Differential effects of glucose-dependent insulinotropic polypeptide receptor/glucagon-like peptide-1 receptor heteromerization on cell signaling when expressed in HEK-293 cells

Abstract

The incretin hormones: glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are important regulators of many aspects of metabolism including insulin secretion. Their receptors (GIPR and GLP-1R) are closely related members of the secretin class of G-protein-coupled receptors. As both receptors are expressed on pancreatic β-cells there is at least the hypothetical possibility that they may form heteromers. In the present study, we investigated GIPR/GLP-1R heteromerization and the impact of GIPR on GLP-1R-mediated signaling and vice versa in HEK-293 cells. Real-time fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) saturation experiments confirm that GLP-1R and GIPR form heteromers. Stimulation with 1 μM GLP-1 caused an increase in both FRET and BRET ratio, whereas stimulation with 1 μM GIP caused a decrease. The only other ligand tested to cause a significant change in BRET signal was the GLP-1 metabolite, GLP-1 (9-36). GIPR expression had no significant effect on mini-G_s recruitment to GLP-1R but significantly inhibited GLP-1 stimulated mini-G_q and arrestin recruitment. In contrast, the presence of GLP-1R improved GIP stimulated mini-G_s and mini-G_q recruitment to GIPR. These data support the hypothesis that GIPR and GLP-1R form heteromers with differential consequences on cell signaling.

Keywords: BRET4; FRET3; G-protein-coupled receptor1; GIP6; GLP-15; heteromerization2.

FIGURE 1

GLP‐1R and GIPR heteromerization. (A) BRET saturation experiments between GLP‐1R‐Nluc and GIPR‐SYFP2 in the absence of ligand (black) or presence of 1 μM GLP‐1 (red) or 1 μM GIP (blue). Data are pooled results from at least three independent experiments performed in triplicate. (B). Agonist‐induced changes in FRET between GLP‐1R‐mTurquoise and GIPR‐SYFP2. Perfusion with 1 μM GLP‐1 (black) resulted in an increase in the FRET signal, whereas treatment with 1 μM GIP (red) decreased the FRET signal. Similar results were obtained in cells transfected with GLP‐1R‐SYFP2 and GIPR‐mTurquoise. Traces are the mean ± SEM displayed as error bars of at least three independent experiments.

FIGURE 2

Agonist induced changes in BRET between GLP‐1R and GIPR. (A) HEK‐293 cells expressing a fixed ratio of GIPR‐SYFP2 and GLP‐1R‐Nluc were stimulated with 1 μM of one of the panel ligands shown. Stimulation GLP‐1 or GLP‐1 (9–36) resulted in a significant (p < .001) increase in BRET ratio, whereas stimulation with GIP resulted in a significant (p < .05) decrease in BRET ratio. The mean ± SEM is shown from at least three independent experiments. (B) Peptide ligands used in this study. X = aminoisobutyric acid. GLP‐1, Ex‐4(9 = 39), and GLP‐1 (9–36) are C‐terminally amidated.

FIGURE 3

Venus‐mG_s recruitment to GLP‐1R and GIPR. Venus‐mG_s recruitment to Nluc‐labeled receptor assessed by BRET. (A) Venus‐mG_s recruitment to either GLP‐1R‐Nluc or GIPR‐Nluc, stimulated by either GLP‐1 or GIP. (B) Maximum Venus‐mG_s recruitment to GLP‐1R‐Nluc or GIPR‐Nluc. (C) Venus‐mG_s recruitment to GLP‐1R‐Nluc in the presence of unlabeled GLP‐1R or GIPR stimulated by either GLP‐1 or GIP (D). Maximum Venus‐mG_s recruitment to GLP‐1R‐Nluc in the presence or absence of unlabeled GLP‐1R or GIPR. (E) Venus‐mG_s recruitment to GIPR‐Nluc in the presence of unlabeled GLP‐1R or GIPR stimulated by either GLP‐1 or GIP (F). Maximum Venus‐mG_s recruitment to GIPR‐Nluc in the presence or absence of unlabeled GLP‐1R or GIPR. BRET ratios were expressed as fold‐change over non‐stimulated. The mean ± SEM displayed as error bars, from at least three independent experiments.

FIGURE 4

Venus‐mG_q recruitment to GLP‐1R and GIPR. Venus‐mG_q recruitment to Nluc‐labeled receptor assessed by BRET. (A) Venus‐mG_q recruitment to either GLP‐1R‐Nluc or GIPR‐Nluc stimulated by either GLP‐1 or GIP. (B) Maximum Venus‐mG_q recruitment to GLP‐1R‐Nluc or GIPR‐Nluc. (C) Venus‐mG_q recruitment to GLP‐1R‐Nluc in the presence of unlabeled GLP‐1R or GIPR stimulated by either GLP‐1 or GIP (D). Maximum Venus‐mG_q recruitment to GLP‐1R‐Nluc in the presence or absence of unlabeled GLP‐1R or GIPR. (E) Venus‐mG_q recruitment to GIPR‐Nluc in the presence of unlabeled GLP‐1R or GIPR stimulated by either GLP‐1 or GIP (F). Maximum Venus‐mG_q recruitment to GIPR‐Nluc in the presence or absence of unlabeled GLP‐1R or GIPR. BRET ratios were expressed as fold‐change over non‐stimulated. The mean ± SEM displayed as error bars, from at least three independent experiments.

FIGURE 5

Visualization of the cellular location of SYFP2‐ and mTurquoise‐labeled receptors transiently expressed in HEK‐293 cells by confocal microscopy. (A) Representative live cell images of HEK‐293 cells transiently co‐transfected with plasma membrane‐targeted mCherry‐CAAX (red) and SYFP2‐labeled receptor (green) and mTurquoise‐labeled receptor (turquoise). GLP‐1R‐SYFP2 appears to be expressed primarily at the plasma membrane, whereas GIPR‐SYFP2 appears to be located not only at the plasma membrane but also in intracellular compartments. Co‐expression of GIPR‐mTurquoise had no effect on cell surface expression of GLP‐1R‐SYFP2 and GLP‐1R‐mTurquoise did not improve the expression of GIPR‐SYFP2 and the plasma membrane. The images are representative of at least three independent experiments. Scale bar = 5 μm. (B). Colocalization of the SYFP2‐labeled receptors with plasma membrane‐targeted mCherry. GLP‐1R‐SYFP2 colocalizes with membrane‐targeted mCherry to a significantly (****p < .001) greater extent than GIPR‐SYFP2. Co‐expression of GIPR‐mTurquoise had no significant effect on the colocalization of GLP‐1R‐SYFP2 with mCherry‐CAAX. The presence of GLP‐1R‐mTurquoise did not improve the colocalization of GIPR‐SYFP2 with mCherry‐CAAX. Data are the mean ± SEM from values measured in n = 19

FIGURE 6

Arrestin3 recruitment to GLP‐1R and GIPR. Arrestin3‐YFP recruitment to Nluc‐labeled receptor assessed by BRET. (A) Arrestin3‐YFP recruitment to either GLP‐1R‐Nluc or GIPR‐Nluc stimulated by either GLP‐1 or GIP. (B) Maximum Arrestin3‐YFP recruitment to GLP‐1R‐Nluc or GIPR‐Nluc. (C) Arrestin3‐YFP recruitment to GLP‐1R‐Nluc in the presence of unlabeled GLP‐1R or GIPR stimulated by either GLP‐1 or GIP (D). Maximum Arrestin3‐YFP recruitment to GLP‐1R‐Nluc in the presence or absence of unlabeled GLP‐1R or GIPR. BRET ratios were expressed as fold‐change over non‐stimulated. The mean ± SEM displayed as error bars, from at least three independent experiments.