Endogenous G protein-coupled receptor kinase 6 regulates M3 muscarinic acetylcholine receptor phosphorylation and desensitization in human SH-SY5Y neuroblastoma cells

We have previously shown that overexpression of G protein-coupled receptor kinase 6 (GRK6) enhanced the phosphorylation and desensitization of the endogenously expressed M, muscarinic acetylcholine (mACh) receptor in human SH-SY5Y neuroblastoma cells. In this study we have examined the potential role of endogenous GRK6 in the regulation of M-3 mACh receptor by blocking its action through the introduction of a kinase-dead, dominant-negative GRK6 ((K215R)GPK6). (K215R)GRK6 expression inhibited methacholine-stimulated M-3 mACh receptor phosphorylation by 50% compared with plasmid transfected control cells. Guanosine-5'-O-(3-[S-35]thio)triphosphate binding and immunoprecipitation studies, conducted after agonist pretreatment (3 min), indicated that M-3 mACh receptor-Galpha(q/11), uncoupling was attenuated by 50% in cells expressing (K215R)GRK6 when compared with control cells. In contrast, expression of the related dominant-negative kinase (K215R)GRK5 had no effect on M-3 mACh receptor phosphorylation or uncoupling. Time course studies also showed that agonist-stimulated [H-3]inositol phosphate accumulations were more sustained in cells expressing (K215R)GRK6 compared with control and (K215R)GRK5-expressing cells, whereas (K215R)GRK6 expression had no effect on the phospholipase C response to direct stimulation of G proteins with AlF4-. The ability of (K215R)GRK6 to inhibit agonist-mediated M-3 mACh receptor phosphorylation and G protein uncoupling suggests that endogenous GRK6 mediates, at least in part, M-3 mACh receptor desensitization in the SH-SY5Y cell line.