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Glucagon·Glucagon-like Peptide I Receptor Chimeras Reveal Domains That Determine Specificity of Glucagon Binding

1995; Elsevier BV; Volume: 270; Issue: 13 Linguagem: Inglês

10.1074/jbc.270.13.7474

1083-351X

Joseph J. Buggy, James N. Livingston, Daniel U. Rabin, H Yoo-Warren,

Receptor Mechanisms and Signaling

The binding of glucagon to its hepatic receptor triggers a G-protein-mediated signal that ultimately leads to an increase in hepatic glucose production (gluconeogenesis) and glycogen breakdown (glycogenolysis). In order to elucidate the structural domain(s) of the human glucagon receptor (hGR) involved in the selective binding of glucagon, a series of chimeras was constructed in which various domains of the hGR were replaced by homologous regions from the receptor for the glucoincretin hormone, glucagon-like peptide I (GLP-IR). hGR and GLP-IR are quite similar (47% amino acid identity) yet have readily distinguishable ligand binding characteristics; glucagon binds to the recombinant hGR expressed in COS-7 cells with a Kd that is 1000-fold lower than the Kd for glucagon binding to GLP-IR. In the present study, chimeric receptors were transiently expressed in COS-7 cells and analyzed for glucagon binding. Expression of each receptor chimera was confirmed by immunofluorescence staining using a hGR-specific monoclonal antibody. This report identifies several non-contiguous domains of the hGR that are important for high affinity glucagon binding. Most notable are the membrane-proximal half of the amino-terminal extension, the first extracellular loop, and the third, fourth, and sixth transmembrane domains. The binding of glucagon to its hepatic receptor triggers a G-protein-mediated signal that ultimately leads to an increase in hepatic glucose production (gluconeogenesis) and glycogen breakdown (glycogenolysis). In order to elucidate the structural domain(s) of the human glucagon receptor (hGR) involved in the selective binding of glucagon, a series of chimeras was constructed in which various domains of the hGR were replaced by homologous regions from the receptor for the glucoincretin hormone, glucagon-like peptide I (GLP-IR). hGR and GLP-IR are quite similar (47% amino acid identity) yet have readily distinguishable ligand binding characteristics; glucagon binds to the recombinant hGR expressed in COS-7 cells with a Kd that is 1000-fold lower than the Kd for glucagon binding to GLP-IR. In the present study, chimeric receptors were transiently expressed in COS-7 cells and analyzed for glucagon binding. Expression of each receptor chimera was confirmed by immunofluorescence staining using a hGR-specific monoclonal antibody. This report identifies several non-contiguous domains of the hGR that are important for high affinity glucagon binding. Most notable are the membrane-proximal half of the amino-terminal extension, the first extracellular loop, and the third, fourth, and sixth transmembrane domains. INTRODUCTIONGlucagon is a 29-amino acid polypeptide hormone that is secreted by the A cells of the islets of Langerhans and plays an important role in the regulation of nutrient homeostasis(1Unger R.H. Orci L. Diabetes Mellitus Theory and Practice. Elsevier Science Publishing Co., Inc., New York1990Google Scholar). The first step in glucagon action is the binding of the hormone to the glucagon receptor (GR) 1The abbreviations used are: GRglucagon receptorGLP-Iglucagon-like peptide IhGRhuman glucagon receptorhGLP-IRhuman islet GLP-I receptorGPCRG-protein coupled receptorGLP-IRglucagon-like peptide I receptorTMDtransmembrane domain. on the surface of liver cells (2Unger R.H. Foster D. Wilson J.D. Foster D.W. Williams‘ Textbook of Endocrinology. W. B. Saunders Co., Philadelphia1992: 1255-1333Google Scholar). Upon glucagon binding, the receptor interacts with the membrane-associated G-protein subunit Gs to ultimately stimulate adenylyl cyclase activity, leading to an increase in intracellular cAMP. Elevation of this second messenger ultimately leads to an increase in gluconeogenesis and glycogenolysis(3Smith R.J. DeGroot L.J. Endocrinology. W. B. Saunders Co., Philadelphia1989: 1333-1345Google Scholar).The glucoincretin hormone known as glucagon-like peptide-I (GLP-I) is secreted in the gut and potentiates the glucose-induced secretion of insulin by the islets of Langerhans(4Dupre J. Samols E. The Endocrine Pancreas. Raven Press, Ltd., New York1991: 253-281Google Scholar, 5Ebert R. Creutzfeld W. Diabetes Metab. Rev. 1987; 3: 1-26Crossref PubMed Scopus (78) Google Scholar). Like glucagon, GLP-I exerts its effects by binding to its cognate receptor generating a rise in intracellular cAMP concentration(6Thorens B. Porret A. Buhler L. Deng S.P. Morel P. Widmannj C. Diabetes. 1992; 42: 1678-1682Crossref Scopus (488) Google Scholar). Both hormones are of considerable importance in the control of blood glucose homeostasis.The cDNAs encoding both the human glucagon receptor (hGR) and the human islet GLP-I receptor (hGLP-IR) have been recently cloned(6Thorens B. Porret A. Buhler L. Deng S.P. Morel P. Widmannj C. Diabetes. 1992; 42: 1678-1682Crossref Scopus (488) Google Scholar, 7Lok S. Kuijper J.L. Jelinek L.J. Kramer J.M. Whitmore T.E. Sprecher C.A. Mathewes S. Grant F.J. Biggs S.H. Rosenberg G.B. Sheppard P.O. O'Hara P.J. Foster D.C. Kindsvogel W.K. Gene (Amst.). 1994; 140: 203-209Crossref PubMed Scopus (117) Google Scholar). Both polypeptides show sequence similarity to a well established family of receptors with seven-transmembrane domains that are known to couple to intracellular effectors through heterotrimeric guanine nucleotide binding proteins (G proteins)(8Dohlman H.G. Thorner J. Caron M.G. Lefkowitz R.J. Annu. Rev. Biochem. 1991; 60: 653-688Crossref PubMed Scopus (1130) Google Scholar). All members of this family of G-protein coupled receptors (GPCRs) share the same basic structure and membrane topology. Within the large family of GPCRs are grouped subfamilies based on primary sequence homology(9Strader C.D. Fong T.M. Tota M.R. Underwood D. Annu. Rev. Biochem. 1994; 63: 101-132Crossref PubMed Scopus (991) Google Scholar). The glucagon and GLP-I peptide receptors are grouped into a subfamily that includes the receptors for secretin, calcitonin, vasoactive intestinal peptide, parathyroid hormone, and growth hormone-releasing factor(9Strader C.D. Fong T.M. Tota M.R. Underwood D. Annu. Rev. Biochem. 1994; 63: 101-132Crossref PubMed Scopus (991) Google Scholar). The hGR and hGLP-IR share 47% amino acid identity and 66% similarity, with the transmembrane segments having the highest degree of similarity (see Fig. 1). Despite this homology, glucagon binds to the hGR with a Kd approximately 1000-fold lower than to the hGLP-IR.Despite a large body of information regarding the ligand binding and signaling characteristics of GPCRs in general, there has been very little structure-function analysis reported about the glucagon-secretin subfamily. In order to understand the nature of the interaction of glucagon with its receptor, we constructed a number of hGR•hGLP-IR chimeras in which the various domains of the hGR were sequentially replaced by homologous domains from hGLP-IR. Since both receptors are highly conserved throughout their entire length, it was possible to swap domains without introducing any gaps or deletions in the primary amino acid sequence. We report here the identification of several, non-continuous domains that are required for high affinity glucagon binding.EXPERIMENTAL PROCEDURESMaterialsThe cDNA that encodes the hGR was identified using the previously described rat GR cDNA (10Jelinek L.J. Lok S. Rosenberg G.B. Smith R.A. Grant F.J. Biggs S. Bensch P.A. Kuijper J.L. Sheppard P.O. Sprecher C.A. O'Hara P.J. Foster D. Walker K.M. Chen L.H.J. McKernan P.A. Kindsvogel W. Science. 1993; 259: 1614-1616Crossref PubMed Scopus (365) Google Scholar) to probe a human liver cDNA library (Clontech). A clone of approximately 1.7 kilobases, consisting of 70 bases of the 5′-untranslated region, the full coding sequence, and 230 bases of 3′-untranslated segment was obtained. Nucleotide sequence of the clone was identical to the corresponding portion of the published hGR (GenBank accession number L20316(7Lok S. Kuijper J.L. Jelinek L.J. Kramer J.M. Whitmore T.E. Sprecher C.A. Mathewes S. Grant F.J. Biggs S.H. Rosenberg G.B. Sheppard P.O. O'Hara P.J. Foster D.C. Kindsvogel W.K. Gene (Amst.). 1994; 140: 203-209Crossref PubMed Scopus (117) Google Scholar)). Isolation of the hGLP-IR cDNA was described previously(11Dillon J.S. Tanizawa Y. Wheeler M.B. Leng X.H. Ligon B.B. Rabin C.U. Yoo-Warren H. Permutt M.A. Boyd A.E. Endocrinology. 1993; 133: 1907-1910Crossref PubMed Scopus (104) Google Scholar).Restriction endonucleases were from Boehringer Mannheim. Polymerase chain reaction reagents were from Invitrogen Corp. and New England Biolabs (Vent polymerase). 125I-Labeled glucagon was obtained from DuPont, and unlabeled glucagon was purchased from Sigma. COS-7 cells were obtained from ATCC.Construction and Expression of ChimerasThe cDNAs encoding the wild type hGR and hGLP-IR were subcloned into an expression vector, pcDNA3 (Invitrogen). Chimeras were constructed using a polymerase chain reaction strand overlap extension (12Horton R.M. Cai Z. Ho S.N. Pease L.R. BioTechniques. 1990; 8: 528PubMed Google Scholar) technique using Vent DNA polymerase (New England Biolabs). The chimeras were engineered precisely through the use of specific oligonucleotide primers (Midland certified reagent company). The region surrounding the chimera splice junctions was subcloned as a cassette into either the hGR or hGLP-IR backbone in vector pcDNA3. The constructions were verified by restriction endonuclease mapping and sequence analysis.The wild type, mutant, and chimeric receptors were expressed transiently by DEAE-mediated transfection of COS-7 cells(13Sambrook J. Fritsch E.F. Maniatus T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar).Glucagon Binding AnalysisBinding assays were performed in duplicate or triplicate on at least two separate occasions on whole cells grown in a 24-well chamber dish. Approximately 105 cells were incubated in 50 mM HEPES (pH 7.3), 150 mM NaCl, 1 mM EDTA, 1.0% bovine serum albumin (Sigma), and 1 mg/ml bacitracin (Aldrich) in a 200-μl volume. Binding was carried out for 90 min at 4°C in the presence of 150,000 cpm of 125I-labeled glucagon and various amounts of unlabeled ligand. Nonspecific binding was determined as total counts bound to COS-7 cells transfected with vector pcDNA3. Bound tracer ligand was determined using an automatic γ counter (Wallac model 1470 Wizard).Immunofluorescence AssaysCells were grown on a 4-well glass chamber slide (Nunc). Cells were blocked with a solution of RPMI 1640 medium (JRH Biosciences), 1.0% bovine serum albumin (Sigma) for 1 h at 25°C. The cells were then incubated in fresh blocking medium containing 10 μg of mouse monoclonal anti-human GR antibody for 1 h at 4°C. This antibody was raised by immunizing mice with Chinese hamster ovary cells stably transfected with hGR. The antibody has been shown to specifically block glucagon binding to hGR (data not shown; characterization of this monoclonal antibody will be reported separately). Cells were washed with blocking solution, and fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Sigma) was added in blocking buffer at 4°C for 1 h. Cells were washed, coverslipped, and viewed by fluorescence microscopy (Leitz Diaplan system).RESULTSLigand Binding Properties of Chimeric ReceptorsTen chimeric hGR•hGLP-IR genes were constructed (Fig. 2). The recombinant chimeras were transiently expressed in COS-7 cells and analyzed for ligand binding. The affinity for 125I-labeled glucagon was ascertained using an equilibrium whole cell binding assay (Fig. 3 and Table 1). With few exceptions, all of the chimeras reported here segregate neatly into two categories: 1) they bind glucagon with wild-type affinity, or 2) they bind glucagon with an affinity similar to that of glucagon binding to GLP-IR.Figure 2Schematic representation of the hGR•hGLP-IR chimeras. Amino acids indicated below each chimera refer to the residues of the hGR present in each construct. Dark line, hGR; gray line, hGLP-IR. Numbering corresponds to that used in Fig. 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Competition curves of glucagon binding to chimeric receptors. 125I-Labeled glucagon was used as a radioligand tracer to analyze the binding of glucagon to various chimeras present in transfected COS-7 cells. Shown is percent maximum binding versus nM unlabeled glucagon for each construct. A, competition curves comparing hGR (○), GLP-IR (•), CH-1 (□), CH-2 (■), CH-3 (×), CH-4 (▵), and CH-5 (▲). B, competition curves comparing hGR (○), GLP-IR (•]), CH-6 (□), and CH-7 (▿). C, competition curves comparing hGR (○), GLP-IR (•), CH-8 (▲), CH-9 (■), and CH-10 (▵). Each point is the average of triplicate measures. Standard error in each case is within ±6%.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Tabled 1View Large Image Figure ViewerDownload Hi-res image Download (PPT) Open table in a new tab Replacement of more than half of the amino-terminal extension (chimera CH-1) with the homologous region from hGLP-IR had little or no effect on ligand binding. However, replacement of the entire amino-terminal extension and the first intracellular loop, including transmembrane domains (TMDs) I and II (chimera CH-2) abolishes high affinity binding (Fig. 3A). Furthermore, chimera CH-3, in which the first intracellular loop plus TMDs I and II are replaced, yields a construct with a high binding affinity. Taken together, binding data from CH-1, CH-2, and CH-3 suggest that the membrane-proximal region of the amino-terminal extension is in fact important for glucagon binding or recognition, whereas the membrane-distal half of the amino-terminal extension, the first intracellular loop, and TMD I and II are not involved in a major way.Substitution of the first extracellular loop plus TMDs III and IV (chimera CH-4) yields a construct that has no high affinity glucagon binding activity (Fig. 3A). However, chimera CH-5, in which the majority of the second intracellular loop was replaced, binds glucagon with high affinity. Data from CH-4 and CH-5 together suggest that the first extracellular loop and TMDs III and IV are involved in glucagon-specific binding, but the second intracellular loop is not.In order to determine whether the domains surrounding the second extracellular loop are important, CH-6 and CH-7 were constructed. CH-6, in which TMD V plus the third intracellular loop have been replaced, binds glucagon with high affinity, as does CH-7 in which the second extracellular loop has also been switched (Fig. 3B). These results indicate that the second extracellular loop, TMD V, and the third intracellular loop are not involved in high affinity glucagon binding.CH-8, CH-9, and CH-10 were designed to address the role of the carboxyl-terminal region of the receptor in ligand binding. Chimera CH-8 is able to bind glucagon with high affinity, which indicates that TMD VII plus the entire intracellular carboxyl-terminal tail is not involved in ligand selection (Fig. 3C). CH-10, in which the third extracellular loop has effectively been converted into GLP-IR, binds glucagon but with somewhat reduced affinity relative to the wild-type receptor, even though this chimera differs in only two adjacent amino acids: Ser-379 to Phe and Ala-380 to Ile. CH-9, however, binds glucagon with an affinity comparable with that of the GLP-IR. These data indicate that TMD VI is important for glucagon binding.In summary, the regions which are not involved in the selective binding of glucagon include the membrane-distal half of the amino-terminal extension, all three intracellular loops, the second extracellular loop, TMDs I, II, V, and VII, and the entire carboxyl-terminal tail. The first and third extracellular loops appear to be involved, as do TMDs III, IV, and VI, as well as the membrane-proximal half of the amino-terminal extension. These results are summarized under “Discussion” and in Fig. 5.Figure 5Regions of the hGR implicated in high affinity glucagon-specific binding. The composite diagram indicates regions of the receptor that are involved in the specificity of glucagon binding. White circles, individual residues that are not involved in the selective binding of glucagon or amino acid residues that are identical between the two receptors. Black circles, residues that have been shown to be required for glucagon binding or non-identical residues between the two receptors.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Receptor ExpressionTo confirm that chimeric receptors that fail to bind glucagon are expressed on the cell surface, we used a hGR-specific monoclonal antibody to assay expression by direct immunofluorescence. Fig. 4 shows the immunofluorescence staining patterns of COS-7 cells transiently expressing wild-type hGR, CH-2, CH-4, CH-9, and CH-10. The antibody detected the three chimeras and the wild-type receptor in approximately 40% of the total cell population, which is consistent with the known transfection frequency. Cells had a typical pattern of membrane staining.Figure 4Cellular distribution of the hGR chimeras in transfected COS-7 cells. Transfected viable COS-7 cells were immunolabeled with a fluorescent tag (fluorescein isothiocyanate). Left panel, fluorescence staining; right panel, phase-contrast micrographs. In control experiments using only preimmune sera, no staining could be detected. A, hGR; B, CH-2; C, CH-4; D, CH-9; E, CH-10; F, hGLP-IR.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONThe construction of chimeric receptors is a useful starting point for identifying structural domains involved in the selective binding of a ligand to its receptor. The strategy in such experiments is to exchange homologous domains between two similar, yet functionally distinct receptors and correlate gain or loss of function with the exchanged domain. This approach eliminates any gross structural alterations such as altered folding or membrane insertion that can occur with other methods such as substitution or deletion mutagenesis. However, while the design of chimeras is a useful method for identifying amino acid residues involved in ligand-specific selectivity, this approach does not necessarily identify domains that may be important for ligand binding but are common to both receptors. A composite diagram indicating the regions of the hGR that are involved in the selective binding of glucagon is shown in Fig. 5. The residues in Fig. 5 shown as “not involved” in high affinity glucagon-specific binding are determined by two criteria: 1) chimera data and 2) sequence identity between the hGR and GLP-IR.The general picture that has emerged of the peptide ligand binding site on GPCRs implicates the extracellular domains, and less often, a few select transmembrane segments. This is true even of very short peptide ligands such as the tripeptide fMet-Leu-Phe, which binds to the N-formyl peptide receptor via multiple, discontinuous regions, especially in the extracellular domains and near the top of the transmembrane regions(14Quehenberger O. Prossnitz E.R. Cavanagh S.L. Cochrane C.G. Ye R.D. J. Biol. Chem. 1993; 268: 18167-18175Abstract Full Text PDF PubMed Google Scholar). Our structure-function analysis of the hGR indicates that it conforms well to this paradigm. Most GPCRs that bind large peptides utilize residues within the amino-terminal extension(9Strader C.D. Fong T.M. Tota M.R. Underwood D. Annu. Rev. Biochem. 1994; 63: 101-132Crossref PubMed Scopus (991) Google Scholar, 15Fong T.M. Yu H. Huang R.C. Strader C.D. Biochemistry. 1992; 31: 11806-11811Crossref PubMed Scopus (113) Google Scholar). Our findings are consistent with this idea. Specifically, we find that residues between Ser-80 and Gln-142 are necessary for glucagon binding specificity. In addition, a recent study by Carruthers et al.(16Carruthers C.J.L. Unson C.G. Kim H.N. Sakmar T.P. J. Biol. Chem. 1994; 269: 29321-29328Abstract Full Text PDF PubMed Google Scholar) demonstrated by site-directed mutagenesis that a highly conserved aspartic acid residue located within the amino-terminal extension of the rat GR (corresponding to Asp-63 in Fig. 1) is crucial for ligand binding. This conserved aspartic acid residue is probably of general structural importance for the binding of a number of peptide ligands within the family of GPCRs and is not involved in ligand selectivity.Huang et al.(17Huang R.C. Yu H. Strader C.D. Fong T.M. Biochemistry. 1994; 33: 3007-3013Crossref PubMed Scopus (135) Google Scholar) have demonstrated that residues lying within TMD II and VII of the neurokinin-1 receptor are important for the specificity of binding of its peptide ligand, substance P; in contrast, we find that residues within TMD II and TMD VII do not appear to be involved in glucagon-specific binding. A two-amino acid change within extracellular loop 3 of the hGR (CH-11) leads to a slight but significant decrease in glucagon-specific binding, similar to the interleukin-8 receptor, which utilizes two residues within its third extracellular loop in the specific binding of interleukin-8(18Hebert C.A. Chuntharapai A. Smith M. Colby T. Kim J. Horuk R. J. Biol. Chem. 1993; 268: 18549-18553Abstract Full Text PDF PubMed Google Scholar).Early biochemical measurements of the site(s) of glucagon interaction with its hepatic receptor demonstrated that both the glucagon binding function of the receptor as well as the region functionally coupled to adenylyl cyclase are contained within a 21-kDa peptide fragment(19Iyengar R. Herberg J.T. J. Biol. Chem. 1984; 259: 5222-5229Abstract Full Text PDF PubMed Google Scholar). These experiments involved the proteolytic treatment of the receptor following covalent cross-linking with monoiodoglucagon. A subsection of the receptor (approximately the carboxyl-terminal two-thirds of the receptor, including all of the hydrophobic membrane binding regions) was identified as the fragment that binds glucagon. This conclusion is consistent with the data presented here.More recently, effort has been directed toward a structural analysis of glucagon, specifically the residues involved in receptor binding (20Hjorth S.A. Adelhorst K. Pederson B.B. Kirk O. Schwartz T.W. J. Biol. Chem. 1994; 269: 30121-30124Abstract Full Text PDF PubMed Google Scholar, 21Unson C.G. Andreu D. Gurzenda E.M. Merrifield R.B. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4083-4087Crossref PubMed Scopus (92) Google Scholar, 22Unson C.G. Merrifield R.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 454-548Crossref PubMed Scopus (49) Google Scholar). Hjorth et al.(20Hjorth S.A. Adelhorst K. Pederson B.B. Kirk O. Schwartz T.W. J. Biol. Chem. 1994; 269: 30121-30124Abstract Full Text PDF PubMed Google Scholar) studied chimeric glucagon•GLP-I peptide hormones' abilities to bind to the rat GR and GLP-IR. They determined that the epitope involved in the selective recognition of the GR is located near the amino terminus of glucagon, and the GLP-I selective recognition epitope is located in the far carboxyl-terminal end of GLP-I. While such analyses do not predict the receptor domains involved in binding, they provide some information on receptor-ligand interaction. A deeper understanding requires the complementary analysis of the receptor itself. Our future effort will be directed toward a detailed structural characterization of the residues of the receptor involved in glucagon recognition and specific contact. INTRODUCTIONGlucagon is a 29-amino acid polypeptide hormone that is secreted by the A cells of the islets of Langerhans and plays an important role in the regulation of nutrient homeostasis(1Unger R.H. Orci L. Diabetes Mellitus Theory and Practice. Elsevier Science Publishing Co., Inc., New York1990Google Scholar). The first step in glucagon action is the binding of the hormone to the glucagon receptor (GR) 1The abbreviations used are: GRglucagon receptorGLP-Iglucagon-like peptide IhGRhuman glucagon receptorhGLP-IRhuman islet GLP-I receptorGPCRG-protein coupled receptorGLP-IRglucagon-like peptide I receptorTMDtransmembrane domain. on the surface of liver cells (2Unger R.H. Foster D. Wilson J.D. Foster D.W. Williams‘ Textbook of Endocrinology. W. B. Saunders Co., Philadelphia1992: 1255-1333Google Scholar). Upon glucagon binding, the receptor interacts with the membrane-associated G-protein subunit Gs to ultimately stimulate adenylyl cyclase activity, leading to an increase in intracellular cAMP. Elevation of this second messenger ultimately leads to an increase in gluconeogenesis and glycogenolysis(3Smith R.J. DeGroot L.J. Endocrinology. W. B. Saunders Co., Philadelphia1989: 1333-1345Google Scholar).The glucoincretin hormone known as glucagon-like peptide-I (GLP-I) is secreted in the gut and potentiates the glucose-induced secretion of insulin by the islets of Langerhans(4Dupre J. Samols E. The Endocrine Pancreas. Raven Press, Ltd., New York1991: 253-281Google Scholar, 5Ebert R. Creutzfeld W. Diabetes Metab. Rev. 1987; 3: 1-26Crossref PubMed Scopus (78) Google Scholar). Like glucagon, GLP-I exerts its effects by binding to its cognate receptor generating a rise in intracellular cAMP concentration(6Thorens B. Porret A. Buhler L. Deng S.P. Morel P. Widmannj C. Diabetes. 1992; 42: 1678-1682Crossref Scopus (488) Google Scholar). Both hormones are of considerable importance in the control of blood glucose homeostasis.The cDNAs encoding both the human glucagon receptor (hGR) and the human islet GLP-I receptor (hGLP-IR) have been recently cloned(6Thorens B. Porret A. Buhler L. Deng S.P. Morel P. Widmannj C. Diabetes. 1992; 42: 1678-1682Crossref Scopus (488) Google Scholar, 7Lok S. Kuijper J.L. Jelinek L.J. Kramer J.M. Whitmore T.E. Sprecher C.A. Mathewes S. Grant F.J. Biggs S.H. Rosenberg G.B. Sheppard P.O. O'Hara P.J. Foster D.C. Kindsvogel W.K. Gene (Amst.). 1994; 140: 203-209Crossref PubMed Scopus (117) Google Scholar). Both polypeptides show sequence similarity to a well established family of receptors with seven-transmembrane domains that are known to couple to intracellular effectors through heterotrimeric guanine nucleotide binding proteins (G proteins)(8Dohlman H.G. Thorner J. Caron M.G. Lefkowitz R.J. Annu. Rev. Biochem. 1991; 60: 653-688Crossref PubMed Scopus (1130) Google Scholar). All members of this family of G-protein coupled receptors (GPCRs) share the same basic structure and membrane topology. Within the large family of GPCRs are grouped subfamilies based on primary sequence homology(9Strader C.D. Fong T.M. Tota M.R. Underwood D. Annu. Rev. Biochem. 1994; 63: 101-132Crossref PubMed Scopus (991) Google Scholar). The glucagon and GLP-I peptide receptors are grouped into a subfamily that includes the receptors for secretin, calcitonin, vasoactive intestinal peptide, parathyroid hormone, and growth hormone-releasing factor(9Strader C.D. Fong T.M. Tota M.R. Underwood D. Annu. Rev. Biochem. 1994; 63: 101-132Crossref PubMed Scopus (991) Google Scholar). The hGR and hGLP-IR share 47% amino acid identity and 66% similarity, with the transmembrane segments having the highest degree of similarity (see Fig. 1). Despite this homology, glucagon binds to the hGR with a Kd approximately 1000-fold lower than to the hGLP-IR.Despite a large body of information regarding the ligand binding and signaling characteristics of GPCRs in general, there has been very little structure-function analysis reported about the glucagon-secretin subfamily. In order to understand the nature of the interaction of glucagon with its receptor, we constructed a number of hGR•hGLP-IR chimeras in which the various domains of the hGR were sequentially replaced by homologous domains from hGLP-IR. Since both receptors are highly conserved throughout their entire length, it was possible to swap domains without introducing any gaps or deletions in the primary amino acid sequence. We report here the identification of several, non-continuous domains that are required for high affinity glucagon binding.