Cloning and Functional Expression of a Human Glucagon-like Peptide-1 Receptor

doi:10.1006/bbrc.1993.2226

Biochemical and Biophysical Research Communications

Volume 196, Issue 1, 15 October 1993, Pages 141-146

https://doi.org/10.1006/bbrc.1993.2226 Get rights and content

Abstract

A human glucagon-like 1 peptide receptor has been cloned from the gastric tumor cell line HGT-1. The cDNA clone encodes a protein of 463 amino acids and is a member of the superfamily of seven transmemhrane domain G protein coupled receptors. Transfection of the human GLP-1 receptor into COS-7 cells confers upon them high affinity binding for [¹²⁵I] GLP-1(7-36) amide. In membranes prepared from COS-7 cells transfected with the human GLP-1 receptor, the binding of [¹²⁵I] GLP-1 (7-36) amide is inhibited with the rank order of potency GLP-1 (7-36) amide > glucagon > secretin, characteristic of a GLP-1 receptor. The human GLP-1 receptor is functionally coupled to increases in intracellular cAMP in these cells: incubation of COS-7 cells expressing the human GLP-1 receptor with GLP-1 (7-36) amide gives rise to a 4-fold increase in cyclic AMP over basal levels, with an EC₅₀ of 25pM. Glucagon is also a full agonist but is 200-fold less potent than GLP-1 (7-36) amide in stimulating the human GLP-1 receptor.

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Cited by (54)

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.
Discovery and pharmacology of the covalent GLP-1 receptor (GLP-1R) allosteric modulator BETP: A novel tool to probe GLP-1R pharmacology
2020, Advances in Pharmacology
Citation Excerpt :
These include parenterally administered multi-functional agents (peptide-based GLP-1R agonists that also activate other receptors) and orally bioavailable non-peptide agonists or positive allosteric modulators that may enhance the actions of endogenous GLP-1R ligands (Mendez et al., 2019; Willard, Briere, & Sloop, 2018). The human GLP-1R cDNA was cloned in 1992 leading to the ability to generate cell lines heterologously expressing GLP-1R and allowing the pharmacology of peptide ligands to be determined unambiguously (Graziano, Hey, Borkowski, Chicchi, & Strader, 1993; Thorens, 1992). In this era, the ligand identification strategies used by the pharmaceutical industry were primarily rational design, natural products, and low capacity small molecule screening.
The glucagon-like peptide-1 receptor (GLP-1R) is a significant therapeutic target for small molecule drug discovery given the therapeutic impact of peptide agonists in the diabetes sphere. We review the discovery and subsequent characterization of the small molecule GLP-1R allosteric modulator 4-(3-(Benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine (BETP). BETP is a covalent modulator of the GLP-1R, and we discuss the pharmacological implications and possible structural basis of this novel mode of action. We highlight the insights into class B G-protein coupled receptor pharmacology and biology provided by studies conducted with BETP. These include the descriptions of exquisite allosteric modulator probe dependence and biased signaling in vitro and in vivo. We conclude with an analysis of the utility of BETP as a chemical probe for the GLP-1R.
Glucagon-like peptide 1 (GLP-1)
2019, Molecular Metabolism
Citation Excerpt :
Under physiological conditions, most studies report no meaningful cross-reactivity among the peptide ligands and the receptors of this family [359,360]. Cloning of rat and human pancreatic GLP-1R cDNA documented that ligand-induced activation of a single unique GLP-1R increases intracellular levels of cAMP and also that GLP-1, but not glucagon, GIP, VIP, or secretin, activates GLP-1R [360–364]. In the pancreas, glucagon has physiologically relevant cross-reactivity with GLP-1R, with an EC50 of 36.4 ± 0.22 nM, but there is no affinity of GLP-1 to the glucagon receptor [365].
The glucagon-like peptide-1 (GLP-1) is a multifaceted hormone with broad pharmacological potential. Among the numerous metabolic effects of GLP-1 are the glucose-dependent stimulation of insulin secretion, decrease of gastric emptying, inhibition of food intake, increase of natriuresis and diuresis, and modulation of rodent β-cell proliferation. GLP-1 also has cardio- and neuroprotective effects, decreases inflammation and apoptosis, and has implications for learning and memory, reward behavior, and palatability. Biochemically modified for enhanced potency and sustained action, GLP-1 receptor agonists are successfully in clinical use for the treatment of type-2 diabetes, and several GLP-1-based pharmacotherapies are in clinical evaluation for the treatment of obesity.
In this review, we provide a detailed overview on the multifaceted nature of GLP-1 and its pharmacology and discuss its therapeutic implications on various diseases.
Since its discovery, GLP-1 has emerged as a pleiotropic hormone with a myriad of metabolic functions that go well beyond its classical identification as an incretin hormone. The numerous beneficial effects of GLP-1 render this hormone an interesting candidate for the development of pharmacotherapies to treat obesity, diabetes, and neurodegenerative disorders
GLP-1: Molecular mechanisms and outcomes of a complex signaling system
2019, Neurochemistry International
Meal ingestion provokes the release of hormones and transmitters, which in turn regulate energy homeostasis and feeding behavior. One such hormone, glucagon-like peptide-1 (GLP-1), has received significant attention in the treatment of obesity and diabetes due to its potent incretin effect. In addition to the peripheral actions of GLP-1, this hormone is able to alter behavior through the modulation of multiple neural circuits. Recent work that focused on elucidating the mechanisms and outcomes of GLP-1 neuromodulation led to the discovery of an impressive array of GLP-1 actions. Here, we summarize the many levels at which the GLP-1 signal adapts to different systems, with the goal being to provide a background against which to guide future research.
Geniposide acutely stimulates insulin secretion in pancreatic β-cells by regulating GLP-1 receptor/cAMP signaling and ion channels
2016, Molecular and Cellular Endocrinology
Citation Excerpt :
So far, the GLP-1R peptide agonists, exenatide (exendin-4) and liraglutide, are widely approved medicines for the treatment of type 2 diabetes mellitus (Lovshin and Drucker, 2009). However, despite decades of research following the molecular identification and cloning of the GLP-1R (Graziano et al., 1993; Thorens, 1992), no orally available small molecule GLP-1R activator has been developed for therapeutic use. Our present study and others have shown that geniposide is not only a GLP-1R activator but also an orally-available small molecular compound (Kojima et al., 2011; Koo et al., 2006).
Geniposide, an iridoid glycoside, has antidiabetic effects. The present study aimed to evaluate whether geniposide has direct effects on insulin secretion from rat pancreatic islets. The results demonstrated that geniposide potentiated insulin secretion via activating the glucagon-like-1 receptor (GLP-1R) as well as the adenylyl cyclase (AC)/cAMP signaling pathway. Inhibition of protein kinase A (PKA) suppressed the insulinotropic effect of geniposide. Geniposide also inhibited voltage-dependent potassium (Kv) channels, and this effect could be attenuated by inhibition of GLP-1R or PKA. Current-clamp recording showed that geniposide prolonged action potential duration. These results collectively imply that inhibition of Kv channels is linked to geniposide-potentiated insulin secretion by acting downstream of the GLP-1R/cAMP/PKA signaling pathway. Moreover, activation of Ca²⁺ channels by geniposide was observed, indicating that the Ca²⁺ channel is also an important player in the geniposide effects. Together, these findings provide new insight into the mechanism underlying geniposide-regulated insulin secretion.
GLP-1 mimetic drugs and the risk of exocrine pancreatic disease: Cell and animal studies
2016, Pancreatology
Glucagon Like Peptide 1 (GLP-1) mimetic drugs or degradation inhibitors mimic the action of native GLP-1 as a incretin hormone and have become a common second line of therapy for Type 2 diabetes. However, an important clinical issue is whether these drugs increase the incidence of pancreatitis and pancreatic cancer.
This paper reviews the physiology of GLP-1 including its synthesis, secretion and action of the peptide. Reported effects of the mimetic drugs on the exocrine pancreas in animal studies are also reviewed.
GLP-1 is synthesized in a specific class of enteroendocrine cell, the L-cell, by post-translational processing of proglucagon. It is released in response to the presence of nutrients in the small intestine and stimulates vagal afferent nerve endings as well as entering the blood where it is rapidly degraded by dipeptidyl peptidase IV. Its actions are mediated by specific G-protein coupled receptors. The major target tissues are the pancreatic islet beta cells, the brain and the heart but GLP-1 also affects gastrointestinal motility and secretion including the exocrine pancreas where its major systemic action is to inhibit secretion. In some animal, as well as human studies, the GLP-1 mimetic drugs are associated with pancreatitis or precursor lessions to pancreatic cancer but a mechanism is not clear. The most common occurrence of pathology in rodents is when the drugs are combined with a high fat diet.
There is nothing in the physiology of GLP-1 or animal toxicology studies to support a mechanism of action or a major concern about the action of GLP-1 mimetic drugs on the exocrine pancreas. Further studies are warranted using animal models of disease and high fat diets.

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Regular ArticleCloning and Functional Expression of a Human Glucagon-like Peptide-1 Receptor

Abstract

Regular Article
Cloning and Functional Expression of a Human Glucagon-like Peptide-1 Receptor