G Protein‐Coupled Receptor Kinase 6 (GRK6) Regulation of Insulin Processing and Secretion

We performed exome sequencing in Type 2 Diabetes (T2D) patients belonging to a T2D family containing two affected and five unaffected individuals revealing an autosomal dominant inheritance pattern. A mutation in the GRK6 gene, coding for G protein-coupled receptor kinase 6, was identified that segregates with the phenotype. GRK6 belongs to a family of serine/threonine kinases that phosphorylate agonist occupied G protein-coupled receptors effectively modulating downstream signaling pathways including those involved in metabolism. These receptors like the Glucagon-like peptide-1 receptor play a critical role in the regulation of insulin processing and secretion and are the target of several approved therapeutics. Therefore, we wanted to determine if GRK6 regulates insulin dynamics potentially contributing to T2D. To start, we performed biochemical assays to unveil the properties of the novel GRK6 mutant. The GRK6 mutant was able to phosphorylate all substrates more effectively than wild type GRK6 but displayed a cytosolic distribution not expected for GRK6. We then utilized pharmacological inhibition and genetic knockdown of GRK6 in MIN6 cells to determine how removal of GRK6 impacts insulin levels after glucose treatment. In MIN6 cells, small molecule inhibition of GRK6 increased insulin secretion but reduced insulin processing. GRK6 knockdown revealed these same processing defects as GRK6 suppression in knockdown cell lines had reduced levels of cellular insulin. Furthermore, these cell lines had attenuated insulin secretion but enhanced proinsulin secretion consistent with decreased processing and the patient phenotype. The altered insulin profile appears to be caused by changes in the proprotein convertases, the enzymes responsible for proinsulin to insulin conversion, as pharmacological inhibition and knockdown resulted in less expression and activity of these enzymes. To support this, we performed rescue experiments where GRK6 re-expression in knockdown cells restored insulin secretion after glucose treatment. Taken together, our data show that GRK6 regulates insulin processing and secretion in a glucose dependent manner and provides a foundation for the contribution of GRK6 to T2D.