CA2168448A1 - Human glucagon-like 1 peptide receptor - Google Patents

Abstract

The human GLP-1 receptor is cloned and used in an in vitro assay to screen for compounds that specifically bind to the human GLP-1 receptor, including compounds effective to reduce symptoms of diabetes. The invention includes the assay, the cloned human receptor used in the assay, an isolated human GLP-1 receptor free of other human proteins, and compounds identified through the use of this novel, cloned receptor, which selectively bind the human GLP-1 receptor.

Description

WO 9S/04821 216 8 ~ 4 8 PCT/US94/08913 -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.

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 2168~48 200-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 -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.

2168~

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 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.

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.

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]

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.

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 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.

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 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.

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 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~

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:

GGATCCACCT CCTGCCACAG A~ll~ll'-l'G CAACCGGACC TTCGATGAAT ACGCCTGCTG 240 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 AGGCTGGGGT GTTCCCCTGC 'l~'l"l'l'~'l~l~'l' CCCCTGGGGC ATTGTCAAGT ACCTCTATGA 900 CAll~ l GCCATTGGGG TGAACTTCCT CAl~lll~ll CGGGTCATCT GCATCGTGGT 1020 CTTCCAGGGG CTGATGGTGG CCATATTATA CTG~ll'l~GlC AACAATGAGG TCCAGCTGGA 1260 GTGGCGAGAG GAGAGGAAAA ACGATCGCTG TGAAAATGAG GAGGATTGCT 'l'Cll~lGAAA 1560 WO 95/04821 PCTrUS94/08913 21684~8 (2) INFORMATION FOR SEQ ID NO:2:
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(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 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 Lys Asp Asn Ser Ser Leu Pro Trp Arg Asp Leu Ser Glu Cys Glu Glu Ser Lys Arg Gly Glu Arg Ser Ser Pro Glu Glu Gln Leu Leu Phe Leu Tyr Ile Ile Tyr Thr Val Gly Tyr Ala Leu Ser Phe Ser Ala Leu Val Ile Ala Ser Ala Ile Leu Leu Gly Phe Arg His Leu His Cys Thr Arg Asn Tyr Ile His Leu Asn Leu Phe Ala Ser Phe Ile Leu Arg Ala Leu Ser Val Phe Ile Lys Asp Ala Ala Leu Lys Trp Met Tyr Ser Thr Ala 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 Ser Cys Arg Leu Val Phe Leu Leu Met Gln Tyr Cys Val Ala Ala Asn Tyr Tyr Trp Leu Leu Val Glu Gly Val Tyr Leu Tyr Thr Leu Leu Ala Phe Ser Val Leu Ser Glu Gln Trp Ile Phe Arg Leu Tyr Val Ser Ile Gly Trp Gly Val Pro Leu Leu Phe Val Val Pro Trp Gly Ile Val Lys Tyr Leu Tyr Glu Asp Glu Gly Cys Trp Thr Arg Asn Ser Asn Met Asn Tyr Trp Leu Ile Ile Arg Leu Pro Ile Leu Phe Ala Ile Gly Val Asn Phe Leu Ile Phe Val Arg Val Ile Cys Ile Val Val Ser Lys Leu Lys Ala Asn Leu Met Cys Lys Thr Asp Ile Lys Cys Arg Leu Ala Lys Ser Thr Leu Thr Leu Ile Pro Leu Leu Gly Thr His Glu Val Ile Phe Ala Phe Val Met Asp Glu His Ala Arg Gly Thr Leu Arg Phe Ile Lys Leu Phe Thr Glu Leu Ser Phe Thr Ser Phe Gln Gly Leu Met Val Ala Ile Leu Tyr Cys Phe Val Asn Asn Glu Val Gln Leu Glu Phe Arg Lys Ser Trp Glu Arg Trp Arg Leu Glu His Leu His Ile Gln Arg Asp Ser Ser Met Lys Pro Leu Lys Cys Pro Thr Ser Ser Leu Ser Ser Gly Ala Thr Ala Gly Ser Ser Met Tyr Thr Ala Thr Cys Gln Ala Ser Cys Ser (2) INFORMATION FOR SEQ ID NO: 3:
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(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

(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
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(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

Claims (9)

WHAT IS CLAIMED IS:

1. An isolated and purified DNA molecule consisting essentially of DNA encoding a human glucagon-like 1 peptide receptor.

2. The DNA molecule of Claim 1, wherein the DNA
coding for the receptor is operably linked to regulatory sequences such that the receptor may be expressed upon introduction into a prokaryotic or eukaryotic cell.

3. The human glucagon-like 1 peptide receptor encoded by the DNA molecule of Claim 1.

4. A cell containing the DNA molecule of Claim 1, the cell expressing a cloned human glucagon-like 1 peptide receptor.

5. A method for identifying compounds which specifically bind to a human glucagon-like 1 peptide receptor comprising the steps of:
(a) cloning the human glucagon-like 1 peptide receptor;
(b) splicing the the cloned glucagon-like 1 peptide receptor into an expression vector to produce a construct such that the glucagon-like 1 peptide receptor is operably linked to transcription and translation signals sufficient to induce expression 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 human 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 glucagon-like 1 peptide receptor, and subsequently adding test compounds at a range of concentrations so as to compete the quantifiable compound from the receptor; and (e) calculating the relative binding of the test compound to the cells or membranes of Step (d).

6. The compounds identified by the method of Claim 5.

7. A method of alleviating the effects of diabetes which comprises administering a pharmaceutically effective amount of a compound which specifically binds to the human GLP-1 receptor.

8. The DNA molecule of Claim 1 having the nucleic acid sequence shown in Figure 1.

9. A purified human glucagon-like 1 peptide receptor encoded by the DNA molecule of Claim 8, the purified receptor having the amino acid sequence shown in Figure 1.