β-arrestin and myosin VI cooperatively control glucose-dependent insulinotropic peptide receptor (GIPR) internalization and signaling dynamics

Figures

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  Figure 1. Myosin VI and β-arrestin dually regulate GIPR internalization.A, schematic representation of GIPR regulation by myosin VI and β-arrestin. GIPR C-tail has a PDZ-binding motif (PBM) that binds PDZ adaptor GIPC, which in turn recruits cytoskeletal motor myosin VI. GIPR also recruits β-arrestin through multiple phosphorylation sites in its C-tail. Δβ-arr indicates β-arrestin half knockdown in HEK293 cells. TIP is a chemical inhibitor of myosin VI activity. B, internalization of GIPR with N-terminal FLAG tag, visualized using anti-FLAG antibody and secondary AlexaFlour 546. HEK293 wild-type (WT) and Δβ-arr cells expressing GIPR were stimulated with GIP (1 μM) for 15 min at 37 °C. Cells were treated with TIP (100 μM) to block myosin VI activity. Scale bar, 10 μm. C and D, number of puncta/cell (C) and cytosol-to-cell surface intensity (D) for GIPR internalization in HEK293 WT or Δβ-arr cells under basal, GIP or GIP + TIP conditions. Data is represented as mean ± SD for four biological replicates. One-way ANOVA with Tukey’s post hoc test, ∗∗∗∗p < 0.0001, ∗∗p < 0.01.

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  Figure 2. Time-course of GIPR internalization. Live-cell imaging of GIPR in HEK293 WT or Δβ-arr cells. FLAG-tagged GIPR was labeled with anti-FLAG antibody and secondary AlexaFlour 546. Cells were stimulated with GIP ± TIP. Scale bar, 10 μm.

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  Figure 3. Myosin VI and β-arrestin regulate GIPR signaling.A and B, cAMP levels in HEK293 WT (A) or Δβ-arr cells (B) expressing GIPR and stimulated with GIP (1 μM) ± TIP (10 μM) for 15 min at room temperature. C and D, pERK signaling for GIPR expressed in HEK293 WT (C) or Δβ-arr (D), in response to GIP (1 μM) ± TIP (10 μM) for 15 min at room temperature. Mean ± SD for at least three biological replicates. Unpaired t test, ∗∗∗∗p < 0.001, ∗∗∗p < 0.001, ∗p < 0.05. E, schematic representation for regulation of GIPR signaling by myosin VI and β-arrestin. Inhibition of myosin VI results in elevated Gs signaling (cAMP production) from the plasma membrane. MAPK signaling (pERK) from endosomal compartments decreases with ablation of myosin VI activity or knockdown of β-arrestin.

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  Figure 4. GIPC recruitment to GIPR is independent of β-arrestin.A and B, merged images for colocalization of GIPR with GIPC or β-arr2 in HEK293 WT (A) or Δβ-arr cells (B), under basal condition or GIP stimulation ± TIP for 15 min at 37 °C. HEK293 cells expressing FLAG-GIPR with GIPC or β-arr2 were visualized by immunostaining the receptor with anti-FLAG antibody and secondary AlexaFlour 647. Insets display magnified view of the areas marked with dashed squares. Scale bar, 10 μm. Refer to Figure. S5 for images of the individual channels. C and D, colocalization analysis of GIPR to GIPC or β-arr2 in HEK293 WT (S) and Δβ-arr (D) cells under basal condition or GIP ± TIP stimulation. Signal to noise ratio above 1 indicates colocalization. Mean ± SD for at least three biological replicates. Two-way ANOVA with Sidak’s post hoc test, ∗∗∗∗p < 0.001, ∗∗∗p < 0.001, ∗p < 0.05.

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  Figure 5. GIPR internalization is dependent on PBM sequence and phosphorylation in the C-tail.A, schematic representation of GIPR variants used in internalization assay. GIPR ΔPBM was created by removing the last five residues of the C-tail. Phosphorylation sites highlighted in gray were mutated to alanine to generate GIPR phosphodeficient construct. B, representative images of HEK293 WT cells expressing GIPR WT, ΔPBM and phosphodeficient, under basal and GIP (1 μM) stimulation at 37 °C. Refer Figure. S7 for GIPR phosphomimetic data. For surface labeling of the receptor, cells were incubated with anti-FLAG antibody and AlexaFlour 546/647. Scale bar, 10 μm. C and D, number of puncta/cell (C) and ratio for cytosol-to-cell membrane intensity (D) for GIPR WT, ΔPBM, phosphodeficient under basal and GIP-stimulated cells. Data is represented as mean ± SD for three biological replicates. One-way ANOVA with Tukey’s post hoc test. ∗∗∗∗p < 0.0001, ∗∗p < 0.01. E, model schematic for regulation of GIPR internalization and signaling by cooperative engagement of GIPC-myosin VI and β-arrestin.

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  Figure 6. β-arrestin enhances GIPC engagement of the GIPR C-tail and myosin VI activity.A, schematic of bimolecular FRET experiment between mCer-GIPC and mCit-GIPR C-tail. Wild-type (wt), phosphodeficient (pd), and phosphomimetic (pm) versions of the GIPR C-tail were used (Fig. S9A). Purified β-arrestin (β-arr2) was included with GIPR pm condition. (B) FRET ratio (525/475 nm) for bimolecular FRET between mCer-GIPC (30 nM) and mCit-GIPR C-tail pm (300 nM) ± β-arr2 (300 nM). Data is represented as mean ± SD for three biological replicates. One-way ANOVA with Tukey’s post hoc test. ∗∗p < 0.01, ∗p < 0.05. C, schematic for surface motility assay to determine myosin VI velocity. D, myosin VI speed with GIPC (2 μM), GIPR C-tail pm (100 μM) and β-arr2 (1 μM). Data is represented as mean ± SD for three biological replicates. One-way ANOVA with Tukey’s post hoc test. ∗∗∗p < 0.001, ∗∗p < 0.01. Refer to Fig. S9 for FRET and motility data of GIPR C-tail WT and pn.

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  Figure 7. Myosin VI desensitizes incretin signaling at GIPR.A, schematic depiction of glucose-stimulated insulin secretion at GIPR. In the presence of glucose, GIP stimulation of GIPR leads to cAMP production, resulting in insulin secretion from pancreatic beta cells. B, cAMP response in INS cells, under GIP stimulation ± TIP for 15 min. Unpaired t test. ∗p < 0.05. C, glucose-dependent insulin secretion by INS cells, in response to GIP stimulation ± TIP for 30 min at 2.5 mM (G2.5) or 11 mM glucose (G11) at 37 °C. Data represented as fold-increase over the respective 2.5 mM glucose condition. Mean ± SD for three biological replicates. One-way ANOVA with Tukey’s post hoc test. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001.