Cloning and Expression of a Human Glucagon Receptor

doi:10.1006/bbrc.1994.1046

Biochemical and Biophysical Research Communications

Volume 198, Issue 1, 15 January 1994, Pages 328-334

https://doi.org/10.1006/bbrc.1994.1046 Get rights and content

Abstract

A human glucagon receptor has been cloned from human liver tissue. The 1578-bp cDNA clone encodes a protein of 477 amino acids with 82% identity to the rat glucagon receptor. The predicted secondary structure and homology to known proteins places this receptor within the superfamily of seven transmembrane domain G protein coupled receptors. Transfection of the human glucagon receptor into COS-7 cells confers upon them high affinity binding for [¹²⁵I] glucagon. In membranes prepared from COS-7 cells transfected with the human glucagon receptor, the binding of [¹²⁵I] glucagon is inhibited with the rank order of potency glucagon > oxyntomodulin > glucagon-like peptide 1 (7-36) amide ⪢ glucagon-like peptide 2 = gastric inhibitory peptide = secretin.

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Cited by (74)

Cardiovascular effects of GLP-1 receptor agonism
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Once released, GLP-1 rapidly circulates and is available to interact with its receptors in a number of target tissues, including the pancreas and brain. The receptor for GLP-1was cloned from a cDNA library derived from rat β-cells from pancreatic islets (Thorens, 1992), closely followed by the cloning of the human β-cell homolog (Thorens et al., 1993) and its cloning from a number of other sources (Dillon et al., 1993; Dillon, Wheeler, Leng, Ligon, & Boyd, 1997; Graziano, Hey, Borkowski, Chicchi, & Strader, 1993; Lankat-Buttgereit & Göke, 1997; Lankat-Buttgereit, Göke, Fehmann, Richter, & Göke, 1994; MacNeil, Occi, Hey, Strader, & Graziano, 1994; Thorens et al., 1993; Volz et al., 1995). Previous evidence had demonstrated that a primary effect of ligand binding to GLP-1R was activation of adenylate cyclase and production of cAMP (Drucker, Philippe, Mojsov, Chick, & Habener, 1987), and heterologous expression of cloned GLP-1Rs demonstrated high-affinity activation of adenylate cyclase by GLP-1.
Glucagon-like peptide-1 (GLP-1) receptor agonists are extensively used in type 2 diabetic patients for the effective control of hyperglycemia. It is now clear from outcomes trials that this class of drugs offers important additional benefits to these patients due to reducing the risk of developing major adverse cardiac events (MACE). This risk reduction is, in part, due to effective glycemic control in patients; however, the various outcomes trials, further validated by subsequent meta-analysis of the outcomes trials, suggest that the risk reduction in MACE is also dependent on glycemic-independent mechanisms operant in cardiovascular tissues. These glycemic-independent mechanisms are likely mediated by GLP-1 receptors found throughout the cardiovascular system and by the complex signaling cascades triggered by the binding of agonists to the G-protein coupled receptors. This heterogeneity of signaling pathways underlying different downstream effects of GLP-1 agonists, and the discovery of biased agonists favoring specific signaling pathways, may have import in the future treatment of MACE in these patients. We review the evidence supporting the glycemic-independent evidence for risk reduction of MACE by the GLP-1 receptor agonists and highlight the putative mechanisms underlying these benefits. We also comment on the different signaling pathways which appear important for mediating these effects.
Glucagon Receptor Antagonism Ameliorates Progression of Heart Failure
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Mice were treated with a fully human monoclonal glucagon receptor antagonistic antibody REMD2.59 following myocardial infarction or pressure overload. REMD2.59 treatment blunted cardiac hypertrophy and fibrotic remodeling, and attenuated contractile dysfunction at 4 weeks after myocardial infarction. In addition, REMD2.59 treatment at the onset of pressure overload significantly suppressed cardiac hypertrophy and chamber dilation with marked preservation of cardiac systolic and diastolic function. Initiation of REMD2.59 treatment 2 weeks after pressure overload significantly blunted the progression of cardiac pathology. These results provide the first in vivo proof-of-concept evidence that glucagon receptor antagonism is a potentially efficacious therapy to ameliorate both onset and progression of heart failure.
Targeting the glucagon receptor family for diabetes and obesity therapy
2012, Pharmacology and Therapeutics
Citation Excerpt :
Thus it will also be important to design glucagon antagonists that achieve the glucose-lowering benefits while avoiding the potential side effects of lipogenesis. The GCGR cDNA has been cloned in several species including rats (Jelinek et al., 1993; Svoboda et al., 1993), mice (Burcelin et al., 1995) and humans (Lok et al., 1994; MacNeil et al., 1994; Menzel et al., 1994). The human GCGR gene has a protein coding region spanning over 5.5 kb and containing 13 exons (Lok et al., 1994).
Diabetes is a debilitating disease characterized by chronic hyperglycemia and is often associated with obesity. With diabetes and obesity incidence on the rise, it is imperative to develop novel therapeutics that will not only lower blood glucose levels, but also combat the associated obesity. The G protein-coupled receptors (GPCRs) for glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1) and glucagon are emerging as targets to treat both hyperglycemia and obesity. GIP is rapidly released from intestinal K-cells following food intake and stimulates glucose-dependent insulin secretion from β-cells and the storage of fat in adipocytes. Both GIP receptor agonists and antagonists have been demonstrated to display therapeutic potential to treat diabetes and obesity. Similar to GIP, GLP-1 is released from intestinal L-cells following food intake and potentiates glucose-dependent insulin secretion from β-cells. In addition, GLP-1 reduces glucagon levels, suppresses gastric emptying and reduces food intake. As such, GLP-1 receptor agonists effectively lower blood glucose levels and reduce weight. Finally, glucagon is released from α-cells and raises blood glucose levels during the fasting state by stimulating gluconeogenesis and glycogenolysis in the liver. Thus, molecules that antagonize the glucagon receptor may be used to treat hyperglycemia. Given the structural similarity of these peptides and their receptors, molecules capable of agonizing or antagonizing combinations of these receptors have recently been suggested as even better therapeutics. Here we review the biology of GIP, GLP-1 and glucagon and examine the various therapeutic strategies to activate and antagonize the receptors of these peptides.
Glucagon-like peptide-1 inhibits insulinotropic effects of oxyntomodulin and glucagon in cattle
2012, Domestic Animal Endocrinology
Citation Excerpt :
These results suggested that GLP-1, which was coinjected in the present study, could not bind to the GLUR and, accordingly, suggested that the first-phase insulin responses to OXM and glucagon were mainly through GLP-1R. These considerations were supported by the previous findings, which showed that greater than 50 nM of GLP-1 was required to displace [125I] glucagon binding to GLUR [30] and that OXM and glucagon bound to GLP-1R with an IC50 of ∼1.5 and ∼15 nM, respectively [31]. Our findings that GLP-1 inhibited the first-phase insulin responses to OXM and glucagon were corroborated by the results that exendin-4 (5-39), a GLP-1 antagonist, inhibited the insulinotropic effects of OXM and glucagon (Fig. 4A and 4B).
Oxyntomodulin (OXM), glucagon, and glucagon-like peptide-1 (GLP-1), peptide hormones derived from the glucagon gene, play an important role in glucose homeostasis. The insulinotropic action of these three homologous peptides has been well documented in monogastric animals. However, information on the relationships among these peptides in insulin-releasing action, specifically in ruminants, is still insufficient. In this regard, we carried out two experiments in cattle. In experiment 1, effects of glucagon and GLP-1 on plasma insulin and glucose were investigated in 10-mo-old Holstein steers (347 ± 8 kg, n = 8) under normoglycemic conditions. Peptides were administered intravenously at dose rates of 0.12, 0.25, 0.50, and 1.25 nmol/kg body weight (BW). In experiment 2, the relationships among OXM, glucagon, and GLP-1 in the insulinotropic and glucoregulatory actions were elucidated in 3-mo-old Holstein steers (94 ± 2 kg, n = 8) using agonist-antagonist strategy. In agonist strategy, these three peptides were administered alone or coadministered at dose rates of 10 μg of OXM/kg BW, 4 μg of glucagon/kg BW, and 2 μg of GLP-1/kg BW. In antagonist strategy, 2 μg of each peptide was administered alone or in combination with 10 μg of [des His1, des Phe6, Glu9] glucagon amide (a glucagon receptor antagonist) or exendin-4 (5-39) amide (a GLP-1 receptor antagonist). Our results showed that OXM, glucagon, and GLP-1 had insulinotropic actions in ruminants under normoglycemic conditions. Our results also showed that the insulin-releasing effects of OXM and glucagon were mediated through both GLP-1 receptors (GLP-1R) and glucagon receptors. These insulinotropic effects of OXM and glucagon through GLP-1R were inhibited by GLP-1. Our findings expand the relationships among OXM, glucagon, and GLP-1 in the insulinotropic and glucoregulatory actions.
Molecular evolution of mammalian incretin hormone genes
2009, Regulatory Peptides
Incretin hormones are encoded by two different genes in the human genome, the proglucagon (GCG) and the glucose-dependent insulinotropic polypeptide (GIP) genes. To better understand the evolution of incretin hormones, as well as the potential for the evolution of species-specific functions for these peptides, we have identified and characterized the genes for these hormones from the genomes of 35 mammalian species, as well as from the genomes of a few non-mammalian vertebrates. Both proglucagon and GIP were found to be single-copy genes in mammals, and exist in stable genomic neighborhoods with conserved flanking gene order. The exon–intron structure of the genes has been conserved within mammals, although variation in the rate of protein sequence evolution for the peptide hormones was observed. Glucagon and GLP-1 sequences are largely invariant among mammals, except the glucagon sequences from hystricomorph rodents. Previous work has shown that the change in glucagon sequences in hystricomorph rodents is associated with the evolution of species-specific functions. GLP-2 sequences have evolved most rapidly, while GIP evolved at an intermediate rate, although both show punctuated rates with a common very rapid phase of evolution on the early mammalian lineage. These observations suggest that GIP and GLP-2 are more likely to have evolved species-specific functions in subgroups of mammals.
Cloning, tissue distribution, and functional characterization of chicken glucagon receptor
2008, Poultry Science
Citation Excerpt :
Moreover, 4 putative N-linked glycosylation sites (N-X-S or N-X-T, where X represents any AA except proline) are also found in the large extracellular domain (Figure 2). Alignment of chicken GCGR with other species shows that chicken GCGR shares high AA sequence identity with that of human (70%; MacNeil et al., 1994), rat (69%; Jelinek et al., 1993), mouse (69%; Burcelin et al., 1995), Xenopus (64%; Sivarajah et al., 2001), and a comparatively lower identity to goldfish (53%; Chow et al., 2004), with the greatest degree of identity observed in the 7 TMD. In addition, 6 cysteine residues critical for the formation of 3 intramolecular disulfide bonds in the extracellular domain, together with the Arg-Leu-Ala-Lys (RLAK) motif responsible for G-protein coupling in the third intracellular loop, are strictly positioned and conserved among all species (Unson, 2002).
Glucagon has been reported to play an important role in hepatic glucose metabolism of vertebrates including birds. However, the molecular mechanism of its actions in birds remains largely unknown. In present study, the full-length cDNA of chicken glucagon receptor (GCGR) was first cloned from brain tissue using reverse transcription-PCR. This putative chicken GCGR (named cGCGR-s) is 496 amino acids (AA) long and shares high AA sequence identity with that of human (70%), rat (69%), mouse (69%), and Xenopus (64%), and a comparatively lower identity with goldfish (53%). In addition, a full-length cDNA encoding a novel glucagon receptor variant (named cGCGR-v1) of 554 AA was identified in this study. Sequence analysis revealed that this receptor variant arises from the retention of intron 4 (174 bp) and thus causes a 58-AA insertion at the large N-terminal extracellular domain. Using the pGL3-CRE-luciferase reporter system, we demonstrated that human glucagon could potently activate chicken GCGR-s and GCGR-v1 expressed in Chinese hamster ovary cells, confirming that both cGCGR-s and cGCGR-v1 are functional and able to couple to the intracellular cyclic adenosine mono-phosphate-protein kinase A signaling pathway. Using a reverse transcription-PCR assay, we further examined the expression of glucagon receptor in adult chicken tissues, including different regions of the brain. Glucagon receptor was shown to be highly expressed in liver and moderately or weakly expressed in other tissues examined. In the central nervous system, the greatest expression was consistently detected in the hypothalamus. Taken together, our data not only suggest that glucagon receptor plays a critical role in mediating the actions of glucagon in liver, but also imply that glucagon may have important roles in nonhepatic tissues, such as in the hypothalamus of brain in chickens.

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Regular ArticleCloning and Expression of a Human Glucagon Receptor

Abstract

Regular Article
Cloning and Expression of a Human Glucagon Receptor