Proc. Nati. Acad. Sci. USAVol. 89, pp. 8641-8645, September 1992Cell Biology

Expression cloning of the pancreatic J8 cell receptor for thegluco-incretin hormone glucagon-like peptide 1

(insulin secretion/non-insulin-dependent diabetes mellitus/entero-insular axis/G proteins/cAMP)

BERNARD THORENSInstitute of Pharmacology and Toxicology, University of Lausanne, 27, Bugnon, 1005 Lausanne, Switzerland

Communicated by Roger H. Unger, June 1, 1992

ABSTRACT Glucagon-like peptide 1 (GLP-1) is a hor-mone derived from the preproglucagon molecule and is se-creted by intestinal L cells. It is the most potent stimulator ofglucose-induced insulin secretion and also suppresses in vivoacid secretion by gastric glands. A cDNA for the GLP-1receptor was isolated by transient expression ofa rat pancreaticislet cDNA library into COS cells; this was followed by bindingof radiolabeled GLP-1 and screening by photographic emulsionautoradiography. The receptor transfected into COS cellsbinds GLP-1 with high affinity and is coupled to activation ofadenylate cyclase. The receptor binds specifically GLP-1 anddoes not bind peptides ofrelated structure and similar function,such as glucagon, gastric inhibitory peptide, vasoactive intes-tinal peptide, or secretin. The receptor is 463 amino acids longand contains seven transmembrane domains. Sequence homol-ogy is found only with the receptors for secretin, calcitonin, andparathyroid hormone, which form a newly characterized fam-ily of G-coupled receptors.

Glucose-induced insulin secretion is modulated by a numberof hormones and neurotransmitters. In particular, two guthormones, glucagon-like peptide 1 (GLP-1) and gastric in-hibitory peptide (GIP), potentiate the effect of glucose oninsulin secretion and are thus called gluco-incretins (1, 2).GLP-1 is a gluco-incretin in rat and in man (1-3). It is part ofthe preproglucagon molecule (4) that is proteolytically pro-cessed in intestinal L cells to GLP-1-(1-37) and GLP-1-(7-36)amide or GLP-1-(7-37) (5, 6). Only the truncated forms ofGLP-1 are biologically active and both have identical effectson insulin secretion in ,. cells (7, 8). They are the most potentgluco-incretins so far described and are active at concentra-tions as low as 1-10 pM. The stimulatory effect of thesegluco-incretin hormones requires the presence of glucose ator above the normal physiological concentration of about 5mM and is mediated by activation of adenylate cyclase anda rise in the intracellular concentration of cAMP (9, 10).GLP-1 has also a stimulatory effect on insulin gene transcrip-tion (9). In rat, non-insulin-dependent diabetes mellitus(NIDDM) is associated with a reduced stimulatory effect ofGLP-1 on glucose-induced insulin secretion (11). In man, inone study, GLP-1 levels were elevated in NIDDM patients inthe basal state and after glucose ingestion; however, follow-ing a glucose load there was only a very small rise in plasmainsulin concentration (12). This suggests that diabetic 3 cells,in addition to being glucose-unresponsive, may also have adecreased sensitivity to the incretin effect of GLP-1. How-ever, a recent study (13) showed that GLP-1 infusion couldameliorate postprandial insulin secretion and glucose dis-posal in NIDDM patients, and this hormone, or derivativesthereof, has been proposed as a new therapeutic agent tostimulate insulin secretion by P cells of type II diabetic

patients. Thus, as a further step in understanding the complexmodulation ofinsulin secretion by gut hormones, the functionof these hormones in the general control of glucose homeo-stasis, as well as the possible dysfunction of these mecha-nisms in diabetes, a cDNA for the 3 cell GLP-1 receptor wasisolated and characterized.*

MATERIAL AND METHODSIodination, Binding Analysis, and cAMP Measurements.

GLP-1-(7-36) amide and the other peptides used were fromPeninsula Laboratories. Iodination was performed by theiodine monochloride method (14), and the labeled peptidewas purified by passage over Sephadex G-10 followed byCM-Sepharose. The specific activity was determined by theself-displacement technique (15) and was routinely between500 to 1000 cpm/fmol. Analysis of binding data was per-formed with the LIGAND program (16). The concentration ofcAMP produced in transfected COS cells was determined bya radioimmunoassay as per the manufacturer's protocol(Amersham).

Islet Preparation, cDNA Library Construction, and Emul-sion Autoradiography Screening. Pancreatic islets were iso-lated from adult male Sprague-Dawley rats according toGotoh et al. (17). Poly(A)+ RNA was prepared and a cDNAlibrary was constructed as described (18, 19). Briefly, firststrand cDNA was primed with oligo(dT) (Promega) andsynthesized by avian myeloblastosis virus reverse transcrip-tase (Molecular Genetic Resources, Tampa, FL); secondstrand cDNA synthesis was performed using DNA polymer-ase I (Boehringer Mannheim) and RNase H (GIBCO/BRL)(20). After addition ofBstXI adapters (Invitrogen, San Diego)the cDNAs were size-selected on a potassium acetate gradi-ent (18) and cDNAs with sizes >1.5 kilobases (kb) werepooled and ligated into the pcDNA-1 vector (Invitrogen).Colonies were grown (5000-8000 per plate) and pooled, andplasmid DNA was prepared (21). These cDNA pools weretransfected (22) into COS cells growing on glass slides. Twodays later, the transfected cells were incubated for 1 hr at23°C in the presence of :0.5 nM iodinated GLP-1. After fourwashes in Hanks' balanced salt solution, cells were fixed with1% glutaraldehyde and covered with NTB-2 photographicemulsion (IBI-Kodak). After 2 days of exposure in the dark,the emulsion was developed and the slides were screened bydark-field microscopy (23). Positive pools were subfraction-ated until isolation of a single clone.

Northern Blot Analysis. TotalRNA was isolated by the acidguanidinium thiocyanate method (24), denatured with glyoxal(25), separated on 1% agarose gel, and transferred to Nylon

Abbreviations: GLP-1, glucagon-like peptide 1; GIP, gastric inhibi-tory peptide; NIDDM, non-insulin-dependent diabetes mellitus;VIP, vasoactive intestinal peptide; GSa, a subunit of stimulatory Gprotein.*The sequence reported in this paper has been deposited in theGenBank data base (accession no. M97797).

8641

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Proc. Natl. Acad. Sci. USA 89 (1992)

membranes (26). Hybridization was performed with the ran-dom primed labeled (27) 1.6-kb pGLPR-1 insert.

RESULTS AND DISCUSSIONA rat pancreatic islet cDNA library was constructed in thepcDNA-1 expression vector as described in Material andMethods. Plasmid DNA was prepared from pools of 5000-8000 bacterial clones and transfected into COS cells. Thepresence of GLP-1 receptor expressed in COS cells wasassessed by binding ofthe radioiodinated peptide followed byphotographic emulsion autoradiography and screening bydark-field microscopy. A 1.6-kb cDNA clone (pGLPR-1) waspurified by subfractionation of an original positive pool andwas used to isolate, by DNA hybridization screening, twoadditional clones from primary positive pools. These plas-mids (pGLPR-16 and -87) had inserts of 3.0 and 1.8 kb,respectively. Transfection of these clones into COS cellsgenerated high-affinity (Kd = 0.6 nM) binding sites for GLP-1(Fig. 1A). This value is comparable to binding ofGLP-1 to therat insulinoma cell line INS-1 (28) (Kd = 0.12 nM; Fig. 1B).In both cases a single high-aimity binding component was

A25

20~~~~~20 -

10~~~15-4 0.15 & .~~~~KD =0.65 nM

0.05

5 0.0010 20 40 60 80 100 120

Bound (pM)0O 0

2 4 6 8 10C GLP-1 (nM)0

_B

C,

2.0

1.5 0.10'

1.0 LL 0.06 ~~~~~~KD=0.12 nM

0.020.5{0

2 4 6 8 10 12 14Bound (pM)

0.01 3 5

GLP-1 (nM)

FIG. 1. Binding of 1251-labeled GLP-1 to COS cells transfectedwith plasmid pGLPR-16 (A) and to INS-1 cells (B). Specific bindingreaches saturation at 1-10 nM GLP-1. (Insets) Scatchard analysis ofGLP-1 binding. B/F, bound/free.

1 400)C

1 20C

D0100

- 80

_- 600

C 40

*f 20

0

I O-1I1 1 0- 9 I ° 7 1 o-S

Peptide concentration (M)

FIG. 2. Displacement of 125I-labeled GLP-1 binding to trans-fected COS cells. Transfected cells were incubated with 20 pM125I-labeled GLP-1 and increasing concentrations of unlabeled pep-tides. Each point was measured in duplicate, and the experimentswere repeated three times with similar results for GLP-1, GIP, andglucagon and once for VIP and secretin.

detected. The binding to GLP-1 receptor-transfected COScells reached a plateau between 1 and 10 nM, concentrationsat which no binding could be detected on vector-transfectedCOS cells. At concentrations >10 nM a second, high-capacity, low-affinity binding component was detected. Al-though specifically displaceable by unlabeled GLP-1, thisbinding was also present in COS cells transfected with theexpression vector alone and was therefore not further char-acterized.

Binding of GLP-1 to the receptor expressed in COS cellswas displaced by unlabeled GLP-1 with a 50%o displacementachieved at 0.5-1 nM (Fig. 2). Other peptide hormones ofrelated structure and similar function such as secretin, GIP,and vasoactive intestinal peptide (VIP) (1) did not displacebinding. Glucagon could displace the binding by 50o but onlyat a concentration of 1 ,.M (Fig. 2). The addition ofGLP-1 totransfected COS cells stimulated the production of cAMP,indicating that the receptor was functionally coupled toactivation of adenylate cyclase (Fig. 3).The complete 1.6-kb cDNA clone as well as parts of the

1.8- and 3.0-kb cDNAs were sequenced (Fig. 4). Relative tothe presented nucleotide sequence, clones pGLPR-1,

1 5 -

0T-1 0o

E

50

4U

0 -

M pGLPR-1-transfectedcells

RL pcDNA-l-transfected cells

I~~mm0 0.1

GLP-1 (nM)1

FIG. 3. Stimulation ofcAMP formation in transfected COS cells.COS cells were transfected with the pcDNA-1 vector alone (o) or thepGLPR-1 plasmid (m) and incubated in the absence or the presenceof GLP-1 at the indicated concentration. cAMP production wasmeasured in triplicate with a radioimmunoassay.

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Cell Biology: Thorens Proc. Natl. Acad. Sci. USA 89 (1992) 8643

1 TCCTCkCCCGC&CATGCCGCCCCCASCTCG=GCCCCTGCTGCTG=MC-CCCGCTTCCCA:GGGGTCCAGGTCCCTCTC WhA12 1 _ TCACGAAGCGXCC C -AlAGGTCTCTTCTCAGAACC LNlTWTAK241 GCCCCCATC CTG;CCCA361 CCAOCCTGCTTCC TGTC1TAC AC YTC481 m C TGC TG CCAA LCGTCT601 _G =TGC=C I =CCTOiCA!GCAAS721 C CTC841 AGC I I CTCG =1 _CGACTTGCCCATTCTC961 T C ATCATGTTMTCA!C TCCTGAC

1081 XTCCGCTTC _SCDCC_T CA CGACTATC TCATC G1201 C C _TTTTGTGAB;T. ACATC1321 CC _ __CAOpC CTTGC1441 TOCTOC _1561 TlCCTCC TGCC G -ACCTCTAATAATC1681 MlClR CTTCTTGGCAAGGCAG C CAACCCCGGATC1801 TCTCGCAAT TGG C CAlGG TGG OCCCLI=]Y AGCC:ATCsS1921_lTTC =LTCCTCCTGM2041 C_ _ _TTCTGG A l CAC2161 TTCTTGCCTTTAACmMTGClUTCTUUl~lXCCG=TACTCCCA _lTAeGAACTCAlnl GTAA2281 A AAGTCTAGACAY C:CAGUGT :MMTC2401 _TCAT C ATTACTC TGCCC T CATCTCC CACATT2521 T ACCTTTATC TC C ACTACACACACCGMI2641 CCCTCTCCCCCACC TGCCCACCT CCAaC T------TTCCTCC2761 AC TCCGATATAGCTC TGTCA2881 ATCCTAGC_ _3 _ _ GCITCTC ATGAGCTTATCTCTCCCTCATCCCGTGAJTGGACCTCCTAGTSTCACA3001CTWACW_ 3066

FIG. 4. Nucleotide sequence of the GLP-1 receptor. The nucleotide sequence from position 1 to position 1546 was obtained from clonepGLPR-1 and from position 1399 to position 3066 from clone pGLPR-16. Positions of the translation initiation and termination codons areunderlined.

pGLPR-16, and pGLPR-87 started at positions 1, 6, and 15, nucleotides present at the 3' end of pGLPR-87 (beyondrespectively. They ended at positions 1546 (pGLPR-1), 1760 position 1760), which was not found in the longest clone(pGLPR-87), and 3066 (pGLPR-16). No differences in nucle- sequenced. These nucleotides may represent a unique se-otide sequence were observed between the three different quence present in the smaller 2.7-kb mRNA generated byclones. The only divergence found was a stretch of 25 differential splicing in the 3' untranslated region or may

GLPR M1AUTP.SLLR-----LRLLLLGAUGRRGPRPQGA-------------------TUSLSETUQKUREYRHQCQRFLTE--------- 52SECR flLSTIRPR-LSLLL-----LRLLLLTKRAHTU----GU-------------------PPRLCDURRULLEERAHCLQQLSKEK------- S.PTHR 11GRPR ISHSLRLLLCCSULSSUYVRLUOlDDU ITKEEQI ILLRHAQAQCEQELKEULRUPELAESRKDUfSRSRKTKKEKPREKLYPQAEE 90CTR1 llRFTLTRUCLTLFIFLNRPLPULPDSADGAHTPTLEPEPFLYY----------- ILGKOQR .L-------LEAQHRC---------Y---D 58

GLPR - APLLATGLF qRTFDDYVAPDGPPGSFUNU UYLPURSSULQGHUYRF' AEGIULHKDHSSLPURDLSECEESKQGERNSP 137SECR -KGALGPETRSG- C :GLUDN11ShJPSSAPARTUEU CKSLLSLSNK-NGSLFRH QDOG-U----SETFPRPOLACGUHIHNNSFHERR 135PTHR SRUDSLD .)PEUDHIU PAGUPGK~UUAU Ij'YFYDFNHH--GRAYRR:i1SHGSUELUPG11HRTUAHYSECUKFLTHETRER 178

* .* .V. * . .. ..*e * ..**e. ...

GLPR EEQLLSLYI IYTUGYALSFSALUIASAILUSFRHLHCTRNYIHLHLFRSFILRRLSUFIKDAALKUflYSTARQQHQUDG-LLSY--QDS- 223SECR HAYLLKLKUnlYTUGVSSSLAR1LLUALS ILCSFRRLHCTRHYIH11HLFUSFILRRLSNF IKDAUL---FSSDD---------UTYCOAHK- 212PTHR EU-FDRLGflIYTUGVSISLGSLTUAULILGYFRRLHCTRHYIHnHLFUSFflLRRUSIFIKDAULYSGUSTOEIERITEEELRAFTEPPPA 267CTRIlNAYI--LVYLAIUGHSLSILTLLISLGIF1FLRSISCQRUTLHKNtFLTYULHSI II IUHLUUI -----------UPHGELUK-RDPPI- 219

* .**.. *. * . *...* ** .* *. ..*. . .

III lUGLPR -----LGCRLUFLLnQYCUAANYYULLUEGUYLYTLLAFSUFSEQR IFKLYLS IGUGUPLLFU IPUG IUKYLYEDEGCUTRHSHN11NYUL 308SECR -----UGCKLUW IFFQYCIn1ANYAULLUEGLYLHTLLRISFFSERKYLQAFULLGUGSPA IFUALRUA ITRHFLENTGCUD INHARNSUUUU 297PTHR DKRGFUGCRUAUTUFLYFLTTIYYU I LUEGLYLHSL I F1AFFSEKKYLUGFTLFGUGLPRUFURUUUTURATLANTECUDLSSGNKKU- 356CTR1 -------CKULHFFHQYlnfSCIYFUnLCEGUYLHTL IUUSUFAEGQRLUUYHULGUGFPL IPTTAHR ITRRNLFHNDCU-LSUDTNLLY I 301

.. * ** *.* **.**. .*. . *.* . . .*** *.*....*U Ul

GLPR IRLP ILFAIGUNFLUFIRU ICIUIARKLKAflNCKTD IKC---RLRKSTLTL IPLLGTHEU IFAFUnDEHARGTLRFUKLFTELSFTSFQG 395SECR IRGPUILSILINFIFFINILRILnRKLRTQETRGSEThH-VKRLRKSTLLLIPLFGIHYIUFAFSHEDRnE-----UQLFFELALGSFQG 381PTHR IQUPILAAIUUIfFILFINI I RULATKLRETNAGRCDTRQQYRKLLKSTLULlPLFGUHYIUFlATPYTEUSG ILUQUQ11HYEn1LFHSFQG 416CTR1 IHGPUn1ARLUUHFFFLLNILRULUKKLKESQE---RESHllVLKAURATLILUPLLGUQFUULPURPSTPLLGK IYD---YUUHSL IHFQG 385

*. * ...*....** . .. ....** *.**.*...........***

UlIGLPR fl1UAULYCFUNNEUQ llEFRKSUERURLE-RLHIQRDSSllKPLKC1---------------------------------------------- 38SECR LUUAULYCFLNGEUQLEUQKKURQUHLQ-EFPLRPUAFHRSFSH ---------------------------------------------- 121PTHR FFUAI IYCFCHGEUQAEIKKSUSRUTLRLDFKRKARSGSSTYSYGPn1USHTSUTNUGPRGGLALSLSPRLAPGRGASANGHHQLPGYUKH 536CTRI FFURIIYCFCNHEUQGRLKRQUNQ------YQAQRUAGRRS----TRRARNARATAAAARALRETU---------------EIPUYICH 119

**..*** **** *

GLPR --------PTSSUSSGATU-----GSSUYAATC-----------QHSCS 163SECR --------ATNGPTHSTKR-----STEQSRSIPA-----------RRSII 119PTHR GS I SEHSLPSSGPEPGTKDDGYLHGSGLYEPRUGEQPPPLLEEERETUR 585CTRl QEPREEP---RGEEPUUEUEG-----------UEUIRlEULEQE--TSA 182

FIG. 5. Comparison of the amino acid sequence of the GLP-1 receptor (GLPR) with the sequence of the secretin receptor (SECR),parathyroid hormone receptor (PITHR), and calcitonin receptor (CTR1). GLP-1 receptor has three N-glycosylation sites in the extracellulardomain (arrowheads) following the initial leader sequence. Four cysteines are conserved at identical places in the four receptors (boxes). Notethe otherwise very divergent sequence in this part of each molecule as well as in the carboxyl-terminal cytoplasmic tail. Sequence identitiesare denoted by asterisks and homologies are indicated by dots. Locations of the transmembrane domains are indicated by horizontal bars abovethe sequences.

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originate from a cloning artefact. Sequence analysis revealeda major open reading frame coding for a 463-amino acidpolypeptide (Fig. 5). Hydropathy plot analysis indicated thepresence of an amino-terminal hydrophobic region mostprobably representing a leader sequence. This hydrophobicsegment is followed by a hydrophilic domain of about 120amino acids, which contains three N-linked glycosylationsites. Seven hydrophobic segments are present, which mayform transmembrane domains. Search for sequence identitiesshowed the GLP-1 receptor to be homologous to the secretinreceptor (29) (40% identity), the parathyroid hormone recep-tor (30) (32.4% identity), and the calcitonin receptor (31)(27.5% identity) (Fig. 5). These four receptors do not shareany significant sequence homology with the other membersof the G-coupled receptor family (29-31). The sequence ofthe extracellular domain is unique to each receptor; fourcysteines are, however, perfectly conserved (boxes in Fig. 5).A fifth cysteine at position 126 of the GLP-1 receptor is alsoconserved in the parathyroid and calcitonin receptors and ata similar location in the secretin receptor (position 123). Thehighest sequence identity between the four proteins resides inthe transmembrane domains. The carboxyl-terminal, cyto-plasmic ends of each receptor are also very different.These receptors all stimulate the production of cAMP in

response to ligand binding (29-31) and are presumably cou-pled to the cyclase through the a subunit of stimulatory Gprotein (Gsa). In this respect, it is interesting to note that asequence motif present in the third cytoplasmic loop of theGLP-1 receptors (RLAK, present just before the sixth trans-membrane domain) is very similar to a motif of the P2-adrenergic receptor (KALK) present at the same location andwhose basic amino acids have been shown to be important inthe coupling of the receptor to Ga (32). Moreover, in the032-adrenergic receptor, this motif is preceded by a basicamino acid located 12 amino acids toward the amino-terminalend. This basic amino acid is also required at this particulardistance for efficient coupling to Gsa. In the GLP-1 receptora lysine residue is also present at a similar location. Thissuggests that, despite the very low overall sequence identity,a structural feature may have been conserved in the thirdcytoplasmic loop between the two receptors, which, one mayspeculate, may be required for the coupling of receptor to theGsa protein.

Determination of the tissue distribution of the GLP-1receptor was performed by Northern blot analysis. TwomRNAs of 2.7 and 3.6 kb could be detected in pancreaticislets as well as in a rat insulinoma cell line (INS-1), instomach, and in lung (Fig. 6). The difference in the structureof the two mRNAs is not known. However, the differencebetween the different clones lies in the length of the 3'untranslated region, suggesting that different poly(A) sitesmay be responsible for the different sizes of the mRNAs. NoGLP-1 receptor mRNA could be detected in brain, liver,thymus, muscle, intestine, and colon. The presence of theGLP-1 receptor has been reported in stomach, where thepeptide inhibits acid secretion by parietal cells in in vivoexperiments (33) but stimulates acid secretion on isolatedparietal glands (34). Binding sites for GLP-1 have also beenreported in lung membrane preparations (35) but the role ofthe hormone in lung physiology is not known.The characterization of the GLP-1 receptor is of consid-

erable physiological and pathological importance because itwill help study a fundamental aspect ofthe entero-insular axis(36): the potentiating effect of gut hormones on glucose-induced insulin secretion and their role in the control ofglucose homeostasis. Investigation of the regulated expres-sion and desensitization of the receptor in the normal stateand during the development of diabetes will contribute to abetter understanding of the modulation of insulin secretion innormal and pathological situations. Availability of the recep-

I I I I

- 28S

lose

4 1454 dwi s

FIG. 6. Tissue distribution of the GLP-1 receptor assessed byNorthern blotting ofRNA from different tissues and from the INS-1cell line. Ten micrograms of total RNA was analyzed on each lane.Two major RNA species of 2.7 and 3.6 kb were detected. Positionsof the migration of the rRNAs are indicated.

tor in a purified form may also aid in the design of newderivatives of GLP-1 that could be used to stimulate insulinsecretion by 13 cells from NIDDM patients (13). Also, anti-bodies against this receptor may permit the analysis of thesurface localization of this receptor and its distribution rel-ative to the 13 cell glucose transporter GLUT2 (37, 38). Thiswill permit testing ofthe hypothesis that the (3 cell membranehas a "regulatory" domain that contains hormone receptors(39) and that may be distinct from GLUT2-containing mem-brane domains previously identified (38). Finally, identifica-tion of an additional member of this family of G-coupledreceptors will facilitate the design ofexperiments to probe thestructure-function relationship of these new molecules.

I thank M. Asfari (H6pital Saint-Antoine, Paris) for the INS-1 cellline and E. Grouzman (University Hospital, Lausanne) for determi-nation of cAMP levels. Communication of the sequences for thecalcitonin and PTH receptors prior to publication by Dr. H. F.Lodish and H. Lin is gratefully acknowledged. I also thank Dr. B.Rossier for his support and C. Rapin for technical assistance. Thiswork was supported by Grant 31-30313.90 from the Swiss NationalScience Fundation. The initial part of this work was performed in Dr.H. F. Lodish's laboratory at the Whitehead Institute for BiomedicalResearch.

1. Dupre, J. (1991) in The Endocrine Pancreas, ed. Samols, E.(Raven, New York), pp. 253-281.

2. Ebert, R. & Creutzfeld, W. (1987) Diabetes Metab. Rev. 3,1-26.

3. Kreymann, B., Williams, G., Ghatei, M. A. & Bloom, S. R.(1987) Lancet i1, 1300-1304.

4. Bell, G. I., Sanchez-Pescadore, R., Laybourn, P. L. & Najar-ian, R. C. (1983) Nature (London) 304, 368-371.

5. Mojsov, S., Heinrich, G., Wilson, I. B., Ravazzola, M., Orci,L. & Habener, J. F. (1986) J. Biol. Chem. 261, 11880-11889.

6. Habener, J. F., Drucker, D. J., Mojsov, S., Knepel, W. &Philippe, J. (1991) in The Endocrine Pancreas, ed. Samols, E.(Raven, New York), pp. 53-71.

7. Mojsov, S., Weir, G. C. & Habener, J. F. (1987) J. Clin. Invest.79, 616-619.

8. Weir, G. C., Mojsov, S., Hendrick, G. K. & Habener, J. F.(1989) Diabetes 38, 338-342.

9. Drucker, D. J., Philippe, J., Mojsov, S., Chick, W. L. &Habener, J. F. (1987) Proc. Nat!. Acad. Sci. USA 84, 3434-3438.

10. Goke, R., Trautman, M. E., Haus, E., Richter, G., Fehman,H.-C., Arnold, R. & G6ke, B. (1989) Am. J. Physiol. 257,G397-G401.

11. Suzuki, S., Kawai, K., Ohashi, S., Mukai, H., Murayama, Y.& Yamashita, K. (1990) Diabetes 39, 1320-1325.

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12. Orskov, C., Jeppesen, J., Madsbad, S. & Holst, J. (1991) J.Clin. Invest. 87, 415-423.

13. Nathan, D. M., Schreiber, E., Fogel, H., Mojsov, S. &Habener, J. F. (1992) Diabetes Care 15, 270-276.

14. Contreras, M. A., Bale, W. F. & Spar, I. L. (1983) MethodsEnzymol. 92, 277-292.

15. Calvo, J. C., Radicella, J. P. & Charreau, E. H. (1983) Bio-chem. J. 212, 259-264.

16. McPherson, G. A. (1985) Kinetic, EBDA, Ligand, Lowry: ACollection of Radioligand Analysis Programs (Elsevier,Amsterdam).

17. Gotoh, M., Maki, T., Kiyoizumi, T., Satomi, S. & Monaco,A. P. (1985) Transplantation 43, 725-730.

18. Aruffo, A. & Seed, B. (1987) Proc. Natl. Acad. Sci. USA 84,8573-8577.

19. Lin, H. Y., Kaji, E. H., Winkel, G. K., Ives, H. E. & Lodish,H. F. (1991) Proc. Natl. Acad. Sci. USA 88, 3185-3189.

20. Gubler, U. & Hoffman, B. J. (1983) Gene 25, 263-269.21. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular

Cloning:A Laboratory Manual (Cold Spring Harbor Lab., ColdSpring Harbor, NY).

22. Sompayrac, L. M. & Dana, K. J. (1981) Proc. Natd. Acad. Sci.USA 78, 7575-7578.

23. Gearing, D. P., King, J. A., Gough, N. M. & Nicolas, N. A.(1989) EMBO J. 8, 3667-3676.

24. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 126,156-159.

25. McMaster, G. K. & Carmichael, G. G. (1977) Proc. Natl.Acad. Sci. USA 74, 4835-4838.

26. Thomas, P. (1980) Proc. Natl. Acad. Sci. USA 77, 5201-5205.

27. Feinberg, A. P. & Vogelstein, B. (1983) Anal. Biochem. 132,6-13.

28. Asfari, M., Janjic, D., Meda, P., Li, G., Halban, P. A. &Wollheim, C. B. (1992) Endocrinology 130, 167-178.

29. Ishihara, T., Nakamura, S., Kaziro, Y., Takahashii, T., Taka-hashi, K. & Nagata, S. (1991) EMBO J. 10, 1635-1641.

30. Jdppner, H., Abou-Samra, A.-B., Freeman, M., Kong, X. F.,Schipiani, E., Richards, J., Kolakowski, L. F., Hock, J., Potts,J. T., Kronenberg, H. M. & Segre, G. V. (1991) Science 254,1024-1026.

31. Lin, H. Y., Harris, T. L., Flannery, M. S., Aruffo, A., Kaji,E. H., Gorn, A., Kolakowski, L. F., Lodish, H. F. & Gold-ring, S. R. (1991) Science 254, 1022-1024.

32. Okamoto, T., Murayama, Y., Hayashi, Y., Inagaki, M., Ogata,E. & Nishimoto, I. (1991) Cell 67, 723-730.

33. Schjoldager, B. T. G., Mortensen, P. E., Christiansen, J., Or-skov, C. & Holst, J. J. (1989) Dig. Dis. Sci. 34, 703-708.

34. Schmidtler, J., Schepp, W., Janczewska, I., Weigert, N.,Fuirlingwer, C., Schusdziarra, V. & Classen, M. (1991) Am. J.Physiol. 260, G940-G950.

35. Richter, G., Goke, R., Goke, B. & Arnold, R. (1990) FEBSLett. 267, 78-80.

36. Unger, R. H. & Eisentraut, A. M. (1969) Arch. Int. Med. 123,261-266.

37. Thorens, B., Sarkar, H. K., Kaback, H. R. & Lodish, H. F.(1988) Cell 55, 281-290.

38. Orci, L., Thorens, B., Ravazzola, M. & Lodish, H. F. (1989)Science 245, 295-297.

39. Bonner-Weir, S. (1988) Diabetes 37, 616-621.

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