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.     

Increased density of glucagon receptors in liver from endurance-trained rats

The binding properties of glucagon receptors were determined in plasma membranes isolated from liver of untrained (n = 6) and swimming endurance-trained Sprague-Dawley male rats (n = 7; 3 h/day, 5 days/wk, for 8 wk). Plasma membranes were purified from liver by aqueous two-phase affinity partitioning, and saturation kinetics were obtained by incubation of plasma membranes (10 microg of proteins/150 microl) with (125)I-labeled glucagon at concentrations ranging from 0.15 to 3.0 nM for 30 min at 30 degrees C. Saturating curve analysis indicated no difference in the affinity of glucagon receptors (0.57 +/- 0.06 and 0.77 +/- 0.09 nM in untrained and trained groups, respectively) but a significant higher glucagon receptor density in liver from untrained vs. trained rats (3.09 +/- 0.12 vs. 4.28 +/- 0.19 pmol/mg proteins). These results suggest that the reported increase in liver glucagon sensitivity in endurance-trained subjects (Drouin R, Lavoie C, Bourque J, Ducros F, Poisson D, and Chiasson J-L. Am J Physiol Endocrinol Metab 274: E23-E28, 1998) could be partly due to an increased glucagon receptor density in response to training.     

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.     

The hepatic glucagon receptor. Solubilization, characterization, and development of an affinity adsorption assay for the soluble receptor

The hepatic glucagon receptor was covalently labeled with [125I-Try10]monoiodoglucagon [( 125I]MIG) by use of the heterobifunctional cross-linker hydroxysuccinimidyl p-azidobenzoate. Labeling of the Mr = 63,000 peptide was sensitive to glucagon and GTP at concentrations at which they affect [125I]MIG binding to the receptor. The labeled receptor was solubilized with Lubrol-PX, and the hydrodynamic characteristics of the receptor were determined. The molecular parameters of the solubilized receptor are: S20,w = 4.3 +/- 0.1, Stokes radius = 6.3 +/- 0.1 nm, frictional coefficient f/f0 = 1.8, and a calculated Mr = 119,000. Incubation of liver membranes at 32 degrees C for 15 min prior to the addition of [125I]MIG permitted us to identify the high molecular weight form (Mr = approximately 113,000) of the receptor by direct sodium dodecyl sulfate-gel electrophoretic analysis. The Mr = 63,000 peptide can be adsorbed to wheat germ lectin-Sepharose. The glycoprotein nature of the receptor has been utilized to develop an assay for the detergent-solubilized receptor that uses wheat germ lectin-Sepharose as a solid matrix to adsorb the [125I] MIG-receptor complex. The free hormone remains in the liquid phase and is removed in the supernatant after low speed centrifugation. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) solubilizes receptors with retention of [125I]MIG binding activity. [125I]MIG binding to the CHAPS-solubilized receptor is specifically affected by unlabeled glucagon. Interaction of [125I]MIG with the soluble receptor is insensitive to the presence of GTP. IC50 for glucagon using the soluble receptor was 33-70 nM, irrespective of the presence or absence of GTP, while when the membrane-bound receptor was used, the IC50 in the absence of GTP was 2-4 nM and in the presence of GTP was 35-80 nM. These data allow us to conclude that the hepatic glucagon receptor in the membrane and in the nondenaturing detergent solution is a dimer of the Mr = 63,000 hormone-binding subunit and a glycoprotein. The soluble receptor does not display any functional interaction with the stimulatory regulator.     

Generation of mice expressing the human glucagon receptor with a direct replacement vector

The process of evaluating the in vivo efficacy of non–peptidyl receptor antagonists in animal models is frequently complicated by failure of compounds displaying high affinity against the human receptors to show measurable affinity at the corresponding rodent receptors. In order to generate a suitable animal model in which to evaluate the in vivo activity of non–peptidyl glucagon receptor antagonists, we have utilized a direct targeting approach to replace the murine glucagon receptor with the human glucagon receptor gene by homologous recombination. Specific expression of the human glucagon receptor (GR) in the livers of transgenic mice was confirmed with an RNase protection assay, and the pharmacology of the human GRs expressed in the livers of these mice parallels that of human GR in a recombinant CHO cell line with respect to both binding of ¹²⁵I–glucagon and the ability of glucagon to stimulate cAMP production. L–168,049, a non–peptidyl GR antagonist selective for the human GR shows a 3.5 fold higher affinity for liver membrane preparations of human GR expressing mice (IC₅₀=172±98nM) in the presence of MgCl₂ in marked contrast to the measured affinity of the murine receptor (IC₅₀=611±97nM) for this non–peptidyl antagonist. The human receptors expressed are functional as measured by the ability of glucagon to stimulate cAMP production and the selectivity of this antagonist for the human receptor is further verified by its ability to block glucagon–stimulated cyclase activity with 5 fold higher potency (IC₅₀=97.2±13.9nM) than for the murine receptor (IC₅₀=504±247nM). Thus we have developed a novel animal model for evaluating GR antagonists in vivo. These mice offer the advantage that the regulatory sequences which direct tissue specific and temporal expression of the GR have been unaltered and thus expression of the human gene in these mice remains in the normal chromosomal context.     

Effect of insulin and glucagon on the uptake of carnitine by perfused rat liver

The possible direct effects of insulin and glucagon on carnitine uptake by perfused rat liver were studied with L-[3H]carnitine of an initial concentration of 50 microM in the perfusate. Insulin (10 nM) did not significantly affect the uptake by livers from fed animals. However, insulin could reverse the stimulated transport by livers from 24-h fasted animals, reducing the uptake rate from 852 +/- 54.1 to 480 +/- 39.9 (mean +/- S.E.), P less than 0.01 (rates are expressed as nmol per h per 100 g body wt). Glucagon (50 nM) stimulated the uptake rate when livers were either from fed (551 +/- 40.1 vs. 915 +/- 55.3, P less than 0.01) or from fasted animals (852 +/- 54.1 vs. 1142 +/- 88.1, P less than 0.02). Based on these and earlier observations, we propose that the carnitine concentration in rat liver is controlled by insulin and glucagon via cellular transport processes.     

Effect of prostaglandin E1 on low density lipoprotein apo-B-receptor binding in human, rat and swine liver in vitro.

The effect of PGE1 on low density lipoprotein (LDL) apo-B-receptor binding was examined in human, rat and swine liver. Autologous LDL (for humans and swines) and homologous LDL (for rats) were isolated by ultracentrifugation and labelled with 123I using Iodogen followed by purification with dialysis. LDL-concentrations of 0.1-6 micrograms protein/ml were used for direct binding assays investigating the specific binding of labelled LDL in presence of increasing PGE1-concentrations (100 pM to 100 microM). In separate experiments the effect of PGE1 on displacement of specifically bound 123I-LDL by unlabelled ones was studied. The binding capacities estimated by Scatchard analysis were similar for human and rat liver LDL-apo-B-receptor binding, however, swine liver exhibited a significantly (p less than 0.001) lower binding capacity for 123I-LDL. PGE1 significantly (p less than 0.01-0.001) increased the amount of 123I-LDL specifically bound to the liver apo-B-receptors and the binding affinity in all liver preparations of the 3 species in a dose-dependent manner. PGE1 also significantly increased competition of unlabelled LDL for 123I-LDL bound to its specific apo-B-receptors in a dose-dependent manner (p less than 0.01-0.001) with an ED50 of 123 +/- 64 nM for human liver, 901 +/- 102 nM for rat liver obtained during anaesthesia, 74 +/- 23 nM for rat liver obtained after decapitation and 941 +/- 121 nM for swine liver. In human liver iloprost (ED50 = 876 +/- 53 nM) and PGI2 (ED50 = 52 +/- 12 microM) were less effective than PGE1, PGE2 had no effect on LDL-induced competition. It is concluded that PGE1 renders LDL more sensitive for apo-B-receptor binding suggesting a potential hypolipidemic action of PGE1.     

Quantitation of glucagon by radioreceptorassay

Receptors for glucagon on rat liver membranes were characterized. They bound [125I] glucagon rapidly in a specific and saturable way. Addition of unlabelled glucagon displaced [125I] glucagon from the binding sites in a concentration dependent way. Concentrations from 10(-9) to 10(-8) M of glucagon caused a linear reduction of binding of labelled glucagon. This concentration interval was used for a three-point assay which fulfilled statistical requirements for validity. Individual assays normally resulted in potency estimates of high precision and statistical weight. Mean values for glucagon activity of preparations tested by receptor assay were within the fiducial limits (P = 0.95) for corresponding activity determined by the rabbit blood glucose method. The receptor assay is less time consuming and requires only part of one rat liver while the in vivo assay uses 16 rabbits. Thus, the receptor assay is less resource demanding and should serve well as a screening instrument for control of potency of glucagon preparations.     

Receptor binding of selectively labeled (Tyr-10) and (Tyr-13)-mono-125I-glucagons and competition by homologous 127I-labeled isomers

Two monoiodinated derivatives of glucagon were prepared by lactoperoxidase catalyzed iodination followed by separation on reverse-phase high-performance liquid chromatography. The purified (Tyr-10) and (Tyr-13)-mono-125I-labeled glucagon isomers were characterized and studied with respect to their binding to the receptors of isolated intact rat hepatocytes. The extent of steady-state binding to cellular receptor sites differed for the two labeled glucagon tracers at 37 degrees C as well as at 15 degrees C with (Tyr-10)-mono-125I-glucagon displaying higher receptor binding. The apparent equilibrium constants, Kd,app at 37 degrees C are 3.6 +/- 0.4 nM (mean +/- S.E. of three independent experiments) for the tyrosine-13-labeled tracer and 5.9 +/- 0.6 nM for the tyrosine-10-labeled glucagon with native glucagon as competitor. Since the observed Kd in the competition assay is a function of the true Kd values of the monoiodinated radioactive glucagon isomers and native glucagon, the dissociation constants were also measured with chemically identical tracer and competitor. Under these conditions, we obtained Kd values of 1.3 +/- 0.2 nM for the tyrosine-10-labeled analog and 2.0 +/- 0.2 nM for the tyrosine-13-labeled glucagon isomers confirming the higher receptor binding affinity of (Try-10)-mono-125I-glucagon. All competition curves fit the mathematical expression for a model of non-cooperative binding to a single class of receptors.     

Genetic obesity and dietary sucrose decrease hepatic glucagon and insulin receptors in LA/N-corpulent rats

A catabolic and hypolipemic effect of glucagon has been described in normal animals. We therefore studied the role of glucagon in genetically obese, hyperlipemic rats. Twelve genetically obese hyperlipemic LA/N-cp/cp (corpulent) rats and 12 lean littermates were fed either 54% starch or 54% sucrose for 12 weeks. Plasma glucagon and insulin levels and glucagon and insulin binding to liver membranes were measured. Comparing all corpulent and lean animals regardless of diet, a significant (P less than 0.0001) phenotypical effect (cp/cp greater than lean) was observed in plasma insulin levels (464 +/- 54 vs 70.3 +/- 7.6 muu/ml, mean +/- SEM). Insulin binding (2.68 vs 16.1%/50 micrograms protein) and glucagon binding (25.6 vs 47.3%/50 micrograms protein) were both significantly lower (P less than 0.0001) in corpulent rats as compared to their lean littermates. Sucrose feeding had marginal effect on plasma insulin or insulin binding. It, however, decreased glucagon binding in corpulent rats but not in their controls. A significant negative correlation was observed between plasma insulin and insulin binding, while a positive correlation was seen for plasma glucagon and glucagon binding. A significant negative correlation was observed between plasma glucagon and lipogenic enzymes (glucose-6-phosphate dehydrogenase and malic enzyme) in liver and between glucagon binding and these enzymes. We propose that in these genetically obese rats, in addition to hyperinsulinemia, impaired glucagon activity as manifested by decreased glucagon binding to target cells may be an important contributor to the hyperlipemia and obesity. A further decrease in glucagon binding in rats fed sucrose indicates that sucrose, per se, may be an additional contributory factor.     

Modulation of glucagon effects by changes in extracellular pH and calcium

We have examined the influence of extracellular pH and calcium concentration on the action of glucagon on isolated rat hepatocytes, perfused liver or plasma membrane preparations. Incubation of rat hepatocytes with 10 nM glucagon at pH 7.4 caused an immediate increase in cAMP concentrations (8-fold), and this rise was almost 50% lower at acidic extracellular pH (6.9). This effect of pH could not be explained by an alteration of the hormone binding to its receptor for glucagon concentrations higher than 1 nM. The effect of acidosis on cAMP production was still present with non-hormonal effectors, such as 10 microM Gpp[NH]p, 30 microM forskolin or 10 mM NaF. This suggests a direct action of acidosis on the regulatory component Ns and/or on the catalytic subunit of adenylate cyclase. Acidic pH also depressed mitochondrial processes responsive to glucagon (NAD(P)H fluorescence, glutamine breakdown). Whatever the experimental model, calcium appeared to be required for maximal stimulation of cAMP production by glucagon. On perfused rat liver, glycogenolysis was depressed in the absence of extracellular calcium in the perfusate. In isolated hepatocytes, the stimulation of phosphorylase alpha activity by glucagon was modulated by extracellular calcium concentrations lower than 0.2 mM. This suggests that, although glucagon action is chiefly cAMP-mediated, its effect on calcium mobilization (affecting various cellular process, including cAMP production itself) should also be taken into account. This work also confirmed the importance of calcium in the stimulation of mitochondrial metabolism of glutamine by glucagon.     

Characterization of angiotensin II receptor subtypes in rat liver

Radioligand binding studies identified two classes of 125I-angiotensin II-binding sites in rat liver membranes. High affinity binding sites (Kd = 0.35 +/- 0.13 nM, N = 372 +/- 69 fmol/mg of protein) were inactivated by dithiothreitol (0.1-10 mM) without any apparent change in low affinity binding sites (Kd = 3.1 +/- 0.8 nM, N = 658 +/- 112 fmol/mg of protein). Dithiothreitol inactivation was readily reversible but could be made permanent by alkylation of membrane proteins with iodoacetamide. Angiotensin II stimulation of glycogen phosphorylase in isolated rat hepatocytes (maximal stimulation 780%, EC50 = 0.4 nM) was completely inhibited by 10 mM dithiothreitol, a concentration which also abolished high affinity site binding; phosphorylase stimulation by glucagon and norepinephrine under these conditions was unaltered. Angiotensin II inhibition of glucagon-stimulated adenylate cyclase activity in hepatocytes required higher angiotensin II concentrations (EC50 = 3 nM) than phosphorylase stimulation and was not affected by dithiothreitol. Fractional occupancy of high affinity binding sites by 125I-angiotensin II correlated closely with angiotensin II-mediated phosphorylase stimulation, whereas occupancy of low affinity sites paralleled inhibition of adenylate cyclase activity. These data indicate that the physiologic effects of angiotensin II in rat liver are mediated by two distinct receptors, apparently not interconvertible, and provide the first evidence for angiotensin II receptor subtypes with differing biochemical features and mechanisms of action.     

Substituted imidazoles as glucagon receptor antagonists.

A modestly active, nonselective triarylimidazole lead was optimized for binding affinity with the human glucagon receptor. This led to the identification of a 2- and/or 4-alkyl or alkyloxy substituent on the imidazole C4-aryl group as a structural determinant for significant enhancement in binding with the glucagon receptor (e.g., 41, IC(50)=0.053 microM) and selectivity (>1000x) over p38MAP kinase in this class of compounds.     

Hepatic glucagon clearance during insulin induced hypoglycemia

Studies concerning the importance of glucagon secretion in hypoglycemic counterregulation have assumed that peripheral levels of glucagon are representative of rates of pancreatic glucagon secretion. The measurement of peripheral levels of this hormone, however, may be a poor reflection of secretion rates because of glucagon's metabolism by the liver. Therefore, in order to understand the relationship between pancreatic glucagon secretion and levels of glucagon in the peripheral blood during hypoglycemia, we evaluated hepatic glucagon metabolism during insulin induced hypoglycemia. Four dogs received an insulin infusion to produce glucose levels less than 50 mg/dl for 45 minutes. In response to this, the delivery of glucagon to the liver increased from 36.7 +/- 5.9 ng/min in the baseline to 322.6 +/- 6.3 ng/min during hypoglycemia. Hepatic glucagon uptake increased proportionally from 13.6 +/- 7.2 ng/min to 103.1 +/- 28.3 ng/min and the percentage of delivered hormone that was extracted did not change (30.8 +/- 13.8% vs 32.9 +/- 11.6%). The absolute amount of glucagon metabolized by the liver was dependent on the rate of delivery and was not directly affected by plasma glucose level per se. To directly study the effect of hypoglycemia on hepatic glucagon metabolism, five dogs were given an exogenous infusion of somatostatin followed by an infusion of glucagon and then administered insulin to produce hypoglycemia. The percent of glucagon extracted by the liver (19.5 +/- 4.9% and 21.3 +/- 6.4%) was not affected by a fall in the plasma glucose level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct observation (NMR) of the efficacy of glucagon receptor antagonists in murine liver expressing the human glucagon receptor

The demonstration of pharmacodynamic efficacy of novel chemical entities represents a formidable challenge in the early exploration of synthetic lead classes. Here, we demonstrate a technique to validate the biological efficacy of novel antagonists of the human glucagon receptor (hGCGR) in the surgically removed perfused liver prior to the optimization of the pharmacokinetic properties of the compounds. The technique involves the direct observation by (13)C NMR of the biosynthesis of [(13)C]glycogen from [(13)C]pyruvate via the gluconeogenic pathway. The rapid breakdown of [(13)C]glycogen (glycogenolysis) following the addition of 50 pM exogenous glucagon is then monitored in real time in the perfused liver by (13)C NMR. The concentration-dependent inhibition of glucagon-mediated glycogenolysis is demonstrated for both the peptidyl glucagon receptor antagonist 1 and structurally diverse synthetic antagonists 2-7. Perfused livers were obtained from a transgenic mouse strain that exclusively expresses the functional human glucagon receptor, conferring human relevance to the activity observed with glucagon receptor antagonists. This technique does not provide adequate quantitative precision for the comparative ranking of active compounds, but does afford physiological evidence of efficacy in the early development of a chemical series of antagonists.     

Potent, orally absorbed glucagon receptor antagonists

The SAR of 2-pyridyl-3,5-diaryl pyrroles, ligands of the human glucagon receptor and inhibitors of p38 kinase, were investigated. This effort resulted in the identification of 2-(4-pyridyl)-5-(4-chlorophenyl)-3-(5-bromo-2-propyloxyphenyl)pyrr ole 49 (L-168,049), a potent (Kb = 25 nM), selective antagonist of glucagon.     

Dominant role of glucagon in the initial induction of phosphoenolpyruvate carboxykinase mRNA in cultured hepatocytes from fetal rats.

The injection of streptozotocin to 18-day-old rat fetuses induced, 2 days later, a 50% fall in plasma insulin and a twofold increase in plasma glucagon concentrations and liver cAMP levels. Phosphoenolpyruvate carboxykinase mRNA that were undetectable in the fetal rat liver, accumulated 48 h after streptozotocin injection, their concentration being 30% of that found in the liver of 1-day-old newborn rats in whom liver phosphoenolpyruvate carboxykinase gene expression is maximal. Physiological concentrations of glucagon (0.7 +/- 0.2 nM) induced, within 2 h, phosphoenolpyruvate carboxykinase mRNA accumulation in cultured hepatocytes from 20-day-old fetuses. The addition of insulin (0.01-100 nM) inhibits, by no more than 30%, the glucagon-induced phosphoenolpyruvate carboxykinase mRNA accumulation. Exposure of fetal hepatocytes to insulin for 24 h did not change the glucagon dose/response curve and did not lead to a more efficient inhibition of the glucagon-induced phosphoenolpyruvate carboxykinase mRNA accumulation, despite a clear stimulatory effect on the rate of lipogenesis. In contrast, when hepatocytes were cultured in the presence of dexamethasone, the glucagon-induced phosphoenolpyruvate carboxykinase mRNA accumulation can be totally inhibited by pharmacological concentrations of insulin (10 nM). From these in-vivo and in-vitro studies, it is concluded that, under physiological conditions, the postnatal rise in plasma glucagon concentration is more important than the fall in the plasma insulin concentration for the primary induction of liver phosphoenolpyruvate carboxykinase gene expression.     

Guanine nucleotide regulation of the interconversion of the two-state hepatic glucagon receptor system of rat

To investigate whether guanine nucleotides regulate interconversion of the two-state hepatic glucagon receptor we have utilized kinetic assays of glucagon binding to partially purified rat liver plasma membranes. Dissociation of glucagon at 30 degrees C exhibited biexponential character in either the absence or presence of GTP, indicating that the system previously seen in intact hepatocytes is independent of intracellular modulators. In each case the receptors underwent a time-dependent conversion from a low affinity to a high affinity state. However, GTP decreased the fraction of receptors in the high affinity state. The rank order for stabilizing the low affinity state was Gpp(NH)p greater than GTP greater than GDP much greater than GMP = no nucleotides. Data from competition binding assays with increasing concentrations of GTP allow calculation of equilibrium constants which are 3.32 nM for glucagon and receptor in the absence of GTP, 18.6 nM for glucagon and receptor in the presence of GTP, 1.55 microM for the association of receptor and GTP presumably linked to an N protein, and 8.86 microM for the association of the glucagon-receptor complex and GTP again presumably linked to an N protein, Glucagon binding to receptor is noncooperative in both the absence and presence of GTP, distinguishing this system from the beta-adrenergic system. With GTP, binding to the low affinity state is favored because of the relative affinities reported. Therefore, GTP regulates the activation by slowing the conversion of the receptor from a low affinity to high affinity form.     

Structure-function relationships of S-carboxymethyl methionine27 glucagon

Carboxymethylation of glucagon and subsequent purification of the hormone has provided a derivative modified by the addition of bulk to the methionine at position 27 without a net charge alteration in the side chain. Unreacted glucagon was removed after methylation of the methionine which provides a positively charged chromatographic handle. The derivative has a half-maximum concentration for binding of 5.3 nM and is a full agonist. These findings along with those provided by methylation of the methionine indicate that a positive charge rather than bulk on the methionine side chain disrupts the binding of hormone to its receptor. The S-carboxymethyl derivative lacks the concentration-dependent aggregation characteristic of glucagon at pH 10.2 as does the S-methyl derivative but increases its helical content in 30% 2-chloroethanol to the same extent as native and S-methyl hormone. Full activity of the S-carboxymethyl methionine27 glucagon does not favor the existence of the globular structure proposed by Korn and Ottensmeyer [(1983) J. Theor. Biol. 105, 403] as the binding species whereas multiple considerations do favor a flexible hormone with nucleation followed by conformational changes for complete binding and activation. Isotopic enrichment using labeled iodoacetate is feasible and can provide more definitive structural information.