G protein-coupled receptor kinases 3 and 6 use different pathways to desensitize the endogenous M-3 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 ace tylcholine receptor (mACh) endogenously expressed in SH-SY5Y cells. Overexp ression of GRK3 or GRK6 enhanced M-3 mACh receptor phosphorylation after hi gh-concentration methacholine (100 muM, 1 min) addition. However, GRK6 was more potent, increasing receptor phosphorylation even after low (3 muM, 1 m in) agonist stimulation. Compared with plasmid-transfected control cells ex pressing equivalent M-3 mACh receptor number, GRK3- or GRK6-overexpressing cells exhibited a reduced phospholipase C activity reflected by a lower acc umulation of total [H-3]inositol phosphates and Ins(1,4,5)P-3 mass. In addi tion, 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 alph a (q/11) indicated that acute methacholine-stimulated receptor/G alpha (q/1 1) coupling was unaffected by GRK overexpression. In contrast, agonist pret reatment of cells for 3 min caused M-3 mACh receptor uncoupling from G alph a (q/11), which was markedly enhanced by GRK6 overexpression, particularly at lower agonist pretreatment concentrations. However, the increased M-3 mA Ch receptor phosphorylation seen in clones overexpressing GRK3 was not acco mpanied by increased receptor-G alpha (q/11) uncoupling. Overall, these dat a suggest that GRK3 and GRK6 use different pathways to desensitize the M3 m ACh receptor. GRK6 seems to act as a classical GRK, inducing increased rece ptor 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 phosp holipase C activity.