Regulator of G‐protein signaling complex Gβ5‐R7 strongly potentiates insulin secretion stimulated by muscarinic cholinergic receptor M3.
Authors: Slepak Vladlen Z. , Wang Qiang , Pronin Alexey 
Published: June 2021

Insulin secretion by pancreatic beta cells is regulated by glucose and other nutrients as well as hormonal and neurotransmitter inputs. Muscarinic receptor M3 (M3R) is responsible for cholinergic regulation of beta cells. In many signaling pathways regulated by G protein‐coupled receptors (GPCR) RGS (regulator of G‐protein signaling) proteins are responsible for timely signal shut‐off, and are referred to as G protein inhibitors. RGS proteins have not been extensively investigated in the pancreas, and in this study we investigated the R7 subfamily of RGS proteins in beta cells. It is established that R7 proteins form obligatory heterodimers with the unique G‐protein subunit Gβ5 (gene: Gnb5). This protein‐protein interaction is essential for stability of Gβ5‐R7 complexes, so that the knockout of the Gnb5 gene results in proteolityc degradation of all R7 subunits.

We performed a series of experiments in vivo using Gnb5−/− mice, ex vivo on isolated pancreatic islets, and in vitro on cell line MIN6. This cell line can secrete insulin in response to cholinergic stimulation. We inactivated Gnb5 in MIN6 cells using CRISPR/Cas9 approach and/or overexpressed the Gβ5‐RGS7 complex in wild‐type or the knockout cells. As the read‐out for the functional activity downstream of M3R we measured insulin secretion, analyzed the levels of cAMP, Ca2+ and DAG by real time fluorescence microscopy and studied protein phosphorylation. We also tested gene expression by RT‐PCR and in situ mRNA hybridization using RNAScope technology (Fig 1).

The main result of our studies is that the Gnb5 knockout in mice or MIN6 cells causes about 90% reduction in the ability of muscarinic agonists to stimulate glucose‐dependent insulin secretion in both isolated islets and MIN6 cells. These results are consistent with the approximately 50% reduction in serum insulin in the Gnb5−/− mice we reported previously. In contrast, overexpression of the Gβ5‐RGS7 complex in either wild‐type and Gnb5 knockout MIN6 cells promotes insulinotropic activity of M3R. The positive effect of Gβ5‐R7 on M3R function is unexpected because RGS proteins are generally known to be negative regulators of GPCR signaling. Like the knockout of RGS4 previously described by Wess and colleagues, the knockout of Gnb5 should have potentiated M3R‐stimulated insulin release. Interestingly, Gnb5 knockout enhances M3R signaling in another biological system endogenously expressing M3R and Gβ5‐R7, the sphincter muscle closing the eye pupil. Examination of the mechanism underlying the effect of the Gnb5 knockout in beta cells showed that cAMP and diacylglycerol responses to cholinergic stimulation were unaffected. We did not detect a significant change in the amplitude of free Ca2+ fluxes induced by cholinergic stimulation in islets from the Gnb5−/− mice, but frequency of Ca2+ oscillations was lowered by >30%. The knockout of Gnb5 in MIN6 cells strongly enhanced M3R‐stimulated phosphorylation of ERK1/2 and several substrates of protein kinase C, some of which we identified by a combination of western blot analysis and mass‐spectrometry.

Thus, Gβ5‐R7 is essential for coupling of M3R to insulin secretion through a mechanism involving protein kinase network. Our results also show the effect of Gβ5‐R7 on M3R is be opposite in different cell types.

Support or Funding Information

This work was supported, in part, by the National Institutes of Health Grants RO1DK105427 and RO1DK111538.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.