Abstract

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 μM, 1 min) addition. However, GRK6 was more potent, increasing receptor phosphorylation even after low (3 μM, 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 Gα_q/11 indicated that acute methacholine-stimulated receptor/Gα_q/11 coupling was unaffected by GRK overexpression. In contrast, agonist pretreatment of cells for 3 min caused M₃ mACh receptor uncoupling from Gα_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-Gα_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 Gα_q/11. Conversely, GRK3 may cause desensitization independently of receptor phosphorylation, possibly via Gβγ binding and/or direct Gα_q binding via its regulator of G protein signaling domain to inhibit phospholipase C activity.