Willets J M, Challiss R A, Kelly E, Nahorski S R
Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK.
Mol Pharmacol. 2001 Aug;60(2):321-30. doi: 10.1124/mol.60.2.321.
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.
我们研究了G蛋白偶联受体激酶（GRK）3和GRK6对SH-SY5Y细胞内源性表达的M3型毒蕈碱型乙酰胆碱受体（mACh）磷酸化及调控的影响。在添加高浓度乙酰甲胆碱（100μM，1分钟）后，GRK3或GRK6的过表达增强了M3 mACh受体的磷酸化。然而，GRK6的作用更强，即使在低浓度（3μM，1分钟）激动剂刺激后也能增加受体磷酸化。与表达等量M3 mACh受体的质粒转染对照细胞相比，过表达GRK3或GRK6的细胞表现出磷脂酶C活性降低，这表现为总[3H]肌醇磷酸和Ins(1,4,5)P3量的积累减少。此外，在过表达GRK3而非GRK6的细胞中，直接刺激G蛋白激活磷脂酶C（通过AlF4(-)）受到抑制。鸟苷-5'-O-(3-[35S]硫代)三磷酸结合及Gα(q/11)的免疫沉淀表明，急性乙酰甲胆碱刺激的受体/Gα(q/11)偶联不受GRK过表达的影响。相反，细胞用激动剂预处理3分钟会导致M3 mACh受体与Gα(q/11)解偶联，GRK6过表达会显著增强这种解偶联，尤其是在较低的激动剂预处理浓度下。然而，在过表达GRK3的克隆中观察到的M3 mACh受体磷酸化增加并未伴随着受体-Gα(q/11)解偶联增加。总体而言，这些数据表明GRK3和GRK6通过不同途径使M3 mACh受体脱敏。GRK6似乎作为一种经典的GRK发挥作用，诱导受体磷酸化增加并伴有受体与Gα(q/11)解偶联。相反，GRK3可能独立于受体磷酸化引起脱敏，可能是通过Gβγ结合和/或通过其G蛋白信号调节域直接结合Gα(q)来抑制磷脂酶C活性。
