Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO 9S/04821 216 8 ~ 4 8 PCT/US94/08913
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TITLE OF THE INVENTION
HUMAN GLUCAGON-LIKE 1 P~ E RECEPTOR
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Sequence of the hllm~n GLP-l receptor. The 463 residue
long amino acid sequence deduced from the cDNA sequence is shown in
single letter code. The 337 base pair (bp) fragment (hGLP-l fragment
1) isolated by degenerate PCR is underlined. Three consensus sequences
for N-linked glycoslylation are marked with diamonds.
Figure 2. Comparison of the human and rat GLP-l receptor cDNA
sequences. Comparison of the human (top) and rat GLP-l receptors
was made using the GCG (Genetics Computer Group) Gap program.
15 The 7 putative transmembrane domains are boxed.
Figure 3. Displacement of [125I] GLP-1 (7-36) amide binding to
transfected COS-7 cells. COS-7 cells (7x106 cells) were transfected
with 20 ,ug of human GLP-l receptor cDNA in pcDNAI/neo and
20 membranes prepared and frozen from the cells. 27 llg of membrane
protein was incubated with 50 pM [125I] GLP-l (7-36) amide and the
indicated concentrations of ligand. Data shown are means +/- S.E.M. of
duplicate determinations and are representative of two experiments.
Symbols; squares, GLP-l (7-36) amide; triangles, glucagon; diamonds,
2s gastric inhibitory peptide; circles, secretin.
Figure 4. cAMP accumulation in transfected COS-7 cells. COS-7 cells
(7x106 cells) were transfected with 100 ,ug of h~lm~n GLP-l receptor
cDNA. Cells were harvested and cAMP accllmlll~tion was determined
30 as outlined in the Examples. Data shown are the mean +/- S.E.M. from
triplicate determinations from a single experiment and are
representative of three experiments. Symbols, triangles, GLP- 1 7-36
amide; squares, glucagon.
WO 95/04821 PCT/US94/08913
21684 18
BACKGROUND OF THE INVENTION-
This is a continll~tion of U.S. Serial No. 08/104, 517 filed
August 9, 1993, now pending.
Glucagon-like 1 peptide (GLP-1) is one of several
5 hormones shown to potentiate glucose-induced insulin secretion. Such
hormones, known as incretins, are produced in the gut, released in
response to a meal, and their interaction with specific receptors on
pancreatic islets causes insulin to be secreted in a glucose-dependent
manner (H.-C. Fehm~nn, J. F. Habener, Trends in Endocrinol. and Met.
3, 158-163 (1992)). GLP-1 is produced by postranslational processing
of the proglucagon gene in intestinal L cells, through a biologically
inactive 37 amino acid form [GLP-1 (1-37)] to either of two
biologically active forms, GLP-1 (7-37) and GLP-1 (7-36) amide.
These biologically active forms of GLP-1 are the most potent incretins
known, with effects on glucose-mediated insulin secretion being seen at
concentrations as low as 10 pM. Infusion of GLP-1 (7-36) amide into
patients with type II diabetes leads to increased secretion of insulin
which occurs in an glucose-dependent fashion (D. M. Nathan, E.
Schreiber, H. Fogel, S. Mojsov, J. F. Habener, Diabetes Care 15, 270-
20 276 (1992); M. Gutniak, C. Orskov, J. J. Holst, B. Ahren, E. S, NewEngl. J. Med. 326, 1316-1322 (1992)). These data suggest that
compounds that act via the GLP-1 receptor may be therapeutic in the
treatment of type II diabetes.
GLP-1 receptors have also been described in lung (G.
Richter, R. Goke, B. Goke, A. R., FEBS Lett. 267, 78-80 (1990)),
adipose (C. Ruiz-Grande, C. Alarcon, E. Merida, I. Vaverde, Peptides
13, 13-16 (1992)); brain (N. S. Hoosein, G. R.S, FEBS. Lett. 178, 83-
86 (1984)); and a gastric tumor cell line, HGT-1 (A. B. Hansen, C. P.
Gespach, G. E. Rosselin, J. J. Holst, FEBS Lett. 236, 119-122 (1988)).
30 A cDNA encoding a GLP-1 receptor has recently been cloned from rat
pancreatic islets (B. Thorens, Proceedings of the National Academy of
Sciences U.S.A 89, 8641-8645 (1992)). This receptor has seven
putative transmembrane domains and belongs to the superfamily of G
protein coupled receptors. The GLP-1 receptor is most homologous to
wo g~ 216 8 1 g 8 PCT/US94/08913
other members of a recently defined subclass of G protein coupled
receptors that includes the receptors for glucagon and secretin. The
GLP-1 receptor acts via stim~ tion of adenylyl cyclase to raise
intracellular levels of cAMP (Fehm~nn et al., supra).
The present invention pertains to the cloning, expression,
and ph~rm~cological characterization of a hllm~n GLP-l receptor from
the gastric tumor cell line HGT-l.
SUMMARY OF THE INVENTION
The human glucagon-like 1 peptide (GLP-1) is cloned,
expressed and used in an in vitro assay to screen for compounds that
bind to the receptor, including compounds which specifically stimulate
or inhibit the activity of the receptor. The invention includes the assay,
the cloned receptor used in the assay, an isolated human GLP-1
receptor, cells expressing the cloned receptor, and compounds identified
through the use of the cloned GLP-1 receptor which selectively bind to
the human GLP-l receptor, including specific agonists or antagonists of
the receptor.
The human glucagon-like 1 peptide receptor of the present
application was cloned from the gastric tumor cell line HGT-1. The
cDNA clone encodes a protein of 463 amino acids. The predicted
secondary structure places this receptor within the superfamily of seven
transmembrane domain G protein coupled receptors. Transfection of
the hllm~n GLP-1 receptor into COS-7 cells confers upon them high
affinity binding for [125I] GLP-l (7-36) amide. In membranes
prepared from COS-7 cells transfected with the hllm~n GLP-1 receptor,
the binding of [125I] GLP-l (7-36) amide is inhibited with the rank
order of potency GLP-1 (7-36) amide > glucagon > secretin; this is
characteristic of a GLP-1 receptor. The human GLP-l receptor
expressed in COS-7 cells is functionally coupled to increases in
intracellular cAMP. Incubation of COS-7 cells expressing the hllm~n
GLP-1 receptor with GLP-1 (7-36) amide gives rise to a 4-fold increase
in cyclic AMP over basal levels, with an EC50 of 25 pM. Glucagon is
WO 95/04821 PCT/US94/08913
2168~48
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200-fold less potent than GLP-1 as an agonist at the expressed human
receptor.
DETAILED DESCRIPTION OF THE INVENTION
The human glucagon-like 1 peptide receptor (GLP-1) was
identified, cloned and expressed in cell cultures by the instant inventors.
A partial coding region for this receptor was generat~ed by polymerase
chain reaction technology (PCR). Degenerate oligonucleotides encoding
amino acids present in the rat GLP-1 receptor were used to prime PCR
reactions using human HGT-1 cDNA as a template. The predicted sized
products were cloned and sequenced. Translation of the amplified
cDNA yielded an open reading frame encoding a protein approximately
91% homologous to the rat GLP-l receptor. This partial sequence was
used to obtain a larger cDNA clone from a hllm~n HGT-1 library. The
lS rem~ining receptor cDNA was obtained by a modification of the PCR-
RACE (Rapid Amplification of cDNA ends) protocol (M. A. Frohman,
M. K. Dush, G. R. Martin, Proc. Natl. Acad. Sci. U.S.A. 85, 8998-9002
(1988)). A cDNA library was made from HGT-1 cDNA and the
plasmid pcDNA I as outlined in Examples. PCR was performed using
the HGT-1 library and primers to both the partial fragment of the
hllm~n GLP-1 receptor and pcDNA I. A series of overlapping cDNA
fragments were obtained and sequenced.
The HGT-1 cDNA library and primers 5'
TGGTGGATTCCTGAACTCC 3' (SEQ ID NO: 3) and 5'
CCTGTGGTTTCACAAGAAGC 3' (SEQ ID NO: 4) were used in a
PCR reaction to generate the complete receptor sequence (Figure 1).
The open reading frame included in this sequence encodes a 463 amino
acid protein that is approximately 91% identical to the rat GLP-1
receptor sequence (Figure 2).
The cloned human GLP-1 receptor, when expressed in
m~mm~ n cell lines including but not limited to, COS-7, CHO or L
cells, is used to discover ligands that bind to the receptor and alter or
stimulate its function. In addition, the cloned GLP-1 receptor enables
quantitation of mRNA levels in human tissues, including the pancreas
WO 95/04821 21 6 ~ 4 4 8 PCT/US94/08913
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and gastrointestinal system, by RNase protection assays. For these
purposes, a complete coding sequence of the receptor is provided.
The specificity of binding of compounds showing affinity
for the GLP-1 receptor is shown by measuring the affinity of the
5 compounds to membranes obtained from cells tranfected with the cloned
GLP-l receptor and membranes from tissues known to express GLP-l
receptors. Expression of the cloned GLP-l receptor, screening for
compounds that inhibit the binding of radiolabeled GLP-l (7-36) amide
or compounds that stimulate cAMP production in these cells provides a
rational way for selection of compounds and discovery of new
compounds with predictable pharmacological activities.
Once the hllm~n receptor is cloned and expressed in a non-
hllm~n cell line, such as COS-7 cells or CHO cells, the recombinant
GLP-1 receptor is free of other hllm~n proteins. The membranes from
15 the recombinant cells expressing human GLP-1 receptor are then
isolated according to methods well known in the art and may be used in
a variety of membrane associated receptor binding assays. One example
of such an assay is described by Strader et al., (Proc. Natl. Acad. Sci.
USA 84, 4384-4388, 1987). Generally, a compound of interest is used
20 to compete with the binding of a known, quantifiable GLP-1 receptor
ligand. Thus, radiolabeled [125I] GLP (7-36) amide or [3H]-GLP may
be used for this purpose. Because of the ease of 125I detection, ~125I]
GLP (7-36) amide is preferred for this purpose. By increasing the
amount of unlabeled test compound, the labeled compound is competed
2s off the receptor. From these experiments, IC50 values for each test
compound and receptor subtype is determined.
In addition, agonist ligands that activate the receptor may
be detected by measuring the ability of added compounds to increase
cAMP production mediated by the receptor expressed in COS-7 or CHO
cells. cAMP can be measured directly by radioimmunoassay or by
stim~ tion of adenylylcyclase in membranes prepared from the cells
(Salomon, Y., Landos, C. and Rodbell, M. 1974. Anal. Biochemistry,
Vol. 58, 541-548) by methods that are well-known in the art.
WO 95/04821 PCT/US94/08913
2168~
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Thus, according to this invention, a method is provided for
identifying compounds specific for the human GLP-1 receptor
comprising the following steps:
a. Cloning the human glucagon-like 1 peptide (GLP-1)
receptor;
b. Splicing the cloned GLP-1 receptor into an
expression vector to produce a construct such that the
GLP-1 receptor is operably linked to transcription
and translation signals sufficient to induce expression
o of the receptor upon introduction of the construct
into a prokaryotic or eukaryotic cell;
c. Introducing the construct into a prokaryotic or
eukaryotic cell which does not express a hllm~n
GLP-1 receptor in the absence of the introduced
construct;
d. Incubating cells or membranes isolated from cells
produced in Step c with a quantifiable compound
known to bind to human GLP-1 receptors, and
subsequently ~(ltling test compounds at a range of
concentrations so as to compete the quantifiable
.compound from the receptor, such that an IC50 for
the test compound is obtained as the concentration of
test compound at which 50% of the qll~ntifi~ble
compound becomes displaced from the receptor; and
e. Incubating cells or membranes from cells produced
in Step c with test compounds in range of
concentrations such that an ED50 for the test
compound is obtained. The ED50 is defined as the
concentration of compound which increases
3 intracellular cyclic AMP to 50% of the maximal
quantity of cAMP produced by interaction of the test
compound with the human GLP-1 receptor.
Whereas GLP-1 has been shown to increase secretion of
insulin in diabetic patients, an agonist of the human GLP-1 receptor
WO 95/04821 2 t 6 8 ~ ~ 8 PCT/US94/08913
discovered as described above would be useful in the treatment of
diabetes.
The following examples are provided to further define the
invention without, however, limiting the invention to the particulars of
5 these examples.
EXAMPLE 1
Culture of HGT-1 and COS-7 cells
HGT-1 cells (cell line Cl.19A, a human gastric carcinoma
cell line) were obtained from Dr. C. L. Laboisse and cultured as
described (Laboisse et al., Cancer Research 42, 1541-1528 (1982)).
COS-7 cells were cultured in a manner identical to HGT-1 cells.
EXAMPLE 2
Cloning of a partial cDNA human GLP-1 receptor clone
Poly A+ RNA was isolated from HGT-1 cells using the
Fast-Track system (Invitrogen). cDNA was prepared from 5 ,ug of
HGT-1 poly A+ RNA by simultaneous priming with random
20 hexanucleotides and oligo dT primers using the Riboclone cDNA
synthesis system (Promega). HGT-1 cDNA and degenerate primers
based on the rat cDNA sequence (B. Thorens, Proceedings of the
National Academy of Sciences U.S.A 89, 8641-8645 (1992)) were
mixed in a PCR reaction to amplify a partial fragment of the human
25 GLP-1 receptor cDNA (Figure 1, underlined). The protocol is
described below:
Degenerate PCR
30 5 ,ul lOx PCR buffer from Boehringer Mannheim Biochemicals (BMB).
4 ,ul 2.5 ~lM each stock dATP, dCTP, dGTP and dTTP 2 ~l HGT-1
cDNA
1 ~l 20,uM primer [5' ATG CA(AG) TA(CT) TG(CT) GTN GC 3';
SEQ ID NO:5]
WO 95/04821 PCT/US94/08913
2168448
1 ~120 uM primer [5' AT(AG) TCN GT(AC) TT(AG) CAC AT 3';
SEQ ID NO:6]
0.25 ,ul (2 units) Amplitaq DNA polymerase (Cetus)
36.75 ~1 water
Reaction conditions: 40 cycles at 95C, 1 min.; 45C, 0.5 min.; 72C,
1 min.
The predomin~nt PCR product, a 337 base pair (bp) DNA
fragment (hGLP- fragment 1) was cloned into plasmid pCR II using the
TA cloning kit (Invitrogen) and transformed into Escherichia coli.
INVaF'. Plasmid DNA was isolated and DNA sequence determined by
the dideoxy chain termination method.
EXAMPLE 3
PCR amplification, cloning, and sequencing of a cDNA encoding the
complete human GLP-l receptor
Poly A+ RNA was isolated from HGT-1 cells using the
20 Fast-Track system (Invitrogen). cDNA was prepared from 5 ,ug of
HGT-1 poly A+ RNA by simultaneous p~ lg with random
hexanucleotides and oligo dT primers using the Riboclone cDNA
synthesis system (Promega).
The cDNA was ligated with non-palindromic BST XI
25 linkers (Invitrogen). Excess linkers were removed by gel-filtration
over a cDNA sizing column (Gibco-BRL). For the PCR-RACE
protocols ~e cDNA was ligated into plasmid pcDNA I (Invitrogen)
after restriction with BST XI. A series of primers were made to
sequences in hGLP-fragment 1 (Example 2) and to sequences in pcDNA
30 - I. PCR was performed as follows:
5 ~1 lOx PCR buffer (BMB)
4 ,ul 2.5 ~M each stock dATP, dCTP, dGTP and dTTP
2 ,ul HGT-1 cDNA
WO 95/~1 21 6 ~ 4 ~ 8 PCT/US94/08913
1 ,ul 20 ~M primer from hGLP-fragment 1
1 ,ul 20 ,uM primer from pcDNA 1
0.25 ,ul (2 units) Amplitaq DNA polymerase
36.75 ~l water
Reaction conditions: 35 cycles at 95C, l min.; 55C, 0.5 min.; 72C,
l min.
Aliquots of the PCR reactions were cloned into plasmid
pCR II using the TA cloning kit (Invitrogen) and transformed into
E. coli. INVaF'. Human GLP-l receptor specific fragments were
identified by filter hybridization using [32p] labeled hGLP-fragment l
as a probe. The following hybridization conditions were employed:
5 SX SSC (lX SSC is O.lS M sodium chloride, O.OlS M sodium citrate)
SX Denharts solution (1% Ficoll, 1% polyvinylpyrrolidone)
100 ,ug/ml salmon sperm DNA
50% form~mide
Hybridize overnight at 42C
Filters were washed 2 times in lX SSC, 0.1% SDS at room
temperature for lO min. each, then 2 times in O.lX SSC, 0.1% SDS for
20 min. Positive clones were identified by autoradiography. DNA
sequence of positive clones was obtained by the dideoxy chain
25 termination method. Using this procedure clones cont~ining DNA
sequence from either the 5' or 3' untranslated regions were obtained.
A single molecule encoding the entire hGLP-l receptor
sequence was obtained by amplification using the polymerase chain
reaction. The following conditions were employed:
5 ,ul lOx PCR Pfu polymerase buffer (Stratagene).
4 ,ul 2.5 ~M each stock dATP, dCTP, dGTP and dTTP
2 ~ll HGT-l cDNA
1 ,ul 20 uM primer 5' TGGTGGATTCCTGAACTCC 3'; SEQ ID NO:7
WO 95tO4821 PCT/US94/08913
216~8
- 10-
1 ,ul 20 uM primer 5' CCTGTGGITTCACAAGAAGC 3'; SEQ ID
NO:8
1 ,ul (5 units) Pfu polymerase (Stratagene)
5 ~ll dimethylsulfoxide
5 29 ,ul water ~
Reaction mixtures were heated at 96C for 5 min. and then 35 thermal
cycles performed: 1 min. at 95C, 0.5 min. at 55C, 2 min. at 72C.
Aliquots of the PCR reactions were blunt end cloned into
plasmid pCR-Script SK+ (Stratagene) and transformed into E. coli
XL-1 Blue. Human GLP-1 receptor specific fragments were identified
by filter hybridization using [32p] labeled hGLP-fragment 1 as a probe.
The hybridization conditions employed were those described in
5 Example 3.
Filters were washed 2 times in lX SSC, 0.1% SDS at room
temperature for 10 min. each, then 2 times in O.lX SSC, 0.1% SDS for
20 min. Positive clones were identified by autoradiography. DNA
sequence of positive clones was obtained by the dideoxy chain
20 termination method. The DNA sequence obtained is shown in Figure 1.
EXAMPLE 4
Expression of the cloned human GLP-1 receptor
COS-7 cells were transfected by electroporation with the
human GLP-1 receptor cDNA subcloned into the eukaryotic expression
vector pcDNA Vneo (Invitrogen). Cells were harvested after 60-72 h.
Membranes cont~ining the expressed receptor protein were prepared as
described (C. D. Strader et al., Proc. Natl. Acad. Sci. U.S.A. 84, 4384- :
30 4388 (1987). Membranes prepared from the COS-7 cells transfected
with the vector containing the human GLP-1 receptor cDNA
specifically bound the GLP-1 receptor agonist [125n GLP-1 (7-36)
amide (Figure 3). Membranes prepared from cells transfected with the
vector alone did not specifically bind [125I] GLP-1 (7-36) amide,
wo gStO~l 21 6 8 4 1 8 PCT/US94/08913
_
proving the expression of the hllm~n GLP-l receptor. As shown in
Figure 3, GLP-1 (7-36) amide inhibits the binding of [125I] GLP-l (7-
36) amide to the receptor with an IC50 of 4 nM. Glucagon, gastric
inhibitory peptide, and secretin inhibit [125I] GLP-1 (7-36) amide
5 binding with a potency at least a 100-fold lower, consistent with the
identification of the receptor as a GLP-1 receptor.
Binding reactions were perfor~ned in a final volume of 200
,ul of PBS (10 mM sodium phosphate, 1 mM potassium phosphate, 2.7
mM potassium chloride, 137 mM sodium chloride, pH 7.0 ) under the
o following conditions:
10-25 ,ug COS-7 membranes prepared from transfected cells
0.1% bovine serum albumin
50 pM [125I] GLP-1 (7-36) amide
0-1 ,uM GLP-1 (7-36) amide (or the other compounds listed in the
legend to Figure 3)
Membranes were incubated at room temperature with
shaking for one hour. Membranes were harvested on GF/C filters
20 (Wh~tm~n) that had been presoaked in 0.5% polyethylenimine/0.1%
BSA. The filters were washed three times with ice-cold PBS and bound
radioactivity determined by gamma counting. Data were analyzed using
the Inplot program (Graphpad Software).
The human GLP-1 receptor is functionally coupled to
2s adenylyl cyclase in transiently transfected COS-7 cells (Figure 4).
Incubation of COS-7 cells expressing the hllm~n GLP-1 receptor with
GLP-1 (7-36) amide leads to a 4-fold increase in cyclic AMP (cAMP)
over basal levels. Under identical assay conditions mock transfected
COS cells show no significant increase in cAMP over basal levels.
30 GLP-1 (7-36) amide stimulates cAMP accumulation with an EC50 of 25
pM. Glucagon also stimulates cAMP accumulation in COS-7 cells
transfected with the human GLP-1 receptor but with a 200-fold
decrease in potency compared with GLP-1 (7-36) arnide (Figure 4).
The decreased potency for glucagon is consistent with its acting via the
WO 95/04821 ` PCT/US94/08913
2 1 6 8 4 ~ 8
- 12 -
hl1m~n GLP-1 receptor. Stim~ tion of cAMP accumulation in intact
COS-1 cells that had been transfected with the hGLP-1 receptor assays
were carried out in 120 ,ul volume of ACC (75 mM Tris pH 7.4, 250
mM sucrose, 12.5 mM magnesium chloride, 1.5 mM ethylene~ mine-
tetraacetic acid (EDTA), 0.1 mM of the phosphodiesterase inhibitor Ro-
201724) cont~ining the following additions:
50,000 COS-7 cells transfected with the human GLP-l
receptor expression construct; and
o 0-100 ~M GLP-1 (7-36) amide or glucagon
Reaction mixtures were incubated for 45 min. at room temperature with
~h~king. Reactions were termin~ted by boiling for 3-5 min. cAMP was
determined by radioimmunoassay.
EXAMPLE 5
Screening Assay: Glucagon-like peptide-1 receptor mediated increase in
intracellular cAMP
Transfected cells expressing recombinant human GLP-l
receptor may be used to identify compounds that are agonists for it.
This is done by incubating cells with test compounds in range of
concentrations such that an ED50 for the test compound is obtained.
The ED50 is defined as the concentration of compound which increases
25 intracellular cyclic AMP (cAMP) to 50% of the m~xim~l quantity of
cAMP produced by interaction of the test compound with the hllm~n
GLP-1 receptor. These reactions are carried out in 120 ,ul volume of
ACC cont~inin~ the following additions:
50,000 COS-7 cells transfected with the hllm~n GLP-1
receptor; and
Various concentration of test compounds.
wo 95/04821 2 1~ ~ g ~ 8 PCT/US94/08913
- 13 -
Reaction mixtllres were incubated for 45 min. at room temperature with
sh~king Reactions were termin~ted by boiling for 3-S min. cAMP was
determined by radioimml-noassay.
W 0 95/04821 PCTrUS94/089l3
21~8~
- 14 -
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANTS: Graziano, Michael P.
Borkowski, Doreen A.
Chicchi, Gary G.
Hey, Patricia J.
Strader, Catherine D.
(ii) TITLE OF INVENTION: Human Glucagon Like 1 Peptide Receptor
(iii) NUMBER OF SEQUENCES: 8
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Carty, Christine E.
(B) STREET: P.O. Box 2000, 126 E. Lincoln Ave.
(C) CITY: Rahway
(D) STATE: New Jersey
(E) COUNTRY: USA
(F) ZIP: 07065
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Carty, Christine E.
(B) REGISTRATION NUMBER: 36,099
(C) REFERENCE/DOCKET NUMBER: 19065
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (908) 594-6734
(B) TELEFAX: (908) 594-4720
(C) TELEX: 138825
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1567 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
WO 95/04821 ~16 ~ 4 4 8 PCT~US94/08913
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
TGGTGGAATT CCTGAACTCC CCGCCATGGC CGGCGCCCCC GGCCCGCTGC GCCTTGCGCT 60
GCTGCTGCTC GGGATGGTGG GCAGGGCCGG CCCCCGCCCC CAGGGTGCCA CTGTGTCCCT 120
CTGGGAGACG GTGCAGAAAT GGCGAGAATA CCGACGCCAG TGCCAGCGCT CCCTGACTGA 180
GGATCCACCT CCTGCCACAG A~ll~ll'-l'G CAACCGGACC TTCGATGAAT ACGCCTGCTG 240
GCCAGATGGG GAGCCAGGCT CGTTCGTGAA TGTCAGCTGC CCCTGGTACC TGCCCTGGGC 300
CAGCAGTGTG CCGCAGGGCC ACGTGTACCG GTTCTGCACA GCTGAAGGCC TCTGGCTGCA 360
GAAGGACAAC TCCAGCCTGC CCTGGAGGGA CTTGTCGGAG TGCGAGGAGT CCAAGCGAGG 420
GGAGAGAAGC TCCCCGGAGG AGCAGCTCCT GTTCCTCTAC ATCATCTACA CGGTGGGCTA 480
CGCACTCTCC ll~l~lGCTC TGGTTATCGC CTCTGCGATC CTCCTCGGCT TCAGACACCT 540
GCACTGCACC AGGAACTACA TCCACCTGAA ~l~lllGCA TCCTTCATCC TGCGAGCATT 600
GTC~ llC ATCAAGGACG CAGCCCTGAA GTGGATGTAT AGCACAGCCG CCCAGCAGCA 660
CCAGTGGGAT GGGClC~l~l' CCTACCAGGA ~ lGAGC TGCCGCCTGG 'l'~l"l"l'~"lGCT 720
CATGCAGTAC l~l~l~GCGG CCAATTACTA CTGGCTCTTG GTGGAGGGCG TGTACCTGTA 780
CACACTGCTG GCCTTCTCGG TCTTATCTGA GCAATGGATC TTCAGGCTCT ACGTGAGCAT 840
AGGCTGGGGT GTTCCCCTGC 'l~'l"l'l'~'l~l~'l' CCCCTGGGGC ATTGTCAAGT ACCTCTATGA 900
GGACGAGGGC TGCTGGACCA GGAACTCCAA CATGAACTAC TGGCTCATTA TCCGGCTGCC 960
CAll~ l GCCATTGGGG TGAACTTCCT CAl~lll~ll CGGGTCATCT GCATCGTGGT 1020
ATCCAAACTG AAGGCCAATC TCATGTGCAA GACAGACATC AAATGCAGAC TTGCCAAGTC 1080
CACGCTGACA CTCATCCCCC TGCTGGGGAC TCATGAGGTC ATCTTTGCCT TTGTGATGGA 1140
CGAGCACGCC CGGGGGACCC TGCGCTTCAT CAAGCTGTTT ACAGAGCTCT CCTTCACCTC 1200
CTTCCAGGGG CTGATGGTGG CCATATTATA CTG~ll'l~GlC AACAATGAGG TCCAGCTGGA 1260
ATTTCGGAAG AGCTGGGAGC GCTGGCGGCT TGAGCACTTG CACATCCAGA GGGACAGCAG 1320
CATGAAGCCC CTCAAGTGTC CCACCAGCAG CCTGAGCAGT GGAGCCACGG CGGGCAGCAG 1380
CATGTACACA GCCACTTGCC AGGCCTCCTG CAGCTGAGAC TCCAGCGCCT GCCCTCCCTG 1440
GGGTCCTTGC TGCAGGCCGG GTGGCCAATC CAGGAGAAGC AGCCTCCTAA TTTGATCACA 1500
GTGGCGAGAG GAGAGGAAAA ACGATCGCTG TGAAAATGAG GAGGATTGCT 'l'Cll~lGAAA 1560
CCACAGG 1567
WO 95/04821 PCTrUS94/08913
21684~8
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(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 463 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Ala Gly Ala Pro Gly Pro Leu Arg Leu Ala Leu Leu Leu Leu Gly
1 5 10 15
Met Val Gly Arg Ala Gly Pro Arg Pro Gln Gly Ala Thr Val Ser Leu
Trp Glu Thr Val Gln Lys Trp Arg Glu Tyr Arg Arg Gln Cys Gln Arg
Ser Leu Thr Glu Asp Pro Pro Pro Ala Thr Asp Leu Phe Cys Asn Arg
Thr Phe Asp Glu Tyr Ala Cys Trp Pro Asp Gly Glu Pro Gly Ser Phe
Val Asn Val Ser Cys Pro Trp Tyr Leu Pro Trp Ala Ser Ser Val Pro
Gln Gly His Val Tyr Arg Phe Cys Thr Ala Glu Gly Leu Trp Leu Gln
100 105 110
Lys Asp Asn Ser Ser Leu Pro Trp Arg Asp Leu Ser Glu Cys Glu Glu
115 120 125
Ser Lys Arg Gly Glu Arg Ser Ser Pro Glu Glu Gln Leu Leu Phe Leu
130 135 140
Tyr Ile Ile Tyr Thr Val Gly Tyr Ala Leu Ser Phe Ser Ala Leu Val
145 150 155 160
Ile Ala Ser Ala Ile Leu Leu Gly Phe Arg His Leu His Cys Thr Arg
165 170 175
Asn Tyr Ile His Leu Asn Leu Phe Ala Ser Phe Ile Leu Arg Ala Leu
180 185 190
Ser Val Phe Ile Lys Asp Ala Ala Leu Lys Trp Met Tyr Ser Thr Ala
195 200 205
WO 95/04821 2 ~ 6 ~ 4 ~ 8 PCT/US94/08913
- Ala Gln Gln His Gln Trp Asp Gly Leu Leu Ser Tyr Gln Asp Ser Leu
210 215 220
Ser Cys Arg Leu Val Phe Leu Leu Met Gln Tyr Cys Val Ala Ala Asn
225 230 235 240
Tyr Tyr Trp Leu Leu Val Glu Gly Val Tyr Leu Tyr Thr Leu Leu Ala
245 250 255
Phe Ser Val Leu Ser Glu Gln Trp Ile Phe Arg Leu Tyr Val Ser Ile
260 265 270
Gly Trp Gly Val Pro Leu Leu Phe Val Val Pro Trp Gly Ile Val Lys
275 280 285
Tyr Leu Tyr Glu Asp Glu Gly Cys Trp Thr Arg Asn Ser Asn Met Asn
290 295 300
Tyr Trp Leu Ile Ile Arg Leu Pro Ile Leu Phe Ala Ile Gly Val Asn
305 310 315 320
Phe Leu Ile Phe Val Arg Val Ile Cys Ile Val Val Ser Lys Leu Lys
325 330 335
Ala Asn Leu Met Cys Lys Thr Asp Ile Lys Cys Arg Leu Ala Lys Ser
340 345 350
Thr Leu Thr Leu Ile Pro Leu Leu Gly Thr His Glu Val Ile Phe Ala
355 360 365
Phe Val Met Asp Glu His Ala Arg Gly Thr Leu Arg Phe Ile Lys Leu
370 375 380
Phe Thr Glu Leu Ser Phe Thr Ser Phe Gln Gly Leu Met Val Ala Ile
385 390 395 400
Leu Tyr Cys Phe Val Asn Asn Glu Val Gln Leu Glu Phe Arg Lys Ser
405 410 415
Trp Glu Arg Trp Arg Leu Glu His Leu His Ile Gln Arg Asp Ser Ser
420 425 430
Met Lys Pro Leu Lys Cys Pro Thr Ser Ser Leu Ser Ser Gly Ala Thr
435 440 445
Ala Gly Ser Ser Met Tyr Thr Ala Thr Cys Gln Ala Ser Cys Ser
450 455 460
(2) INFORMATION FOR SEQ ID NO: 3:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
WO 9~/04821 PCT/US94/08913
21~84~8
- 18 -
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
TGGTGGATTC CTGAACTCC 19
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
CCTGTGGTTT CACAAGAAGC 20
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
ATGCAAGTAC TTGCTGTGCN 20
(2) INFORMATION FOR SEQ ID NO:6:
W 0 95/04821 216 ~ 4 ~ ~ PCT~US94/08913
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
- (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
ATAGTCNGTAC TTAGCACAT 20
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l9 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOln~llCAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
TGGTGGATTC CTGAACTCC l9
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
~'W~'lW'l'l"l' CACAAGAAGC 20
