Photoaffinity labeling of the glucagon receptor with a new glucagon analog

The flash excitation of the pyridoxal 5'-phosphate cofactor of glycogen phosphorylase b by an ultraviolet laser produces a transient state from a proton transfer of the bound cofactor. The rate of decay of this transient state is sensitive to the ionization state of the cofactor. This proved a useful probe for the ionization state of the 5'-phosphate group of the cofactor on the binding by the enzyme of various substrates. The decay rate data show, for the binding of glucose 1-phosphate, a partially negative 5'-HPO4- and evidence for a PO4-PO4 interaction. The data is interpreted in terms of a dynamic shift of substrates at the active site.     

Biaryl amide glucagon receptor antagonists

Biaryl amides derived from a reported series of ureas 1 were evaluated and found to be potent human glucagon receptor antagonists. The benzofuran analogue 6i was administered in Sprague-Dawley rats and blocked the effects of an exogenous glucagon challenge.     

Partial purification and characterization of the glucagon receptor

Specific labeling of liver plasma membrane glucagon receptors has been achieved by the photoincorporation of a 125I-labeled photoderivative of glucagon, NE-4-azidophenylamidinoglucagon. Identification of glucagon receptors was facilitated by irradiating membranes in the presence of excess unlabeled glucagon. Isoelectric focusing of radioiodinated membrane proteins revealed one major band of glucagon displaceable material which had an isoelectric point of 5.85. When this material was isolated and run on SDS-polyacrylamide gels a major labeled band of Mr55000 was obtained which had properties consistent with those of the glucagon receptor. These studies indicate that a purification of the glucagon receptor of greater than 700-fold can be attained through the use of isoelectric focusing and SDS-polyacrylamide electrophoresis.     

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.     

Identification of the glucagon receptor by covalent labeling with a radiolabeled photoreactive glucagon analogue

The photoreactive 125I-labeled glucagon-NAPS [125I-labeled 2-[2-nitro-4-azidophenyl)sulfenyl]-Trp25-glucagon] was used to label the glucagon receptor sites in rat liver plasma membranes. The proteins labeled were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with or without reduction with dithiothreitol. The photoaffinity peptide specifically labeled a number of bands with apparent molecular weights greater than 200000 and probably at least two protein bands in the molecular weight range 52000-70000. The relative amounts of radioactivity associated with these bands and their relative mobilities differed in samples from reduced and unreduced membranes. Their relative mobilities also differed with percent acrylamide cross-linking, suggesting a glycoprotein nature and the presence of intramolecular disulfide bonds. A nonspecifically labeled band with an apparent molecular weight of 27000-28000 also displayed a similar behavior. Photolabeling in the presence of 0.1 mM guanosine 5'-triphosphate (GTP) decreased the amount of radiolabeling of these bands, suggesting their involvement in the glucagon stimulation of adenylate cyclase. The photolabeled receptor in the membranes, solubilized with Lubrol-PX and fractionated on an Ultrogel AcA22 column, eluted with an apparent molecular weight of 200000-250000. Addition of GTP to the solubilized glucagon receptor of nonirradiated membranes caused complete dissociation of the complex. Gel electrophoresis of the partially purified radiolabeled receptor identified the same protein components observed in photolabeled membranes. These results indicate that the glucagon receptor is an oligomer probably composed of at least two different subunits that are linked together or greatly stabilized by disulfide bonds. They also show that 125I-labeled glucagon-NAPS can be used effectively to covalently label the putative glucagon receptor and thus aid in its further characterization.     

The lipid environment of the glucagon receptor regulates adenylate cyclase activity.

1. The lipids composition of rat liver plasma membranes was substantially altered by introducing synthetic phosphatidylcholines into the membrane by the techniques of lipid substitution or lipid fusion. 40-60% of the total lipid pool in the modified membranes consisted of a synthetic phosphatidylcholine. 2. Lipid substitution, using cholate to equilibrate the lipid pools, resulted in the irreversible loss of a major part of the adenylate cyclase activity stimulated by F-, GMP-P(NH)P or glucagon. However, fusion with presonicated vesicles of the synethic phosphatidylcholines causes only small losses in adenylate cyclase activity stimulated by the same ligands. 3. The linear form of the Arrhenius plots of adenylate cyclase activity stimulated by F- or GMP-(NH)P was unaltered in all of the membrane preparations modified by substitution or fusion, with very similar activation energies to those observed with the native membrane. The activity of the enzyme therefore appears to be very insensitive to its lipid environment when stimulated by F- or gmp-p(nh)p. 4. in contrast, the break at 28.5 degrees C in the Arrhenius plot of adenylate cyclase activity stimulated by glucagon in the native membrane, was shifted upwards by dipalmitoyl phosphatidylcholine, downwards by dimyristoyl phosphatidylcholine, and was abolished by dioleoyl phosphatidylcholine. Very similar shifts in the break point were observed for stimulation by glucagon or des-His-glucagon in combination with F- or GMP-P(NH)P. The break temperatures and activation energies for adenylate cyclase activity were the same in complexes prepared with a phosphatidylcholine by fusion or substitution. 5. The breaks in the Arrhenius plots of adenylate cyclase activity are attributed to lipid phase separations which are shifted in the modified membranes according to the transition temperature of the synthetic phosphatidylcholine. Coupling the receptor to the enzyme by glucagon or des-His-glucagon renders the enzyme sensitive to the lipid environment of the receptor. Spin-label experiments support this interpretation and suggest that the lipid phase separation at 28.5 degrees C in the native membrane may only occur in one half of the bilayer.     

Molecular determinants of glucagon receptor signaling

A 29-amino acid polypeptide hormone, glucagon has been one of the most prolific models in the study of hormone action. The key biologic function of glucagon is to counterbalance the actions of insulin and maintain a normal level of serum glucose. Diabetes mellitus can thus be considered a bihormonal disorder with an excess of glucagon contributing to the hyperglycemic state. The effects of glucagon are mediated by the glucagon receptor, which is itself a prototypical member of a distinct category called family B receptors within the G protein-coupled superfamily of seven-helical transmembrane receptors (GPCRs). At the structural level, the peptide ligands of family B receptors are highly homologous, in particular in the N-terminal region of the molecules. The mechanism by which highly homologous peptide ligands selectively recognize their receptors involves distinct molecular interactions that are gradually being elucidated. This review focuses on structural determinants of the glucagon receptor that are important for its activity with respect to interaction with its ligand and G proteins. Information about the glucagon receptor is presented within the context of what is known about other members of the family B GPCRs.     

Fluorescent glucagon derivatives. II. The use of fluorescent glucagon derivatives for the study of receptor disposition in membranes

When monolayer cultured hepatocytes were incubated with 1 nM [125I]glucagon at 30 degrees C, equilibrium was reached after 10 min, whereas at 4 degrees C, equilibrium was reached after 60 min. At the higher temperature, 11.2% of the bound ligand was broken down after 60 min, at the lower temperature, the amount of degradation was negligible. At 30 degrees C, acid-washing did not remove specifically bound ligand; thus, it was assumed that the ligand was internalised at this temperature, since some of the specifically bound ligand could be washed off at lower temperatures. This was confirmed in experiments when monolayer cultures of hepatocytes were incubated with fluorescein-labelled derivatives of glucagon. The distribution of specific binding on the cell surface was studied at both 30 and 4 degrees C using video intensification microscopic techniques. In keeping with studies using radiolabelled glucagon, more fluorescence was detected following incubation at 4 degrees C than at 30 degrees C and it could be removed by washing the cells. Video intensification microscopy indicated that at the lower temperature, the bound ligand was distributed all over the cell surface. At the higher temperature, ligand-derived fluorescence could only be detected in mobile intracellular vesicles.     

The glucagon receptor is involved in mediating the body weight-lowering effects of oxyntomodulin

Oxyntomodulin (OXM) is a peptide secreted postprandially from the L-cells of the gut that has a weak affinity for both the glucagon-like peptide-1 receptor (GLP1R) and the glucagon receptor (GCGR). Peripheral administration of OXM in humans and rodents causes weight loss reducing food intake and increasing energy expenditure. It has been suggested that OXM modulates energy intake solely through GLP1R agonism. Because glucagon decreases food intake in rodents and humans, we examined whether activation of the GCGR is involved in the body weight-lowering effects of OXM. We identified an equipotent GLP1R-selective peptide agonist that differs from OXM by only one residue (Q3→E, OXMQ3E), but has no significant GCGR agonist activity in vitro and ~100-fold reduced ability to stimulate liver glycogenolysis. Chronic treatment of obese mice with OXM and OXMQ3E demonstrated that OXM exhibits superior weight loss and lipid-lowering efficacy, and antihyperglycemic activity that is comparable to the corresponding GLP1R-selective agonist. Studies in Glp1r(-/-) mice and coadministration of OXM and a GCGR antagonist revealed that the antiobesity effect of OXM requires activation of both GLP1R and GCGR. Our data provide new insight into the mechanism of action of OXM and suggest that activation of GCGR is involved in the body weight-lowering action of OXM.     

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.     

Purification of the photoaffinity-labeled glucagon receptor by gel electrophoretic methods

Rat liver plasma membrane glucagon receptor has been purified with a yield of 0.01% to an estimated homogeneity of 32-60%, using a 2-stage electrophoretic procedure. SDS-solubilized membrane proteins labeled by the photoaffinity-agent, Ne-4-azidophenylamidinoglucagon (APA-glucagon), were separated by polyacrylamide gel electrophoresis in SDS-containing buffers. Gel slices corresponding to the molecular weight of the receptor were excised, electrophoretically extracted and concentrated. The concentrate was subjected to isoelectric focusing on Sephadex to yield a purified product in which the photoaffinity-labeled receptor, with a molecular weight of 56K and a pI' of 5.9, is the sole major component.     

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.     

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.     

Negative allosteric modulation of the glucagon receptor by RAMP2

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