Rapid communication
A human glucagon-like peptide-1 receptor polymorphism results in reduced agonist responsiveness

https://doi.org/10.1016/j.regpep.2005.05.001Get rights and content

Abstract

Glucagon-like peptide-1 (GLP-1) and its cognate receptor play an important physiological role in maintaining blood glucose homeostasis. A GLP-1 receptor (GLP-1R) polymorphism in which threonine 149 is substituted with a methionine residue has been recently identified in a patient with type 2 diabetes but was not found in non-diabetic control subjects. We have functionally assessed the recombinant GLP-1R variant after transient expression in COS-7 and HEK 293 cells. Compared to the wild type receptor, the variant GLP-1R showed (i) similar expression levels, (ii) 60-and 5-fold reduced binding affinities, respectively, for two GLP-1R full agonists, GLP-1 and exendin-4, and (iii) markedly decreased potencies of these peptides in triggering cAMP-mediated signaling (despite conserved efficacies). In contrast to full agonists, the efficacy of the primary GLP-1 metabolite/GLP-1R partial agonist, GLP-1 (9-36) amide, was essentially abolished by the T149M substitution. By hydropathy analysis, the polymorphism localizes to transmembrane domain 1, suggesting this receptor segment as a novel determinant of agonist affinity/efficacy. These findings reveal that naturally occurring sequence variability of the GLP-1R within the human population can result in substantial loss-of-function. A genetic link between the T149M variant and increased susceptibility to type 2 diabetes remains to be established.

Introduction

Glucagon-like peptide-1 (GLP-1, 7–36 amide) is an enteroendocrine hormone that plays an important physiological role in maintaining blood glucose homeostasis [1], [2]. This hormone is produced by L-cells primarily localized in the ileal/colonic mucosa. Following food ingestion, GLP-1 is secreted into the circulation and acts on multiple target tissues to attenuate the postprandial increase in blood glucose levels. In the endocrine pancreas, GLP-1 enhances glucose-induced insulin secretion as well as stimulates the growth of insulin-producing beta cells [3], [4]. This peptide also has additional peripheral and central functions, including delay of gastric emptying and induction of satiety [5].

GLP-1 acts via a cognate G-protein-coupled receptor (GLP-1R) that falls within the class B subfamily. Stimulation of the receptor with endogenous agonist (GLP-1) triggers cAMP production as the primary signal transduction pathway [6]. Given the physiological importance of GLP-1, derivatives of this peptide which extend its extremely short biological half life provide a promising new class of drugs for the treatment of diabetes [7], [8]. One of these stable peptides, exendin-4, was identified as a naturally occurring receptor agonist that was isolated from the salivary gland of the lizard reptile, Heloderma suspectum [9]. As a complementary strategy to enhance GLP-1R activity, drugs are also under development which inhibit the rapid enzymatic degradation of endogenously produced GLP-1 [10].

In addition to drug discovery efforts, studies of the GLP-1R have been directed at identifying polymorphisms in the GLP-1R gene that may be a contributing factor in the pathogenesis of diabetes. Although conclusive evidence for this link remains to be established, it is of note that a recent study of 791 Japanese individuals with type 2 diabetes identified a patient with a heterozygous GLP-1R missense polymorphism that resulted in substitution of threonine 149 by methionine (T149M). In contrast to several other GLP-1R polymorphisms that were found in this study [11], the T149M substitution was not observed in a control population of 318 non-diabetic individuals, and did not correspond to any sequence variation that occurs in known mammalian homologues of this receptor.

In the current study, we demonstrate that the T149M substitution, when introduced into the human GLP-1R by recombinant DNA techniques, causes a significant loss of function vs. the wild type protein. This finding warrants further investigation, to address the question whether this receptor polymorphism plays a role in the development of diabetes.

Section snippets

Generation of mutant GLP-1R cDNA

Experiments were performed using the human GLP-1 receptor cDNA in the expression vector pcDNA1 [12], [13]. The T149M substitution was introduced using a quick-change mutagenesis kit (Stratagene, La Jolla, CA). Dideoxynucleotide sequencing confirmed that the deduced amino acid sequence of the protein coding region was otherwise identical to the published receptor cDNA (Swiss-Prot Database, P43220).

Functional characterization of the recombinant GLP-1R

COS-7 cells, which lack endogenous GLP-1Rs, were transiently transfected with receptor cDNA using

Results

Properties of the T149M variant and the wild type receptor were compared using several well-established GLP-1R peptide ligands (Fig. 1). Among these, GLP-1 and exendin-4 are known full GLP-1R agonists, whereas the amino-terminally truncated derivative exendin (9-39) has been used as a pharmacological antagonist of this receptor [12], [16]. GLP-1 (9-36)amide is the primary endogenous metabolite of GLP-1, resulting from rapid enzymatic cleavage of its two amino-terminal residues [10]. GLP-1

Discussion

The T149M substitution in the human GLP-1R induces a marked decrease in the affinity and potency of both GLP-1 and exendin-4. In contrast, this polymorphism does not alter the affinity of corresponding amino-terminally truncated peptides, GLP-1 (9-36)amide and exendin (9-39) (see Fig. 1 for sequence comparisons). It thus appears that ligand sensitivity to the GLP-1R polymorphism depends on an intact amino terminus (of GLP-1 or exendin-4). As a possible explanation for this requirement, one may

Acknowledgement

The excellent technical assistance by Ci Chen is greatly appreciated. This work was supported by grants from the National Institutes of Health, National Institute of Diabetes, Digestive and Kidney Diseases (DK56674, DK46767 and P30 DK34928).

References (27)

  • V. Behar et al.

    Photoaffinity cross-linking identifies differences in the interactions of an agonist and an antagonist with the parathyroid hormone/parathyroid hormone-related protein receptor

    J Biol Chem

    (2000)
  • T.J. Kieffer et al.

    The glucagon-like peptides

    Endocr Rev

    (1999)
  • D.J. Drucker

    Glucagon-like peptides

    Diabetes

    (1998)
  • Cited by (83)

    • GLP-1 receptor agonists in the treatment of type 2 diabetes – state-of-the-art

      2021, Molecular Metabolism
      Citation Excerpt :

      Along these lines, certain polymorphisms regarding the TCF7L2 gene (probably involved in determining β cell mass and the expression of GLP-1 receptors) impair insulin responses to exogenous GLP-1 [216]. One study described a modification of the in vitro effects of GLP-1 RAs for the GLP-1 receptor variant T149M (methionine instead of threonine in position 149) on β cells [217]. However, a preliminary clinical study did not describe differences in pharmacological effects in response to short-term treatment with exenatide [218].

    View all citing articles on Scopus
    View full text