleotide according to the invention, possibly linked
to other expression sequences and incorporated into a
vector (plasmids, viruses, liposomes, cationic
vesicles,...) and host cells transformed by such vector.
[0028] The present invention is also related to the
recombinant, preferably human receptor according to the
invention, produced by such host cells according to the
method well known by the person skilled in the art, as well
as a functional assay (diagnostic kit) comprising all the
means and media for the identification of the receptor, its
nucleotide sequence, as well as agonist, reverse agonist,
antagonist and inhibitor of said receptor or its nucleotide
sequence. Said diagnostic kit comprises preferably the
following elements . the receptor, its encoding nucleotide
sequence, antibodies directed against said receptor or its
nucleotide sequence, as well as possible agonist, reverse
agonist, antagonist or inhibitor compounds of said
receptor. Said diagnostic kit comprises means and media for
performing said diagnostic preferably through a measure of
dosage/activity of said receptor, by genetic analysis of
the receptor nucleotide sequence, preferably by RT/PCR or
by immuno-analysis, preferably by the use of antibodies
directed against said receptor.
[0029] The present invention is also related to a
transgenic non-human mammal comprising a partial or total
deletion of the genetic sequence encoding the receptor
according to the invention, preferably a non human mammal
comprising an homologous recombination "knock-out" of the
nucleotide sequence (polynucleotide) according to the
invention or a transgenic non human mammal overexpressing
above natural level said polynucleotide sequence.
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[0030] Said transgenic non-human mammal can be
obtained by methods well known by the person skilled in the
art, for instance by the one described in the document
W098/20112 using classical techniques based upon the
transfection of embryonic stem cells, preferably according
to the method described by Carmeliet et al., Nature,
Vol. 380, p. 435-439, 1996.
[0031] Preferably, in said transgenic non human
mammal overexpressing, the polynucleotide according to the
invention or active portions thereof has been previously
incorporated in a DNA construct with an inducible promoter
allowing its overexpression and possibly with tissues and
other specific regulatory elements.
[0032] Another aspect of the present invention is
related to a method and kit for performing said method for
the screening (detection and possibly recovering) of
compounds or a natural extract which are unknown (not yet
described in the state of the art) or not known to be
agonists, reverse agonists, antagonists or inhibitors of
natural compounds to the receptor according to the
invention, said method comprising .
- contacting a cell or cell extract from the cell
transfected with a vector expressing the polynucleotide
encoding the receptor according to the invention or
active portions) thereof,
- possibly isolating a membrane fraction from the cell
extract or the complete cell with a compound or
molecules present in said natural extract under
conditions permitting binding of said compound or said
mixture of molecules to said receptor, possibly by the
activation of a functional response and
- detecting the presence (and possibly the binding) of
said compound or said mixture of molecules to said
receptor by means of a bioassay, (preferably a
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modification in the production of a second messenger or
an increase in the receptor activity) in the presence of
another compound working as an agonist, reverse agonist,
antagonist or inhibitor to the receptor according to the
invention and thereby possibly recovering and
determining whether said compound or mixture of
molecules is (are) able to work as agonist, reverse
agonist, antagonist, or inhibitor of the compound to its
receptor.
[0033] Preferably, the second messenger assay
comprises the measurement of intra-cellular cAMP,
intracellular inositol phosphates, intra-cellular
diacylglycerol concentrations, arachinoid acid
concentration, MAP kinase(s) or tyrosine kinase(s) pathways
activation or intra-cellular calcium mobilisation.
[0034] Preferably, said bioassay is validated by the
addition of angiopeptin and any other suitable related
peptides to the receptor according to the invention by a
method well-known by the person skilled in the art and
described hereafter.
[0035] The screening method according to the
invention could be performed by well known methods to the
person skilled in the art, preferably by high-throughput
screening, diagnostic and dosage devices based upon the
method described in the International patent application
WO00/02045 performed upon various solid supports such as
micro-titer plates or biochips (microarrays) according to
known techniques by the person skilled in the art.
[0036] The present invention is also related .to the
known or unknown compound or molecules characterised and
possibly recovered by said method for its (their) use as a
medicament in therapy and is related to the pharmaceutical
composition comprising a sufficient amount of said compound
or molecules) and a pharmaceutically acceptable carrier or
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diluent for the preparation of a medicament in the
prevention and/or the treatment of various diseases.
[0037] In the pharmaceutical composition, the
carrier or the adequate pharmaceutical carrier or diluant
can be any solid, liquid or gaseous support which is non
toxic and adapted for the administration (in vivo or ex
vivo) to the patient, including the human, through various
administration roots such as oral administration,
intravenous administration, intradermal administration,
etc.
[0038] Said pharmaceutical composition may comprise
also various vesicles or adjuvants well known by the person
skilled in the art, able to modulate the immune response of
the patient. The percentage of active compound-molecules/
pharmaceutical carriers can vary, the range being only
limited by the tolerance and the efficiency of the- active
compounds to the patient. Said ranges of administration are
also limited by the frequency of administration and the
possible side effects of the compound or molecules.
[0039] A further aspect of the present invention is
related to said unknown compound or molecules) identified
by said screening method, to the pharmaceutical composition
comprising it and to their use in the treatment of viral
infections or diseases induced by various viruses or
bacteria, the treatment or prevention of disturbances of
cell migration, diseases or perturbations of the immune
system, including cancer, development of tumours and tumour
metastasis, inflammatory and neo-plastic processes,
bacterial and fungal infections, for wound and bone healing
and dysfunction of regulatory growth functions, pains,
diabetes, obesity, anorexia, bulimia, acute heart failure,
hypotension, hypertension, urinary retention, osteoporosis,
angina pectoris, myocardial infarction, restenosis,
atherosclerosis, diseases characterised by excessive smooth
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muscle cell proliferation, aneurysms, wound healing,
diseases characterised by loss of smooth muscle cells or
reduced smooth muscle cell proliferation, stroke, ischemia,
ulcers, allergies, benign prostatic hypertrophy, migraine,
5 vomiting, psychotic arid neurological disorders, including
anxiety, schizophrenia, maniac depression, depression,
delirium, dementia and severe mental retardation,
degenerative diseases, neurodegenerative diseases such as
Alzheimer's disease or Parkinson's disease, and
10 dyskinasias, such as Huntington's disease or Gilles de la
Tourett's syndrome and other related diseases.
[0040] Among the mentioned diseases the preferred
applications are related to therapeutic agents targeting
7TM receptor that can play a function in preventing,
improving or correcting dysfunctions or diseases,
including, but not limited to fertility, fetal development,
infections such as bacterial, fungal, protozoan and viral
infections, particularly infections caused by HIV1 and
HIV2, pain, cancer, anorexia, bulimia, asthma, Parkinson's
disease, acute heart failure, hypertension, urinary
retention, osteoporosis, angina pectoris, myocardial
infarction, ulcers, asthma, allergies, benign prostatic
hypertrophy, psychotic and neurological disorders including
anxiety, depression, migraine, vomiting, stroke,
schizophrenia, manic depression, delirium, dementia, severe
mental retardation and dyskinesias, such as Huntington's
disease or Gilles de la Tourette's syndrome.
[0041] This invention relates to the use of a human
G protein-coupled receptor as a screening tool to identify
agonists or antagonists of the aequorin luminescence
resulting from expression of this receptor.
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Example l: Cloning of human GPCRxII receptor
[0042] In order to identify and clone novel human GPCR
(G-protein coupled receptor) the following approach was
used. Sequences of the following GPCR: GPR8, ChemR23, HM74
and GPR14 were used as queries to search for homologies in
public high-throughput genomic sequence databases (NCBI).
[0043] Using the above strategies, a novel human
sequence of GPCR was identified. We called this new GPCR:
GPCRxll (SEQ ID number 1 and 2).
[0044] Tn order to clone the GPCRxII sequence we
performed a polymerase chain reaction (PCR) on total human
genomic DNA. Primers were synthetized based upon the
GPCRxll human sequence and were as follows:
SEQ ID 23 GPCRxII fw: 5'-ccggaattcaccatggatccaaccaccccg-3'
SEQ ID 24 GPCRxII rv: 5'-ctagtctagactctacaccagactgcttctc-3'
[0045] Amplification resulted in a fragments of 0.99
kilobase containing the entire coding sequence of the
GPCRxIl gene. This fragment was subcloned into the pCDNA3
(Invitrogen) vector for DNA sequencing analysis.
[0046] Nucleotide and deduced amino acid sequence of
human GPCRxII (SEQ ID NO 1)
2 1 M D P T T P A W G T E S T T V
5
15
1 ATGGAT CCAACCACC CCGGCCTGG GGAACAGAA AGTACAACA GTG
45
16 N G N D Q A L L L L C G K E T
30
46 AATGGA AATGACCAA GCCCTTCTT CTGCTTTGT GGCAAGGAG ACC
90
31 L I P V F L I L F I A L V G L
91 CTGATC CCGGTCTTC CTGATCCTT TTCATTGCC CTGGTCGGG CTG
135
40 46 V G N G F V L W L L G F R M R
60
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l36 GTA GGAAACGGG TTTGTGCTC TGGCTCCTG GGCTTCCGC ATGCGC
180
61 R N A F S V Y V L S L A G A D
75
181 AGG AACGCCTTC TCTGTCTAC GTCCTCAGC CTGGCCGGG GCCGAC
225
7 F L F L C F Q I I N C L V Y L
6
90
226 TTC CTCTTCCTC TGCTTCCAG ATTATAAAT TGCCTGGTG TACCTC
270
9 S N F F C S I S I N F P S F F
1
105
271 AGT AACTTCTTC TGTTCCATC TCCATCAAT TTCCCTAGC TTCTTC
315
106 T T V M T C A Y L A G L S M L
120
316 ACC ACTGTGATG ACCTGTGCC TACCTTGCA GGCCTGAGC ATGCTG
360
121 S T V S T E R C L S V L W P I
135
361 AGC ACCGTCAGC ACCGAGCGC TGCCTGTCC GTCCTGTGG CCCATC
405
136 W Y R C R R P R H L S A V V C
150
406 TGG TATCGCTGC CGCCGCCCC AGACACCTG TCAGCGGTC GTGTGT
450
151 V L L W A L S L L L S I L E G
165
451 GTC CTGCTCTGG GCCCTGTCC CTACTGCTG AGCATCTTG GAAGGG
495
166 K F C G F L F S D G D S G W C
lso
496 AAG TTCTGTGGC TTCTTATTT AGTGATGGT GACTCTGGT TGGTGT
540
181 Q T F D F I T A A W L I F L F
195
541 CAG ACATTTGAT TTCATCACT GCAGCGTGG CTGATTTTT TTATTC
585
196 M V L C G S S L A L L V R I L
210
586 ATG GTTCTCTGT GGGTCCAGT CTGGCCCTG CTGGTCAGG ATCCTC
630
211 C G S R G L P L T R L Y L T I
225
631 TGT GGCTCCAGG GGTCTGCCA CTGACCAGG CTGTACCTG ACCATC
675
226 L L T V L V F L L C G L P F G
240
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720
676 CTG CTC ACA GTG CTG GTG TTC CTC CTC TGC GGC CTG CCC TTT GGC
241 I Q W F L I L W I W K D S D V
255
721 ATT CAG TGG TTC CTA ATA TTA TGG ATC TGG AAG GAT TCT GAT GTC
765
256 L F C H I H P V S V V L S S L
270
766 TTA TTT TGT CAT ATT CAT CCA GTT TCA GTT GTC CTG TCA TCT CTT
810
271 N S S A N P I I Y F F V G S F
285
811 AAC AGC AGT GCC AAC CCC ATC ATT TAC TTC TTC GTG GGC TCT TTT
855
286 R K Q W R L Q Q P I L K L A L
300
856 AGG AAG CAG TGG CGG CTG CAG CAG CCG ATC CTC AAG CTG GCT CTC
900
301 Q R A L Q D I A E V D H S E G
315
901 CAG AGG GCT CTG CAG GAC ATT GCT GAG GTG GAT CAC AGT GAA GGA
945
316 C F R Q G T P E M S R S S L V
330
946 TGC TTC CGT CAG GGC ACC CCG GAG ATG TCG AGA AGC AGT CTG GTG
990
331
331
991 TAG
993
[0047] Amino acid sequence of human GPCRxII (330 amino
acids) (SEQ ID N0:2). The seven predicted transmembrane
domaines are underlined.
MDPTTPAWGTESTTVNGNDQALLLLCGKETLIPVFLILFIALVGLVGNGFVLWLLGFRM
RRNAFSVYVLSLAGADFLFLCFQIINCLVYLSNFFCSISINFPSFFTTVMTCAYLAGLS
MLSTVSTERCLSVLWPIWYRCRRPRHLSAVVCVLLWALSLLLSILEGKFCGFLFSDGDS
GWCQTFDFITAAWLIFLFMVLCGSSLALLVRILCGSRGLPLTRLYLTILLTVLVFLLCG
LPFGIQWFLILWIWKDSDVLFCHIHPVSWLSSLNSSANPIIYFFVGSFRKQWRLQQPI
LKLALQRALQDTAEVDHSEGCFRQGTPEMSRSSLV
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[0048] At the amino acid sequence level, the human
GPCRxII is 37% identical to the rat RTA receptor. The gene
coding GPCRxl1 is located on chromosome 11.
Alignment of GPCRxII (fig. l)
[0049] Alignment of the amino acid sequence of GPCRxl1
with RTA and other RTA related sequences were performed
using ClustalX algorithm. Then, the dendrogram was
constucted using TreeView algorithm.
GPRI
l vt~ ~
Tissular distribution of GPCRxl2
[0050] Reverse transcription-polymerase chain
reaction (RT-PCR) experiments were carried out using a
panel of polyA~ RNA (Clontech). The primers were as
follows: GPCRxll sense primer (SEQ ID NO 25: 5'-
TTCTCTGTCTACGTCCTCAG-3') and GPCRxII antisense primer (SEQ
ID NO 26: 5'-GTCCTGTCATCTCTTAACAG-3'). The expected size of
the amplified DNA band was 586 bp. Approximately 75 ng of
poly A+ RNA was reverse transcribed with superscript II
(Life Technologies) and used for PCR. PCR was performed
under the following conditions: denaturation at 94°C for 3
mina 38 cycles at 94°C for 1 min, 58°C for 2 min and 72°C
for 2 min. Aliquots (10 ~.l) of the PCR reaction were
analysed by 1o agarose gel electrophoresis.
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(0051] GPCRxIl mRNA was assayed by RT-PCR in 16 human
tissues. A strong band of expected sire (586 bp) was
detected in testis, at lower levels in uterus and thymus,
while not in pituitary gland, spinal cord, pancreas, small
5 intestine, placenta, stomach, liver, lung, spleen, brain,
heart, kidney and skeletal muscle.
Functional assay for GPCRxII
(0052] GPCRxII expressing clones have been obtained
by transfection of CHO-K1 cells coexpressing mitochondrial
10 apoaequorin and Galphal6, limit dilution and selection by
northern blotting. Positive clones were used for screening
with a reference peptidic library containing 250 peptides
and neuropeptides at a concentration of 100 nM. A specific
activity was obtained with angiopeptin (D-NaI-Cys-Tyr-D-
15 trp-Lys-Val-Cys-Thr-NH2 with a disulfide bridge between the
two cysteines) and confirmed by a dose respone curve (see
figure 1). Additional related peptides were tested using
the same cells. Amongst the different peptides tested,
somatostatin analog (D2-NaI-Cys-Tyr-D-trp-Lys-Val-Cys-D2-
NaI-NH2) exhibited similar affinity. Somatostatin 14 has
no activity on GPCRxll.
Material. All chemicals were obtained from Sigma, unless
stated. The cell culture media were from Gibco BRL and the
peptides from bachem
Aequorin assays. CHO-K1 cell lines expressing GPCRxll
receptors, Galphal6 and mitochondrial apoaequorin were
established. A functional assay based on the luminescence
of mitochondrial aequorin following intracellular Caz+
release (1) was performed as described (2). Briefly, cells
were collected from plates with PBS containing 5 mM EDTA,
pelleted and resuspended at 5 X 106 cells/ml in DMEM-F12
medium, incubated with 5 ~.M Coelenterazine H (Molecular
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Probes) for 4 hours at room temperature. Cells were then
washed in DMEM-F12 medium and resuspended at a
concentration of 0.5 X 106 cells/ml. Cells were then mixed
with the peptides and the light emission recorded during 30
sec. using a Microlumat luminometer (Perkin Elmer). Results
are expressed as Relative Light Units (RLU).
Antibodies
[0053] Antibodies directed against GPCRxl1 have been
produced by repeated injections of plasmid encoding GPCRxll
to mice. Serum has been collected following 5 injections
and used for flow cytometry analysis with cells transfected
with GPCRxll. Several sera were positive and can be used
for immunohistochemistry and other related applications
Example 2 Cloning of the other sequences related to
G-protein-coupled receptors
[0054] In order to identify and clone novel human DNA
sequences related to GPCR, the following approche was used.
Sequences of the following GPCR: GPR8, ChemR23, HM74 and
GPR14 were used as queries to search for homologies in
public high-throughput genomic sequence databases (NCBI).
[0055] Using the above strategies, ten novel human
sequences of GPCR were identified. None of these clones
contain introns .
GPCRx2, SEQ ID NO 3
GPCRxS, SEQ ID NO 5
GPCRx7, SEQ ID NO 7
GPCRx9, SEQ ID NO 9
GPCRxI4, SEQ ID NO 11
GPCRxI6, SEQ ID NO 13
GPCRxl7, SEQ ID NO 15
GPCRxI8, SEQ ID NO 17
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GPCRxI9, SEQ ID NO 19
GPCRx20, SEQ ID NO 21
[0056] In order to clone these GPCRx sequences, a
polymerase chain reaction (PCR) was performed on total
human genomic DNA. Primers were synthetized based upon the
human sequences described above and were as follows:
SEQ ID NO 27 GPCRx2 fw: 5'-ccggaattcaccatggagtcctcacccatc-3'
SEQ ID NO 28 GPCRx2 rv: 5'-ctagtctagacatcatgactccagccggg-3'
SEQ ID NO 29 GPCRx5 fw: 5'-ccggaattcaccatggatccaaccatctcaacc-3'
SEQ ID NO 30 GPCRx5 rv: 5'-ctagtctagatcactgctccaatctgcttc-3'
SEQ ID NO 31 GPCRx7 fw:
5'-ccggaattcaccatgaaccagactttgaatagcagtgg-3'
SEQ ID NO 32 GPCRx7 rv:
5'-ctagtctagatctcaagcccccatctcattggtgccc-3'
SEQ ID NO 33 GPCRx9 fw: 5'-ccggaattcaccatggaagctgacctgg-3'
SEQ ID NO 34 GPCRx9 rv: 5'-ctagtctagactcacgtggggcctgcgcc-3'
SEQ ID NO 35 GPCRxI4 fw: 5'-ccggaattcgccatgtacaacgggtcg-3'
SEQ ID NO 36 GPCRxI4 rv: 5'-ctagtctagattcagtgccactcaacaatg-3'
[0057] Amplification resulted in a fragments of
approximately 1 - 1.5 kilobase containing the entire coding
sequence of the human genes. These fragments obtained were
subcloned into the pCDNA3 (Invitrogen) vector for DNA
sequencing analysis.
Tissue distribution of identified (GPCRx) receptors
[0058] To determine the tissue distribution of different
GPCRx mRNA, reverse transcriptase-polymerase chaine
reaction (RT-PCR) were performed with 200 ng of mRNA
isolated from human tissues (Clontech). The oligo(dT)
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primer was used in the reverse transcription step. Then,
different GPCRx cDNA were amplified with specifics primers.
GPCRx GPCRx GPCRx GPCRx GPCRx GPCRx GPCRxGPCRx GPCRx
2 7 9 14 16 17 18 19 20
Li _ _ _ _ _ _ _ _ +
Lu .+. _ + + - ++ - - ++
/
_
Sp _ - ++ + - _ _ _ +
Te - + - ++ - ++ - -I-/- +
Br ++ - - - - - ++ - ++
He _ _ _ _ _ _ _ - ++
Ki +/ - - + - ++ - - +
_
Sk.m _ _ _ _ _ + _ - ++
Pi.G _ - - - - - ++ +/- +
Sp.C ++ _ - - - ++ +/- +/- +~-
Th +/ - + - - +-I- - - ++
_
Pa _ _ _ _ - ++ .+/ _ -
_
S +/ - + - - ++ - - +
. _
In
Ut _ _ _ _ _ .+.+ _ +/ +,
_
P1 - - - ++ ++ - - - +
St - - + +/- - ..~+ - - +
Table l: Tissue distribution of GPCRxs: The presence or
absence of differents GPCRx was determined by RT-PCR
analysis. ++, strong signal; +, signal clearly detected;
+/-, weak signal; -, signal not detected. The tissues are
the following: Li, liver; Lu, lung; Sp, Spleen; Te, testis;
Br, Brain; He, Heart; Ki, Kidney; Sk.M, Skeletal muscle;
Pi.G, Pituitary gland; Sp.C, spinal cord; Th, Thymus; Pa,
Pancreas; S.In, Small intestine; Ut, Uterus; Pl, Plancenta;
St, Stomach.
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Reference
1. Stables, J., A. Green, F. Marshall, N. Fraser, E.
Knight, M. Sautel, G. Milligan, M. Lee, and S. Rees.
1997. A bioluminescent assay for agonist activity at
potentially any G-protein-coupled receptor. Anal.
Biochem. 252:115-126.
2. Blanpain, C., I. Migeotte, B. Lee, J. Vakili, B.J.
Doran~, C. Govaerts, G. Vassart, R.W. Doms, and M.
Parmentier. 1999 CCRS binds multiple CC-chemokines: MCP
3 acts as a natural antagonist. Blood 94:1899-1905.
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Nucleotide sequence human 3 and
and of GPCRx2 4
deduced (SEQ
amino ID
acid NO:
respectively)
1 M E S S P I P Q S S G N 5 S T 15
1 ATGGAG TCCTCACCC ATCCCC CAGTCATCA GGGAACTCT TCCACT 45
16 L G R V P Q T P G P S T A S G 30
46 TTGGGG AGGGTCCCT CAAACC CCAGGTCCC TCTACTGCC AGTGGG 90
31 V P E V G L 12 D V A S E S V A 45
9l GTCCCG GAGGTGGGG CTACGG GATGTTGCT TCGGAATCT GTGGCC 135
46 L F F M L L L D L T A V A G N 60
136 CTCTTC TTCATGCTC CTGCTG GACTTGACT GCTGTGGCT GGCAAT 180
61 A A V M A V T A K T P A L R K 75
181 GCCGCT GTGATGGCC GTGATC GCCAAGACG CCTGCCCTC CGAAAA 225
76 F V F V F H L C L V D L L A A 90
226 TTTGTC TTCGTCTTC CACCTC TGCCTGGTG GACCTGCTG GCTGCC 270
91 L T L M P L A M L S S S A L F 105
271 CTGACC CTCATGCCC CTGGCC ATGCTCTCC AGCTCTGCC CTCTTT 315
106 D H A L F G E V A C R L Y L F 120
316 GACCAC GCCCTCTTT GGGGAG GTGGCCTGC CGCCTCTAC TTGTTT 360
121 L S V C F V S L A I L S V S A 135
361 CTGAGC GTGTGCTTT GTCAGC CTGGCCATC CTCTCGGTG TCAGCC 405
136 I N V E R Y Y Y V V H P M R Y 150
406 ATCAAT GTGGAGCGC TACTAT TACGTAGTC CACCCCATG CGCTAC 450
151 E V R M T L G L V A S V L V G 165
451 GAGGTG CGCATGACG CTGGGG CTGGTGGCC TCTGTGCTG GTGGGT 495
166 V W V K A L A M A S V P V L G 180
496 GTGTGG GTGAAGGCC TTGGCC ATGGCTTCT GTGCCAGTG TTGGGA 540
181 R V S W E E G A P S V P P G C 195
541 AGGGTC TCCTGGGAG GAAGGA GCTCCCAGT GTCCCCCCA GGCTGT 585
196 S L Q W S H S A Y C Q L F V V 210
586 TCACTC CAGTGGAGC CACAGT GCCTACTGC CAGCTTTTT GTGGTG 630
211 V F A V L Y F L L P L L L I L 225
631 GTCTTT GCTGTCCTT TACTTT CTGTTGCCC CTGCTCCTC ATACTT 675
226 V V Y C S M F R V A R V A A M 240
676 GTGGTC TACTGCAGC ATGTTC CGAGTGGCC CGCGTGGCT GCCATG 720
241 Q H G P L P T W M E T P R Q R 255
721 CAGCAC GGGCCGCTG CCCACG TGGATGGAG ACACCCCGG CAACGC 765
256 S E S L S S R S T M V T S S G 270
766 TCCGAA TCTCTCAGC AGCCGC TCCACGATG GTCACCAGC TCGGGG 810
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271 A P Q T T P H R T F G G G K A 285
811 GCCCCC CAGACCACCCCA CACCGG ACGTTTGGG GGAGGGAAA GCA 855
286 A V V L L A V G G Q F L L C W 300
856 GCAGTG GTTCTCCTGGCT GTGGGG GGACAGTTC CTGCTCTGT TGG 900
301 L P Y F S F H L Y V A L S A Q 315
901 TTGCCC TACTTCTCTTTC CACCTC TATGTTGCC CTGAGTGCT CAG 945
316 P I S T G Q V E S V V T W I G 330
946 CCCATT TCAACTGGGCAG GTGGAG AGTGTGGTC ACCTGGATT GGC 990
331 Y F C F T S N P F F Y G C L N 345
991 TACTTT TGCTTCACTTCC AACCCT TTCTTCTAT GGATGTCTC AAC 1035
346 R Q I R G E L S K Q F V C F F 360
1036CGGCAG ATCCGGGGGGAG CTCAGC AAGCAGTTT GTCTGCTTC TTC 1080
361 K P A P E E E L R L P S R E G 375
1081AAGCCA GCTCCAGAGGAG GAGCTG AGGCTGCCT AGCCGGGAG GGC 1125
376 S I E E N F L Q F L Q G T G C 390
1126TCCATT GAGGAGAACTTC CTGCAG TTCCTTCAG GGGACTGGC TGT 1170
391 P S E S W V S R P L P S P K Q 405
1171CCTTCT GAGTCCTGGGTT TCCCGA CCCCTACCC AGCCCCAAG CAG 1215
406,E P P A V D F R I P G Q I A E 420
1216GAGCCA CCTGCTGTTGAC TTTCGA ATCCCAGGC CAGATAGCT GAG 1260
421 E T S E F L E Q Q L T S D I I 435
1261GAGACC TCTGAGTTCCTG GAGCAG CAACTCACC AGCGACATC ATC 1305
436 M S D S Y L R P A A S P R L E 450
1306ATGTCA GACAGCTACCTC CGTCCT GCCGCCTCA CCCCGGCTG GAG 1350
451 S * 452
1351TCATGA 1356
Amino acid sequence of human GPCRx2 (451 amino acids) (SEQ ID N~: 4). The
seven
predicted transmembrane domaines are underlined.
MESSPIPQSSGNSSTLGRVPQTPGPSTASGVPEVGLRDVASESVALFFMLLLDLTAVAGNAAVMAVIAKTPALRKFV_F
'VF
HLCLVDLLAALTLMPLAMLSSSALFDHALFGEVACRLYLFLSVCFVSLAILSVSAINVERYYYVVHPMRYEVRMTLGLV
A
SVLVGVWKALAMASVPVLGRVSWEEGAPSVPPGCSLQWSHSAYCQLFWVFAVLYFLLPLLLILVWCSMFRVARVAAM
QHGPLPTWMETPRQRSESLSSRSTMVTSSGAPQTTPHRTFGGGKAAWLLAVGGQFLLCWLPYFSFHLWALSAQPISTG
QVESWTWIGYFCFTSNPFFYGCLNRQIRGELSKQFVCFFKPAPEEELRLPSREGSIEENFLQFLQGTGCPSESWVSRPL
PSPKQEPPAVDFRIPGQIAEETSEFLEQQLTSDIIMSDSYLRPAASPRLES
At the amino acid sequence level, the human GPCRx2 is 23o identical to the
human
histamine H2 receptor.
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Nucleotide acidsequence human 5 and
and of GPCRx5 6
deduced (SEQ
amino ID
NO:
respectively)
1 M D P T I S T L D T E L T P T 15
1 ATG GATCCAACC ATCTCA ACCTTGGACACA GAACTGACA CCAATC 45
Z6 N G T E E T L C Y IC Q T L S L 30
46 AAC GGAACTGAG GAGACT CTTTGCTACAAG CAGACCTTG AGCCTC 90
31 T V L T C I V S L V G L T G N 45
91 ACG GTGCTGACG TGCATC GTTTCCCTTGTC GGGCTGACA GGAAAC 135
46 A V V L W L L G C R M R R N A 60
136 GCA GTTGTGCTC TGGCTC CTGGGCTGCCGC ATGCGCAGG AACGCC 180
61 F S I Y I L N L A A A D F L F 75
181 TTC TCCATCTAC ATCCTC AACTTGGCCGCA GCAGACTTC CTCTTC 225
76 L S G R L I Y S L L S F I S I 90
226 CTC AGCGGCCGC CTTATA TATTCCCTGTTA AGCTTCATC AGTATC 270
91 P H T I S IC I L Y P V M M F S 105
271 CCC CATACCATC TCTAAA ATCCTCTATCCT GTGATGATG TTTTCC 315
106 Y F A G L S F L S A V S T E R 120
316 TAC TTTGCAGGC CTGAGC TTTCTGAGTGCC GTGAGCACC GAGCGC 360
121 C L S V L W P I W Y R C H R P 135
361 TGC CTGTCCGTC CTGTGG CCCATCTGGTAC CGCTGCCAC CGCCCC 405
136 T H L S A V V C V L L W A L S 150
406 ACA CACCTGTCA GCGGTG GTGTGTGTCCTG CTCTGGGCC CTGTCC 450
151 L L R S I L E W M L C G F L F 165
451 CTG CTGCGGAGC ATCCTG GAGTGGATGTTA TGTGGCTTC CTGTTC 495
166 S G A D S A W C Q T S D F I T 180
496 AGT GGTGCTGAT TCTGCT TGGTGTCAAACA TCAGATTTC ATCACA 540
l81 V A W L I F L C V V L C G S S 195
541 GTC GCGTGGCTG ATTTTT TTATGTGTGGTT CTCTGTGGG TCCAGC 585
196 L V L L I R I L C G S R K I P 210
586 CTG GTCCTGCTG ATCAGG ATTCTCTGTGGA TCCCGGAAG ATACCG 630
211 L T R L Y V T I L L T V L V F 225
631 CTG ACCAGGCTG TACGTG ACCATCCTGCTC ACAGTACTG GTCTTC 675
226 L L C G L P F G I Q F F L F L 240
676 CTC CTCTGTGGC CTGCCC TTTGGCATTCAG TTTTTCCTA TTTTTA 720
241 W I H V D R E V L F C H V H L 255
721 TGG ATCCACGTG GACAGG GAAGTCTTATTT TGTCATGTT CATCTA 765
256 V S I F L S A L N S S A N P I 270
766 GTT TCTATTTTC CTGTCC GCTCTTAACAGC AGTGCCAAC CCCATC 810
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271I Y F F V G S F R Q R Q N R Q 285
811ATTTAC TTCTTC GTGGGCTCC TTTAGGCAG CGTCAAAAT AGGCAG 855
286N L K L V L Q R A L Q D A S E 300
856AACCTG AAGCTG GTTCTCCAG AGGGCTCTG CAGGACGCG TCTGAG 900
301V D E G G G Q L P E E I L E L 315
901GTGGAT GAAGGT GGAGGGCAG CTTCCTGAG GAAATCCTG GAGCTG 945
316S G S R L E Q * 323
946TCGGGA AGCAGA TTGGAGCAG TGA 969
Amino acid sequence of human GPCRx5 (322 amino acids) (SEQ ID N0:6) . The
seven
predicted transmembrane domaines are underlined.
MDPTISTLDTELTPINGTEETLCYKQTLSLTVLTCIVSLVGLTGNAVVLWLLGCRMRRNAFSIYILNLAAADFLFLSGR
L
IYSLLSFISIPHTISKILYPVMMFSYFAGLSFLSAVSTERCLSVLWPIWYRCHRPTHLSAWCVLLWALSLLRSILEWML
CGFLFSGADSAWCQTSDFITVAWLIFLCVVLCGSSLVLLIRILCGSRKIPLTRLYVTILLTVLVFLLCGLPFGIQFFLF
L
WIHVDREVLFCHVHLVSIFLSALNSSANPIIYFFVGSFRQRQNRQNLKLVLQRALQDASEVDEGGGQLPEEILELSGSR
L
EQ
At the amino acid sequence level, the human GPCRxS is 31% identical to the
human
mas receptor.
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Nucleotide acidsequence human 7 and
and of GPCRx7 8
deduced (SEQ
amino ID
NO:
respectively)
1 M N Q T L N S S G T V E S A L 15
1 ATGAAC CAGACTTTG AATAGCAGT GGGACCGTG GAGTCAGCC CTA 45
16 N Y S R G S T V H T A Y L V L 30
46 AACTAT TCCAGAGGG AGCACAGTG CACACGGCC TACCTGGTG CTG 90
31 S S L A M F T C L C G M A G N 45
91 AGCTCC CTGGCCATG TTCACCTGC CTGTGCGGG ATGGCAGGC AAC 135
46 S M V I W L L G F R M H R N P 60
136 AGCATG GTGATCTGG CTGCTGGGC TTTCGAATG CACAGGAAC CCC 180
61 F C I Y I L N L A A A D L L F 75
181 TTCTGC ATCTATATC CTCAACCTG GCGGCAGCC GACCTCCTC TTC 225
76 L F S M A S T L S L E T Q P L 90
226 CTCTTC AGCATGGCT TCCACGCTC AGCCTGGAA ACCCAGCCC CTG 270
91 V N T T D K V H E L M K R L M 105
271 GTCAAT ACCACTGAC AAGGTCCAC GAGCTGATG AAGAGACTG ATG 315
106 Y F A Y T V G L S L L T A 2 S 120
316 TACTTT GCCTACACA GTGGGCCTG AGCCTGCTG ACGGCCATC AGC 360
121 T Q R C L S V L F P I W F K C 135
361 ACCCAG CGCTGTCTC TCTGTCCTC TTCCCTATC TGGTTCAAG TGT 405
136 H R P R H L S A W V C G L L W 150
406 CACCGG CCCAGGCAC CTGTCAGCC TGGGTGTGT GGCCTGCTG TGG 450
151 T L C L L M N G L T S S F C S 165
451 ACACTC TGTCTCCTG ATGAACGGG TTGACCTCT TCCTTCTGC AGC 495
166 K F L K F N E D R C F R V D M 180
496 AAGTTC TTGAAATTC AATGAAGAT CGGTGCTTC AGGGTGGAC ATG 540
181 V Q A A L I M G V L T P V M T 195
541 GTCCAG GCCGCCCTC ATCATGGGG GTCTTAACC CCAGTGATG ACT 585
196 L S S L T L F V W V R R S S Q 210
586 CTGTCC AGCCTGACC CTCTTTGTC TGGGTGCGG AGGAGCTCC CAG 630
211 Q W R R Q P T R L F V V V L A 225
631 CAGTGG CGGCGGCAG CCCACACGG CTGTTCGTG GTGGTCCTG GCC 675
226 S V L V F L I C S L P L S I Y 240
676 TCTGTC CTGGTGTTC CTCATCTGT TCCCTGCCT CTGAGCATC TAC 720
241 W F V L Y W L S L P P E M Q V 255
721 TGGTTT GTGCTCTAC TGGTTGAGC CTGCCGCCC GAGATGCAG GTC 765
256 L C F S L S R L S S S V S S S 270
766 CTGTGC TTCAGCTTG TCACGCCTC TCCTCGTCC GTAAGCAGC AGC 810
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271A N P V I Y F L V G S R R S H 285
811GCCAAC CCCGTC ATCTACTTC CTGGTGGGC AGCCGGAGG AGCCAC 855
286R L P T R S L G T V L Q Q A L 300
856AGGCTG CCCACC AGGTCCCTG GGGACTGTG CTCCAACAG GCGCTT 900
301R E E P E L E G G E T P T V G 315
901CGCGAG GAGCCC GAGCTGGAA GGTGGGGAG ACGCCCACC GTGGGC 945
316T N E M G A * 322
946ACCAAT GAGATG GGGGCTTGA 966
Amino acid sequence of human GPCRx7 (321 amino acids) (SEQ ID N0:8). The seven
predicted transmembrane domaines are underlined.
MNQTLNSSGTVESALNYSRGSTVHTAYLVLSSLAMFTCLCGMAGNSMVIWLLGFRMHRNPFCIYILNLAAADLLFLFSM
A
STLSLETQPLVNTTDKVHELMKRLMYFAYTVGLSLLTAISTQRCLSVLFPIWfKCHRPRHLSAWVCGLLWTLCLLMNGL
T
SSFCSKFLKFNEDRCFRVDMVQAALIMGVLTPVMTLSSLTLFVWVRRSSQQWRRQPTRLFVVVLASVLVFLICSLPLSI
Y
WFVLYWLSLPPEMQVLCFSLSRLSSSVSSSANPVIYFLVGSRRSHRLPTRSLGTVLQQALREEPELEGGETPTVGTNEM
G
A
At the amino acid sequence level, the human GPCRx7 is 29o identical to the rat
RTA receptor.
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Nucleotide acidsequence human 9 and
and of GPCRx9 10
deduced (SEQ
amino ID
NO:
respectively)
1 M E A D L G A T G H R P R T E 15
1 ATG GAAGCTGAC CTGGGTGCC ACTGGCCACAGG CCCCGCACA GAG 45
16 L D D E D S Y P Q G G W D T V 30
46 CTT GATGATGAG GACTCCTAC CCCCAAGGTGGC TGGGACACG GTC 90
31 F L V A L L L L G L P A N G L 45
91 TTC CTGGTGGCC CTGCTGCTC CTTGGGCTGCCA GCCAATGGG TTG 135
46 M A W L A G S Q A R H G A G T 60
136 ATG GCGTGGCTG GCCGGCTCC CAGGCCCGGCAT GGAGCTGGC ACG l80
61 R L A L L L L S L A L S D F L 75
181 CGT CTGGCGCTG CTCCTGCTC AGCCTGGCCCTC TCTGACTTC TTG 225
76 F L A A A A F Q I L E I R H G 90
226 TTC CTGGCAGCA GCGGCCTTC CAGATCCTAGAG ATCCGGCAT GGG 270
91 G H W P L G T A A C R F Y Y F 105
271 GGA CACTGGCCG CTGGGGACA GCTGCCTGCCGC TTCTACTAC TTC 315
106 L W G V S Y S S G L F L L A A 120
316 CTA TGGGGCGTG TCCTACTCC TCCGGCCTCTTC CTGCTGGCC GCC 360
121 L S L D R C L L A L C P H W Y 135
361 CTC AGCCTCGAC CGCTGCCTG CTGGCGCTGTGC CCACACTGG TAC 405
136 P G H R P V R L P L W V C A G 150
406 CCT GGGCACCGC CCAGTCCGC CTGCCCCTCTGG GTCTGCGCC GGT 450
151 V W V L A T L F S V P W L V F 165
451 GTC TGGGTGCTG GCCACACTC TTCAGCGTGCCC TGGCTGGTC TTC 495
166 P E A A V W W Y D L V I C L D 180
496 CCC GAGGCTGCC GTCTGGTGG TACGACCTGGTC ATCTGCCTG GAC 540
181 F W D S E E L S L R M L E V L 195
541 TTC TGGGACAGC GAGGAGCTG TCGCTGAGGATG CTGGAGGTC CTG 585
196 G G F L P F L L L L V C H V L 210
586 GGG GGCTTCCTG CCTTTCCTC CTGCTGCTCGTC TGCCACGTG CTC 630
211 T Q A T A C R T C H R Q Q Q P 225
631 ACC CAGGCCACA GCCTGTCGC ACCTGCCACCGC CAACAGCAG CCC 675
226 A A C R G F A R V A R T I L S 240
676 GCA GCCTGCCGG GGCTTCGCC CGTGTGGCCAGG ACCATTCTG TCA 720
241 A Y V V L R L P Y Q L A Q L L 255
721 GCC TATGTGGTC CTGAGGCTG CCCTACCAGCTG GCCCAGCTG CTC 765
256 Y L A F L W D V Y S G Y L L W 270
766 TAC CTGGCCTTC CTGTGGGAC GTCTACTCTGGC TACCTGCTC TGG 810
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271 E A L V Y S 77 Y L I L L N S C 285
811 GAG CTGGTC TCCGAC TACCTGATC CTACTCAAC TGC 855
GCC TAC AGC
286 L S P F L C L M A S A D L R T 300
856 CTC CCCTTC TGCCTC ATGGCCAGT GCCGACCTC ACC 900
AGC CTC CGG
301 L L R S V L S S F A A A L C E 315
901 CTG CGCTCC CTCTCG TCCTTCGCG GCAGCTCTC GAG 945
CTG GTG TGC
316 E R P G S F T P T E P Q T Q L 330
946 GAG CCGGGC TTCACG CCCACTGAG CCACAGACC CTA 990
CGG AGC CAG
331 D S E G P T L P E P M A E A Q 345
99I GAT GAGGGT ACTCTG CCAGAGCCG ATGGCAGAG CAG 1035
TCT CCA GCC
346 S Q M D P V A Q P Q V N P T L 360
1036 TCA ATGGAT GTGGCC CAGCCTCAG GTGAACCCC CTC 1080
CAG CCT ACA
361 Q P R S D P T A Q P Q L N P T 375
1081 CAG CGATCG CCCACA GCTCAGCCA CAGCTGAAC ACG 1125
CCA GAT CCT
376 A Q P Q S D P T A Q P Q L N L 390
1126 GCC CCACAG GATCCC ACAGCCCAG CCACAGCTG CTC 1170
CAG TCG AAC
391 M A Q P Q S D S V A Q P Q A D 405
1171 ATG CAGCCA TCAGAT TCTGTGGCC CAGCCACAG GAC 1215
GCC CAG GCA
406 T N V Q T P A P A A S S V P S 420
1216 ACT GTCCAG CCTGCA CCTGCTGCC AGTTCTGTG AGT 1260
AAC ACC CCC
421 P C D E A S P T P S S H P T P 435
1261 CCC GATGAA TCCCCA ACCCCATCC TCGCATCCT CCA 1305
TGT GCT ACC
436 G A L E D P A T P P A S E G E 450
1306 GGG CTTGAG CCAGCC ACACCTCCT GCCTCTGAA GAA 1350
GCC GAC GGA
451 S P S S T P P E A A P G A G P 465
1351 AGC AGCAGC CCGCCA GAGGCGGCC CCGGGCGCA CCC 1395
CCC ACC GGC
466 T * 467
1396 ACG 1401
TGA
Amino f humanGPCRx9 (466amino cids) . The
acid a (SEQ six
sequence ID
o N0:10)
predictedtransmembrane
domaines
are
underlined.
MEADLGATGHRPRTELDDEDSYPQGGWDTVFLVALLLLGLPANGLMAWLAGSQARHGAGTRLALLLLSLALSDFLFLAA
A
A_FQILEIRHGGHWPLGTAACRFYYFLWGVSYSSGLFLLAALSLDRCLLALCPHWYPGHRPVRLPLWVCAGVWVLATLF
SV
PWLVFPEAAVWWYDLVICLDFWDSEELSLRMLEVLGGFLPFLLLLVCHVLTQATACRTCHRQQQPAACRGFARVARTIL
S
AYVVLRLPYQLAQLLYLAFLWDVYSGYLLWEALVYSDYLILLNSCLSPFLCLMASADLRTLLRSVL
SSFAAALCEERPGS
FTPTEPQTQLDSEGPTLPEPMAEAQSQMDPVAQPQVNPTLQPRSDPTAQP
QLNPTAQPQSDPTAQP QLNLMAQPQSDSVA
QPQADTNVQTPAPAASSVPSPCDEASPTPS SHPTPGALEDPATPPASEGE
SPSSTPPEAAPGAGPT
At the no acidsequence el,the human is 33aidentical the human
ami lev GPCRx9 to
ChemR23
receptor.
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Nucleotide acid GPCRxI4 (SEQID 11
and sequence NO: and
deduced of
amino human
12 respectively)
1 M Y N G S C C R I E G D T I S 15
1 ATGTAC AACGGGTCG TGCTGC CGCATCGAGGGG GACACCATC TCC 45
16 Q V M P P L L I V A F V L G A 30
46 CAGGTG ATGCCGCCG CTGCTC ATTGTGGCCTTT GTGCTGGGC GCA 90
31 L G N G V A L C G F C F H M K 45
91 CTAGGC AATGGGGTC GCCCTG TGTGGTTTCTGC TTCCACATG AAG 135
46 T W K P S T V Y L F N L A V A 60
136 ACCTGG AAGCCCAGC ACTGTT TACCTTTTCAAT TTGGCCGTG GCT 180
61 D F L L M I C L P F R T D Y Y 75
181 GATTTC CTCCTTATG ATCTGC CTGCCTTTTCGG ACAGACTAT TAC 225
76 L R R R H W A F G D I P C R V 90
226 CTCAGA CGTAGACAC TGGGCT TTTGGGGACATT CCCTGCCGA GTG 270
91 G L F T L A M N R A G S I V F 105
271 GGGCTC TTCACGTTG GCCATG AACAGGGCCGGG AGCATCGTG TTC 315
106 L T V V A A D R Y F K V V H P 120
316 CTTACG GTGGTGGCT GCGGAC AGGTATTTCAAA GTGGTCCAC CCC 360
121 H H A V N T I S T R V A A G I 135
361 CACCAC GCGGTGAAC ACTATC TCCACCCGGGTG GCGGCTGGC ATC 405
136 V C T L W A L V I L G T V Y L 150
406 GTCTGC ACCCTGTGG GCCCTG GTCATCCTGGGA ACAGTGTAT CTT 450
151 L L E N H L C V Q E T A V S C 165
451 TTGCTG GAGAACCAT CTCTGC GTGCAAGAGACG GCCGTCTCC TGT 495
166 E S F I M E S A N G W H D I M 180
496 GAGAGC TTCATCATG GAGTCG GCCAATGGCTGG CATGACATC ATG 540
181 F Q L E F F M P L G I I L F C 195
541 TTCCAG CTGGAGTTC TTTATG CCCCTCGGCATC ATCTTATTT TGC 585
196 S F K I V W S L R R R Q Q L A 210
586 TCCTTC AAGATTGTT TGGAGC CTGAGGCGGAGG CAGCAGCTG GCC 630
211 R Q A R M K K A T R F I M V V 225
631 AGACAG GCTCGGATG AAGAAG GCGACCCGGTTC ATCATGGTG GTG 675
226 A I V F I T C Y L P S V S A R 240
676 GCAATT GTGTTCATC ACATGC TACCTGCCCAGC GTGTCTGCT AGA 720
241 L Y F L W T V P S S A C D P S 255
721 CTCTAT TTCCTCTGG ACGGTG CCCTCGAGTGCC TGCGATCCC TCT 765
256 V H G A L H I T L S F T Y M N 270
766 GTCCAT GGGGCCCTG CACATA ACCCTCAGCTTC ACCTACATG AAC 810
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271 S M L D P L V Y Y F S S P S F 285
811 AGC ATG CTG GAT CCC CTG GTG TAT TAT TTT AGC CCC TCC 855
TCA TTT
286 P K F Y N K L K I C S L K P K 300
856 CCC AAA TTC TAC AAC AAG CTC AAA ATC TGC CTG AAA CCC 900
AGT AAG
301 Q P G H S K T Q R P E E M P I 315
901 CAG CCA GGA CAC TCA AAA ACA CAA AGG CCG GAG ATG CCA 945
GAA ATT
316 S N L G R R S C I S V A N S F 330
946 TCG AAC CTC GGT CGC AGG AGT TGC ATC AGT GCA AAT AGT 990
GTG TTC
331 ~Q S Q S D G Q W D P H I V E W 345
991 CAA AGC CAG TCT GAT GGG CAA TGG GAT CCC ATT GTT GAG 1035
CAC TGG
346 H * 347
1036 CAC TGA 1041
Amino acid sequence of human GPCRxI4 (346 (SEQ ID N0:12).The
amino acids) seven
predicted transmembrane domaines are underlined.
MYNGSCCRIEGDTISQVMPPLLIVAFVLGALGNGVALCGFCFHMKTWKPSTVYLFNLAVADFLLMICLPFRTDYYLRRR
H
WAFGDIPCRVGLFTLAMNRAGSIVFLTWAADRYFKVVHPHHAVNTISTRVAAGIVCTLWALVILGTVYLL
LENHLCVQE
TAVSCESFIMESANGWHDIMFQLEFFMPLGIILFCSFKIVWSLRRRQQLARQARMKKATRFIMVVAIVFITCYLPSVSA
R
LYFLWTVPSSACDPSVHGALHITLSFTYMNSMLDPLVYYFSSPSFPKFYNKLKICSLKPKQPGHSKTQRPEEMPISNLG
R
RSCISVANSFQSQSDGQWDPHIVEWH
At the amino acid sequence level, the human 50o identical the
GPCRxI4 is to
human HM74 receptor.
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Nucleotide acidsequence human ID 13
and of GPCRxI6 N0: and
deduced (SEQ
amino
14 respectively). This tidesequence located on the
chromosome4.
nucleo is
1 M G P G E A L L A G L L V M V 15
1 ATGGGC CCCGGC GAGGCGCTG CTGGCGGGT CTCCTGGTG ATGGTA 45
16 L A V A L L S N A L V L L C C 30
46 CTGGCC GTGGCG CTGCTATCC AACGCACTG GTGCTGCTT TGTTGC 90
31 A Y S A E L R T R A S G V L L 45
91 GCCTAC AGCGCT GAGCTCCGC ACTCGAGCC TCAGGCGTC CTCCTG 135
46 V N L S L G H L L L A A L D M 60
136 GTGAAT CTGTCT CTGGGCCAC CTGCTG'CTGGCGGCGCTG GACATG 180
61 P F T L L G V M R G R T P S A 75
181 CCCTTC ACGCTG CTCGGTGTG ATGCGCGGG CGGACACCG TCGGCG 225
76 P G A C Q V I G F L D T F L A 90
226 CCCGGC GCATGC CAAGTCATT GGCTTCCTG GACACCTTC CTGGCG 270
91 S N A A L S V A A h 5 A D Q W 105
27l TCCAAC GCGGCG CTGAGCGTG GCGGCGCTG AGCGCAGAC CAGTGG 315
7.06 L A V G F P L R Y A G R L R P 120
316 CTGGCA GTGGGC TTCCCACTG CGCTACGCC GGACGCCTG CGACCG 360
121 R Y A G L L L G C A W G Q S L 135
361 CGCTAT GCCGGC CTGCTGCTG GGCTGTGCC TGGGGACAG TCGCTG 405
136 A F S G A A L G C S W L G Y S 150
406 GCCTTC TCAGGC GCTGCACTT GGCTGCTCG TGGCTTGGC TACAGC 450
151 S A F A S C S L R L P P E P E 165
451 AGCGCC TTCGCG TCCTGTTCG CTGCGCCTG CCGCCCGAG CCTGAG 495
166 R P R F A A F T A T L H A V G 180
496 CGTCCG CGCTTC GCAGCCTTC ACCGCCACG CTCCATGCC GTGGGC 540
181 F V L P L A V L C L T S L Q V 195
541 TTCGTG CTGCCG CTGGCGGTG CTCTGCCTC ACCTCGCTC CAGGTG 585
196 H R V A R R H C Q R M D T V T 210
586 CACCGG GTGGCA CGCAGACAC TGCCAGCGC ATGGACACC GTCACC 630
211 M K A L A L L A D L H P R Y W 225
631 ATGAAG GCGCTC GCGCTGCTC GCCGACCTG CACCCCAGG TATTGG 675
226 P S A C R Q A Q A R D L G A P 240
676 CCCAGT GCATGC CGACAGGCC CAGGCCAGG GACTTGGGC GCTCCC 720
241 W A V G L R S L W A S P P L L 255
721 TGGGCA GTTGGC TTGAGGAGC CTGTGGGCA TCACCACCG TTACTC 765
256 C P E F T S H S T A P A R C S 270
766 TGCCCA GAGTTC ACCAGCCAC AGCACTGCC CCTGCACGC TGCTCA 810
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271Q G F P V G S L V Q T L R G P 2B5
811CAG GGGTTTCCT GTTGGTTCATTG GTGCAGACA CTGCGG GGGCCT 855
286L P P G I C A H S A Q G A L R 300
856CTG CCTCCTGGG ATATGTGCTCAC AGTGCACAG GGAGCT TTGCGC 900
301R A V G C A S P G G V P R A L 3l5
901AGA GCTGTGGGG TGTGCTTCTCCG GGAGGGGTT CCGCGG GCTCTG 945
316L W A A R H T P P V H G C G S 330
946CTG TGGGCGGCC AGACACACCCCT CCTGTGCAT GGCTGT GGGTCT 990
331E A S A C F C P L L T Q C P C 345
991GAG GCATCTGCT TGTTTCTGCCCA CTGCTGACC CAGTGC CCTTGC 1035
346M D L G F K S * 352
1036ATG GACTTGGGC TTCAAG'PCTTGA 1059
Amino acid sequence of human GPCRxl6 (352 amino acids) (SEQ ID N0: 14). The
six
predicted transmembrane domaines are underlined.
MGPGEALLAGLLVMVLAVALLSNALVLLCCAYSAELRTRASGVLLVNLSLGHLLLAALDMPFTLLGVMRGRTPSAPGA_
CQ
VIGFLDTFLASNAALSVAALSADQWLAVGFPLRYAGRLRPRYAGLLLGCAWGQSLAFSGAALGCSWLGYSSAFASCSLR
L
PPEPERPRFAAFTATLHAVGFVLPLAVLCLTSLQVHRVARRHCQRMDTVTMKALALLADLHPRYWPSACRQAQARDLGA
P
WAVGLRSLWASPPLLCPEFTSHSTAPARCSQGFPVGSLVQTLRGPLPPGICAHSAQGALRRAVGCASPGGVPRALLWAA
R
HTPPVHGCGSEASACFCPLLTQCPCMDLGFKS
At the amino acid sequence level, the human GPCRxI6 is 50o identical to the
rat
GPR 26 receptor.
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Nucleotide and deduced amino acid sequence of human GPCRxI7 (SEQ TD N0: 1S and
16 respectively). This nucleotide sequence is located on the chromosome 2.
1 M T P N S T G E V P S P I P IC 15
1 ATGACG CCCAACAGC ACTGGCGAG GTGCCCAGC CCCATTCCC AAG 45
16 G A L G L S L A L A S L I I T 30
46 GGGGCT TTGGGGCTC TCCCTGGCC CTGGCAAGC CTCATCATC ACC 90
31 A N L L L A L G I A G T A A C 45
91 GCGAAC CTGCTCCTA GCCCTGGGC ATCGCTGGG ACCGCCGCC TGC 135
46 A A T C W L L L P E P T A G W 60
136 GCAGCC ACCTGCTGG CTGCTTCTT CCTGAGCCT ACTGCTGGC TGG 180
61 A A H G S G I A T L P G L W N 75
181 GCTGCT CACGGGTCT GGCATTGCC ACATTGCCA GGGCTGTGG AAC 225
76 Q S R R G Y W S C L L V Y L A 90
226 CAGAGT CGCCGGGGT TACTGGTCC TGCCTCCTC GTCTACTTG GCT 270
91 P N F S F L S L L A N L L L V 105
271 CCCAAC TTCTCCTTC CTCTCCCTG CTTGCCAAC CTCTTGCTG GTG 315
106 H G E R Y M A V L R P L Q P P 120
316 CACGGG GAGCGCTAC ATGGCAGTC CTGAGGCCA CTCCAGCCC CCT 360
121 G S I R L A L L L T W A G P L 135
361 GGGAGC ATTCGGCTG GCCCTGCTC CTCACCTGG GCTGGTCCC CTG 405
136 L F A S L P A L G W N H W T P 150
406 CTCTTT GCCAGTCTG CCCGCTCTG GGGTGGAAC CACTGGACC CCT 450
151 G A N C S S Q A I F P A P Y L 165
451 GGTGCC AACTGCAGC TCCCAGGCT ATCTTCCCA GCCCCCTAC CTG 495
166 Y L E V Y G L L L P A V G A A 180
496 TACCTC GAAGTCTAT GGGCTCCTG CTGCCCGCC GTGGGTGCT GCT 540
181 A F L 5 V R V L A T A H R Q L 195
541 GCCTTC CTCTCTGTC CGCGTGCTG GCCACTGCC CACCGCCAG CTG 585
196 Q D I C R L E R A V C R D E P 210
586 CAGGAC ATCTGCCGG CTGGAGCGG GCAGTGTGC CGCGATGAG CCC 630
211 5 A L A R A L T W R Q A R A Q 225
631 TCCGCC CTGGCCCGG GCCCTTACC TGGAGGCAG GCAAGGGCA CAG 675
226 A G A M L L F G L C W G P Y V 240
676 GCTGGA GCCATGCTG CTCTTCGGG CTGTGCTGG GGGCCCTAC GTG 720
241 A T' L L L S V L A Y E Q R P P 255
721 GCCACA CTGCTCCTC TCAGTCCTG GCCTATGAG CAGCGCCCG CCA 765
256 L G P G T L L 5 L L S L G S A 270
766 CTGGGG CCTGGGACA CTGTTGTCC CTCCTCTCC CTAGGAAGT GCC 810
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271S A A A V P V A M G L G D Q R 285
811AGTGCA GCG GTG CCCGTAGCC ATGGGGCTG GGCGATCAG CGC 855
GCA
286Y T A P W R Q P P K G A C R G 300
856TACACA GCC TGG AGGCAGCCG CCCAAAGGT GCCTGCAGG GGC 900
CCC
301C G E E P P G T V P A P A L P 315
901TGTGGG GAA CCT CCCGGGACA GTCCCGGCC CCAGCATTG CCT 945
GAG
316T T Q A A K A V S T W T * 327
946ACCACC CAA GCC AAAGCAGTG TCGACCTGG ACTTGA 984
GCA
Amino acid sequence of human GPCRxI7 (327 amino acids) (SEQ TD N0:16). The
seven
predicted transmembrane domaines are underlined.
MTPNSTGEVPSPIPKGALGLSLALASLIITANLLLALGIAGTAACAATCWLLLPEPTAGWAAHGSGIATLPGLWNQSRR
G
YWSCLLVYLAPNFSFLSLLANLLLVHGERYMAVLRPLQPPGSIRLALLLTWAGPLLFASLPALGWNHWTPGANCSSQAI
F
PAPYLYLEVYGLLLPAVGAAAFLSVRVLATAHRQLQDICRLERAVCRDEPSALARALTWRQARAQAGAMLLFGLCWGPY
V
ATLLLSVLAYEQRPPLGPGTLLSLLSLGSASAAAVPVAMGLGDQRYTAPWRQPPKGACRGCGEEPPGTVPAPALPTTQA
A
At the amino acid sequence level, the human GPCRxI7 is 28's identical to the
human EDGE receptor
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Nucleotide acidsequence human ID 17
and of GPCRxl8 NO: and
deduced (SEQ
amino
18 respectively). This tidesequence located on the chromosome
2.
nucleo is
1 M G D E L A P C P V G T T A W 15
1 ATG GGGGATGAG CTGGCACCT TGCCCTGTG GGCACTACA GCTTGG 45
16 P A L I Q L I S K T P C M P Q 30
46 CCG GCCCTGATC CAGCTCATC AGCAAGACA CCCTGCATG CCCCAA 90
31 A A S N T S L G L G D L R V P 45
91 GCA GCCAGCAAC ACTTCCTTG GGCCTGGGG GACCTCAGG GTGCCC 135
46 S S M L Y W L F L P S S L L A 60
136 AGC TCCATGCTG TACTGGCTT TTCCTTCCC TCAAGCCTG CTGGCT 180
61 A A T L A V S P L L L V T I L 75
181 GCA GCCACACTG GCTGTCAGC CCCCTGCTG CTGGTGACC ATCCTG 225
76 R N Q R L R Q E P H Y L L P A 90
226 CGG AACCAACGG CTGCGACAG GAGCCCCAC TACCTGCTC CCGGCT 270
91 N I L L S D L A Y I L L H M L 105
271 AAC ATCCTGCTC TCAGACCTG GCCTACATT CTCCTCCAC ATGCTC 315
106 I S S S 5 L G G W E L G R M A 120
316 ATC TCCTCCAGC AGCCTGGGT GGCTGGGAG CTGGGCCGC ATGGCC 360
121 C G I L T D A V F A A C T 5 T 135
361 TGT GGCATTCTC ACTGATGCT GTCTTCGCC GCCTGCACC AGCACC 405
136 I L S F T A I V L H T Y L A V 150
406 ATC CTGTCCTTC ACCGCCATT GTGCTGCAC ACCTACCTG GCAGTC 450
151 I H P L R Y L S F M S H G A A 165
451 ATC CATCCACTG CGCTACCTC TCCTTCATG TCCCATGGG GCTGCC 495
166 W K A V A L I W L V A C C F P 180
496 TGG AAGGCAGTG GCCCTCATC TGGCTGGTG GCCTGCTGC TTCCCC 540
181 T F L I W L S K W Q D A Q L E 195
541 ACA TTCCTTATT TGGCTCAGC AAGTGGCAG GATGCCCAG CTGGAG 585
196 E Q G A S Y I L P P S M G T Q 210
586 GAG CAAGGAGCT TCATACATC CTACCACCA AGCATGGGC ACCCAG 630
211 P G C G L L V I V T Y T S I L 225
631 CCG GGATGTGGC CTCCTGGTC ATTGTTACC TACACCTCC ATTCTG 675
226 C V L F L C T A L I A N C F W 240
676 TGC GTTCTGTTC CTCTGCACA GCTCTCATT GCCAACTGT TTCTGG 720
241 R I Y A E A K T S G I W G Q G 255
721 AGG ATCTATGCA GAGGCCAAG ACTTCAGGC ATCTGGGGG CAGGGC 765
256 Y S R A R G T L L I H S V L I 270
766 TAT TCCCGGGCC AGGGGCACC CTGCTGATC CACTCAGTG CTGATC 810
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271 T L Y V S T G V V F S L D M V 285
811 ACA TTG TAC GTG AGC ACA GGG GTG GTG TTC CTG GAC ATG 855
TCC GTG
286 L T R Y H H I D S G T H T W L 300
856 CTG ACC AGG TAC CAC CAC ATT GAC TCT GGG CAC ACA TGG 900
ACT CTC
301 L A A N S E V L M M L P R A M 315
901 CTG GCA GCT AAC AGT GAG GTA CTC ATG ATG CCC CGT GCC 945
CTT ATG
316 L T Y L Y L L R Y R Q L L G M 330
946 CTC ACA TAC CTG TAC CTG CTC CGC TAC CGG CTG TTG GGC 990
CAG ATG
331 V R G H L P S R R H Q A I F T 345
991 GTC CGG GGC CAC CTC CCA TCC AGG AGG CAC GCC ATC TTT 1035
CAG ACC
346 I S * 347
1036 ATT TCC TAG 1044
Amino acid sequence of human GPCRxI8 (347 (SEQ ID N0:18).The
amino acids) seven
predicted transmembrane domaines are underlined.
MGDELAPCPVGTTAWPALIQLISKTPCMPQAASNTSLGLGDLRVPSSMLYWLFLPSSLLAAATLAVSPLLLVTILRNQR
L
RQEPHYLLPANILLSDLAYILLHMLISSSSLGGWELGRMACGILTDAVFAACTSTILSFTAIVLHTYLAVT
HPLRYLSFM
SHGAAWKAVALIWLVACCFPTFLIWLSKWQDAQLEEQGASYILPPSMGTQPGCGLLVIVTYTSILCVLFLC
TALIANCFW
RIYAEAKTSGIWGQGYSRARGTLLIHSVLITLYVSTGWFSLDMVLTRYHHIDSGTHTWLLAANSEVLMMLPRAMLTYLY
LLRYRQLLGMVRGHLPSRRHQAIFTIS
At the amino acid sequence level, the human 25% identical the
GPCRxIB is to
rabbit 5HT1D-(3 receptor.
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Nucleotide acid sequence) GPCR.xl9
and sequence of
deduced (partial human
amino
(SEQ 9 respective ly).This tidesequenc e locatedon
ID NO: and nucleo is the
1 20
chromosome .
16
1 G P H R S Q R S H L C F R A K 15
1 GGCCCCCAT AGGAGC CAACGAAGTCAT CTTTGC TTCAGAGCTAAA 45
16 P V F L L S T A N I L T V I T 30
46 CCAGTTTTT CTTCTC TCCACAGCAAAT ATCTTG ACAGTGATCATC 90
31 L S Q L V A R R Q K S S Y N Y 45
91 CTCTCCCAG CTGGTG GCAAGAAGACAG AAGTCC TCCTACAACTAT 135
46 L L A L A A A D I L V L F F I 60
136 CTCTTGGCA CTCGCT GCTGCCGACATC TTGGTC CTCTTTTTCATA 180
61 V F V D F L L E D F I L N M Q 75
181 GTGTTTGTG GACTTC CTGTTGGAAGAT TTCATC TTGAACATGCAG 225
76 M P Q V P D K I I E V L E F S 90
226 ATGCCTCAG GTCCCC GACAAGATCATA GAAGTG CTGGAATTCTCA 270
91 S I H T S I W I T V P L T I D 105
271 TCCATCCAC ACCTCC ATATGGATTACT GTACCG TTAACCATTGAC 3l5
106 R Y I A V C H P L K Y H T V S 120
316 AGGTATATC GCTGTC TGCCACCCGCTC AAGTAC CACACGGTCTCA 360
121 Y P A R T R K V I V S V Y I T 135
361 TACCCAGCC CGCACC CGGAAAGTCATT GTAAGT GTTTACATCACC 405
136 C F L T S I P Y Y W W P N I W 150
406 TGCTTCCTG ACCAGC ATCCCCTATTAC TGGTGG CCCAACATCTGG 450
151 T E D Y I S T S V H H V L I W 165
451 ACTGAAGAC TACATC AGCACCTCTGTG CATCAC GTCCTCATCTGG 495
166 I H C F T V Y L V P C S I F F 180
496 ATCCACTGC TTCACC GTCTACCTGGTG CCCTGC TCCATCTTCTTC 540
181 I L N S I I V Y K L R R K S N 195
541 ATCTTGAAC TCAATC ATTGTGTACAAG CTCAGG AGGAAGAGCAAT 585
196 F R L R G Y S T G K T T A I L 210
586 TTTCGTCTC CGTGGC TACTCCACGGGG AAGACC ACCGCCATCTTG 630
211 F T I T S I F A T L W A P R I 225
631 TTCACCATT ACCTCC ATCTTTGCCACA CTTTGG GCCCCCCGCATC 675
226 I M I L Y H L Y G A P I Q N R 240
676 ATCATGATT CTTTAC CACCTCTATGGG GCGCCC ATCCAGAACCGC 720
241 W L V H I M S D I A N M L A L 255
721 TGGCTGGTA CACATC ATGTCCGACATT GCCAAC ATGCTAGCCCTT 765
256 L N T A I N F F L Y C F I S K 270
766 CTGAACACA GCCATC AACTTCTTCCTC TACTGC TTCATCAGCAAG 810
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271 R F R T M A A A T L K A F F K 285
811 CGG TTC CGC ACC ATG GCA GCC GCC ACG CTC AAG GCT TTC TTC AAG 855
286 C Q K Q P V Q F Y T N H N F S 300
856 TGC CAG AAG CAA CCT GTA CAG TTC TAC ACC AAT CAT AAC TTT TCC 900
301 I T S S P W I 5 P A N S H C I 315
901 ATA ACA AGT AGC CCC TGG ATC TCG CCG GCA AAC TCA CAC TGC ATC 945
316 K M L V Y Q Y D K N G K P I K 330
946 AAG ATG CTG GTG TAC CAG TAT GAC AAA AAT GGA AAA CCT ATA AAA 990
331 V S P * 333
991 GTA TCC CCG TGA 1002
Partial amino acid sequence of human GPCRxI9 (333 amino acids) (SEQ ID N0:20).
The seven predicted transmembrane domaines are underlined.
GPHRSQRSHLCFRAKPVFLLSTANILTVIILSQLVARRQKSSYNYLLALAAADILVLFFIVFVDFLLEDFILNMQMPQV
P
DKIIEVLEFSSIHTSIWITVPLTIDRYIAVCHPLKYHTVSYPARTRKVIVSVYITCFLTSIPYYWWPNIWTEDYISTSV
H
HVLIWIHCFTVYLVPCSIFFILNSIIVYKLRRKSNFRLRGYSTGKTTAILFTITSIFATLWAPRIIMILYHLYGAPIQN
_R
WLVHIMSDIANMLALLNTAINFFLYCFISKRFRTMAAATLKAFFKCQKQPVQFYTNHNFSITSSPWISPANSHCIKMLV
Y
QYDKNGKPIKVSP
At the amino acid sequence level, the human GPCRxI9 is 25o identical to the C.
EleganS F21C10.9 G-protein coupled receptor.
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Nucleotide acid GPCRx20 (SEQ 21
and sequence ID and
deduced of N0:
amino human
22 respectively) . s cated thechromosome 5.
This lo on
nucleotide
sequence
i
l M L A A A F A D S N S S S M N 15
1 ATGCTG GCAGCTGCC TTTGCAGAC TCTAACTCC AGCAGCATG AAT 45
16 V S F A H L H F A G G Y L P S 30
46 GTGTCC TTTGCTCAC CTCCACTTT GCCGGAGGG TACCTGCCC TCT 90
31 D S Q D W R T I T P A L L V A 45
91 GATTCC CAGGACTGG AGAACCATC ATCCCGGCT CTCTTGGTG GCT 135
46 V C L V G F V G N L C V T G I 60
136 GTCTGC CTGGTGGGC TTCGTGGGA AACCTGTGT GTGATTGGC ATC 180
61 L L H N A W K G K P S M I H S 75
181 CTCCTT CACAATGCT TGGAAAGGA AAGCCATCC ATGATCCAC TCC 225
76 L I L N L S L A D L S L L L F 90
226 CTGATT CTGAATCTC AGCCTGGCT GATCTCTCC CTCCTGCTG TTT 270
91 S A P I R A T A Y S K S V W D 105
271 TCTGCA CCTATCCGA GCTACGGCG TACTCCAAA AGTGTTTGG GAT 315
106 L G W F V C K S S D W F I H T 120
316 CTAGGC TGGTTTGTC TGCAAGTCC TCTGACTGG TTTATCCAC ACA 360
121 C M A A K S L T I V V V A K V 135
361 TGCATG GCAGCCAAG AGCCTGACA ATCGTTGTG GTGGCCAAA GTA 405
136 C F M Y A S D P A K Q V S I H 150
406 TGCTTC ATGTATGCA AGTGACCCA GCCAAGCAA GTGAGTATC CAC 450
151 N Y T I W S V L V A I W T V A 165
451 AACTAC ACCATCTGG TCAGTGCTG GTGGCCATC TGGACTGTG GCT 495
I66 S L L P L P E W F F S T I R H 180
496 AGCCTG TTACCCCTG CCGGAATGG TTCTTTAGC ACCATCAGG CAT 540
181 H E G V E M C L V D V P A V A 195
541 CATGAA GGTGTGGAA ATGTGCCTC GTGGATGTA CCAGCTGTG GCT 585
196 E E F M S M F G K L Y P L L A 210
586 GAAGAG TTTATGTCG ATGTTTGGT AAGCTCTAC CCACTCCTG GCA 630
211 F G L P L F F A S F Y F W R A 225
631 TTTGGC CTTCCATTA TTTTTTGCC AGCTTTTAT TTCTGGAGA GCT 675
226 Y D Q C K K R G T K T Q N L R 240
676 TATGAC CAATGTAAA AAACGAGGA ACTAAGACT CAAAATCTT AGA 720
241 N Q I R S K Q V T V M L L S I 255
721 AACCAG ATACGCTCA AAGCAAGTC ACAGTGATG CTGCTGAGC ATT 765
256 A I I S A L L W L P E W V A W 270
766 GCCATC ATCTCTGCT CTCTTGTGG CTCCCCGAA TGGGTAGCT TGG 810
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271 L W V W H L K A A G P A P P Q 285
811 CTG TGG GTA TGG CAT CTG AAG GCA GGC GCC CCA CCA 855
GCT CCG CAA
286 G F I A L S Q V L M F S I S S 300
856 GGT TTC ATA GCC CTG TCT CAA TTG ATG TCC ATC TCT 900
GTC TTT TCA
301 A N P L I F L V M S E E F R E 315
901 GCA AAT CCT CTC ATT TTT CTT ATG TCG GAG TTC AGG 945
GTG GAA GAA
316 G L K G V W K W M I T K K P P 330
946 GGC TTG AAA GGT GTA TGG AAA ATG ATA AAA AAA CCT 990
TGG ACC CCA
331 T V S E S Q E T P A G N S E G 345
991 ACT GTC TCA GAG TCT CAG GAA CCA GCT AAC TCA GAG 1035
ACA GGC GGT
346 L P D K V P S P E S P A S T P 360
1036 CTT CCT GAC AAG GTT TCT GAA TCC GCA TCC ATA 1080
CCA CCA CCA CCA
361 E K E K P S S P S S G K G K T 375
1081 GAA AAA GAG AAA CCC TCT TCC TCT AAA GGG AAA 1125
AGC CCC GGC ACT
376 E K A E I P I L P D V E Q F W 390
1126 GAG AAG GCA GAG ATT ATC CCT GAC GAG CAG TTT 1170
CCC CTT GTA TGG
391 H E R D T V P S V Q D N D P I 405
1171 CAT GAG AGG GAC ACA CCT GTA CAG AAT GAC CCT 1215
GTC TCT GAC ATC
406 P W E H E D Q E T G E G V K * 419
2216 CCC TGG GAA CAT GAA CAA ACA GGG GGT GTT AAA 1260
GAT GAG GAA TAG
Amino acid sequence of human amino acids)(SEQ ID N0:22).The seven
GPCRx20 (419
predicted transmembrane domaines
are underlined.
MLAAAFADSNSSSMNVSFAHLHFAGGYLPSDSQDWRTITPALLVAVCLVGFVGNLCVIGILLHNAWKGKPS
MIHSlILNL
SLADLSLLLFSAPIRATAYSKSVWDLGWFVCKSSDWFIHTCMAAKSLTIVWAKVCFMYASDPAKQVSIHN
YTIWSVLVA
IWTVASLLPLPEWFFSTIRHHEGVEMCLVDVPAVAEEFMSMFGKLYPLLAFGLPLFFASFYFWRAYDQCKKRGTKTQNL
R
NQTRSKQVTVMLLSIAIISALLWLPEWVAWLWVWHLKAAGPAPPQGFIALSQVLMFSISSANPLIFLVMSE
EFREGLKGV
WKWMITKKPPTVSESQETPAGNSEGLPDKVPSPESPASTPEKEKPSSPSSGKGKTEKAEIPILPDVEQFWHERDTVPSV
Q
DNDPIPWEHEDQETGEGVK
At the amino acid sequence the 20s identical the
level, human to
GPCRx20
is
mouse galanin 2 receptor.
