A Novel GLP1 Receptor Interacting Protein ATP6ap2 Regulates Insulin Secretion in Pancreatic Beta Cells

Abstract

GLP1 activates its receptor, GLP1R, to enhance insulin secretion. The activation and transduction of GLP1R requires complex interactions with a host of accessory proteins, most of which remain largely unknown. In this study, we used membrane-based split ubiquitin yeast two-hybrid assays to identify novel GLP1R interactors in both mouse and human islets. Among these, ATP6ap2 (ATPase H(+)-transporting lysosomal accessory protein 2) was identified in both mouse and human islet screens. ATP6ap2 was shown to be abundant in islets including both alpha and beta cells. When GLP1R and ATP6ap2 were co-expressed in beta cells, GLP1R was shown to directly interact with ATP6ap2, as assessed by co-immunoprecipitation. In INS-1 cells, overexpression of ATP6ap2 did not affect insulin secretion; however, siRNA knockdown decreased both glucose-stimulated and GLP1-induced insulin secretion. Decreases in GLP1-induced insulin secretion were accompanied by attenuated GLP1 stimulated cAMP accumulation. Because ATP6ap2 is a subunit required for V-ATPase assembly of insulin granules, it has been reported to be involved in granule acidification. In accordance with this, we observed impaired insulin granule acidification upon ATP6ap2 knockdown but paradoxically increased proinsulin secretion. Importantly, as a GLP1R interactor, ATP6ap2 was required for GLP1-induced Ca(2+) influx, in part explaining decreased insulin secretion in ATP6ap2 knockdown cells. Taken together, our findings identify a group of proteins that interact with the GLP1R. We further show that one interactor, ATP6ap2, plays a novel dual role in beta cells, modulating both GLP1R signaling and insulin processing to affect insulin secretion.

Keywords: cyclic AMP (cAMP); insulin secretion; receptor; receptor-interacting protein (RIP); yeast two-hybrid.

FIGURE 1.

Membrane-based split ubiquitin yeast two-hybrid. Left panel, membrane-bound ubiquitin protein was split into two halves: C terminus (Cub) and N terminus (Nub, NubG is point mutation of Nub to avoid self-activation). Cub is associated with a TF that was fused to the bait GLP1R as GLP1R-Cub, and NubG was fused to the prey interactors as interactor-NubG. Right panel, if the bait interacts with the prey, the resulting proximity of the ubiquitin halves induced by the interaction will enable the reconstitution of Cub and NubG to form a functional pseudoubiquitin protein. Reconstitution recruits ubiquitin-specific proteases that cleave TF downstream of Cub, allowing the TF to translocate into the nucleus to initiate the transcription of reporter genes, which serve as readout of MYTH. As a result, the MYTH system does not rely on protein expression within the nucleus as does the traditional yeast two-hybrid system and can be used to study membrane-bound proteins such as GLP1R and its interactors.

FIGURE 2.

Interactor networks of GLP1R identified from human and mouse islet MYTH screens. A and B, mouse islet (A) and human islet (B) cDNA libraries. Each interactor is represented by a separate dot. Pink dots represent interactors identified in mouse islets, and blue dots represent interactors identified in human islets. Interactors common to both mouse and human islets are identified in red.

FIGURE 3.

The expression of selected interactors in MIN6 (A), INS1 832/3 cells (B), and mouse islets (C) presented as the percentage of β-actin in the cell. The values are represented by the averages ± S.E. from triplicates in three independent experiments.

FIGURE 4.

ATP6ap2 expression in the pancreas and islets. A, immunohistochemistry showing ATP6ap2 localization in the mouse pancreatic sections. The right panels show the enlarged images of the left panels. Bar, 100 μm. B, immunofluorescence showing ATP6ap2 localization in dispersed human islets. Bar, 20 μm. Representative images are from three independent experiments.

FIGURE 5.

ATP6ap2 expression and GSIS in human islets from normal and diabetic donors. A, representative images and quantitative analysis (quantification of fluorescence intensity and distribution of fluorescence intensity within cell population) of ATP6ap2 expression in human islets from normal and diabetic donors. Bar, 20 μm. B, ATP6ap2 expression in human dispersed islets from diabetic donors. Arrow indicates reduced ATP6ap2 expression in insulin-positive cells. Bar, 10 μm. Quantitative analysis of ATP6ap2 expression in both insulin- and glucagon-positive cells is shown (normal donors, n = 3; diabetic donors, n = 2). C, glucose-stimulated insulin secretion in human islets from diabetic donors.

FIGURE 6.

Effect of ATP6ap2 overexpression on insulin secretion and cAMP. A, overexpression of ATP6ap2, detected by anti-FLAG and anti-ATP6ap2. B, immunoprecipitation showing the interaction between GLP1R and ATP6ap2 in INS-1 cells. GLP1R overexpression alone was used as negative control to validate the specificity of immunoprecipitation. Representative images are from three independent experiments. C, effect of ATP6ap2 on glucose-stimulated insulin secretion in INS-1 cells. D, effect of ATP6ap2 overexpression on cAMP accumulation stimulated by GLP1 in CHO cells with GLP1R overexpressed.

FIGURE 7.

Effect of ATP6ap2 in INS-1 cells. A, qPCR showing the efficiency of siATP6ap2 in knocking down ATP6ap2 in INS-1 cells. B, effect of ATP6ap2 knockdown on insulin secretion in INS-1 cells. C, insulin content in INS-1 cells transfected with siATP6ap2. D, insulin secretion in INS-1 cells transfected with scrambled siRNA (Control) and siATP6ap2 at different glucose concentration. E, insulin secretion in cells transfected with siATP6ap2 at 11.5 mm glucose in the presence of incremental doses of GLP1. F, cAMP accumulation in INS-1 cells transfected with siATP6ap2 at different doses of GLP1. G, cAMP accumulation in siATP6ap2 transfected cells that were treated with forskolin, GIP, and GLP1, respectively. The values are represented by the averages ± S.E. from triplicates in at least three independent experiments. *, p < 0.05 (n = 4–6).

FIGURE 8.

Effect of knocking down ATP6ap2 on insulin granules. A, representative images of electron microscopy showing insulin granules in INS-1 cells transfected with siATP6ap2. The lower panel is shown in higher magnification. Bar, 500 nm. B, granule numbers in INS-1 cells transfected with siATP6ap2. C, percentages of dense (mature) and gray/empty (immature) granules in INS-1 cells transfected with siATP6ap2. Representative images are from at least three independent experiments. *, p < 0.05 (n = 3–5).

FIGURE 9.

Effect of knocking down ATP6ap2 on insulin granule acidification and proinsulin processing. A, ratio of proinsulin versus insulin in cells transfected with siATP6ap2. B, expression of PC1/3, PC2, and CPE in INS-1 cells transfected with siATP6ap2. The expression was presented as ratio over control. C, representative images and quantitative analysis of LysoTracker staining of INS1 cells transfected with scramble siRNA (control), siATP6ap2, and treated with bafilomycin A (10 nm). The values are represented by the averages ± S.E. from triplicates in three independent experiments. *, p < 0.05 (n = 3).

FIGURE 10.

Effect of ATP6ap2 knockdown on intracellular Ca²⁺ in dispersed mouse islets. A, representative images (5 min after GLP1stimulation) and quantitative analysis of Fluo4 in dispersed mouse islets transfected with scramble siRNA (control, upper panels) or siATP6ap2 (lower panels). Nuclear staining in blue and Fluo4 in green for merged images. B, qPCR showing the efficiency of ATP6ap2 knockdown in dispersed mouse islets. The values are represented by the averages ± S.E. from triplicates in three independent experiments. *, p < 0.05 (n = 3).