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. 2010 Jan;332(1):274-80.
doi: 10.1124/jpet.109.160531. Epub 2009 Oct 19.

Pharmacological Characterization of Human Incretin Receptor Missense Variants

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Free PMC article

Pharmacological Characterization of Human Incretin Receptor Missense Variants

Jean-Philippe Fortin et al. J Pharmacol Exp Ther. .
Free PMC article

Abstract

Glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are gut-derived incretin hormones that regulate blood glucose levels. In addition to their widely accepted insulinotropic role, there is evidence that GLP-1 modulates feeding behavior and GIP regulates lipid metabolism, thereby promoting postprandial fat deposition. In this study, we investigated whether naturally occurring polymorphisms in the GLP-1 receptor (GLP-1R) and the GIP receptor (GIP-R) affect the pharmacological properties of these proteins. After transient expression of the receptors in human embryonic kidney 293 cells, basal and ligand-induced cAMP production were assessed by use of luciferase reporter gene assays. Our data reveal that the wild-type GIP-R displays a considerable degree of ligand-independent activity. In comparison, the GIP-R variants C46S, G198C, R316L, and E354Q show a marked decrease in basal signaling that may, at least in part, be explained by reduced cell surface expression. When stimulated with GIP, the C46S and R316L mutants display significantly reduced potency (>1000 and 25- fold, respectively) compared with wild type. Complementary competition binding assays further demonstrate that the C46S variant fails to bind radio-iodinated GIP, whereas all other GIP-R mutants maintain normal ligand affinity. In contrast to the GIP-R, the wild-type GLP-1R lacks constitutive activity. Furthermore, none of the 10 GLP-1R missense mutations showed an alteration in pharmacological properties versus wild type. The extent to which abnormalities in GIP-R function may lead to physiological changes or affect drug sensitivity in selected populations (e.g., obese, diabetic individuals) remains to be further investigated.

Figures

Fig. 1.
Fig. 1.
Localization of the GLP-1R and GIP-R missense mutations within the receptor protein. Residues flanking the GIPR epitope tag are also shown. Schematic illustration of the location of amino acid substitutions within the 7-transmembrane domain structure of the human GLP-1R (A) and GIP-R (B). Respective residues in the wild-type proteins are indicated by the single letter code.
Fig. 2.
Fig. 2.
All GLP-1R variants show a pharmacological response to GLP-1 and exendin-4 that is similar to wild type. HEK293 cells were transiently transfected with a receptor-encoding cDNA and a CRE-Luc reporter gene construct. Forty-eight hours after transfection, cells were stimulated for 4 h with media containing either no peptide (basal) or increasing concentrations of GLP-1 (A) or exendin-4 (B). After stimulation, luciferase activity was quantified as described under Materials and Methods. All activity values were normalized relative to the GLP-1- or exendin-4-induced maximal stimulation (A or B) at the wild-type GLP-1R. Average values for basal and GLP-1/exendin-4-induced maximum luciferase activity were 3.51 ± 0.62 × 104 and 2.38 ± 0.34 × 106/2.34 ± 0.21 × 106 cps, respectively. Data represent the mean ± S.E.M. from at least three independent experiments, each performed in triplicate. Representative variants are shown.
Fig. 3.
Fig. 3.
Selected GIP-R mutations alter GIP-induced signaling. HEK293 cells were transiently transfected with the empty vector pcDNA1.1 or a receptor-encoding cDNA, together with a CRE-Luc reporter gene construct. Forty-eight hours after transfection, cells were stimulated for 6 h with media containing either no peptide (basal) or increasing concentrations of GIP. After stimulation, luciferase activity was quantified as described in Materials and Methods. All activity values were normalized relative to the GIP-stimulated maximum at the wild-type GIP-R. Average values for basal and GIP-induced maximum luciferase activity were 1.50 ± 0.22 × 106 and 5.62 ± 0.70 × 106 cps, respectively. Data represent the mean ± S.E.M. from at least three independent experiments, each performed in quadruplicate.
Fig. 4.
Fig. 4.
Effect of GIP-R mutations on GIP binding affinity. 125I-GIP radioligand binding with increasing concentrations of unlabeled GIP was evaluated in HEK293 cells transiently expressing either the wild-type or a mutant GIP-R. The cells were incubated in the presence of radioligand with indicated concentrations of unlabeled GIP for 8 h at 4°C. Data represent the mean ± S.E.M. from at least three independent experiments, each performed in quadruplicate.
Fig. 5.
Fig. 5.
Selected GIP-R missense mutations alter basal signaling and cell surface expression. A, basal activity of multiple GIP-Rs increases as a function of cDNA concentration. HEK 293 cells were transfected with increasing amounts of plasmid encoding either the wild-type or a mutant GIPR, together with a CRE-Luc reporter gene construct. After 48 h, ligand-independent luciferase activity was measured as described under Materials and Methods. B, cell surface expression of HA-tagged GIP-Rs increases as a function of cDNA concentration. HEK 293 cells were transfected with increasing amounts of plasmid encoding either the wild type or a mutant HA-tagged GIP-R. After 48 h, surface expression was measured by ELISA as described in Materials and Methods. C, surface expression (corresponding to data in B) and basal activity (corresponding to data in A) of wild-type and mutant GIPRs show a linear correlation. The slope of the correlation lines for most mutants approximates the wild-type value, with the exception of the G198C, R316L, and E354Q variants (see Table 2). Basal signaling and expression data are shown as a percentage of the maximal value observed at the wild-type GIP-R (transfection of 2 ng of cDNA/well). Each data point represents the mean ± S.E.M. from at least three independent experiments, each performed in triplicate.

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