Note: Descriptions are shown in the official language in which they were submitted.
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human G-protein coupled receptor
Description
The present invention relates to novel identified polynucleotides,
polypeptides encoded by
them and to the use of such polynucleotides and polypeptides, and to their
production. More
particularly, the polynucleotides and polypeptides of the present invention
relate to a G-protein
coupled receptor (GPCR), hereinafter referred to as IGS3. The invention also
relates to inhibiting
or activating the action of such polynucleotides and polypeptides, to a vector
containing said
polynucleotides, a host cell containing such vector and transgenic animals
where the IGS3-gene
is either overexpressed, misexpressed, underexpressed and/or suppressed (knock-
out animals).
The invention further relates to a method for screening compounds capable to
act as an agonist
or an antagonist of said G-protein coupled receptor IGS3.
BACKGROUND OF THE INVENTION
It is well established that many medically significant biological processes
are mediated by
proteins participating in signal transduction pathways that involve G-proteins
and/or second
messengers; e.g., cAMP (Lefkowitz, Nature, 1991, 351:353-354). Herein these
proteins are
referred to as proteins participating in pathways with G-proteins. Some
examples of these
proteins include the GPC receptors, such as those for adrenergic agents and
dopamine
(Kobilka, B.K., et al., Proc. Natl. Acad. Sci., USA, 1987, 84:46-50; Kobilka,
B.K., et al., Science,
1987, 238:650-656; Bunzow, J.R., et al., Nature, 1988, 336:783-787), G-
proteins themselves,
effector proteins, e.g., phospholipase C, adenylate cyclase, and
phosphodiesterase, and
actuator proteins, e.g., protein kinase A and protein kinase C (Simon, M.I.,
et al., Science, 1991,
252:802-8).
For example, in one form of signal transduction, upon hormone binding to a
GPCR the
receptor interacts with the heterotrimeric G-protein and induces the
dissociation of GDP from the
guanine nucleotide-binding site. At normal cellular concentrations of guanine
nucleotides, GTP
fills the site immediately. Binding of GTP to the a subunit of the G-protein
causes the
dissociation of the G-protein from the receptor and the dissociation of the G-
protein into a and (3y
subunits. The GTP-carrying form then binds to activated adenylate cyclase.
Hydrolysis of GTP
to GDP, catalyzed by the G-protein itself (a subunit possesses an intrinsic
GTPase activity),
returns the G-protein to its basal, inactive form. The GTPase activity of the
a subunit is, in
essence, an internal clock that controls an on/off switch. The GDP bound form
of the a subunit
has high affinity for /3y and subsequent reassociation of aGDP with py returns
the system to the
CONFIRMATION COPY
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basal state. Thus the G-protein serves a dual role, as an intermediate that
relays the signal from
receptor to effector (in this example adenylate cyclase), and as a clock that
controls the duration
of the signal.
The membrane bound superfamily of G-protein coupled receptors has been
characterized
as having seven putative transmembrane domains. The domains are believed to
represent
transmembrane a-helices connected by extracellular or cytoplasmic loops. G-
protein coupled
receptors include a wide range of biologically active receptors, such as
hormone, viral, growth
factor and neuroreceptors.
The G-protein coupled receptor family includes dopamine receptors which bind
to
neuroleptic drugs used for treating CNS disorders. Other examples of members
of this family
include, but are not limited to calcitonin, adrenergic, neuropeptideY,
somastotatin, neurotensin,
neurokinin, capsaicin, VIP, CGRP, CRF, CCK, bradykinin, galanin, motilin,
nociceptin,
endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin, histamine,
thrombin, kinin,
follicle stimulating hormone, opsin, endothelial differentiation gene-1,
rhodopsin, odorant, and
cytomegalovirus receptors.
Most G-protein coupled receptors have single conserved cysteine residues in
each of the
first two extracellular loops which form disulfide bonds that are believed to
stabilize functional
protein structures. The 7 transmembrane regions are designated as TM1, TM2,
TM3, TM4, TMS,
TM6 and TM7. The cytoplasmic loop which connects TM5 and TM6 may be a major
component
of the G-protein binding domain.
Most G-protein coupled receptors contain potential phosphorylation sites
within the third
cytoplasmic loop and/or the carboxy terminus. For several G-protein coupled
receptors, such as
the (3-adrenoreceptor, phosphorylation by protein kinase A and/or specific
receptor kinases
mediates receptor desensitization.
Recently, it was discovered that certain GPCRs, like the calcitonin-receptor
like receptor,
might interact with small single pass membrane proteins called receptor
activity modifying
proteins (RAMP's). This interaction of the GPCR with a certain RAMP is
determining which
natural ligands have relevant affinity for the GPCR-RAMP combination and
regulate the
functional signaling activity of the complex (McLathie, L.M. et al., Nature
(1998) 393:333-339).
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For some receptors, the ligand binding sites of G-protein coupled receptors
are believed to
comprise hydrophilic sockets formed by several G-protein coupled receptor
transmembrane
domains, said sockets being surrounded by hydrophobic residues of the G-
protein coupled
receptors. The hydrophilic side of each G-protein coupled receptor
transmembrane helix is
postulated to face inward and form a polar ligand-binding site. TM3 has been
implicated in
several G-protein coupled receptors as having a ligand-binding site, such as
the TM3 aspartate
residue. TM5 serines, a TM6 asparagine and TM6 or TM7 phenylalanines or
tyrosines are also
implicated in ligand binding.
G-protein coupled receptors can be intracellularly coupled by heterotrimeric G-
proteins to
various intracellular enzymes, ion channels and transporters (see, Johnson et
al., Endoc. Rev.,
1989, 10:317-331 ). Different G-protein a-subunits preferentially stimulate
particular effectors to
modulate various biological functions in a cell. Phosphorylation of
cytoplasmic residues of G-
protein coupled receptors has been identified as an important mechanism for
the regulation of
G-protein coupling of some G-protein coupled receptors. G-protein coupled
receptors are found
in numerous sites within a mammalian host.
Receptors - primarily the GPCR class - have led to more than half of the
currently known
drugs (brews, Nature Biotechnology, 1996, 14: 1516). This indicates that these
receptors have
an established, proven history as therapeutic targets. The new IGS3 GPCR
described in this
invention clearly satisfies a need in the art for identification and
characterization of further
receptors that can play a role in diagnosing, preventing, ameliorating or
correcting dysfunctions,
disorders, or diseases, hereafter generally referred to as "the Diseases". The
Diseases include,
but are not limited to, psychiatric and CNS disorders, including
schizophrenia, episodic
paroxysmal anxiety (EPA) disorders such as obsessive compulsive disorder
(OCD), post
traumatic stress disorder (PTSD), phobia and panic, major depressive disorder,
bipolar disorder,
Parkinson's disease, general anxiety disorder, autism, delirium, multiple
sclerosis, Alzheimer
disease/dementia and other neurodegenerative diseases, severe mental
retardation,
dyskinesias, Huntington's disease, Tourett's syndrome, tics, tremor, dystonia,
spasms, anorexia,
bulimia, stroke, addiction/dependency/craving, sleep disorder, epilepsy,
migraine; attention
deficit/hyperactivity disorder (ADHD); cardiovascular diseases, including
heart failure, angina
pectoris, arrhythmias, myocardial infarction, cardiac hypertrophy,
hypotension, hypertension -
e.g. essential hypertension, renal hypertension, or pulmonary hypertension,
thrombosis,
arteriosclerosis, cerebral vasospasm, subarachnoid hemorrhage, cerebral
ischemia, cerebral
infarction, peripheral vascular disease, Raynaud's disease, kidney disease -
e.g. renal failure;
dyslipidemias; obesity; emesis; gastrointestinal disorders, including
irritable bowel syndrome
(IBS), inflammatory bowel disease (IBD), gastroesophagal reflux disease
(GERD), motility
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disorders and conditions of delayed gastric emptying, such as post operative
or diabetic
gastroparesis, and diabetes, ulcers - e.g. gastric ulcer; diarrhoea; other
diseases including
osteoporosis; inflammations; infections such as bacterial, fungal, protozoan
and viral infections,
particularly infections caused by HIV-1 or HIV-2; pain; cancers; chemotherapy
induced injury;
tumor invasion; immune disorders; urinary retention; asthma; allergies;
arthritis; benign prostatic
hypertrophy; endotoxin shock; sepsis; complication of diabetes mellitus; and
gynaecological
disorders.
SUMMARY OF THE INVENTION
In one aspect, the invention relates to IGS3 polypeptides, polynucleotides and
recombinant materials and methods for their production. Another aspect of the
invention relates
to methods for using such IGS3 polypeptides, polynucleotides and recombinant
materials. Such
uses include, but are not limited to, use as a therapeutic target and for
treatment of one of the
Diseases as mentioned above.
In still another aspect, the invention relates to methods to identify agonists
and antagonists
using the materials provided by the invention, and treating conditions
associated with IGS3
imbalance with the identified compounds. Yet another aspect of the invention -
relates to
diagnostic assays for detecting diseases associated with inappropriate IGS3
activity or levels. A
further aspect of the invention relates to animal-based systems which act as
models for
disorders arising from aberrant expression or activity of IGS3.
BRIEF DESCRIPTION OF THE FIGURE
Figure 1. Schematic representation of the relative positions of the different
DNA clones
that were isolated to generate the consensus IGS3 cDNA sequence. HNT1370
represents the
"founding" genomic clone. ~,-IGS3.1A,B etc. indicate separate (nearly)
overlapping sequence
contigs obtained from sequence analysis of DNA from lambda clone IGS3.1. PCR
primers that
have been described in this document are indicated (1P#). CONSENSUS denotes
the contig that
was obtained after merging all obtained sequences. The part of the CONSENSUS
contig that
was fully validated by sequence analysis of at least three independent clones
is represented by
IGS3DNA (SEQ ID NO: 1 ). The 330 amino acids long open reading frame present
in IGS3DNA
is indicated with "**". The position of EST AF003828 is indicated with "_- .
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Table 1: IGS3-DNA of SEO ID NO: 1
5'-
TTAATCTCTTCAAGCCTCTGATTTCCTCTCCTGTAAAACAGGGGCGGTAATTACCACATA
5 ACAGGCTGGTCATGAAAATCAGTGAACATGCAGCAGGTGCTCAAGTCTTGTTTTTGTTTC
CAGGGGCACCAGTGGAGGTTTTCTGAGCATGGATCCAACCACCCCGGCCTGGGGAACAGA
AAGTACAACAGTGAATGGAAATGACCAAGCCCTTCTTCTGCTTTGTGGCAAGGAGACCCT
GATCCCGGTCTTCCTGATCCTTTTCATTGCCCTGGTCGGGCTGGTAGGAAACGGGTTTGT
GCTCTGGCTCCTGGGCTTCCGCATGCGCAGGAACGCCTTCTCTGTCTACGTCCTCAGCCT
O GGCCGGGGCCGACTTCCTCTTCCTCTGCTTCCAGATTATAAATTGCCTGGTGTACCTCAG
TAACTTCTTCTGTTCCATCTCCATCAATTTCCCTAGCTTCTTCACCACTGTGATGACCTG
TGCCTACCTTGCAGGCCTGAGCATGCTGAGCACCGTCAGCACCGAGCGCTGCCTGTCCGT
CCTGTGGCCCATCTGGTATCGCTGCCGCCGCCCCAGACACCTGTCAGCGGTCGTGTGTGT
CCTGCTCTGGGCCCTGTCCCTACTGCTGAGCATCTTGGAAGGGAAGTTCTGTGGCTTCTT
ATTTAGTGATGGTGACTCTGGTTGGTGTCAGACATTTGATTTCATCACTGCAGCGTGGCT
GATTTTTTTATTCATGGTTCTCTGTGGGTCCAGTCTGGCCCTGCTGGTCAGGATCCTCTG
TGGCTCCAGGGGTCTGCCACTGACCAGGCTGTACCTGACCATCCTGCTCACAGTGCTGGT
GTTCCTCCTCTGCGGCCTGCCCTTTGGCATTCAGTGGTTCCTAATATTATGGATCTGGAA
GGATTCTGATGTCTTATTTTGTCATATTCATCCAGTTTCAGTTGTCCTGTCATCTCTTAA
2O CAGCAGTGCCAACCCCATCATTTACTTCTTCGTGGGCTCTTTTAGGAAGCAGTGGCGGCT
GCAGCAGCCGATCCTCAAGCTGGCTCTCCAGAGGGCTCTGCAGGACATTGCTGAGGTGGA
TCACAGTGAAGGATGCTTCCGTCAGGGCACCCCGGAGATGTCGAGAAGCAGTCTGGTGTA
GAGATGGACAGCCTCTACTTCCATCAGATATATGTG-3'
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Table 2: IGS3-protein of SEQ ID NO: 2
MDPTTPAWGTESTTVNGNDQALLLLCGKETLIPVFLILFIALVGLVGNGFVLWLLGFRMR
RNAFSVYVLSLAGADFLFLCFQIINCLVYLSNFFCSISINFPSFFTTVMTCAYLAGLSML
STVSTERCLSVLWPIWYRCRRPRHLSAWCVLLWALSLLLSILEGKFCGFLFSDGDSGWC
QTFDFITAAWLIFLFMVLCGSSLALLVRILCGSRGLPLTRLYLTILLTVLVFLLCGLPFG
IQWFLILWIWKDSDVLFCHIHPVSWLSSLNSSANPIIYFFVGSFRKQWRLQQPILKLAL
QRALQDIAEVDHSEGCFRQGTPEMSRSSLV
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DESCRIPTION OF THE INVENTION
Structural and chemical similarity, in the context of sequences and motifs,
exists among
the IGS3 GPCR of the invention and other human GPCR's. Therefore, IGS3 is
implied to play a
role among other things in the Diseases mentioned above.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention
belongs. Although any methods and materials similar or equivalent to those
described herein
can be used in the practice or testing of the present invention, the preferred
methods, devices,
and materials are now described. All publications cited in this specification
are herein
incorporated by reference as if each individual publication were specifically
and individually
indicated to be incorporated by reference herein as though fully set forth.
Definitions
The following definitions are provided to facilitate understanding of certain
terms used
frequently herein.
"IGS3" refers, among others, to a polypeptide comprising the amino acid
sequence set
forth in SEQ ID N0:2, or a Variant thereof.
"Receptor Activity" or "Biological Activity of the Receptor" refers to the
metabolic or
physiologic function of said IGS3 including similar activities or improved
activities or these
activities with decreased undesirable side effects. Also included are
antigenic and immunogenic
activities of said IGS3.
"IGS3-gene" refers to a polynucleotide comprising the nucleotide sequence set
forth in
SEQ ID N0:1 or Variants thereof and/or their complements.
"Antibodies" as used herein includes polyclonal and monoclonal antibodies,
chimeric,
single chain, and humanized antibodies, as well as Fab fragments, including
the products of a
Fab or other immunoglobulin expression library.
"Isolated" means altered "by the hand of man" from the natural state and/or
separated
from the natural environment. Thus, if an "isolated" composition or substance
that occurs in
nature has been "isolated", it has been changed or removed from its original
environment, or
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both. For example, a polynucleotide or a polypeptide naturally present in a
living animal is not
"isolated," but the same polynucleotide or polypeptide separated from the
coexisting materials of
its natural state is "isolated", as the term is employed herein.
"Polynucleotide" generally refers to any polyribonucleotide or
polydeoxribonucleotide,
which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides"
include,
without limitation single- and double-stranded DNA, DNA that is a mixture of
single- and double-
stranded regions, single- and double-stranded RNA, and RNA that is a mixture
of single-and
double-stranded regions, hybrid molecules comprising DNA and RNA that may be
single-
stranded or, more typically, double-stranded or a mixture of single- and
double-stranded regions.
In addition, "polynucleotide" may also include triple-stranded regions
comprising RNA or DNA or
both RNA and DNA. The term polynucleotide also includes DNAs or RNAs
containing one or
more modified bases and DNAs or RNAs with backbones modified for stability or
for other
reasons. "Modified" bases include, for example, tritylated bases and unusual
bases such as
inosine. A variety of modifications has been made to DNA and RNA; thus,
"polynucleotide"
embraces chemically, enzymatically or metabolically modified forms of
polynucleotides as
typically found in nature, as well as the chemical forms of DNA and RNA
characteristic of viruses
and cells. "Polynucleotide" also embraces relatively short polynucleotides,
often referred to as
oligonucleotides.
"Polypeptide" refers to any peptide or protein comprising two or more amino
acids joined
to each other by peptide bonds or modified peptide bonds, i.e., peptide
isosteres. "Polypeptide"
refers to short chains, commonly referred to as peptides, oligopeptides or
oligomers, and to
longer chains, generally referred to as proteins, and/or to combinations
thereof. Polypeptides
may contain amino acids other than the 20 gene-encoded amino acids.
"Polypeptides" include
amino acid sequences modified either by natural processes, such as
posttranslational
processing, or by chemical modification techniques which are well known in the
art. Such
modifications are well-described in basic texts and in more detailed
monographs, as well as in
voluminous research literature. Modifications can occur anywhere in a
polypeptide, including the
peptide backbone, the amino acid side-chains and the amino or carboxyl
termini. It will be
appreciated that the same type of modification may be present in the same or
varying degrees at
several sites in a given polypeptide. Also, a given polypeptide may contain
many types of
modifications. Polypeptides may be branched as a result of ubiquitination, and
they may be
cyclic, with or without branching. Cyclic, branched and branched cyclic
polypeptides may result
from posttranslation natural processes or may be made by synthetic methods.
Modifications
include acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a nucleotide or
nucleotide
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derivative, covalent attachment of a lipid or lipid derivative, covalent
attachment of
phosphotidylinositol; cross-linking, cyclization, disulfide bond formation,
demethylation, formation
of covalent cross-links, formation of cystine, formation of pyroglutamate,
formylation, gamma-
carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination,
methylation,
myristoylation, oxidation, proteolytic processing, phosphorylation,
prenylation, racemization,
selenoylation, sulfation, transfer-RNA mediated addition of amino acids to
proteins such as
arginylation, and ubiquitination. See, for instance, PROTEINS - STRUCTURE AND
MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New
York, 1993 and Wold, F., Posttranslational Protein Modifications: Perspectives
and Prospects,
pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.
Johnson, Ed., Academic Press, New York, 1983; Seifter et al., "Analysis for
protein modifications
and nonprotein cofactors", Meth. Enzymol. (1990) 182:626-646 and Rattan et
al., "Protein
Synthesis: Posttranslational Modifications and Aging", Ann. NY Acad. Sci.
(1992) 663:48-62.
"Variant" as the term is used herein, is a polynucleotide or polypeptide that
differs from a
reference polynucleotide or polypeptide respectively, but retains essential
properties such as
essential biological, structural, regulatory or biochemical properties. A
typical variant of a
polynucleotide differs in nucleotide sequence from another, reference
polynucleotide. Changes
in the nucleotide sequence of the variant may or may not alter the amino acid
sequence of a
polypeptide encoded by the reference polynucleotide. Nucleotide changes may
result in amino
acid substitutions, additions, deletions, fusions and truncations in the
polypeptide encoded by
the reference sequence, as discussed below. A typical variant of a polypeptide
differs in amino
acid sequence from another, reference polypeptide. Generally, differences are
limited so that the
sequences of the reference polypeptide and the variant are closely similar
overall and, in many
regions, identical. A variant and reference polypeptide may differ in amino
acid sequence by one
or more substitutions, additions, and deletions in any combination. A
substituted or inserted
amino acid residue may or may not be one encoded by the genetic code. A
variant of a
polynucleotide or polypeptide may be a naturally occurring such as an allelic
variant, or it may be
a variant that is not known to occur naturally. Non-naturally occurring
variants of polynucleotides
and polypeptides may be made by mutagenesis techniques or by direct synthesis.
"Identity" is a measure of the identity of nucleotide sequences or amino acid
sequences.
In general, the sequences are aligned so that the highest order match is
obtained. "Identity" per
se has an art-recognized meaning and can be calculated using published
techniques. See, e.g.:
(COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A.M., ed., Oxford University Press,
New
York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D.W., ed.;
Academic Press, New York, 1993; COMPUTER ANALYSIS OF SEQUENCE DATA, PART 1,
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Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994;
SEQUENCE ANALYSIS
IN MOLECULAR BIOLOGY, von Heinje, G., Academic Press, 1987; and SEQUENCE
ANALYSIS PRIMER, Gribskov, M. and Devereux, J., eds., M Stockton Press, New
York, 1991 ).
While there exist a number of methods to measure identity between two
polynucleotide or
5 polypeptide sequences, the term "identity" is well known to skilled artisans
(Carillo, H., and
Lipton, D., SIAM J. Applied Math. (1988) 48:1073). Methods commonly employed
to determine
identity or similarity between two sequences include, but are not limited to,
those disclosed in
Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego,
1994, and Carillo,
H., and Lipton, D., SIAM J. Applied Math. (1988) 48:1073. Methods to determine
identity and
10 similarity are codified in computer programs. Preferred computer program
methods to determine
identity and similarity between two sequences include, but are not limited to,
GCG program
package (Devereux, J., et al., Nucleic Acids Research (1984) 12(1):387),
BLASTP, BLASTN,
FASTA (Atschul, S.F. et al., J. Molec. Biol. (1990) 215:403). The word
"homology" may
substitute for the words "identity".
As an illustration, by a polynucleotide having a nucleotide sequence having at
least, for
example, 95% "identity" to a reference nucleotide sequence of SEQ ID NO: 1 is
intended that the
nucleotide sequence of the polynucleotide is identical to the reference
sequence except that the
polynucleotide sequence may include up to five nucleotide differences per each
100 nucleotides
of the reference nucleotide sequence of SEQ ID NO: 1. In other words, to
obtain a
polynucleotide having a nucleotide sequence at least 95% identical to a
reference nucleotide
sequence, up to any 5% of the nucleotides in the reference sequence may be
deleted or
substituted with another nucleotide, or a number of nucleotides up to any 5%
of the total
nucleotides in the reference sequence may be inserted into the reference
sequence, or in a
number of nucleotides of up to any 5% of the total nucleotides in the
reference sequence there
may be a combination of deletion, insertion and substitution. These mutations
of the reference
sequence may occur at the 5 or 3 terminal positions of the reference
nucleotide sequence or
anywhere between those terminal positions, interspersed either individually
among nucleotides
in the reference sequence or in one or more contiguous groups within the
reference sequence.
Similarly, by a polypeptide having an amino acid sequence having at least, for
example,
95% "identity" to a reference amino acid sequence of SEQ ID N0:2 is intended
that the amino
acid sequence of the polypeptide is identical to the reference sequence except
that the
polypeptide sequence may include up to five amino acid alterations per each
100 amino acids of
the reference amino acid of SEQ ID NO: 2. In other words, to obtain a
polypeptide having an
amino acid sequence at least 95% identical to a reference amino acid sequence,
up to any 5%
of the amino acid residues in the reference sequence may be deleted or
substituted with another
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amino acid, or a number of amino acids up to any 5% of the total amino acid
residues in the
reference sequence may be inserted into the reference sequence. These
alterations of the
reference sequence may occur at the amino or carboxy terminal positions of the
reference
amino acid sequence or anywhere between those terminal positions, interspersed
either
individually among residues in the reference sequence or in one or more
contiguous groups
within the reference sequence.
Polypeptides of the Invention
In one aspect, the present invention relates to IGS3 polypeptides (including
IGS3
proteins). The IGS3 polypeptides include the polypeptide of SEQ ID N0:2 and
the polypeptide
having the amino acid sequence encoded by the DNA insert contained in the
deposit no. CBS
102196, deposited on September 15, 1999 at the Centraalbureau voor
Schimmelcultures at
Baarn (The Netherlands); as well as polypeptides comprising the amino acid
sequence of SEO
ID N0:2 and the polypeptide having the amino acid sequence encoded by the DNA
insert
contained in the deposit no. CBS 102196 at the Centraalbureau voor
Schimmelcultures at Baarn
(The Netherlands), and polypeptides comprising an amino acid sequence having
at least 80%
identity to that of SEQ ID N0:2 and/or to the polypeptide having the amino
acid sequence
encoded by the DNA insert contained in the deposit no. CBS 102196 at the
Centraalbureau voor
Schimmelcultures at Baarn (The Netherlands) over its entire length, and still
more preferably at
least 90% identity, and even still more preferably at least 95% identity to
said amino acid
sequence. Furthermore, those with at least 97%, in particular at least 99%,
are highly preferred.
Also included within IGS3 polypeptides are polypeptides having the amino acid
sequence which
has at least 80% identity to the polypeptide having the amino acid sequence of
SEO ID NO: 2 or
the polypeptide having the amino acid sequence encoded by the DNA insert
contained in the
deposit no. CBS 102196 at the Centraalbureau voor Schimmelcultures at Baarn
(The
Netherlands) over its entire length, and still more preferably at least 90%
identity, and even still
more preferably at least 95% identity to SEO ID NO: 2. Furthermore, those with
at least 97%, in
particular at least 99% are highly preferred. Preferably IGS3 polypeptides
exhibit at least one
biological activity of the receptor.
In an additional embodiment of the invention, the IGS3 polypeptides may be a
part of a
larger protein such as a fusion protein. It is often advantageous to include
an additional amino
acid sequence which contains secretory or leader sequences, pro-sequences,
sequences which
aid in purification such as multiple histidine residues, sequences which aid
in detection such as
antigenic peptide tags (such as the haemagglutinin (HA) tag), or an additional
sequence for
stability during recombinant production.
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Fragments of the IGS3 polypeptides are also included in the invention. A
fragment is a
polypeptide having an amino acid sequence that is the same as part of, but not
all of, the amino
acid sequence of the aforementioned IGS3 polypeptides. As with IGS3
polypeptides, fragments
may be "free-standing," or comprised within a larger polypeptide of which they
form a part or
region, most preferably as a single continuous region. Representative examples
of polypeptide
fragments of the invention, include, for example, fragments from about amino
acid number 1-20;
21-40, 41-60, 61-80, 81-100; and 101 to the end of IGS3 polypeptide. In this
context "about"
includes the particularly recited ranges larger or smaller by several, 5, 4,
3, 2 or 1 amino acid at
either extreme or at both extremes.
Preferred fragments include, for example, truncation polypeptides having the
amino acid
sequence of IGS3 polypeptides, except for deletion of a continuous series of
residues that
includes the amino terminus, or a continuous series of residues that includes
the carboxyl
terminus or deletion of two continuous series of residues, one including the
amino terminus and
one including the carboxyl terminus. Also preferred are fragments
characterized by structural or
functional attributes such as fragments that comprise alpha-helix and alpha-
helix forming
regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming
regions, coil and coil-
forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic
regions, beta
amphipathic regions, flexible regions, surface-forming regions, substrate
binding region, and
high antigenic index regions. Other preferred fragments are biologically
active fragments.
Biologically active fragments are those that mediate receptor activity,
including those with a
similar activity or an improved activity, or with a decreased undesirable
activity. Also included
are those that are antigenic or immunogenic in an animal, especially in a
human.
Thus, the polypeptides of the invention include polypeptides having an amino
acid
sequence that is at least 80% identical to either that of SEQ ID N0:2 and/or
the polypeptide
having the amino acid sequence encoded by the DNA insert contained in the
deposit no. CBS
102196 at the Centraalbureau voor Schimmelcultures at Baarn (The Netherlands),
or fragments
thereof with at least 80% identity to the corresponding fragment. Preferably,
all of these
polypeptide fragments retain the biological activity of the receptor,
including antigenic activity.
Variants of the defined sequence and fragments also form part of the present
invention.
Preferred variants are those that vary from the referents by conservative
amino acid
substitutions -- i.e., those that substitute a residue with another of like
characteristics. Typical
such substitutions are among Ala, Val, Leu and Ile; among Ser and Thr; among
the acidic
residues Asp and Glu; among Asn and Gln; and among the basic residues Lys and
Arg; or
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13
aromatic residues Phe and Tyr. Particularly preferred are variants in which
several, 5-10, 1-5, or
1-2 amino acids are substituted, deleted, or added in any combination.
The IGS3 polypeptides of the invention can be prepared in any suitable manner.
Such
polypeptides include isolated naturally occurring polypeptides, recombinantly
produced
polypeptides, synthetically produced polypeptides, or polypeptides produced by
a combination of
these methods. Methods for preparing such polypeptides are well known in the
art.
Polynucleotides of the Invention
A further aspect of the invention relates to IGS3 polynucleotides. IGS3
polynucleotides
include isolated polynucleotides which encode the IGS3 polypeptides and
fragments, and
polynucleotides closely related thereto. More specifically, the IGS3
polynucleotide of the
invention includes a polynucleotide comprising the nucleotide sequence
contained in SEQ ID
N0:1, such as the one capable of encoding a IGS3 polypeptide of SEQ ID NO: 2,
polynucleotides having the particular sequence of SEQ ID NO: 1 and
polynucleotides which
essentially correspond to the DNA insert contained in the deposit no. CBS
102196 at the
Centraalbureau voor Schimmelcultures at Baarn (The Netherlands).
IGS3 polynucleotides further include polynucleotides comprising a nucleotide
sequence
that has at least 80% identity over its entire length to a nucleotide sequence
encoding the IGS3
polypeptide of SEQ ID N0:2, polynucleotides comprising a nucleotide sequence
that is at least
80% identical to that of SEQ ID N0:1 over its entire length and a
polynucleotide which
essentially corresponds to the DNA insert contained in the deposit no. CBS
102196 at the
Centraalbureau voor Schimmelcultures at Baarn (The Netherlands).
In this regard, polynucleotides with at least 90% identity are particularly
preferred, and
those with at least 95% are especially preferred. Furthermore, those with at
least 97% are highly
preferred and those with at least 98-99% are most highly preferred, with at
least 99% being the
most preferred. Also included under IGS3 polynucleotides are a nucleotide
sequence which has
sufficient identity to a nucleotide sequence contained in SEO ID NO: 1 or to
the DNA insert
contained in the deposit no. CBS 102196 at the Centraalbureau voor
Schimmelcultures at Baarn
(The Netherlands) to hybridize under conditions useable for amplification or
for use as a probe
or marker. The invention also provides polynucleotides which are complementary
to such IGS3
polynucleotides.
IGS3 of the invention is structurally related to other proteins of the G-
protein coupled
receptor family, as shown by the results of BLAST searches in the public
databases. The amino
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14
acid sequence of Table 2 (SEO ID N0:2) has about 35 % identity (using BLAST,
Altschul S.F. et
al. Nucleic Acids Res. (1997) 25:3389-3402) over most of its length (amino
acid residues 2-306 )
with the protein encoded by the human mas oncogene (Sequence 1 in patent
application WO
8707472). The sequence is 37% identical (amino acid residues 35-315) with the
G-protein
coupled receptor published in patent application WO 9616087 (GENESEQ 96P-
897222 ). The
nucleotide sequence of Table 1 (SEQ ID NO: 1 ) has 52 % and 54 % identity over
most of its
length to the two receptors above (GENESEO 87N-70685 and 96N-T28807
respectively). Also
there is 48% identity to the human Somatostatin-3 receptor in residues 104-
1144 (WO 9313130;
93N-Q45657). Hydropathy analysis (Kyte J. et al., J. Mol. Biol. (1982) 157:
105-132; Klein P.et
al., Biochim. Biophys. Acta (1985) 815: 468-476) of the IGS3 protein sequence
expectedly
showed the presence of 7 transmembrane domains. Thus, IGS3 polypeptides and
polynucleotides of the present invention are expected to have, inter alia,
similar biological
functions/properties to their homologous polypeptides and polynucleotides, and
their utility is
obvious to anyone skilled in the art.
Polynucleotides of the invention can be obtained from natural sources such as
genomic
DNA. In particular, degenerated PCR primers can be designed that encode
conserved regions
within a particular GPCR gene subfamily. PCR amplification reactions on
genomic DNA or cDNA
using the degenerate primers will result in the amplification of several
members (both known and
novel) of the gene family under consideration (the degenerated primers must be
located within
the same exon, when a genomic template is used). (Libert et al., Science,
1989, 244: 569-572).
Polynucleotides of the invention can also be synthesized using well-known and
commercially
available techniques.
The nucleotide sequence encoding the IGS3 polypeptide of SEQ ID N0:2 may be
identical
to the polypeptide encoding sequence contained in SEQ ID N0:1 (nucleotide
number 149 to
1138), or it may be a different nucleotide sequence, which as a result of the
redundancy
(degeneracy) of the genetic code might also show alterations compared to the
polypeptide
encoding sequence contained in SEO ID N0:1, but also encodes the polypeptide
of SEQ ID
N0:2.
When the polynucleotides of the invention are used for the recombinant
production of the
IGS3 polypeptide, the polynucleotide may include the coding sequence for the
mature
polypeptide or a fragment thereof, by itself; the coding sequence for the
mature polypeptide or
fragment in reading frame with other coding sequences, such as those encoding
a leader or
secretory sequence, a pre-, or pro- or prepro- protein sequence, or other
fusion peptide portions.
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For example, a marker sequence which facilitates purification of the fused
polypeptide can be
encoded. In certain preferred embodiments of this aspect of the invention, the
marker sequence
is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and
described in Gentz
et al., Proc. Natl. Acad. Sci USA (1989) 86:821-824, or is an HA tag. The
polynucleotide may
5 also contain non-coding 5' and 3' sequences, such as transcribed, non-
translated sequences,
splicing and polyadenylation signals, ribosome binding sites and sequences
that stabilize mRNA.
Further preferred embodiments are polynucleotides encoding IGS3 variants
comprising
the amino acid sequence of the IGS3 polypeptide of SEO ID N0:2 in which
several, 5-10, 1-5, 1-
10 3, 1-2 or 1 amino acid residues are substituted, deleted or added, in any
combination.
The polynucleotides of the invention can be engineered using methods generally
known
in the art in order to alter IGS3-encoding sequences for a variety of purposes
including, but not
limited to, modification of the cloning, processing, and/or expression of the
gene product. DNA
15 shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example,
oligonucleotide-mediated site-directed mutagenesis may be used to introduce
mutations that
create amino acid substitutions, create new restriction sites, alter
modification (e.g. glycosylation
or phosphorylation) patterns, change codon preference, produce splice
variants, and so forth.
The present invention further relates to polynucleotides that hybridize to the
herein above-
described sequences. In this regard, the present invention especially relates
to polynucleotides
which hybridize under stringent conditions to the polynucleotides described
above. As herein
used, the term "stringent conditions" means hybridization will occur only if
there is at least 80%,
and preferably at least 90%, and more preferably at least 95%, yet even more
preferably at least
97%, in particular at least 99% identity between the sequences.
Polynucleotides of the invention, which are identical or sufficiently
identical to a nucleotide
sequence contained in SEO ID N0:1 or a fragment thereof, may be used as
hybridization probes
for cDNA and genomic DNA, to isolate full-length cDNAs and genomic clones
encoding IGS3
and to isolate cDNA and genomic clones of other genes (including genes
encoding homologs
and orthologs from species other than human) that have a high sequence
similarity to the IGS3
gene. People skilled in the art are well aware of such hybridization
techniques. Typically these
nucleotide sequences are 80% identical, preferably 90% identical, more
preferably 95% identical
to that of the referent. The probes generally will comprise at least 5
nucleotides, and preferably
at least 8 nucleotides, and more preferably at least 10 nucleotides, yet even
more preferably at
least 12 nucleotides, in particular at least 15 nucleotides. Most preferred,
such probes will have
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16
at least 30 nucleotides and may have at least 50 nucleotides. Particularly
preferred probes will
range between 30 and 50 nucleotides.
One embodiment, to obtain a polynucleotide encoding the IGS3 polypeptide,
including
homologs and orthologs from species other than human, comprises the steps of
screening an
appropriate library under stringent hybridization conditions with a labeled
probe having the SEQ
ID NO: 1 or a fragment thereof, and isolating full-length cDNA and genomic
clones containing
said polynucleotide sequence. Such hybridization techniques are well known to
those of skill in
the art. Stringent hybridization conditions are as defined above or
alternatively conditions under
overnight incubation at 42 °C in a solution comprising: 50% formamide,
5xSSC (150mM NaCI,
15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's
solution, 10
dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA,
followed by
washing the filters in 0.1 xSSC at about 65°C.
The polynucleotides and polypeptides of the present invention may be used as
research
reagents and materials for discovery of treatments and diagnostics to animal
and human
disease.
Vectors, Host Cells, Expression
The present invention also relates to vectors which comprise a polynucleotide
or
polynucleotides of the present invention, and host cells which are genetically
engineered with
vectors of the invention and to the production of polypeptides of the
invention by recombinant
techniques. Cell-free translation systems can also be used to produce such
proteins using RNAs
derived from the DNA constructs of the present invention.
For recombinant production, host cells can be genetically engineered to
incorporate
expression systems or portions thereof for polynucleotides of the present
invention. Introduction
of polynucleotides into host cells can be effected by methods described in
many standard
laboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY
(1986)
and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) such as
calcium phosphate
transfection, DEAE-dextran mediated transfection, transvection,
microinjection, cationic lipid-
mediated transfection, electroporation, transduction, scrape loading,
ballistic introduction or
infection.
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17
Representative examples of appropriate hosts include bacterial cells, such as
streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis
cells; fungal cells, such as
yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and
Spodoptera Sf9 cells;
animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes
melanoma cells;
and plant cells.
A great variety of expression systems can be used. Such systems include, among
others,
chromosomal, episomal and virus-derived systems, e.g., vectors derived from
bacterial
plasmids, from bacteriophage, from transposons, from yeast episomes, from
insertion elements,
from yeast chromosomal elements, from viruses such as baculoviruses, papova
viruses, such
as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies
viruses and
retroviruses, and vectors derived from combinations thereof, such as those
derived from plasmid
and bacteriophage genetic elements, such as cosmids and phagemids. The
expression systems
may contain control regions that regulate as well as engender expression.
Generally, any
system or vector suitable to maintain, propagate or express polynucleotides to
produce a
polypeptide in a host may be used. The appropriate nucleotide sequence may be
inserted into
an expression system by any of a variety of well-known and routine techniques,
such as, for
example, those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY
MANUAL (supra).
For secretion of the translated protein into the lumen of the endoplasmic
reticulum, into the
periplasmic space or into the extracellular environment, appropriate secretion
signals may be
incorporated into the desired polypeptide. These signals may be endogenous to
the polypeptide
or they may be heterologous signals, i.e. derived from a different species.
If the IGS3 polypeptide is to be expressed for use in screening assays,
generally, it is
preferred that the polypeptide be produced at the surface of the cell. In this
event, the cells may
be harvested prior to use in the screening assay. In case the affinity or
functional activity of the
IGS3 polypeptide is modified by receptor activity modifying proteins (RAMP),
coexpression of
the relevant RAMP most likely at the surface of the cell is preferred and
often required. Also in
this event harvesting of cells expressing the IGS3 polypeptide and the
relevant RAMP prior to
use in screening assays is required. If the IGS3 polypeptide is secreted into
the medium, the
medium can be recovered in order to recover and purify the polypeptide; if
produced
intracellularly, the cells must first be lysed before the polypeptide is
recovered. Membranes
expressing the IGS3 polypeptide can be recovered by methods that are well
known to a person
skilled in the art. In general, such methods include harvesting of the cells
expressing the IGS3
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18
polypeptide and homogenization of the cells by a method such as, but not
limited to, pottering.
The membranes may be recovered by washing the suspension one or several times.
IGS3 polypeptides can be recovered and purified from recombinant cell cultures
by well-
known methods including ammonium sulfate or ethanol precipitation, acid
extraction, anion or
cation exchange chromatography, phosphocellulose chromatography, hydrophobic
interaction
chromatography, affinity chromatography, hydroxylapatite chromatography and
lectin
chromatography. Most preferably, high performance liquid chromatography is
employed for
purification. Well-known techniques for refolding proteins may be employed to
regenerate active
conformation when the polypeptide is denatured during isolation and or
purification.
Diagnostic Assays
This invention also relates to the use of IGS3 polynucleotides for use as
diagnostic
reagents. Detection of a mutated form of the IGS3 gene associated with a
dysfunction will
provide a diagnostic tool that can add to or define a diagnosis of a disease
or susceptibility to a
disease which results from under-expression, over-expression or altered
expression of IGS3.
Also in this event co-expression of relevant receptor activity modifying
proteins can be required
to obtain diagnostic assays of desired quality. Individuals carrying mutations
in the IGS3 gene
may be detected at the DNA level by a variety of techniques.
Nucleic acids for diagnosis may be obtained from a subject's cells, such as
from blood,
urine, saliva, tissue biopsy or autopsy material. The genomic DNA may be used
directly for
detection or may be amplified enzymatically by using PCR or other
amplification techniques prior
to analysis. RNA or cDNA may also be used in similar fashion. Deletions and
insertions can be
detected by a change in size of the amplified product in comparison to the
normal genotype.
Point mutations can be identified by hybridizing amplified DNA to labeled IGS3
nucleotide
sequences. Perfectly matched sequences can be distinguished from mismatched
duplexes by
RNase digestion or by differences in melting temperatures. DNA sequence
differences may also
be detected by alterations in electrophoretic mobility of DNA fragments in
gels, with or without
denaturing agents, or by direct DNA sequencing. See, e.g., Myers et al.,
Science (1985)
230:1242. Sequence changes at specific locations may also be revealed by
nuclease protection
assays, such as RNase and S1 protection or the chemical cleavage method. See
Cotton et al.,
Proc. Natl. Acad. Sci. USA (1985) 85: 4397-4401. In another embodiment, an
array of
oligonucleotide probes comprising the IGS3 nucleotide sequence or fragments
thereof can be
constructed to conduct efficient screening of e.g., genetic mutations. Array
technology methods
are well known and have general applicability and can be used to address a
variety of questions
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19
in molecular genetics including gene expression, genetic linkage, and genetic
variability. (See for
example: M.Chee et al., Science, Vol 274, pp 610-613 (1996)).
The diagnostic assays offer a process for diagnosing or determining a
susceptibility to
among other things the Diseases as mentioned above, through detection of
mutation in the IGS3
gene by the methods described.
In addition, among other things, the Diseases as mentioned above can be
diagnosed by
methods comprising determining from a sample derived from a subject an
abnormally decreased
or increased level of the IGS3 polypeptide or IGS3 mRNA.
Decreased or increased expression can be measured at the RNA level using any
of the
methods well known in the art for the quantitation of polynucleotides, such
as, for example, PCR,
RT-PCR, RNase protection, Northern blotting and other hybridization methods.
Assay
techniques that can be used to determine levels of a protein, such as an IGS3,
in a sample
derived from a host are well known to those of skill in the art. Such assay
methods include
radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA
assays.
In another aspect, the present invention relates to a diagnostic kit for among
other things
the Diseases or suspectability to one of the Diseases as mentioned above.
The kit may comprise:
(a) an IGS3 polynucleotide, preferably the nucleotide sequence of SEQ ID N0:1,
or a
fragment thereof; and/or
(b) a nucleotide sequence complementary to that of (a); and/or
(c) an IGS3 polypeptide, preferably the polypeptide of SEO ID N0:2, or a
fragment
thereof; and/or
(d) an antibody to an IGS3 polypeptide, preferably to the polypeptide of SEQ
ID NO: 2;
and/or
(e) a RAMP polypeptide required for the relevant biological or antigenic
properties of an
IGS3 polypeptide.
It will be appreciated that in any such kit, (a), (b), (c) (d) or (e) may
comprise a substantial
component.
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Chromosome Assays
The nucleotide sequences of the present invention are also valuable for
chromosome
identification. The sequence is specifically targeted to and can hybridize
with a particular location
5 on an individual human chromosome. The mapping of relevant sequences to
chromosomes
according to the present invention is an important first step in correlating
those sequences with
gene associated disease. Once a sequence has been mapped to a precise
chromosomal
location, the physical position of the sequence on the chromosome can be
correlated with
genetic map data. Such data are found, for example, in V. McKusick, Mendelian
Inheritance in
10 Man (available on line through Johns Hopkins University Welch Medical
Library). The
relationship between genes and diseases that have been mapped to the same
chromosomal
region are then identified through linkage analysis (coinheritance of
physically adjacent genes).
The differences in the cDNA or genomic sequence between affected and
unaffected
individuals can also be determined. If a mutation is observed in some or all
of the affected
15 individuals but not in any normal individuals, then the mutation is likely
to be the causative agent
of the disease.
Antibodies
20 The polypeptides of the invention or their fragments or analogs thereof, or
cells expressing
them if required together with relevant RAMP's, may also be used as immunogens
to produce
antibodies immunospecific for the IGS3 polypeptides. The term "immunospecific"
means that the
antibodies have substantiall greater affinity for the polypeptides of the
invention than their affinity
for other related polypeptides in the prior art.
Antibodies generated against the IGS3 polypeptides may be obtained by
administering the
polypeptides or epitope-bearing fragments, analogs or cells to an animal,
preferably a
nonhuman, using routine protocols. For preparation of monoclonal antibodies,
any technique,
which provides antibodies produced by continuous cell line cultures, may be
used. Examples
include the hybridoma technique (Kohler, G. and Milstein, C., Nature (1975)
256:495-497), the
trioma technique, the human B-cell hybridoma technique (Kozbor et al.,
Immunology Today
(1983) 4:72) and the EBV-hybridoma technique (Cole et al., MONOCLONAL
ANTIBODIES AND
CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985).
The above-described antibodies may be employed to isolate or to identify
clones
expressing the polypeptide or to purify the polypeptides by affinity
chromatography.
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21
Antibodies against IGS3 polypeptides as such, or against IGS3 polypeptide-RAMP
complexes, may also be employed to treat among other things the Diseases as
mentioned
above.
Animals
Another aspect of the invention relates to non-human animal-based systems
which act as
models for disorders arising from aberrant expression or activity of IGS3. Non-
human animal-
based model systems may also be used to further characterize the activity of
the IGS3 gene.
Such systems may be utilized as part of screening strategies designed to
identify compounds
which are capable to treat IGS3 based disorders such as among other things the
Diseases as
mentioned above.
In this way the animal-based models may be used to identify pharmaceutical
compounds,
therapies and interventions which may be effective in treating disorders of
aberrant expression
or activity of IGS3. In addition such animal models may be used to determine
the LDSO and the
EDSO in animal subjects. These data may be used to determine the in vivo
efficacy of potential
IGS3 disorder treatments.
Animal-based model systems of IGS3 based disorders, based on aberrant IGS3
expression or
activity, may include both non-recombinant animals as well as recombinantly
engineered
transgenic animals.
Animal models for IGS3 disorders may include, for example, genetic models.
Animal
models exhibiting IGS3 based disorder-like symptoms may be engineered by
utilizing, for
example, IGS3 sequences such as those described, above, in conjunction with
techniques for
producing transgenic animals that are well known to persons skilled in the
art. For example,
IGS3 sequences may be introduced into, and overexpressed and/or misexpressed
in, the
genome of the animal of interest, or, if endogenous IGS3 sequences are
present, they may
either be overexpressed, misexpressed, or, alternatively, may be disrupted in
order to
underexpress or inactivate IGS3 gene expression.
In order to overexpress or misexpress a IGS3 gene sequence, the coding portion
of the
IGS3 gene sequence may be ligated to a regulatory sequence which is capable of
driving high
level gene expression or expression in a cell type in which the gene is not
normally expressed in
the animal type of interest. Such regulatory regions will be well known to
those skilled in the art,
and may be utilized in the absence of undue experimentation.
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For underexpression of an endogenous IGS3 gene sequence, such a sequence may
be
isolated and engineered such that when reintroduced into the genome of the
animal of interest,
the endogenous IGS3 gene alleles will be inactivated, or "knocked-out".
Preferably, the
engineered IGS3 gene sequence is introduced via gene targeting such that the
endogenous
IGS3 sequence is disrupted upon integration of the engineered IGS3 gene
sequence into the
animal's genome.
Animals of any species, including, but not limited to, mice, rats, rabbits,
squirrels, guinea
pigs, pigs, micro-pigs, goats, and non-human primates, e.~c ., baboons,
monkeys, and
chimpanzees may be used to generate animal models of IGS3 related disorders.
Any technique known in the art may be used to introduce a IGS3 transgene into
animals
to produce the founder lines of transgenic animals. Such techniques include,
but are not limited
to pronuclear microinjection (Hoppe, P.C. and Wagner, T.E., 1989, U.S. Pat.
No. 4,873,191);
retrovirus mediated gene transfer into germ lines (van der Putten et al.,
Proc. Natl. Acad. Sci.,
USA 82:6148-6152, 1985); gene targeting in embryonic stem cells (Thompson et
al., Cell
56:313-321, 1989,); electroporation of embryos (Lo, Mol. Cell. Biol. 3:1803-
1814, 1983); and
sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723, 1989); etc.
For a review of such
techniques, see Cordon, Transgenic Animals, Intl. Rev. Cytol.115:171-229,
1989.
The present invention provides for transgenic animals that carry the IGS3
transgene in all
their cells, as well as animals which carry the transgene in some, but not all
their cells, i.e.,
mosaic animals. (See, for example, techniques described by Jakobovits, Curr.
Biol. 4:761-763,
1994) The transgene may be integrated as a single transgene or in concatamers,
e.g., head-to-
head tandems or head-to-tail tandems. The transgene may also be selectively
introduced into
and activated in a particular cell type by following, for example, the
teaching of Lasko et al.
(Lasko, M..et al., Proc. Natl. Acad. Sci. USA 89:6232-6236, 1992).
The regulatory sequences required for such a cell-type specific activation
will depend
upon the particular cell type of interest, and will be apparent to those of
skill in the art.
When it is desired that the IGS3 transgene be integrated into the chromosomal
site of the
endogenous IGS3 gene, gene targeting is preferred. Briefly, when such a
technique is to be
utilized, vectors containing some nucleotide sequences homologous to the
endogenous IGS3
gene of interest (e.g., nucleotide sequences of the mouse IGS3 gene) are
designed for the
purpose of integrating, via homologous recombination with chromosomal
sequences, into and
disrupting the function of, the nucleotide sequence of the endogenous IGS3
gene or gene allele.
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23
The transgene may also be selectively introduced into a particular cell type,
thus inactivating the
endogenous gene of interest in only that cell type, by following, for example,
the teaching of Gu
et al. (Gu, H. et al.-, Science 265:103-106, 1994). The regulatory sequences
required for such a
cell-type specific inactivation will depend upon the particular cell type of
interest, and will be
apparent to those of skill in the art.
Once transgenic animals have been generated, the expression of the recombinant
IGS3
gene and protein may be assayed utilizing standard techniques. Initial
screening may be
accomplished by Southern blot analysis or PCR techniques to analyze animal
tissues to assay
whether integration of the transgene has taken place. The level of mRNA
expression of the IGS3
transgene in the tissues of the transgenic animals may also be assessed using
techniques which
include but are not limited to Northern blot analysis of tissue samples
obtained from the animal,
in situ hybridization analysis, and RT-PCR. Samples of target gene-expressing
tissue, may also
be evaluated immunocytochemically using antibodies specific for the target
gene transgene
product of interest. The IGS3 transgenic animals that express IGS3 gene mRNA
or IGS3
transgene peptide (detected immunocytochemically, using antibodies directed
against target
gene product epitopes) at easily detectable levels may then be further
evaluated to identify those
animals which display characteristic IGS3 based disorder symptoms.
Once IGS3 transgenic founder animals are produced i.e., those animals which
express
IGS3 proteins in cells or tissues of interest, and which, preferably, exhibit
symptoms of IGS3
based disorders), they may be bred, inbred, outbred, or crossbred to produce
colonies of the
particular animal. Examples of such breeding strategies include but are not
limited to:
outbreeding of founder animals with more than one integration site in order to
establish separate
lines; inbreeding of separate lines in order to produce compound IGS3
transgenics that express
the IGS3 transgene of interest at higher levels because of the effects of
additive expression of
each IGS3 transgene; crossing of heterozygous transgenic animals to produce
animals
homozygous for a given integration site in order to both augment expression
and eliminate the
possible need for screening of animals by DNA analysis; crossing of separate
homozygous lines
to produce compound heterozygous or homozygous lines; breeding animals to
different inbred
genetic backgrounds so as to examine effects of modifying alleles on
expression of the IGS3
transgene and the development of IGS3-like symptoms. One such approach is to
cross the IGS3
transgenic founder animals with a wild type strain to produce an F1 generation
that exhibits
IGS3 related disorder-like symptoms, such as those described above. The F1
generation may
then be inbred in order to develop a homozygous line, if it is found that
homozygous target gene
transgenic animals are viable.
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24
Vaccines
Another aspect of the invention relates to a method for inducing an
immunological
response in a mammal which comprises administering to (for example by
inoculation) the
mammal the IGS3 polypeptide, or a fragment thereof, if required together with
a RAMP
polypeptide, adequate to produce antibody and/or T cell immune response to
protect said animal
from among other things one of the Diseases as mentioned above.
Yet another aspect of the invention relates to a method of inducing
immunological
response in a mammal which comprises delivering the IGS3 polypeptide via a
vector directing
expression of the IGS3 polynucleotide in vivo in order to induce such an
immunological
response to produce antibody to protect said animal from diseases.
A further aspect of the invention relates to an immunological/vaccine
formulation
(composition) which, when introduced into a mammalian host, induces an
immunological
response in that mammal to an IGS3 polypeptide wherein the composition
comprises an IGS3
polypeptide or IGS3 gene. Such immunological/vaccine formulations
(compositions) may be
either therapeutic immunological/vaccine formulations or prophylactic
immunological/vaccine
formulations. The vaccine formulation may further comprise a suitable carrier.
Since the IGS3
polypeptide may be broken down in the stomach, it is preferably administered
parenterally
(including subcutaneous, intramuscular, intravenous, intradermal etc.
injection). Formulations
suitable for parenteral administration include aqueous and non-aqueous sterile
injection
solutions which may contain anti-oxidants, buffers, bacteriostats and solutes
which render the
formulation isotonic with the blood of the recipient; and aqueous and non-
aqueous sterile
suspensions which may include suspending agents or thickening agents. The
formulations may
be presented in unit-dose or multi-dose containers, for example, sealed
ampoules and vials and
may be stored in a freeze-dried condition requiring only the addition of the
sterile liquid carrier
immediately prior to use. The vaccine formulation may also include adjuvant
systems for
enhancing the immunogenicity of the formulation, such as oil-in water systems
and other
systems known in the art. The dosage will depend on the specific activity of
the vaccine and can
be readily determined by routine experimentation.
Screening Assays
The IGS3 polypeptide of the present invention may be employed in a screening
process
for compounds which bind the receptor and which activate (agonists) or inhibit
activation of
(antagonists) the receptor polypeptide of the present invention. Thus,
polypeptides of the
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invention may also be used to assess the binding of small molecule substrates
and ligands in,
for example, cells, cell-free preparations, chemical libraries, and natural
product mixtures. These
substrates and ligands may be natural substrates and ligands or may be
structural or functional
mimetics.
5
IGS3 polypeptides are responsible for biological functions, including
pathologies.
Accordingly, it is desirable to find compounds and drugs which stimulate IGS3
on the one hand
and which can inhibit the function of IGS3 on the other hand. In general,
agonists are employed
for therapeutic and prophylactic purposes for such conditions as among other
things the
10 Diseases as mentioned above.
Antagonists may be employed for a variety of therapeutic and prophylactic
purposes for
such conditions as among other things the Diseases as mentioned above.
15 In general, such screening procedures involve producing appropriate cells,
which express
the receptor polypeptide of the present invention on the surface thereof and,
if essential co-
expression of RAMP's at the surface thereof. Such cells include cells from
mammals, yeast,
Drosophila or E. coli. Cells expressing the receptor (or cell membrane
containing the expressed
receptor) are then contacted with a test compound to observe binding, or
stimulation or inhibition
20 of a functional response.
One screening technique includes the use of cells which express the receptor
of this
invention (for example, transfected CHO cells) in a system which measures
extracellular pH,
intracellular pH, or intracellular calcium changes caused by receptor
activation. In this technique,
25 compounds may be contacted with cells expressing the receptor polypeptide
of the present
invention. A second messenger response, e.g., signal transduction, pH changes,
or changes in
calcium level, is then measured to determine whether the potential compound
activates or
inhibits the receptor.
Another method involves screening for receptor inhibitors by determining
modulation of a
receptor-mediated signal, such as cAMP accumulation and/or adenylate cyclase
activity. Such a
method involves transfecting an eukaryotic cell with the receptor of this
invention to express the
receptor on the cell surface. The cell is then exposed to an agonist to the
receptor of this
invention in the presence of a potential antagonist. If the potential
antagonist binds the receptor,
and thus inhibits receptor binding, the agonist-mediated signal will be
modulated.
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26
Another method for detecting agonists or antagonists for the receptor of the
present
invention is the yeast-based technology as described in U.S. Patent 5,482,835.
The assays may simply test binding of a candidate compound wherein adherence
to the
cells bearing the receptor is detected by means of a label directly or
indirectly associated with
the candidate compound or in an assay involving competition with a labeled
competitor. Further,
these assays may test whether the candidate compound results in a signal
generated by
activation of the receptor, using detection systems appropriate to the cells
bearing the receptor
at their surfaces. Inhibitors of activation are generally assayed in the
presence of a known
agonist and the effect on activation by the agonist by the presence of the
candidate compound is
observed.
Further, the assays may simply comprise the steps of mixing a candidate
compound with
a solution containing an IGS3 polypeptide to form a mixture, measuring the
IGS3 activity in the
mixture, and comparing the IGS3 activity of the mixture to a standard.
The IGS3 cDNA, protein and antibodies to the protein may also be used to
configure
assays for detecting the effect of added compounds on the production of IGS3
mRNA and
protein in cells. For example, an ELISA may be constructed for measuring
secreted or cell
associated levels of IGS3 protein using monoclonal and polyclonal antibodies
by standard
methods known in the art, and this can be used to discover agents which may
inhibit or enhance
the production of IGS3 (also called antagonist or agonist, respectively) from
suitably manipulated
cells or tissues. Standard methods for conducting screening assays are well
known in the art.
Examples of potential IGS3 antagonists include antibodies or, in some cases,
oligonucleotides or proteins which are closely related to the ligand of the
IGS3, e.g., a fragment
of the ligand, or small molecules which bind to the receptor but do not elicit
a response, so that
the activity of the receptor is prevented.
Thus in another aspect, the present invention relates to a screening kit for
identifying
agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for IGS3
polypeptides; or
compounds which decrease, increase and/or otherwise enhance the production of
IGS3
polypeptides, which comprises:
(a) an IGS3 polypeptide, preferably that of SEO ID N0:2;
(b) a recombinant cell expressing an IGS3 polypeptide, preferably that of SEO
ID N0:2;
(c) a cell membrane expressing an IGS3 polypeptide, preferably that of SEQ ID
N0:2; or
(d) antibody to an IGS3 polypeptide, preferably that of SEQ ID NO: 2.
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27
It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise
a substantial
component.
Prophylactic and Therapeutic Methods
This invention provides methods of treating abnormal conditions related to
both an excess
of and insufficient amounts of IGS3 activity.
If the activity of IGS3 is in excess, several approaches are available. One
approach
comprises administering to a subject an inhibitor compound (antagonist) as
hereinabove
described along with a pharmaceutically acceptable carrier in an amount
effective to inhibit
activation by blocking binding of ligands to the IGS3, or by inhibiting
interaction with a RAMP
polypeptide or a second signal, and thereby alleviating the abnormal
condition.
In another approach, soluble forms of IGS3 polypeptides still capable of
binding the ligand
in competition with endogenous IGS3 may be administered. Typical embodiments
of such
competitors comprise fragments of the IGS3 polypeptide.
In still another approach, expression of the gene encoding endogenous IGS3 can
be
inhibited using expression-blocking techniques. Known such techniques involve
the use of
antisense sequences, either internally generated or separately administered.
See, for example,
O'Connor, J Neurochem (1991 ) 56:560 in Oligodeoxynucleotides as Antisense
Inhibitors of
Gene Expression, CRC Press, Boca Raton, Florida USA (1988). Alternatively,
oligonucleotides,
which form triple helices with the gene, can be supplied. See, for example,
Lee et al., Nucleic
Acids Res (1979) 6:3073; Cooney et al., Science (1988) 241:456; Dervan et al,
Science (1991 )
251:1360. These oligomers can be administered per se or the relevant oligomers
can be
expressed in vivo. Synthetic antisense or triplex oligonucleotides may
comprise modified bases
or modified backbones. Examples of the latter include methylphosphonate,
phosphorothioate or
peptide nucleic acid backbones. Such backbones are incorporated in the
antisense or triplex
oligonucleotide in order to provide protection from degradation by nucleases
and are well known
in the art. Antisense and triplex molecules synthesized with these or other
modified backbones
also form part of the present invention.
In addition, expression of the IGS3 polypeptide may be prevented by using
ribozymes
specific to the IGS3 mRNA sequence. Ribozymes are catalytically active RNAs
that can be
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28
natural or synthetic (see for example Usman, N, et al., Curr. Opin. Struct.
Biol (1996) 6(4), 527-
33.) Synthetic ribozymes can be designed to specifically cleave IGS3 mRNAs at
selected
positions thereby preventing translation of the IGS3 mRNAs into functional
polypeptide.
Ribozymes may be synthesized with a natural ribose phosphate backbone and
natural bases, as
normally found in RNA molecules. Alternatively the ribosymes may be
synthesized with non-
natural backbones to provide protection from ribonuclease degradation, for
example, 2'-O-
methyl RNA, and may contain modified bases.
For treating abnormal conditions related to an under-expression of IGS3 and
its activity,
several approaches are also available. One approach comprises administering to
a subject a
therapeutically effective amount of a compound which activates IGS3, i.e., an
agonist as
described above, in combination with a pharmaceutically acceptable carrier, to
thereby alleviate
the abnormal condition. Alternatively, gene therapy may be employed to effect
the endogenous
production of IGS3 by the relevant cells in the subject. For example, a
polynucleotide of the
invention may be engineered for expression in a replication defective
retroviral vector, as
discussed above. The retroviral expression construct may then be isolated and
introduced into a
packaging cell transduced with a retroviral plasmid vector containing RNA
encoding a
polypeptide of the present invention such that the packaging cell now produces
infectious viral
particles containing the gene of interest. These producer cells may be
administered to a subject
for engineering cells in vivo and expression of the polypeptide in vivo. For
overview of gene
therapy, see Chapter 20, Gene Therapy and other Molecular Genetic-based
Therapeutic
Approaches, (and references cited therein) in Human Molecular Genetics,
Strachan T. and Read
A.P., BIOS Scientific Publishers Ltd (1996).
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as dogs, cats, cows,
horses, rabbits,
monkeys, and most preferably, humans.
Formulation and Administration
Peptides, such as the soluble form of IGS3 polypeptides, and agonists and
antagonist
peptides or small molecules, may be formulated in combination with a suitable
pharmaceutical
carrier. Such formulations comprise a therapeutically effective amount of the
polypeptide or
compound, and a pharmaceutically acceptable carrier or excipient. Formulation
should suit the
mode of administration, and is well within the skill of the art. The invention
further relates to
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29
pharmaceutical packs and kits comprising one or more containers filled with
one or more of the
ingredients of the aforementioned compositions of the invention.
Polypeptides and other compounds of the present invention may be employed
alone or in
conjunction with other compounds, such as therapeutic compounds.
Preferred forms of systemic administration of the pharmaceutical compositions
include
injection, typically by intravenous injection. Other injection routes, such as
subcutaneous,
intramuscular, or intraperitoneal, can be used. Alternative means for systemic
administration
include transmucosal and transdermal administration using penetrants such as
bile salts or
fusidic acids or other detergents. In addition, if properly formulated in
enteric or encapsulated
formulations, oral administration may also be possible.
The dosage range required depends on the choice of peptide or compound, the
route of
administration, the nature of the formulation, the nature of the subject's
condition, and the
judgment of the attending practitioner. Suitable dosages are in the range of
0.1-100 ~g/kg of
subject. Wide variations in the needed dosage, however, are to be expected in
view of the
variety of compounds available and the differing efficiencies of various
routes of administration.
For example, oral administration would be expected to require higher dosages
than
administration by intravenous injection. Variations in these dosage levels can
be adjusted using
standard empirical routines for optimization, as is well understood in the
art.
Polypeptides used in treatment can also be generated endogenously in the
subject, in
treatment modalities often referred to as "gene therapy" as described above.
Thus, for example,
cells from a subject may be engineered with a polynucleotide, such as a DNA or
RNA, to encode
a polypeptide ex vivo, and for example, by the use of a retroviral plasmid
vector. The cells are
then introduced into the subject.
The following examples are only intended to further illustrate the invention
in more detail,
and therefore these examples are not deemed to restrict the scope of the
invention in any way.
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EXAMPLE 1. THE CLONING OF GENOMIC DNA ENCODING A NOVEL G PROTEIN-
COUPLED RECEPTOR.
5 Example 1a. Homology PCR cloning of a genomic fragment encoding a novel G-
protein
coupled receptor (GPCR).
A PCR based homology cloning strategy was used to isolate partial genomic DNA
sequences encoding novel G-protein coupled receptors (GPCR). The following
forward (F20)
10 and reverse (R42, R43) degenerate PCR primers were designed in conserved
areas of the
neurotensin receptor gene family (Vita N. et al. [1993] Febs Lett. 317: 139-
142; Vita N. et al.
[1998] Eur. J. Pharmacol. 360: 265-272) at the boundary of intracellular loop
n°1 (11) with
transmembrane domain 2 (TM2) and at the boundary of transmembrane domain 3
with
intracellular loop n°2 (TM3/12) respectively:
F20 (11/TM2):
5'-CTGCACTACCACGTGCTC(A or T)(G or C)(A,C,G or T)(C or T)T(A,C,G or T)GC -3'
(SEQ ID NO: 3)
R42 (TM3/12):
5'-GGGTGGCAGATGGCCA(A or G)(A or G)(C or T)A(A,C,G or T)C(G or T)(C or T)TC( C
or
Inosine)(C,G or T)
(SEQ ID NO: 4)
R43 (TM3/12):
5'-GTGGCAGATGGCCAGGCAGCG(A or G)TC(A,C,G or T)(A or G)C(A or G)CT(A,G or T) -
3'
(SEQ ID NO: 5)
In order to suppress amplification of known members of the neurotensin
receptor family, the 3'
ultimate nucleotide position of primers R42 and R43 was chosen in such a way
that it was either
not complementary to the corresponding position of the human NTR1 cDNA (R42)
or to the
corresponding position of both NTR1 and NTR2 cDNA (R43).
The primary PCR reaction was carried out in a 60p1 volume and contained 100 ng
human genomic DNA (Clontech), 6 p1 GeneAmpT"~ 10 x PCR buffer II (100mM Tris-
HCI pH 8.3;
500 mM KCI, Perkin Elmer), 3.6 p1 25 mM MgCl2, 0.36N1 dNTPs (25mM of each
dNTP), 1.5 units
AmpIiTaq Gold T"' polymerise (Perkin Elmer) and 30 pmoles of each of the
degenerated forward
(F20) and reverse primer (R42). Reaction tubes were heated at 95°C for
10 min and then
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31
subjected to 35 cycles of denaturation (95°C, 1 min), annealing
(55°C, 2 min) and extension
(72°C, 3min). Finally reaction tubes were heated for 10 min at
72°C.
For the semi-nested PCR reaction 1 p1 of a 1/50 dilution of the primary PCR
reaction
was used as a template using the degenerate forward and reverse primers F20
and R43
respectively. The semi-nested PCR reaction was carried out under the same
conditions as the
primary PCR reaction.
Semi-nested PCR reaction products were size fractionated on an agarose gel and
stained with ethidium bromide. A fragment of ~ 220 by was identified, purified
from gel using the
Qiaex-IIT"~ purification kit (Qiagen) and ligated into the pGEM-T plasmid
according to the
procedure recommended by the supplier (pGEM-T kit, Promega). The recombinant
plasmids
thus produced were used to transform competent E. coli SURET"~ 2 bacteria
(Stratagene).
Transformed cells were plated on LB agar plates containing ampicillin (100
Ng/ml). Plasmid DNA
was purified from mini-cultures of individual colonies using a Qiagen-tip 20
miniprep kit (Qiagen).
DNA sequencing reactions were carried out on the purified plasmid DNA with the
ABI PrismT"~
BigDyeT"~ Terminator Cycle Sequencing Ready Reaction kit (PE-ABI), using
insert-flanking.
Sequencing reaction products were purified via EtOH/NaOAc precipitation and
analysed on an
ABI 377 automated sequences.
A computer-assisted homology search of the insert sequence of clone HNT1370
against
public domain sequence databanks (Blastn; Altschul S.F. et al. [1997], Nucleic
Acids Res.
25:3389-3402) revealed strong indications that it encoded (part of) a novel
member of the GPCR
family. Although HNT1370 had been cloned from a ~ 220 by fragment the insert
size was only ~
130 by as a result of a cloning artefact. We refer to this novel GPCR sequence
as IGS3.
Table 3: Overview of oligo primers used.
SEQ ID NO: F20: 5'-CTGCACTACCACGTGCTC(A or T)(G or C)(A,C,G
3 or T)(C or
T)T(A,C,G or T)GC -3'
SEQ ID NO: R42: 5'-GGGTGGCAGATGGCCA(A or G)(A or G)(C or
4 T)A(A,C,G or T)C(G
or T)(C or T)TC( C or Inosine)(C,G or T)
SEQ ID NO: R43: 5'-GTGGCAGATGGCCAGGCAGCG(A or G)TC(A,C,G
5 or T)(A or G)C(A
or G)CT(A,G or T) -3'
SEQ ID NO: IP11969: 5'GGGGCCGACTTCCTCTTCCTCTGCTTCC-3'
6
SEQ ID NO: IP12008: 5'-GCAAGGTAGGCACAGGTCATCACAGTGG-3'
7
SEQ ID NO: IP12936: 5'-ATAAGCTTCTCCCTGGCCCTTAATAAATGAC-3'
8
SEQ ID NO: IP12937: 5'-AGGAATTCAGACAGACAGGGGCAAAGTTG-3'
9
CA 02383177 2002-03-13
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32
Example 1b. Cloning of genomic DNA fragments containing the complete IGS3
coding
sequence.
The complete coding sequence of IGS3 was obtained via hybridization screening
of a
human genomic library. A human genomic DNA library (Clontech #HL1067j),
constructed in the
lambda EMBL3 SP6/T7 phage vector was screened by hybridization using an IGS3
specific
probe. This probe was derived from a 130 by PCR fragment amplified from the
HNT1355
plasmid (which contained an identical insert as HNT1370) using IGS3 specific
primers IP11969
(SEQ ID NO: 6) and IP12008 (SEQ ID NO: 7) (Fig.1 ). The 130 by fragment was
purified from gel
using the Qiaex-IIT"" purification kit (Qiagen) and radiolabelled via random
primed incorporation
of [a 32P]dCTP to a specific activity of > 109 cpm/ug using the Prime-It II
kitT"~ (Stratagene)
according to the instructions provided by the supplier. Aproximately 550,000
plaques were
screened with the 130 by probe according to the Lambda Library User Manual of
Clontech
(PT1010-1 ). Three positive clones (~,-IGS3.1, ~-IGS3.3 and 7~-IGS3.5) were
plaque-purified and
recombinant phage DNA was prepared from small-scale liquid cultures as
described by Maniatis
et al. (Sambrook, J. et al. Molecular Cloning: A Laboratory Manual Second
Edition [1989], CSH
Laboratory Press).
Sequence analysis of the recombinant phage DNA using IGS3 specific primers
showed
that the inserts of all 3 lambda clones contained a long open reading frame
encoding a novel
putative (intron-less) GPCR of 330 amino acids (the postulated start of
translation was preceded
by an in-frame stop codon). The IGS3 coding sequence was subcloned into a
plasmid vector
after PCR amplification. PCR reactions were carried out on the isolated 7~-
IGS3.1, 7~-IGS3.3 and
7~-IGS3.5 phage DNA (500 ng) with the IP12936 (SEO ID NO: 8) and IP12937 (SEQ
ID NO: 9)
oligonucleotide primers using the ExpandT"" High Fidelity PCR system
(Boehringer). PCR
reaction tubes were heated at 95°C for 2 min and then subjected to 35
cycles of denaturation
(95°C, 30 sec), annealing (58°C, 30 sec) and extension
(72°C, 1 min). There was a final
elongation step at 72°C (10 min). A ~ 1,200 by PCR product was purified
from gel and ligated
into the pGEM-T vector. The recombinant DNA was then used to transform E.coli
bacterial strain
DHSaF'. This yielded bacterial clones HB4971, HB4972 (both subcloned from 7~-
IGS3.1 ),
HB4973 and HB4974 (both subcloned from ~-IGS3.3) and HB4975 and HB4976 (both
subcloned from ~-IGS3.5). The inserts of all plasmid clones were completely
sequenced. A meld
of all sequence data yielded a consensus sequence, which confirmed the
existence of a long
open reading frame of 330 amino acids that encoded a putative novel GPCR
receptor (IGS3)
(Fig.1 ). The consensus cDNA and protein sequence of IGS3 are presented here
as IGS3DNA
(SEO ID NO: 1 ) and IGS3PROT (SEQ ID NO: 2) respectively. Homology searches of
DNA
CA 02383177 2002-03-13
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33
databanks with the IGS3DNA sequence showed one EST sequence (accession
n° AF003828)
which partially overlapped with IGS3DNA at the 3' end (Fig.1 ).
The bacterial strain harboring plasmid HNT4971 (containing the IGS3DNA
sequence)
was recloned after replating on LB agar plates containing 100 Ng ampicillin/ml
and deposited
both in the Innogenetics N.V. strain list (ICCG4319) and at the
"Centraalbureau voor
Schimmelculturen (CBS)" in Baarn, The Netherlands (accession n°
102196). Plasmid DNA was
prepared from the recloned isolate and the insert was resequenced and found to
be identical to
the IGS3DNA sequence.
CA 02383177 2002-03-13
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34
PCT
Original (for SUBMISSION) - printed on 15.09.2000 04:00:52 PM
0-1 Form - PCT/R0/134 (EASY)
Indications Relating to Deposited
Microorganisms) or Other Biological
Material (PCT Rule l3bis)
0-1-1 Prepared using PCT-EASY Version 2 . 90
(updated 15.12.1999)
onal Aoolication No.
0-~Aoolicant's or a4ent's file reference I SPW99 . 07
1 The indications made
below relate to
the deposited microorganisms)
or
other biological material
referred to in
the description on:
1-1 page 33
1-2 line 3>
1-3 Identification of
Deposit
1-3-1Name of depositary Centraalbureau voor Schimmelcultures
institution
1-3-2Address of depositaryOOSterStraat 1, PostbuS 273 , NL-3740 AG
institution
Baarn, Netherlands
1-3-3Date of deposit 15 September 1999 (15.09.1999)
1-3-4Accession Number CBS 102196
1~t AdditionallndicationsNONE
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CA 02383177 2002-03-13
WO 01/19983 3r~ PCT/EP00/09116
BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS
FOR THE PURPOSES OF PATENT PROCEDURE
INTERNATIONAL FORM
Duphar International Research B.V. RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
POStbUS 900 issued pursuant to Rule 7.1 by the
13HO DA WEESP INTERNATIONAL DEPOSITARY AUTHORITY
identified at the bottom of this page
The Netherlands
name and address of depositor
I. IDENTIFICATION OF THE MICROORGANISM
Identification reference given by Accession number given by the
the
7EPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY:
E. coli DH5 alpha F' pGEM-ThIGS3 CBS 102196
ICCG 4319
II. SCIENTIFIC DESCRIPTION AND/OR
PROPOSED TAXONOMIC DESIGNATION
The microorganism identified under
I above was accompanied by:
a scientific description
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(mark with a cross where applicable)
III. RECEIPT AND ACCEPTANCE
This International Depositary accepts
the microorganism identified under
I above, which
received by it on 15-09-99 (date
dd-mm-yy of the original deposit)
1
IV. RECEIPT OF REQUEST FOR CONVERSION
The microorganism identified under
I above was received by this International
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Authority on notappliC2ble (date
dd-mm-yy of the original deposit)
and a
request to convert the original deposit
to a deposit under the Budapest
Treaty was received
by it on notappliCable (date dd-mm-yy
of receipt of request for conversion)
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name : Centraalbureau voor Schimmelculturessignature ( s ) of person ( s )
having
the power
to represent the International Depositary
Authorit or_of authorized official(s):
Address: posterstraat 1
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3740 AG BAARN Mrs F.B. Snippe- laus Dr J. Stalper
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o ~ ~
.
1 Where Rule 6.4(d) applies, such date is the date on which the status of
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CA 02383177 2002-03-13
WO 01/19983 36 PCT/EP00/09116
BUDAPEST TREATY ON THE INTERNATIONAL
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date (dd-mm-yy), the said
microorganism was
n3 viable
~
r--,3
no longer viable
lIndicate the date of the original deposit or, where a new deposit or a
transfer has been
made, the most recent relevant date (date of the new deposit or date of the
transfer).
2 In the cases referred to in Rule 10.2(a)(ii) and (iii), refer to the most
recent
viability test.
3
Mark with a cross the applicable box.
Form BP/9 (first page)
CA 02383177 2002-03-13
WO 01/19983 3~ PCT/EP00/09116
i IV. CONDITIONS UNDER WHICH THE VIABILITY HAS BEEN PERFORMED 4 I
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name : Centraalbureau voor Schimmelcultures signature ( s ) of person ( s )
having the power
to represent the International Depositary
Authorit~of authorized official(s):
Addres s : posterstraat 1 ,~ ..--
P.O. Box 273
3740 AG BAARN Mrs F.B. Snippe-Claus Dr .~ ers -
The Netherlands Date (dd-mm-yy) : 17-09-99 p ~, R ftrSe~.
4Fi11 in if the information has been requested and if the results of the test
were
negative.
Form BP/9 (second and last page)
CA 02383177 2002-03-13
WO 01/19983 ~ ~6 PCT/EP00/09116
SEQUENCE LISTING
<110> SOLVAY PHARMACEUTICALS B.V.
<120> Novel Human G-Protein coupled Receptor
<130> SPW 99.07
<140>
<141>
<160> 9
<170> PatentIn Ver. 2.1
<210> 1
<211> 1176
<212> DNA
<213> Homo Sapiens
<220>
<221> CDS
<222> (149)..(1138)
<400> 1
ttaatctctt caagcctctg atttcctctc ctgtaaaaca ggggcggtaa ttaccacata 60
acaggctggt catgaaaatc agtgaacatg cagcaggtgc tcaagtcttg tttttgtttc 120
caggggcacc agtggaggtt ttctgagc atg gat cca acc acc ccg gcc tgg 172
Met Asp Pro Thr Thr Pro Ala Trp
1 5
gga aca gaa agt aca aca gtg aat gga aat gac caa gcc ctt ctt ctg 220
Gly Thr Glu Ser Thr Thr Val Asn Gly Asn Asp Gln Ala Leu Leu Leu
10 15 20
ctt tgt ggc aag gag acc ctg atc ccg gtc ttc ctg atc ctt ttc att 268
Leu Cys Gly Lys Glu Thr Leu Ile Pro Val Phe Leu Ile Leu Phe.Ile
25 30 35 40
gcc ctg gtc ggg ctg gta gga aac ggg ttt gtg ctc tgg ctc ctg ggc 316
Ala Leu Val Gly Leu Val Gly Asn Gly Phe Val Leu Trp Leu Leu Gly
50 55
ttc cgc atg cgc agg aac gcc ttc tct gtc tac gtc ctc agc ctg gcc 364
Phe Arg Met Arg Arg Asn Ala Phe Ser Val Tyr Val Leu Ser Leu Ala
60 65 70
ggg gcc gac ttc ctc ttc ctc tgc ttc cag att ata aat tgc ctg gtg 412
Gly Ala Asp Phe Leu Phe Leu Cys Phe Gln Ile Ile Asn Cys Leu Val
75 80 85
CA 02383177 2002-03-13
WO 01/19983 2~6 PCT/EP00/09116
tac ctc agt aac ttc ttc tgt tcc atc tcc atc aat ttc cct agc ttc 460
Tyr Leu Ser Asn Phe Phe Cys Ser Ile Ser Ile Asn Phe Pro Ser Phe
90 95 100
ttc acc act gtg atg acc tgt gcc tac ctt gca ggc ctg agc atg ctg 508
Phe Thr Thr Val Met Thr Cys Ala Tyr Leu Ala Gly Leu Ser Met Leu
105 110 115 120
agc acc gtc agc acc gag cgc tgc ctg tcc gtc ctg tgg ccc atc tgg 556
0 Ser Thr Val Ser Thr Glu Arg Cys Leu Ser Val Leu Trp Pro Ile Trp
125 130 135
tat cgc tgc cgc cgc ccc aga cac ctg tca gcg gtc gtg tgt gtc ctg 604
Tyr Arg Cys Arg Arg Pro Arg His Leu Ser Ala Val Val Cys Val Leu
140 145 150
ctc tgg gcc ctg tcc cta ctg ctg agc atc ttg gaa ggg aag ttc tgt 652
Leu Trp Ala Leu Ser Leu Leu Leu Ser Ile Leu Glu Gly Lys Phe Cys
155 160 165
ggc ttc tta ttt agt gat ggt gac tct ggt tgg tgt cag aca ttt gat 700
Gly Phe Leu Phe Ser Asp Gly Asp Ser Gly Trp Cys Gln Thr Phe Asp
170 175 180
ttc atc act gca gcg tgg ctg att ttt tta ttc atg gtt ctc tgt ggg 748
Phe Ile Thr Ala Ala Trp Leu Ile Phe Leu Phe Met Val Leu Cys Gly
185 190 195 200
tcc agt ctg gcc ctg ctg gtc agg atc ctc tgt ggc tcc agg ggt ctg 796
Ser Ser Leu Ala Leu Leu Val Arg Ile Leu Cys Gly Ser Arg Gly Leu
205 210 215
cca ctg acc agg ctg tac ctg acc atc ctg ctc aca gtg ctg gtg ttc 844
Pro Leu Thr Arg Leu Tyr Leu Thr Ile Leu Leu Thr Val Leu Val Phe
220 225 230
ctc ctc tgc ggc ctg ccc ttt ggc att cag tgg ttc cta ata tta tgg 892
Leu Leu Cys Gly Leu Pro Phe Gly Ile Gln Trp Phe Leu Ile Leu Trp
235 240 245
atc tgg aag gat tct gat gtc tta ttt tgt cat att cat cca gtt tca 940
Ile Trp Lys Asp Ser Asp Val Leu Phe Cys His Ile His Pro Val Ser
250 255 260
gtt gtc ctg tca tct ctt aac agc agt gcc aac ccc atc att tac ttc 988
Val Val Leu Ser Ser Leu Asn Ser Ser Ala Asn Pro Ile Ile Tyr Phe
265 270 275 280
ttc gtg ggc tct ttt agg aag cag tgg cgg ctg cag cag ccg atc ctc 1036
Phe Val Gly Ser Phe Arg Lys Gln Trp Arg Leu Gln Gln Pro Ile Leu
2g5 290 295
aag ctg get ctc cag agg get ctg cag gac att get gag gtg gat cac 1084
Lys Leu Ala Leu Gln Arg Ala Leu Gln Asp Ile Ala Glu Val Asp His
300 305 310
CA 02383177 2002-03-13
WO 01/19983 3~6 PCT/EP00/09116
agt gaa gga tgc ttc cgt cag ggc acc ccg gag atg tcg aga agc agt 1132
Ser Glu Gly Cys Phe Arg Gln Gly Thr Pro Glu Met Ser Arg Ser Ser
315 320 325
ctg gtg tagagatgga cagcctctac ttccatcaga tatatgtg 1176
Leu Val
330
15
<210> 2
<211> 330
<212> PRT
<213> Homo Sapiens
<400> 2
Met Asp Pro Thr Thr Pro Ala Trp Gly Thr Glu Ser Thr Thr Val Asn
1 5 10 15
Gly Asn Asp Gln Ala Leu Leu Leu Leu Cys Gly Lys Glu Thr Leu Ile
20 25 30
Pro ValPhe LeuIleLeu PheIleAla LeuValGlyLeu ValGlyAsn
35 40 45
Gly PheVal LeuTrpLeu LeuGlyPhe ArgMetArgArg AsnAlaPhe
50 55 60
Ser ValTyr ValLeuSer LeuAlaGly AlaAspPheLeu PheLeuCys
65 70 75 80
Phe GlnIle IleAsnCys LeuValTyr LeuSerAsnPhe PheCysSer
85 90 95
Ile SerIle AsnPhePro SerPhePhe ThrThrValMet ThrCysAla
100 105 110
Tyr LeuAla GlyLeuSer MetLeuSer ThrValSerThr GluArgCys
115 120 125
Leu SerVal LeuTrpPro IleTrpTyr ArgCysArgArg ProArgHis
130 135 140
Leu SerAla ValValCys ValLeuLeu TrpAlaLeuSer LeuLeuLeu
145 150 155 160
Ser IleLeu GluGlyLys PheCysGly PheLeuPheSer AspGlyAsp
165 170 175
CA 02383177 2002-03-13
WO 01/19983 4~6 PCT/EP00/09116
Ser Gly Trp Cys Gln Thr Phe Asp Phe Ile Thr Ala Ala Trp Leu Ile
180 185 190
Phe Leu Phe Met Val Leu Cys Gly Ser Ser Leu Ala Leu Leu Val Arg
195 200 205
Ile Leu Cys Gly Ser Arg Gly Leu Pro Leu Thr Arg Leu Tyr Leu Thr
210 215 220
Ile Leu Leu Thr Val Leu Val Phe Leu Leu Cys Gly Leu Pro Phe Gly
225 230 235 240
Ile Gln Trp Phe Leu Ile Leu Trp Ile Trp Lys Asp Ser Asp Val Leu
245 250 255
Phe Cys His Ile His Pro Val Ser Val Val Leu Ser Ser Leu Asn Ser
260 265 270
Ser Ala Asn Pro Ile Ile Tyr Phe Phe Val Gly Ser Phe Arg Lys Gln
275 280 285
Trp Arg Leu Gln Gln Pro Ile Leu Lys Leu Ala Leu Gln Arg Ala Leu
290 295 300
Gln Asp Ile Ala Glu Val Asp His Ser Glu Gly Cys Phe Arg Gln Gly
305 310 315 320
Thr Pro Glu Met Ser Arg Ser Ser Leu Val
325 330
<210> 3
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Degenerated
primers
<220>
<221> variation
<222> (21)
<223> A,C,G or T
<220>
<221> variation
<222> (24)
<223> A,C,G or T
<400> 3
ctgcactacc acgtgctcws nytngc 26
CA 02383177 2002-03-13
WO 01/19983 5~6 PCT/EP00/09116
<210> 4
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Degenerated
primers
<220>
<221> variation
<222> (21)
<223> A,C,G or T
<220>
<221> variation
<222> (27)
<223> C or Inosine
<400> 4
gggtggcaga tggccarrya nckytcnb 28
<210> 5
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Degenerated
primers
<220>
<221> variation
<222> (25)
<223> A,C,G or T
<400> 5
gtggcagatg gccaggcagc grtcnrcrct d 31
<210> 6
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 6
ggggccgact tcctcttcct ctgcttcc 28
CA 02383177 2002-03-13
WO 01/19983 PCT/EP00/09116
6/6
<210> 7
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400>
gcaaggtagg cacaggtcat cacagtgg 28
<210> 8
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 8
ataagcttct ccctggccct taataaatga c 31
<210> 9
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 9
aggaattcag acagacaggg gcaaagttg 29
