Three Distinct Epitopes on the Extracellular Face of the Glucagon Receptor Determine Specificity for the Glucagon Amino Terminus*

Steffen Runge {ddagger} §, Christian Gram {ddagger} §, Hans Bräuner-Osborne {ddagger}, Kjeld Madsen ¶, Lotte B. Knudsen || and Birgitte S. Wulff {ddagger} **

From the {ddagger}Molecular Pharmacology, Novo Nordisk, DK-2760 Maaloev, Denmark, the §Department of Medicinal Chemistry, Pharmaceutical University of Denmark, DK-2100 Copenhagen, Denmark, and Medicinal Chemistry IV and ||Pharmacological Research 3, Novo Nordisk, DK-2760 Maaloev, Denmark

Received for publication, January 31, 2003 , and in revised form, April 15, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The glucagon and glucagon-like peptide-1 (GLP-1) receptors are homologous family B seven-transmembrane (7TM) G protein-coupled receptors, and they selectively recognize the homologous peptide hormones glucagon (29 amino acids) and GLP-1 (30–31 amino acids), respectively. The amino-terminal extracellular domain of the glucagon and GLP-1 receptors (140–150 amino acids) determines specificity for the carboxyl terminus of glucagon and GLP-1, respectively. In addition, the glucagon receptor core domain (7TM helices and connecting loops) strongly determines specificity for the glucagon amino terminus. Only 4 of 15 residues are divergent in the glucagon and GLP-1 amino termini; Ser2, Gln3, Tyr10, and Lys12 in glucagon and the corresponding Ala8, Glu9, Val16, and Ser18 in GLP-1. In this study, individual substitution of these four residues of glucagon with the corresponding residues of GLP-1 decreased the affinity and potency at the glucagon receptor relative to glucagon. Substitution of distinct segments of the glucagon receptor core domain with the corresponding segments of the GLP-1 receptor rescued the affinity and potency of specific glucagon analogs. Site-directed mutagenesis identified the Asp385 -> Glu glucagon receptor mutant that specifically rescued Ala2-glucagon. The results show that three distinct epitopes of the glucagon receptor core domain determine specificity for the N terminus of glucagon. We suggest a glucagon receptor binding model in which the extracellular ends of TM2 and TM7 are close to and determine specificity for Gln3 and Ser2 of glucagon, respectively. Furthermore, the second extracellular loop and/or proximal segments of TM4 and/or TM5 are close to and determine specificity for Lys12 of glucagon.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Glucagon and GLP-11 evolved from a common ancestor by a gene duplication in early vertebrate evolution, and human tissue-specific processing of their common precursor peptide, preproglucagon, generates glucagon in the pancreatic {alpha}-cells and GLP-1 in the intestinal L-cells (1, 2). Activation of hepatic glucagon receptors (GluR) by glucagon stimulates glycogenolysis and gluconeogenesis. Activation of GLP-1 receptors (GLP-1R) on pancreatic {beta}-cells by GLP-1 stimulates glucose-induced insulin secretion. Given their biological functions in glucose homeostasis, both receptors are promising targets for the treatment of type II diabetes.

GluR and GLP-1R belong to family B of the 7TM GPCRs, which includes the receptors for peptide hormones of the glucagon/PACAP superfamily: glucagon-like peptide-2 (GLP-2), glucose-dependent insulinotropic polypeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal polypeptide (VIP), growth hormone-releasing hormone, and secretin and other peptide hormones such as calcitonin, corticotrophin-releasing factor, and parathyroid hormone (PTH). The fingerprint of family B 7TM GPCRs is six conserved cysteines that form three disulfide bonds in the N-terminal extracellular domain (Nt-domain) (3). The structural constraints imposed by an identical disulfide bond pattern probably guide the Nt-domain of family B 7TM GPCRs into a common structural fold regardless of limited sequence identity (4, 5). The present knowledge about the structure and arrangement of the 7TM helices is based primarily on multiple sequence alignment analyses, although a functional coupling of conserved polar residues of the PTH receptor (PTH1R) suggests the existence of a cooperative helix-helix interface between TM2 and TM7 (6, 7).

Structure-activity analyses of peptides of the glucagon/PACAP superfamily suggest that their entire length is required for optimal biological activity and that the C terminus is primarily involved in receptor binding, whereas the N terminus contains the residues involved in receptor activation (8). The N-terminally modified glucagon analog desHis1Glu9-glucagon is a potent GluR antagonist, which emphasizes the importance of His1 and Asp9 of glucagon in activation of GluR (9). The N termini of GLP-1 and exendin-4 are highly conserved, and the N terminus of exendin-4 is essential for activation of GLP-1R (10). Furthermore, His7, Gly10, Phe12, Thr13, and Asp15 of the GLP-1 N terminus are important for optimal binding and activation of GLP-1R, and they are all conserved in exendin-4 (11). NMR structures of GLP-1, glucagon, and PACAP-(1–38) in lipophilic solvents or dodecylphosphocholine micelles agree that the central and C-terminal parts are {alpha}-helical, often with a central distortion of the helix geometry, whereas the N terminus is a flexible random coil (1214). Interestingly, the N terminus of receptor-bound PACAP-(1–21) forms a specific {beta}-coil structure, and the PACAP N terminus is important for receptor activation (8, 14). The N termini of glucagon and PACAP are highly conserved, and thus the glucagon N terminus may form a PACAP-like structure upon binding to GluR.

The isolated Nt-domain of family B 7TM GPCRs is sufficient for low affinity ligand binding, and it is a critical determinant of ligand selectivity (3, 4, 15). The Nt-domain of GLP-1R binds exendin-4-(9–39) with high affinity, and therefore either the C-terminal extension of exendin-4 (the Trp cage) or divergent residues in exendin-4 and GLP-1 increase the affinity of exendin-4 at the Nt-domain of the GLP-1R relative to GLP-1 (16). Additional interactions with the extracellular loops (ECL) and the extracellular end of the 7TM helices may explain the high affinity ligand binding of intact receptors and provide additional determinants of ligand selectivity (1720).

The molecular information about receptor-ligand complexes of family B 7TM GPCRs is limited. A two-site binding model has been proposed for the ligand interaction with PTH1R, in which the PTH C terminus interacts with the PTH1R Nt-domain and the PTH N terminus interacts with the PTH1R core domain (21). Peptides of the glucagon/PACAP superfamily may follow a similar binding mechanism (14, 22). The conserved Asp198 in the boundary between TM2 and ECL1 of GLP-1R is important to maintain the binding site for the GLP-1 N terminus (23). Specifically, Asp3 in the N terminus of VIP and secretin interacts with positively charged residues in the extracellular end of TM2 of VPAC1 (VIP receptor) and the secretin receptor, respectively (24, 25). The corresponding Gln3 in the N terminus of glucagon most likely interacts with the extracellular end of TM2 of GluR (26). In addition, p-benzoyl-L-phenylalanine in position 22 or 26 of the secretin C terminus cross-links to specific residues in the Nt-domain of the secretin receptor (27).

Family B 7TM GPCRs selectively bind their natural ligands with high affinity, although they may bind homologous ligands with low affinity. GLP-1R binds GLP-1 with high affinity and glucagon with low affinity, and glucagon is a low potency full agonist of GLP-1R (22). The GLP-1R Nt-domain defines almost completely the glucagon/GLP-1 selectivity profile of GLP-1R by selective recognition of the GLP-1 C terminus, and the GLP-1R core domain is not important for glucagon/GLP-1 selectivity (22). In contrast, GluR has a very strong glucagon/GLP-1 selectivity profile and does not cross-react with GLP-1. The GluR Nt-domain selectively recognizes the glucagon C terminus, and the GluR core domain strongly determines specificity for the glucagon N terminus. These results encouraged a search for the selectivity determinants in the GluR core domain. Single-substituted glucagon analogs addressing four divergent residues in the glucagon and GLP-1 N termini were analyzed for their ability to bind and activate GluR. Loss-of-function mutations in glucagon were rescued by gain-of-function mutations in GluR. The results show that three distinct epitopes of the GluR core domain determine specificity for the N terminus of glucagon.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Receptor Constructs—The cDNAs encoding the human GLP-1R and the human GluR were originally obtained from Dr. B. Thorens and Zymogenetics Inc., respectively, and subcloned into the mammalian expression vector pcDNA3.1/V5-His-TOPO® (Invitrogen) (10, 28). Chimeric glucagon/GLP-1 receptors were generated by overlap extension PCR, as previously described (22). Chimera A was composed of amino acid residues 1–144 of GluR and residues 148–463 of GLP-1R (22). Chimeric receptor TM2 (ChTM2) was composed of residues 1–187 and 199–477 of GluR and residues 190–200 of GLP-1R. ChECL2 was composed of amino acid residues 1–273 and 321–474 of GluR and residues 276–322 of GLP-1R. ChECL3 was composed of amino acid residues 1–359 and 386–477 of GluR and amino acid residues 362–387 of GLP-1R. Site-directed mutagenesis of GluR was done using QuikChangeTM (Stratagene). Plasmid DNA was generated and sequenced as previously described (22).

Peptide Synthesis and Radiolabeling—Glucagon, GLP-1-(7–37), Ala2-glucagon, Glu3-glucagon, Val10-glucagon, Ser12-glucagon, Ser8-GLP-1, Gln9-GLP-1, Tyr16-GLP-1, and Lys18-GLP-1 were synthesized and characterized as previously described (22).

The radioligand 125I-Glucagon (2.2 Ci/µmol) was prepared by the chloramine-T method, and 125I-GLP-1 (2.2 Ci/µmol) was prepared by the lactoperoxidase method (29). Both radioligands were purified by reverse-phase high pressure liquid chromatography.

Cell Culture and Transient Receptor Expression—HEK293 cells were maintained in Dulbecco's modified Eagle' medium supplemented with 10% fetal bovine serum (Invitrogen) and penicillin/streptomycin (90 units/ml and 90 µg/ml, respectively). Cells were seeded in T75 flasks, transfected with 9 µg of DNA using the FuGeneTM transfection reagent (Roche Molecular Biochemicals), harvested 24 h after transfection, and used for plasma membrane preparations or applied directly to functional experiments.

Functional Assay—HEK293 cells transiently expressing the desired wild type or chimeric receptor were harvested and resuspended in assay buffer (Flashplate®; PerkinElmer Life Sciences) to a cell density of 1.8 x 106 cells/ml. Peptides were diluted in phosphate-buffered saline with 0.02% Tween 20. Cells (50 µl) and peptides (50 µl) were mixed in 96-well Flashplates® (PerkinElmer Life Sciences), gently agitated for 5 min, and incubated for 25 min at room temperature. The resulting intracellular level of cAMP was measured according to the supplier's manual, and data were analyzed by nonlinear regression analysis using Prism®, (GraphPad Software, Inc.).

Binding Assay—Binding experiments were performed as previously described (22). Briefly, a freshly thawed plasma membrane preparation of HEK293 cells transiently expressing the desired wild type or chimeric receptor was incubated for 2 h at 30 °C in the presence of peptide and tracer. Bound and unbound peptide/radioligand were separated, and filters were washed twice in cold incubation buffer and left to dry. Finally, the amount of bound radioligand was determined using a Packard {gamma}-counter, and data were analyzed by nonlinear regression analysis using Prism®, (GraphPad Software, Inc.).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Glucagon and GLP-1 Receptors—In GLP-1, Ala8, Glu9, Val16 and Ser18 were substituted with the corresponding residues of glucagon. The GLP-1 analogs were analyzed for their ability to bind and activate GLP-1R. In competition binding, GLP-1 displaced 125I-GLP-1 from GLP-1R with an IC50 value of 1.1 nM, and the IC50 values of the GLP-1 analogs were similar to that of GLP-1 (Table I). Whole cells transiently expressing the human GLP-1R gave a functional response with half-maximal stimulation (EC50) of the adenylate cyclase at 12 pM GLP-1, and the GLP-1 analogs were full agonists and equipotent with GLP-1 (Table I). In glucagon, Ser2, Gln3, Tyr10, and Lys12 were substituted with the corresponding residues of GLP-1 (Fig. 1). The glucagon analogs were analyzed for their ability to bind and activate GluR. In competition binding, glucagon displaced 125I-glucagon from GluR with an IC50 value of 2.1 nM, and the glucagon analogs displaced 125I-glucagon with significantly higher IC50 values (Fig. 1A and Table I). Whole cells transiently expressing the human GluR, responded functionally with half-maximal stimulation at 11 pM of glucagon, and the potencies of the glucagon analogs were significantly lower (Fig. 1B and Table I). The results showed that the individual substitutions of Ala8, Glu9, Val16, and Ser18 in the GLP-1 N terminus with the corresponding glucagon residues had only subtle effects on affinity or potency at GLP-1R relative to GLP-1. Conversely, the individual substitutions of Ser2, Gln3, Tyr10, and Ser12 in the glucagon N terminus with the corresponding GLP-1 residues decreased the affinity and potency at GluR relative to glucagon.


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TABLE I
Binding and functional experiments with GLP-1R, GluR, and chimera A

IC50 and EC50 values are given in nM and represent the mean ± S.D. of three or more independent experiments.

 


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FIG. 1.
Binding and functional analyses of glucagon analogs and chimeric glucagon/GLP-1 receptors. GluR (white) and the chimeric glucagon/GLP-1 receptors (white/black) are illustrated in the upper panel. Competition binding analyses using 125I-glucagon as tracer are shown in the middle panel, and functional analyses are shown in the lower panel. Each figure is representative of three or more independent experiments performed in triplicates (binding analyses) or duplicates (functional analyses). The arrows illustrate the loss of binding (A) and loss of function (B) at GluR and gain of binding (C, E, G, and I) and gain of function (D, F, H, and J) at the chimeric receptors. The arrows are colored according the color of the curves. The color codes of glucagon and the glucagon analogs are shown at the bottom, the divergent residues in the N terminus are framed, and a star indicates amino acid identity between glucagon and GLP-1-(7–37).

 

Chimera A—The chimeric receptor, chimera A, consists of the GluR Nt-domain and the GLP-1R core domain (Fig. 1) (22). The single-substituted glucagon analogs were analyzed for their ability to bind and activate chimera A. In competition binding, glucagon displaced 125I-glucagon from chimera A with an IC50 value of 1.2 nM (Fig. 1C and Table I). The IC50 values of Ala2-glucagon and Glu3-glucagon were lower than that of glucagon, whereas the IC50 values of Val10-glucagon and Ser12-glucagon were similar to that of glucagon (Fig. 1C and Table I). In functional experiments with chimera A, all of the glucagon analogs were equipotent with glucagon (Fig. 1D and Table I). The results obtained with GluR and chimera A showed that substitution of the GluR core domain with the GLP-1R core domain increased the affinity and potency of Ala2-glucagon, Glu3-glucagon, Val10-glucagon, and Ser12-glucagon relative to glucagon.

Dissection of the Glucagon Receptor Core Domain—Small segments of the GluR core domain were substituted with the corresponding segments of GLP-1R. Ala2-glucagon, Glu3-glucagon, and Ser12-glucagon were analyzed for their ability to bind and activate three chimeric receptors: ChTM2, ChECL2, and ChECL3 (Fig. 1). In ChTM2, the extracellular end of TM2 of GluR was substituted with the corresponding segment of GLP-1R. In competition binding, glucagon displaced 125I-glucagon from ChTM2 with an IC50 value of 1.4 nM (Fig. 1E and Table II). The IC50 value of Glu3-glucagon was lower than that of glucagon, whereas the IC50 values of Ala2-glucagon and Ser12-glucagon were higher than that of glucagon. On whole cells transiently expressing ChTM2, Glu3-glucagon was equipotent with glucagon, whereas Ala2-glucagon and Ser12-glucagon were less potent than glucagon (Fig. 1F and Table II). The results obtained with GluR and ChTM2 showed that substitution of the extracellular end of TM2 of GluR with the corresponding segment of GLP-1R increased the affinity and potency of Glu3-glucagon relative to glucagon, Ala2-glucagon, and Ser12-glucagon. In addition, the affinity but not the potency of Ala2-glucagon had increased relative to glucagon and Ser12-glucagon.


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TABLE II
Binding and functional experiments with chimeric receptors and GluR point mutants

IC50 and EC50 values are given in nM and represent the mean ± S.D. of three or more independent experiments. The IC50 and EC50 values of glucagon and the rescued glucagon analogs are shown in boldface type and underlined. ND, not determined.

 

In ChECL2, the second extracellular loop and the extracellular ends of TM4 and TM5 were substituted with the corresponding segments of GLP-1R. In competition binding, glucagon displaced 125I-glucagon from ChECL2 with an IC50 value of 3.1 nM (Fig. 1G and Table II). The IC50 value of Ser12-glucagon was similar to that of glucagon, whereas the IC50 values of Ala2-glucagon and Glu3-glucagon were higher than that of glucagon. On whole cells transiently expressing ChECL2, Ser12-glucagon was equipotent with glucagon, whereas Ala2-glucagon and Glu3-glucagon were less potent than glucagon (Fig. 1H and Table II). The results showed that substitution of the second extracellular loop and the extracellular ends of TM4 and TM5 with the corresponding segments of GLP-1R specifically increased the affinity and potency of Ser12-glucagon relative to glucagon, Ala2-glucagon, and Glu3-glucagon.

In ChECL3, the third extracellular loop and the extracellular ends of TM6 and TM7 were substituted with the corresponding segments of GLP-1R. In competition binding, glucagon displaced 125I-glucagon from ChECL3 with an IC50 value of 1.9 nM (Fig. 1I and Table II). The IC50 value of Ala2-glucagon was lower than that of glucagon, whereas the IC50 values of Glu3-glucagon and Ser12-glucagon were higher than that of glucagon. On whole cells transiently expressing ChECL3, Ala2-glucagon was equipotent with glucagon, whereas Glu3-glucagon and Ser12-glucagon were less potent than glucagon (Fig. 1J and Table II). The results showed that substitution of the third extracellular loop and the extracellular ends of TM6 and TM7 with the corresponding segments of GLP-1R specifically increased the affinity of Ala2-glucagon relative to glucagon, Glu3-glucagon, and Ser12-glucagon. In functional experiments, the potency of Ala2-glucagon and to a lesser extent Ser12-glucagon had increased relative to glucagon and Glu3-glucagon.

We were unable to generate a chimeric receptor that specifically increased the affinity and potency of Val10-glucagon; therefore, Val10-glucagon was not analyzed further.

Point Mutations in the Glucagon Receptor—The region defined by the GLP-1R segment of ChECL3 was investigated by site-directed mutagenesis of the corresponding segment of GluR. The divergent residues in this region were substituted individually to the corresponding residues of GLP-1R. Initially, the GluR point mutants were analyzed in binding and functional experiments with glucagon and Ala2-glucagon. In competition binding with the Asp385-Glu mutant, the affinity of Ala2-glucagon was slightly higher than that of glucagon (Fig. 2C and Table II). In functional experiments with whole cells transiently expressing the Asp385-Glu mutant, the potency of Ala2-glucagon was slightly higher than that of glucagon (Fig. 2D and Table II). With the other GluR point mutants, the affinity and potency of Ala2-glucagon were lower than the affinity and potency of glucagon, in a manner similar to GluR (Table II). This is illustrated by the Ser379 -> Phe mutant (Fig. 2, A and B). Subsequently, the Asp385 -> Glu and Ser379 -> Phe mutants were analyzed in binding and functional experiments with Glu3-glucagon and Ser12-glucagon. At both mutants, the affinity and potency of Glu3-glucagon and Ser12-glucagon were significantly lower than the affinity and potency of glucagon. The results showed that the point mutation Asp385 -> Glu in the extracellular end of TM7 specifically increased the affinity and potency of Ala2-glucagon relative to glucagon, Glu3-glucagon, and Ser12-glucagon.



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FIG. 2.
Binding and functional analyses of glucagon analogs and the Ser379 -> Phe and Asp385 -> Glu GluR mutants. The GLP-1R segment of ChECL3 is aligned with the corresponding segment of GluR. Divergent residues are shown in white, and conserved residues are shown in gray. The predicted positions of Ser379 and Asp385 in GluR are illustrated in black. A and C, competition binding analyses using 125I-glucagon as tracer. B and D, functional analyses. The green arrows indicate the rescue of affinity and potency of Ala2-glucagon at the Asp385 -> Glu GluR mutant. The color codes of glucagon and the glucagon analogs are shown at the bottom.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The glucagon analogs bound and activated GluR with lower affinity and potency than glucagon. Substitution of the entire GluR core domain with the GLP-1R core domain rescued the affinity and potency of all of the glucagon analogs relative to glucagon. In addition, the corresponding GLP-1 analogs bound and activated GLP-1R with the same affinity and potency as GLP-1. Apparently, the GluR core domain selectively recognized Ser2, Gln3, Tyr10, and Ser12 of the glucagon N terminus and discriminated the corresponding residues of the GLP-1 N terminus. In contrast, the GLP-1R core domain (GLP-1R and chimera A) potently accommodated both the GLP-1 and glucagon N termini, although the substituted residues in the GLP-1 and glucagon analogs may interact differently with the GLP-1R core domain than the corresponding residues of native GLP-1 and glucagon.

Dissection of the GluR core domain identified three distinct epitopes of the GLP-1R core domain that rescued the affinity and potency of specific glucagon analogs. The extracellular end of TM2 (ChTM2) rescued Glu3-glucagon, ECL2 and the proximal segments of TM4 and TM5 (ChECL2) rescued Ser12-glucagon, and ECL3 and the proximal segments of TM6 and TM7 (ChECL3) rescued Ala2-glucagon. ChTM2, ChECL2, and ChECL3 did not compromise glucagon binding or potency, which confirmed the structural integrity of the chimeric receptors. Two nonexclusive explanations may account for these results: 1) the GLP-1R segments of ChTM2, ChECL2, and ChECL3 interact directly with Glu3, Ser12, and Ala2, respectively, and/or 2) the GLP-1R segments of ChTM2, ChECL2, and ChECL3 are required to maintain a local binding site conformation that accommodates the interaction with Glu3, Ser12, and Ala2, respectively. Nevertheless, it is difficult to explain the results without considering the proximity of the GLP-1R segments in ChTM2, ChECL2, and ChECL3 with Glu3, Ser12, and Ala2 of the glucagon analogs, respectively. In addition, the corresponding segments of GluR define the strong glucagon/GLP-1 selectivity profile of the GluR core domain by selective recognition of the glucagon N terminus.

Most family B 7TM GPCRs have two positively charged residues in TM2, whereas GluR has only one and a neutral hydrophobic residue in place of the other (Lys187 and Ile194 in human GluR). Furthermore, the glucagon/PACAP superfamily peptides have either Asp or Glu in position 3 except glucagon, which has a Gln. The K187R/I194K GluR mutant rescued both affinity and potency of Asp3-glucagon relative to glucagon (26). The additional positive charge of the K187R/I194K GluR mutant and the chimeric receptor ChTM2 probably accommodates the extra negative charge of Asp3-glucagon and Glu3-glucagon, respectively. In fact, Glu3-glucagon bound ChTM2 with higher affinity than glucagon but was equipotent with glucagon in functional experiments with ChTM2. The small discrepancy in affinity versus potency suggests that Glu3 of Glu3-glucagon provides a binding determinant for interaction with ChTM2 that is not equally favorable for activation of ChTM2. However, the discrepancy is small compared with the total rescue of Glu3-glucagon by ChTM2. The corresponding Asp3 of secretin and VIP interact with the conserved Arg and Lys in TM2 of the secretin and VPAC1 receptor, respectively, and the interaction with Arg in the center of TM2 is important for receptor activation (24, 25). Site-directed mutagenesis of a highly conserved His in the cytoplasmic end of TM2 leads to constitutive activity of several family B 7TM GPCRs (3032). Collectively, it appears that TM2 is important for agonist binding and activation of family B 7TM GPCRs. Ile194 in TM2 of GluR may serve as a selectivity determinant that prevents access of homologous peptides to the activation site of GluR.

The combined analyses of GluR mutants and single-substituted glucagon analogs demonstrated the functional significance of the correlated substitution of residues in glucagon (Gln3) and GluR (Ile194). Glucagon is highly conserved during evolution, and specifically position 3 is occupied by Gln in vertebrates except bony fish, where position 3 is occupied by either Gln, Asp, or Glu. In bony fish, glucagon and GLP-1 have overlapping biological activities, and GLP-1 acquired the incretin function after the divergence of bony fish and mammals (33). Specificity of receptor-ligand pairs most likely evolved to ensure distinct physiological functions, and therefore the change of selection pressure on position 3 of glucagon may reflect the divergence of the biological activities of glucagon and GLP-1.

Eight divergent residues in the region defined by the GLP-1R segment of ChECL3 was investigated by site-directed mutagenesis of GluR. Only the substitution of Asp385 with the corresponding Glu of GLP-1R rescued the affinity and potency of Ala2-glucagon without disturbing the pharmacological profile of the other glucagon analogs relative to glucagon. It is difficult to explain the rescue of affinity of Ala2-glucagon by a direct interaction with Glu385 in the Asp385 -> Glu GluR mutants. It seems more likely that the Asp385 -> Glu mutation stabilized a local binding site conformation that preferably interacted with Ala2-glucagon. Nevertheless the results strongly suggest proximity between Ala2 of Ala2-glucagon and the extracellular end of TM7 of the GluR mutant Asp385 -> Glu.

The dissection of the GluR core domain provides three constraints that orient the glucagon N terminus with respect to the structural elements of the GluR core domain. Given the structural integrity of the GluR mutants in this study, we suggest a binding model in which Ser2 of glucagon is close to Asp385 of TM7, Gln3 of glucagon is close to Ile194 on TM2, and Lys12 is close to ECL2 and/or proximal segments of TM4 and/or TM5 (Fig. 3). The proximity of Ser2 with TM7 and Gln3 with TM2 is consistent with the potential helix-helix interface between TM2 and TM7 of PTH1R. Accordingly, the high level of amino acid identity of the predicted 7TM helices of family B 7TM GPCRs probably reflects an arrangement into a conserved helical bundle structure in which TM2 is close to TM7. Proximity of TM2 and TM7 is consistent with a rhodopsin-like arrangement of the 7TM helices, although the orientation may be either clockwise or counterclockwise (34).



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FIG. 3.
Binding model of the glucagon N terminus and the GluR core domain. Ser2 of glucagon is close to the extracellular end of TM7 of GluR, Gln3 is close to the extracellular end of TM2, and Lys12 is close to ECL2 and/or the proximal helices TM4 and/or TM5.

 

The position of ECL2 relative to the extracellular end of TM3 is probably constrained by a conserved disulfide bond of many family A and B 7TM GPCRs, and ECL2 is involved in peptide-agonist binding of family A 7TM GPCRs (3438). ECL2 of the secretin and VPAC2 receptors has been implicated in ligand binding, and specifically four residues in the N-terminal half of ECL2 contribute significantly to the secretin/VIP selectivity profile of the secretin receptor (17, 20). On the basis of the results presented here, we will attempt to identity the specific residue(s) in ECL2, TM4, and/or TM5 of GluR that determines specificity for Lys12 of glucagon.


    FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** To whom correspondence should be addressed: Novo Nordisk, Molecular Pharmacology, Novo Nordisk Park C9 S.27, DK-2760 Maaloev, Denmark. Tel.: 45-44434545; Fax: 45-44434587; E-mail: bsw{at}novonordisk.com.

1 The abbreviations used are: GLP, glucagon-like peptide; GluR, glucagon receptor(s); GLP-1R, GLP-1 receptor; TM, transmembrane; GPCR, G protein-coupled receptor; VIP, vasoactive intestinal polypeptide; PTH, parathyroid hormone; Nt-domain, N-terminal extracellular domain; ECL, extracellular loop(s); ChTM2, chimeric receptor TM2; ChECL2 and -3, chimeric receptor ECL2 and -3, respectively; PACAP, pituitary adenylate cyclase-activating polypeptide. Back


    ACKNOWLEDGMENTS
 
We are grateful to Vivian L. Tychsen for technical assistance and Carsten E. Stidsen and Sanne M. Knudsen for helpful discussions and critical reading of the manuscript.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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