The C-terminal regions of the GLP-1 and GIP receptors are not the key determinants of their differential arrestin recruitment but modulate the rate of receptor endocytosis

Abstract

Introduction: Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are important regulators of metabolism and mediate the incretin effect. This glucose-dependent potentiation of insulin secretion is severely impaired in patients with type-2 diabetes mellitus. While pharmacological doses of GLP-1 can overcome this impairment, the same is not true for GIP. The reasons for this are unclear. However, differences in the signalling profiles of the GLP-1 and GIP receptors (GLP-1R and GIPR) may contribute. GLP-1R and GIPR are closely related G protein-coupled receptors but differ in their ability to recruit arrestin, GIPR being relatively poorer. Furthermore, these receptors have been reported to utilize different mechanisms to undergo agonist-induced internalization. Methods: This study aimed to identify the role of the C-terminal region of the two receptors in their differing signalling behaviour using chimeric receptors where the C-terminal tail of one receptor was replaced with that of the other. Results: Replacement of the C-terminal tail had only limited effects on G protein and arrestin recruitment to either receptor. GIP-stimulated internalisation of GIPR occurred at a significantly (P < 0.001) slower rate than GLP-1-stimulated internalisation of GLP-1R. Replacement of the C-terminal tail of GIPR with that of GLP-1R significantly (P < 0.05) increased the internalization rate but not to the rate of wild-type GLP-1R. The reciprocal substitution significantly (P < 0.005) decreased internalization rate. Conclusion: These data show that the C-terminal region of GLP-1R and GIPR is not the critical determinant of their differing ability to recruit arrestin but modulates receptor endocytosis.

Keywords: G protein-coupled receptor; arrestin; endocytosis; glucagon-like polypeptide-1; glucose-dependent insulinotropic polypeptide.

FIGURE 1

Wild-type and chimeric receptors used in this study. (A) Sequence alignment of the C-terminal regions of GLP-1R and GIPR. Potential phosphorylation sites are shown in bold. Sequence immediately following transmembrane helix 7 was substituted to generate the chimeric receptors. (B) Schematic representation of the wild-type and chimeric receptors used in this study.

FIGURE 2

The replacement of C-terminal region does not impact G-protein recruitment. (A) Concentration-dependent Venus-mG_αs recruitment to NLuc-labeled wild-type or chimeric GLP-1R stimulated by GLP-1. (B) Schematic representation of the chimeric receptors, BRET pairs, and ligands used in (A). (C) Concentration-dependent Venus-mG_αs recruitment to NLuc-labeled wild-type or chimeric GIPR stimulated by GIP. (D) Schematic representation of the chimeric receptors, BRET pairs, and ligands used in (C). (E) Concentration-dependent Venus-mG_αq recruitment to NLuc-labeled wild-type or chimeric GLP-1R stimulated by GLP-1. (F) Schematic representation of the chimeric receptors, BRET pairs, and ligands used in (E). (G) Concentration-dependent Venus-mG_αq recruitment to NLuc-labeled wild-type or chimeric GIPR stimulated by GIP. (H) Schematic representation of the chimeric receptors, BRET pairs, and ligands used in (G) Data represent the mean ± S.E.M displayed as error bars, from at least three independent experiments, each performed in triplicate.

FIGURE 3

The replacement of the C-terminal region of GIPR with that of GLP-1R does not improve arrestin recruitment to GIPR. (A) Concentration-dependent Arr3–NLuc recruitment to SYFP2-labeled wild-type or chimeric receptors stimulated by GLP-1 or GIP. (B) Schematic representation of the chimeric receptors, BRET pairs, and ligands used in (A). (C) Concentration-dependent Arr3–SYFP2 recruitment to RLuc8-labeled wild-type or chimeric receptors stimulated by GLP-1 or GIP. (D) Schematic representation of the chimeric receptors, BRET pairs, and ligands used in (C). Data represent the mean ± S.E.M displayed as error bars, from at least three independent experiments, each performed in triplicate. *P < 0.05. (E) HEK-293 cells were transiently transfected with Arr3–SYFP2 and NLuc-labeled receptors. Confocal images were captured immediately before and 15 min after treatment with 1 μM GLP-1 (for GLP-1R and GLP-1/GIPR) or GIP (for GIPR and GIP/GLP-1R). The images are representative of at least three independent experiments. Scale bar: 5 μm.

FIGURE 4

Cell surface expression of wild-type and chimeric GLP-1R and GIPR (A). Representative live cell images of HEK-293 cells transiently co-transfected with plasma membrane-targeted mCherry-CAAX (red) and SYFP2-labeled receptor (yellow) visualized by confocal microscopy. GLP-1R-SYFP2 and GLP-1/GIPR-SYFP2 appear to be expressed primarily at the plasma membrane, while GIPR-SYFP2 and GIP/GLP-1R-SYFP2 are found not only at the plasma membrane but also in the cytosol. (B) The exchange of the C-terminal region neither affected the surface expression of GLP-1/GIPR nor enhanced the surface expression of GIP/GLP-1R compared to the corresponding wild-type receptor. The mean ± S.E.M displayed as error bars, from at least three independent experiments, and the images are representative of at least three independent experiments. Scale bar: 5 μm.

FIGURE 5

The replacement of the C-terminal region of GIPR with that of GLP-1R increases the rate of agonist-stimulated receptor endocytosis, and the reciprocal substitution decreased the rate of endocytosis. Receptor endocytosis was assessed as a loss of BRET between RLuc8-labeled receptor and Venus-KRAS expressed in HEK-293 cells. GLP-1R and GLP-1/GIPR were stimulated with 1 μM GLP-1, whereas GIPR and GIP/GLP-1R were stimulated with 1 μM GIP. (A) Loss of the BRET signal over time between Venus-KRAS and RLuc8-labeled wild-type and chimeric receptors. (B) Extent of receptor endocytosis expressed as an area under the curve. The replacement of GLP-1R’s C-terminal tail with that of GIPR significantly (P < 0.0001) inhibited receptor endocytosis. In contrast, the reciprocal substitution significantly (P < 0.01) enhanced receptor endocytosis. (C) Rate of receptor endocytosis. GIP-stimulated internalization of GIPR occurred at a significantly (P < 0.01) slower rate than GLP-1-stimulated internalization of GLP-1R. The replacement of the C-terminal tail of GIPR with that of GLP-1R significantly (P < 0.05) increased the rate of internalization, but not to the rate of wild-type GLP-1R. Conversely, the replacement of GLP-1R’s C-terminal tail with that of GIPR significantly (P < 0.001) decreased the rate of internalization. (D) Schematic representation of the chimeric receptors, BRET pairs, and ligands used in (A–C). Data represent the mean ± S.E.M displayed as error bars, from at least three independent experiments, each performed in triplicate.