Development of potent glucagon antagonists: structure-activity relationship study of glycine at position 4.

We examined the functional role of glycine at position 4 in the potent glucagon antagonist [desHis(1), Glu(9)]glucagon amide, by substituting the L- and D-enantiomers of alanine and leucine for Gly(4) in this antagonist. The methyl and isobutyl side-chain substituents were introduced to evaluate the preference shown by the glucagon receptor, if any, for the orientation of the N-terminal residues. The L-amino acids demonstrated only slightly better receptor recognition than the D-enantiomers. These results suggest that the Gly(4) residue in glucagon antagonists may be exposed to the outside of the receptor. The enhanced binding affinities of analogs 1 and 3 compared with the parent antagonist, [desHis(1), Glu(9)]glucagon amide, may have resulted from the strengthened hydrophobic patch in the N-terminal region and/or the increased propensity for a helical conformation due to the replacement of alanine and leucine for glycine. Thus, as a result of the increased receptor binding affinities, antagonist activities of analogs 1-4 were increased 10-fold compared with the parent antagonist, [desHis(1), Glu(9)]glucagon amide. These potent glucagon antagonists have among the highest pA(2) values of any glucagon analogs reported to date.     

Three distinct epitopes on the extracellular face of the glucagon receptor determine specificity for the glucagon amino terminus

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.     

Glucagon antagonists: contribution to binding and activity of the amino-terminal sequence 1-5, position 12, and the putative alpha-helical segment 19-27

Glucagon binding to and recognition by its cell surface receptor is the necessary first step in the cascade of events leading to the activation of adenylate cyclase by the hormone. It has long been presumed that glucagon adopts an ordered conformation upon binding to its membrane-bound receptor. A recent model of this three-dimensional structure based on biophysical data, predicts beta-turns at positions 2-5, 10-13, and 15-18, and an alpha-helical region between residues 19-27. Our approach in the design of antagonists of glucagon was to elucidate the steric and electronic features that stabilize these secondary structures to obtain analogs that bind with high affinity to the receptor but do not activate adenylate cyclase. Nineteen glucagon analogs incorporating structural changes at the amino-terminal sequence 1-5, at positions 9 and 12, and at the carboxyl-terminal helical region were synthesized. Des-His1-[Glu9]glucagon amide was recently shown to be a competitive inhibitor. Our synthetic studies in combination with this modification have resulted in seven new glucagon antagonists. The implications for the structural and conformational properties required for binding and activity of glucagon and the glucagon peptide family are discussed.     

Potentiation of glucose-induced insulin release in islets by desHis1[Glu9]glucagon amide

Glucagon and secretin and some of their hybrid analogs potentiate glucose-induced release of insulin from isolated mouse pancreatic islets. It was recently shown that the synthetic glucagon analog, desHis1[Glu9]glucagon amide, does not stimulate the formation of cyclic adenosine monophosphate in the rat hepatocyte membrane, but binds well to the glucagon receptor and is a good competitive antagonist of glucagon. In the present study the effect of this analog on isolated islets was examined. desHis1-[Glu9]glucagon amide at 3 x 10(-7) M, in the presence of 0.01 M D-glucose, increased the release of insulin by 30% and maintained that level for the full 30-min test period. The rate of insulin release returned to the glucose-induced base line after removal of the peptide. The same insulin level was produced by 3 x 10(-9) M glucagon, and at 3 x 10(-7) M glucagon insulin release was enhanced 290% above the glucose base line.     

Position 9 replacement analogs of glucagon uncouple biological activity and receptor binding.

Recent studies on the glucagon antagonist des-His1-[Glu9]glucagon amide have resulted in pure inhibitors of the hormone, suggesting that the inhibitory properties may be centered around position 9. The present study was designed to investigate the chemical characteristics of substitutions in position 9 of glucagon that determine binding affinity and biological activity. Twenty replacement analogs of position 9 of glucagon were synthesized and assessed for their ability to bind to the glucagon receptor in rat hepatocyte membranes and to activate adenylate cyclase. Any substitution of aspartic acid 9 was accompanied by a severely diminished capacity to transmit the biological signal, while retaining receptor binding affinity. These results are an indication of an uncoupling of receptor binding and biological activity at this locus and define a central role of aspartic acid 9 in glucagon activity. Single replacement or deletion of either His1 or Asp9 in glucagon caused a 20- to 50-fold decrease in cyclase activity, whereas these same changes made in tandem caused virtually complete loss of activity, with decreases of 10(4)-to 10(6)-fold. These observations have led us to speculate that, at the molecular level, the region of glucagon required for transduction of the biological response may be distinct from the binding region and is mediated by a coupled interaction between His1 and Asp9 of the hormone and a complementary functional site of the glucagon receptor.     

Structure-conformation-activity studies of glucagon and semi-synthetic glucagon analogs

Summary Examination of glucagon structure-activity relationships and their use for the development of glucagon antagonists (inhibitors) have been hampered until recently by the lack of high purity of semisynthetic glucagon analogs and inadequate study of full dose-response curves for these analogs in sensitive bioassay systems. Recently a number of highly purified glucagon fragments and semi-synthetic analogs have been prepared and their full dose-response activities examined over a wide concentration range using the hepatic membrane adenylate cyclase assay, the hepatic membrane receptor binding assay, and glycogenolytic activity in isolated rat hepatocytes. The results of these studies have enabled us to identify and dissociate the structural (and in some cases conformational) features of glucagon important for binding from those most responsible for biological activity (transduction). Key findings in these studies were the observation that: (1) the C-terminal region of glucagon is primarily of importance for hormone binding to receptors; (2) glucagon_1–21 and glucagon_1–6 have low potency, but are essentially fully active glucagon derivatives; and (3) highly purified glucagon_2–29 ([1-des-histidine]-glucagon), [1-N-carbamoylhistidine]-glucagon and [1-N-carbamoylhistidine, 12-N-carbamoyllysine]-glucagon are all partial agonists.These and other findings led us to synthesize several semisynthetic analogs of glucagon which were found to possess no intrinsic biological activity in the hepatic adenylate cyclase assay system, but which could block the effect of glucagon (competitive inhibitors) in activating adenylate cyclase in this system. Two of these highly purified analogs [1-des-histidine] [2-N-trinitrophenylserine, 12-homoarginine]-glucagon and [1-N-trinitrophenylhistidine, 12-homoarginine]-glucagon were quite potent glucagon antagonists (inhibitors) with pA₂ values of 7.41 and 8.16 respectively. The latter compound has also been demonstrated to decrease dramatically blood glucose levels of diabetic animals in vivo. These results demonstrate that glucagon is a major contributor to the hyperglycemia of diabetic animals.Examination of the known and calculated conformational properties of glucagon provide insight into the structural and conformational properties of glucagon and its analogs most responsible for its biological activity. Consideration of these features and the mechanism of glucagon action at the membrane receptor level provide a framework for further developing glucagon analogs for theoretical and therapeutic applications.     

Synthetic glucagon antagonists and partial agonists.

This paper reports the synthesis and the biological activities of six new glucagon analogues. In these compounds N-terminal modifications of the glucagon sequence were made, in most cases combined with changes in the C-terminal region which had been shown previously to enhance receptor affinity. The design of these analogues was based on [Lys17,18,Glu21]glucagon,1 a superagonist, which binds five times better than glucagon to the glucagon receptor, and on the potent glucagon antagonist [D-Phe4,Tyr5,Arg12]glucagon, which does not stimulate adenylate cyclase system even at very high concentrations. The N-terminal modifications involved substitution of His1 by the unnatural conformationally constrained residue, 4,5,6,7-tetrahydro-1H-imidazo[c]pyridine-6-carboxylic acid (Tip) and by desaminohistidine (dHis). In addition we prepared two analogues (6 and 7), in which we deleted the Phe6 residue, which was suggested to be part of a hydrophobic patch and involved in receptor binding. The following compounds were synthesized: [Tip1, Lys17,18,Glu21]glucagon (2); [Tip1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon (3); [dHis1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon (4); [dHis1,Asp3,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21+ ++]glucagon (5); des-Phe6-[Tip1,D-Phe4,Tyr5,Arg12,Glu21]glucagon (6); des-Phe6-[Asp3,D-Phe4,Tyr5,Arg12,Glu21]glucagon (7). The binding potencies of these new analogues relative to glucagon (= 100) are 3.2 (2), 2.9 (3), 10.0 (4), 1.0 (5), 8.5 (6), and 1.7 (7). Analogue 2 is a partial agonist (maximum stimulation of adenylate cyclase (AC) approximately 15% and a potency 8.9% that of glucagon, while the remaining compounds 3-7 are antagonists unable to activate the AC system even at concentrations as high as 10(-5) M. In addition, in competition experiments, analogues 3-7 caused a right-shift of the glucagon stimulated adenylate cyclase dose-response curve.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucagon antagonists. Synthesis and inhibitory properties of Asp3-containing glucagon analogs

In an effort to find analogs of glucagon that would bind to the glucagon receptor of the rat liver membrane but would not activate membrane-bound adenyl cyclase, several hybrid molecules were synthesized which contained sequences from both glucagon and secretin. [Asp3, Glu9]Glucagon and [Asp3, Glu9, Arg12]glucagon were inactive in the adenyl cyclase assay even at high concentrations but retained some binding affinity for the receptor. They were able to displace 125I-glucagon completely from its receptor and could completely inhibit the activation of adenyl cyclase by natural or synthetic glucagon. The inhibition index [I/A]50 was approximately 110 for both analogs. [Asp3]Glucagon, [Glu3]glucagon and [Asp3, Lys17, 18, Glu21]glucagon were weak partial agonists, while [Asp3, Glu21]glucagon was inactive and a poor inhibitor. The peptides were synthesized by solid-phase methods and purified to homogeneity by reverse-phase high-performance liquid chromatography on C18 silica columns. These are the first fully synthetic competitive glucagon antagonists to be reported.     

The relation of predicted structure to observed conformation and activity of glucagon analogs containing replacements at positions 19, 22, and 23

Six new analogs of glucagon have been synthesized containing replacements at positions 19, 22, and 23. They were designed to study the correlation between predicted conformation in the 19-27 segment of the hormone and the conformation calculated from circular dichroism measurements and the observed activation of adenylate cyclase in the liver membrane. The analogs were [Val19]glucagon, [Val22]glucagon, [Glu23]glucagon, [Val19,Glu23]glucagon, [Glu22,Glu23]glucagon, and [Ala22,Ala23]glucagon. The structures predicted for the 19-27 segment ranged from strongly alpha helical to weakly beta sheet. The observed conformations varied as functions of amino acid composition, solvent, concentration, pH, and temperature but did not correlate well with prediction. There was, however, a correlation between predicted structure and activation of adenylate cyclase in rat liver membranes.     

Stabilization of soluble active rat liver glucagon receptor

Active glucagon receptor was solubilized with 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate (Chaps) from rat liver plasma membranes but rapidly (less than 8 h) lost activity. Either inclusion of 1X Hanks' balanced salt solution in the 3 mM Chaps solubilization buffer or its addition after solubilization increased the percentage of total binding attributable to specific glucagon binding from approximately 10 to greater than 80%; of great importance, it increased the stability from near zero binding at 8 h to 50% binding at 48 h (4 degrees C). Of the Hanks' solution components, either NaCl (137 mM) or CaCl2 (1.26 mM) was effective in increasing specific binding to approximately 70 and 60% respectively: Mg salts were ineffective. Soluble receptor binding activity was assayed by dextran-coated charcoal adsorption of free hormone. The assay is rapid, simple, and reproducible. It is suitable for monitoring receptor activity during purification and molecular characterization. Competition binding studies gave an IC50 value of 10-20 nM (slope factor approximately 1), with or without GTP. Dissociation assays revealed GTP sensitivity when receptors were solubilized either as glucagon-receptor complexes or free receptor. Active glucagon-receptor complexes could be eluted from wheat germ lectin-agarose: neither concanavalin A-agarose nor soybean agglutinin-agarose bind receptor. A glucagon degrading activity which co-solubilized with the receptor but did not require detergent for extraction was distinguishable from the soluble receptor not only by solubility but also by its heat stability (30 degrees C), its inhibition by bacitracin, its affinity for glucagon, its retention of activity for at least 1 week at 4 degrees C, and its size.     

Conformational analyses by 1H NMR and computer simulations of cyclic hexapeptides related to somatostatin containing acidic and basic peptoid residues.

We report the conformational analysis by 1H NMR in DMSO and computer simulations involving distance geometry and molecular dynamics simulations at 300K of peptoid analogs of the cyclic hexapeptide c-[Phe11-Pro6-Phe7-D-Trp8-Lys9-Thr10]. The analogs c-[Phe11-Nasp6-Phe7-D-Trp8-Lys9-Thr10](1), c-[Phe11-Ndab6Phe7-D-Trp8-Lys9-Thr10] (2) and c-[Phen11-Nlys6-Phe7-D-Trp8-Lys9-Thr10](3) where Nasp denotes N-(2-carboxyethyl) glycine, Ndab N-(2-aminoethyl) glycine and Nlys N-(4-aminobutyl) glycine are subject to conformational studies. The results of free and restrained molecular dynamics simulations at 300K are reported and give insight into the conformational behaviour of these analogs. The compounds show two sets of nuclear magnetic resonance signals corresponding to the cis and trans orientations of the peptide bond between residues 11 and 6. The backbone conformation of the cis isomers that we believe are the bioactive isomers of the three compounds are very similar to each other while there are larger variations amongst the trans isomers. The binding data to the isolated receptors show that the introduction of the Nlys residue in analog 3 leads to an enhancement of binding potency to the hsst5 receptor compared with analog 2 while maintaining identical binding potency to the hsst2 receptor. The Nasp6 analog 1 binds weakly to the hsst2 and is essentially inactive towards the other receptors. Comparison of the conformations and binding activities of these three analogs indicates that the Nlys residue extends sufficiently far to allow binding to a negatively charged binding domain on the hsst5 receptor. According to this model, the Ndab analog 2 cannot extend far enough to allow for binding to the receptor pocket. The loss of activity observed for the Nasp6 compound 1 indicates that the presence of a negatively charged residue in position 6 is unfavorable for binding to the hsst receptors.     

NMR solution structure of the glucagon antagonist [desHis1, desPhe6, Glu9]glucagon amide in the presence of perdeuterated dodecylphosphocholine micelles

Glucagon, a 29-residue peptide hormone, plays an important role in glucose homeostasis and in diabetes mellitus. Several glucagon antagonists and agonists have been developed, but limited structural information is available to clarify the basis of their biological activity. The solution structure of the potent glucagon antagonist, [desHis1, desPhe6, Glu9]glucagon amide, was determined by homonuclear 2D NMR spectroscopy at pH 6.0 and 37 degrees C in perdeuterated dodecylphosphocholine micelles. The overall backbone root-mean-square deviation (rmsd) for the structured portion (residues 7-29, glucagon numbering) of the micelle-bound 27-residue peptide is 1.36 A for the 15 lowest-energy structures, after restrained molecular dynamics simulation. The structure consists of four regions (segment backbone rmsd in A): an unstructured N-terminal segment between residues 2 and 5 (1.68), an irregular helix between residues 7 and 14 (0.79), a hinge region between residues 15 and 18 (0.54), and a well-defined alpha-helix between residues 19 and 29 (0.33). The two helices form an L-shaped structure with an angle of about 90 degrees between the helix axes. There is an extended hydrophobic cluster, which runs along the inner surface of the L-structure and incorporates the side chains of the hydrophobic residues of each of the amphipathic helices. The outer surface contains the hydrophilic side chains, with two salt bridges (D15-R18 and R17-D21) implied from close approach of the charged groups. This result is the first clear indication of an overall tertiary fold for a glucagon analogue in the micelle-bound state. The relationship of the two helical structural elements may have important implications for the biological activity of the glucagon antagonist.     

Design and synthesis of glucagon partial agonists and antagonists

The hyperglycemia and ketosis of diabetes mellitus are generally associated with elevated levels of glucagon in the blood. This suggests that glucagon is a contributing factor in the metabolic abnormalities of diabetes mellitus. A glucagon-receptor antagonist might provide important evidence for glucagons's role in this disease. In this work we describe how we combined structural modifications that led to glucagon analogues with partial agonist activity to give glucagon analogues that can act as competitive antagonists of glucagon-stimulated adenylate cyclase activity. Using solid-phase synthesis methodology and preparative reverse-phase high-performance liquid chromatography, we synthesized the following seven glucagon analogues and obtained them in high purity: [D-Phe4,Tyr5,Arg12]glucagon (2); [D-Phe4,Tyr5,Lys17,18]glucagon (3); [Phe1,Glu3,Lys17,18]glucagon (4); [Glu3,Val5,Lys17,18]glucagon (5); [Asp3,D-Phe4,Ser5,Lys17,18]glucagon (6); I4-[Asp3,D-Phe4,Ser5,Lys17,18]glucagon (7); [Pro3]glucagon (8). Purity was assessed by enzymatic total hydrolysis, by chymotryptic peptide mapping, and by reverse-phase high-performance liquid chromatography. The new analogues were tested for specific binding, for their effect on the adenylate cyclase activity in rat liver membranes, and for their effect on the blood glucose levels in normal rats relative to glucagon. Analogues showing no adenylate cyclase activity were examined for their ability to act as antagonists by displacing glucagon-stimulated adenylate cyclase dose-response curves to the right (higher concentrations). The binding potencies of the new analogues relative to glucagon (= 100) were respectively 1.0 (2), 1.3 (3), 3.8 (4), 0.4 (5), 1.3 (6), 5.3 (7), and 3 (8). Glucagon analogues 3-5 and 8 were all weak partial agonists with EC50 values of 500 (3), 250 (4), 1600 (5), and 395 nM (8), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lysine-50 is a likely site for anchoring the plasminogen N-terminal peptide to lysine-binding kringles.

Interactions between the kringle 4 (K4) domain of human plasminogen (Pgn) and segments of the N-terminal Glu1-Lys77 peptide (NTP) have been investigated via 1H-NMR at 500 MHz. NTP peptide stretches devoid of Lys residues but carrying an internal Arg residue show negligible affinity toward K4 (equilibrium association constant Ka < 0.05 mM(-1)). In contrast, while most fragments containing an internal Lys residue exhibit affinities comparable to that shown by the blocked Lys derivative Nalpha-acetyl-L-lysine-methyl ester (Ka approximately 0.2 mM(-1), peptides encompassing Lys50O consistently show higher Ka values. Among the investigated linear peptides, Nalpha-acetyl-Ala-Phe-Tyr-His-Ser-Ser-Lys5O-Glu-Gln-NH2 (AcAFYHSK5OEQ-NH2) exhibits the strongest interaction with K4 (Ka approximately 1.4 mM(-1)), followed by AcYHSK50EQ-NH2 (Ka approximately 0.9 mM(-1)). Relative to the wild-type sequence, mutated hexapeptides exhibit lesser affinity for K4. When a Lys50 --> Ser mutation was introduced (==> AcYHSS50EQ-NH2), binding was abolished. The Ile27-lle56 construct (L-NTP) contains the Lys50 site within a loop constrained by two cystine bridges. The propensity of recombinant Pgn K1 (rK1) and K2 (rK2) modules, and of Pgn fragments encompassing the intact K4 and K5 domains, for binding L-NTP, was investigated. We find that L-NTP interacts with rK1, rK2, K4, and K5-all lysine-binding kringles-in a fashion that closely mimics what has been observed for the Glul-HSer57 N-terminal fragment of Pgn (CB-NTP). Thus, both the constellation of kringle lysine binding site (LBS) aromatic residues that are perturbed upon complexation of L-NTP and magnitudes of kringle-L-NTP binding affinities (rK1, Ka approximately 4.3 mM(-1); rK2, Ka approximately 3.7 mM(-1; K4, Ka approximately 6.4 mM(1); and K5, Ka approximately 2.1 mM(-1)) are essentially the same as for the corresponding kringle-CB-NTP pairs. Molecular modeling studies suggest that the Glu39-Lys50 stretch in NTP generates an area that complements, both topologically and electrostatically, the solvent-exposed kringle LBS surface.     

Effects of intracerebroventricular injections of des-His1 (Glu9) glucagon amide on the regulatory thermogenesis in muscovy ducklings

Recent investigations have demonstrated a modulatory action of glucagon on shivering via the central nervous system in ducklings. Such an action could be mediated by glucagon receptors that have been recently detected in several brain areas involved in the central control of the involuntary motricity in this avian species. The present study using des-His1 (Glu9) glucagon amide, was performed to investigate the central mechanisms of glucagon on shivering. This glucagon analog was found to be an antagonist of glucagon devoid of adenylate cyclase activity (GR2) by triggering the breakdown of inositol phosphate (GR1) in mammals hepatocytes. The intracerebroventricular administration of des-His1 (Glu9) glucagon amide or glucagon induced a marked inhibition of shivering in ducklings exposed to cold. It seems likely that GR1 receptors contribute to decreased shivering in ducklings exposed to cold. Central glucagon or des-His1 (Glu9) glucagon amide were devoid of thermogenic effect at thermoneutrality.     

Characterization of a novel, non-peptidyl antagonist of the human glucagon receptor

We have identified a series of potent, orally bioavailable, non-peptidyl, triarylimidazole and triarylpyrrole glucagon receptor antagonists. 2-(4-Pyridyl)-5-(4-chlorophenyl)-3-(5-bromo-2-propyloxyphenyl)p yrr ole (L-168,049), a prototypical member of this series, inhibits binding of labeled glucagon to the human glucagon receptor with an IC50 = 3. 7 +/- 3.4 nM (n = 7) but does not inhibit binding of labeled glucagon-like peptide to the highly homologous human glucagon-like peptide receptor at concentrations up to 10 microM. The binding affinity of L-168,049 for the human glucagon receptor is decreased 24-fold by the inclusion of divalent cations (5 mM). L-168,049 increases the apparent EC50 for glucagon stimulation of adenylyl cyclase in Chinese hamster ovary cells expressing the human glucagon receptor and decreases the maximal glucagon stimulation observed, with a Kb (concentration of antagonist that shifts the agonist dose-response 2-fold) of 25 nM. These data suggest that L-168,049 is a noncompetitive antagonist of glucagon action. Inclusion of L-168, 049 increases the rate of dissociation of labeled glucagon from the receptor 4-fold, confirming that the compound is a noncompetitive glucagon antagonist. In addition, we have identified two putative transmembrane domain residues, phenylalanine 184 in transmembrane domain 2 and tyrosine 239 in transmembrane domain 3, for which substitution by alanine reduces the affinity of L-168,049 46- and 4. 5-fold, respectively. These mutations do not alter the binding of labeled glucagon, suggesting that the binding sites for glucagon and L-168,049 are distinct.     

Roles of specific extracellular domains of the glucagon receptor in ligand binding and signaling

To identify structural determinants of ligand binding in the glucagon receptor, eight receptor chimeras and additional receptor point mutants were prepared and studied. Amino acid residues 103-117 and 126-137 in the extracellular N-terminal tail and residues 206-219 and 220-231 in the first extracellular loop of the glucagon receptor were replaced with the corresponding segments of the glucagon-like peptide-1 receptor or the secretin receptor. Specific segments of both the N-terminal tail and the first extracellular loop of the glucagon receptor are required for hormone binding. The 206-219 segment of the first loop appears to be important for both glucagon binding and receptor activation. Functional studies with a synthetic chimeric peptide consisting of the N-terminal 14 residues of glucagon and the C-terminal 17 residues of glucagon-like peptide 1 suggest that hormone binding specificity may involve this segment of the first loop. The binding selectivity may arise in part from aspartic acid residues in this segment. Mutation of R-202 located at the junction between the second transmembrane helix and the first loop resulted in a mutant receptor that failed to bind glucagon or signal. We conclude that high-affinity glucagon binding requires multiple contacts with residues in the N-terminal tail and first extracellular loop domain of the glucagon receptor, with hormone specificity arising primarily from the amino acid 206-219 segment. The data suggest a model whereby glucagon first interacts with the N-terminal domain of the receptor followed by more specific interactions between the N-terminal half of the peptide and the first extracellular loop of the receptor, leading to activation.     

Effects of glucagon-like peptide 1 (7-36) amide and glucagon on amylin release from perfused rat pancreas.

The effects of glucagon-like peptide 1 (7-36) amide [GLP-1 (7-36) amide] and glucagon on the release of islet amyloid polypeptide (IAPP), or amylin, from the isolated perfused rat pancreas were studied. In the presence of 5.6 mM glucose, GLP-1 (7-36) amide and glucagon stimulated the release of amylin from the perfused pancreas. The infusion of GLP-1 (7-36) amide at a concentration of 10(-9) M elicited a biphasic release of amylin similar to that of insulin. The cumulative output of amylin induced by 10(-9)M GLP-1 (7-36) amide was significantly higher than that by 10(-9)M glucagon (p less than 0.01). The amylin/insulin molar ratios induced by GLP-1 (7-36) amide and glucagon were about 1% and did not differ significantly. These findings suggest that GLP-1 (7-36) amide and glucagon stimulate the release of amylin from the pancreas and that the concomitant secretion of amylin and insulin might contribute to glucose homeostasis.