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A High-content In Vitro Pancreatic Islet β-cell Replication Discovery Platform
Published on: July 16, 2016
A Novel GLP1 Receptor Interacting Protein ATP6ap2 Regulates Insulin Secretion in Pancreatic Beta Cells
Feihan F Dai1, Alpana Bhattacharjee1, Ying Liu1
1From the Departments of Physiology and Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
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.
Insights
Researchers identified ATP6ap2 as a novel protein interacting with the GLP1 receptor (GLP1R) in beta cells. ATP6ap2 modulates GLP1R signaling and insulin processing, impacting insulin secretion and cAMP accumulation.
Area of Science:
- Endocrinology
- Molecular Cell Biology
- Diabetes Research
Background:
- Glucagon-like peptide-1 (GLP1) receptor (GLP1R) activation enhances insulin secretion, but its signaling complex involves largely unknown accessory proteins.
- Understanding GLP1R interactors is crucial for elucidating insulin secretion mechanisms.
Purpose of the Study:
- To identify novel proteins interacting with GLP1R in mouse and human islets.
- To investigate the functional role of identified interactors, specifically ATP6ap2, in beta cell function and insulin secretion.
Main Methods:
- Membrane-based split ubiquitin yeast two-hybrid assays were employed to screen for GLP1R interactors in mouse and human islets.
- Co-immunoprecipitation confirmed direct interaction between GLP1R and ATP6ap2 in beta cells.
- siRNA knockdown was used to assess the functional impact of ATP6ap2 on insulin secretion, cAMP accumulation, and calcium influx.
Main Results:
- ATP6ap2 (ATPase H(+)-transporting lysosomal accessory protein 2) was identified as a novel GLP1R interactor in both mouse and human islets and is abundant in alpha and beta cells.
- ATP6ap2 knockdown in beta cells decreased glucose-stimulated and GLP1-induced insulin secretion, accompanied by reduced cAMP accumulation and impaired GLP1R-mediated calcium influx.
- While ATP6ap2 knockdown impaired insulin granule acidification, it paradoxically increased proinsulin secretion, highlighting a dual role.
Conclusions:
- ATP6ap2 is a novel GLP1R-interacting protein with a significant role in beta cell function.
- ATP6ap2 modulates GLP1R signaling pathways, including cAMP production and calcium influx, thereby influencing insulin secretion.
- ATP6ap2 plays a dual role in beta cells, affecting both GLP1R signaling and insulin granule processing, which collectively impacts insulin secretion.
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