We have investigated the effects of G protein-coupled receptor kinase (GRK) 3 and GRK6 on the phosphorylation and regulation of the M3 muscarinic acetylcholine receptor (mACh) endogenously expressed in SH-SY5Y cells. Overexpression of GRK3 or GRK6 enhanced M3 mACh receptor phosphorylation after high-concentration methacholine (100 microM, 1 min) addition. However, GRK6 was more potent, increasing receptor phosphorylation even after low (3 microM, 1 min) agonist stimulation. Compared with plasmid-transfected control cells expressing equivalent M3 mACh receptor number, GRK3- or GRK6-overexpressing cells exhibited a reduced phospholipase C activity reflected by a lower accumulation of total [3H]inositol phosphates and Ins(1,4,5)P3 mass. In addition, direct stimulation of G protein activation of phospholipase C (by AlF4(-)) was inhibited in GRK3- but not GRK6-overexpressing cells. Guanosine-5'-O-(3-[35S]thio)triphosphate binding and immunoprecipitation of Galpha(q/11) indicated that acute methacholine-stimulated receptor/Galpha(q/11) coupling was unaffected by GRK overexpression. In contrast, agonist pretreatment of cells for 3 min caused M3 mACh receptor uncoupling from Galpha(q/11), which was markedly enhanced by GRK6 overexpression, particularly at lower agonist pretreatment concentrations. However, the increased M3 mACh receptor phosphorylation seen in clones overexpressing GRK3 was not accompanied by increased receptor-Galpha(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 Galpha(q/11). Conversely, GRK3 may cause desensitization independently of receptor phosphorylation, possibly via Gbetagamma binding and/or direct Galpha(q) binding via its regulator of G protein signaling domain to inhibit phospholipase C activity.