Regulator of G-protein signaling Gbeta5-R7 is a crucial activator of muscarinic M3 receptor-stimulated insulin secretion

抄録

Background. Muscarinic cholinergic receptor M3 (M3R) stimulates glucose-induced insulin secretion in pancreatic beta cells. RGS (regulator of G-protein signaling) proteins are essential components of GPCR pathways, but they have not been systematically investigated in the pancreas. Here, we focused on the role of the R7 subfamily of RGS proteins in beta cells. A unique feature of R7 proteins is that they form obligatory heterodimers with the atypical G-protein subunit Gbeta5. This interaction is essential for stability of the Gbeta5-R7 complex, so that the knockout of the Gnb5 gene results in degradation of all R7 subunits.

Methods. Experiments were done in vivo on Gnb5-/- mice, iex vivo on isolated pancreatic islets, and iin vitro on insulin-secreting cell line MIN6 where we inactivated Gnb5 by CRISPR/Cas9 and/or overexpressed the Gbeta5-R7 complex. Insulin secretion was measured using ELISA. Second messengers cAMP, Ca²⁺ and DAG were measured by real time fluorescence microscopy using novel biosensors and fura2. Protein phosphorylation was examined using western blot and mass-spectrometry, and gene expression was studied by in situ mRNA hybridization using RNAScope technology (image: expression of M3R (Chrm3) and Gnb5 genes in mouse islet).

Results. Gnb5 knockout in mice or MIN6 cells almost completely eliminated insulinotropic activity of M3R. In contrast, overexpression of Gbeta5-RGS7 promoted M3R-stimulated insulin secretion. This positive effect is surprising because RGS proteins are known as negative regulators of GPCR signaling, and so the knockout should have augmented M3R-stimulated insulin release. Indeed, Gnb5 knockout enhances M3R signaling in another biological system, the pupilary sphincter muscle. 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. There was no reduction in the amplitude of free Ca²⁺ transients induced by cholinergic agonist 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.

Conclusions. Gbeta5-R7 is essential for coupling of M3R to insulin secretion through a mechanism involving protein kinase network.