G protein-coupled receptor kinases 3 and 6 use different pathways to desensitize the endogenous M3 muscarinic acetylcholine receptor in human SH-SY5Y cells

We have investigated the effects of G protein-coupled receptor kinase (GRK) 3 and GRK6 on the phosphorylation and regulation of the M-3 muscarinic acetylcholine receptor (mACh) endogenously expressed in SH-SY5Y cells. Overexpression of GRK3 or GRK6 enhanced M-3 mACh receptor phosphorylation after high-concentration methacholine (100 muM, 1 min) addition. However, GRK6 was more potent, increasing receptor phosphorylation even after low (3 muM, 1 min) agonist stimulation. Compared with plasmid-transfected control cells expressing equivalent M-3 mACh receptor number, GRK3- or GRK6-overexpressing cells exhibited a reduced phospholipase C activity reflected by a lower accumulation of total [H-3]inositol phosphates and Ins(1,4,5)P-3 mass. In addition, direct stimulation of G protein activation of phospholipase C (by AIF(4)(-)) was inhibited in GRK3- but not GRK6-overexpressing cells. Guanosine-5'-O-(3- [S-35]thio)triphosphate binding and immunoprecipitation of G alpha (q/11) indicated that acute methacholine-stimulated receptor/G alpha (q/11) coupling was unaffected by GRK overexpression. In contrast, agonist pretreatment of cells for 3 min caused M-3 mACh receptor uncoupling from G alpha (q/11), which was markedly enhanced by GRK6 overexpression, particularly at lower agonist pretreatment concentrations. However, the increased M-3 mACh receptor phosphorylation seen in clones overexpressing GRK3 was not accompanied by increased receptor-G alpha (q/11) uncoupling. Overall, these data suggest that GRK3 and GRK6 use different pathways to desensitize the M3 mACh receptor. GRK6 seems to act as a classical GRK, inducing increased receptor phosphorylation accompanied by an uncoupling of receptor and G alpha (q/11). Conversely, GRK3 may cause desensitization independently of receptor phosphorylation, possibly via G beta gamma binding and/or direct G alpha (q) binding via its regulator of G protein signaling domain to inhibit phospholipase C activity.