Departments of Pharmacology and Physiology and University of Rochester School of Medicine and Dentistry, Rochester, New York 14642.
Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Geert Grooteplein 28, 6525 GA Nijmegen, The Netherlands.
J Biol Chem. 2014 Apr 18;289(16):11206-11218. doi: 10.1074/jbc.M113.538546. Epub 2014 Mar 4.
Phospholipase Cβ (PLCβ) enzymes are activated by G protein-coupled receptors through receptor-catalyzed guanine nucleotide exchange on Gαβγ heterotrimers containing Gq family G proteins. Here we report evidence for a direct interaction between M3 muscarinic receptor (M3R) and PLCβ3. Both expressed and endogenous M3R interacted with PLCβ in coimmunoprecipitation experiments. Stimulation of M3R with carbachol significantly increased this association. Expression of M3R in CHO cells promoted plasma membrane localization of YFP-PLCβ3. Deletion of the PLCβ3 C terminus or deletion of the PLCβ3 PDZ ligand inhibited coimmunoprecipitation with M3R and M3R-dependent PLCβ3 plasma membrane localization. Purified PLCβ3 bound directly to glutathione S-transferase (GST)-fused M3R intracellular loops 2 and 3 (M3Ri2 and M3Ri3) as well as M3R C terminus (M3R/H8-CT). PLCβ3 binding to M3Ri3 was inhibited when the PDZ ligand was removed. In assays using reconstituted purified components in vitro, M3Ri2, M3Ri3, and M3R/H8-CT potentiated Gαq-dependent but not Gβγ-dependent PLCβ3 activation. Disruption of key residues in M3Ri3N and of the PDZ ligand in PLCβ3 inhibited M3Ri3-mediated potentiation. We propose that the M3 muscarinic receptor maximizes the efficiency of PLCβ3 signaling beyond its canonical role as a guanine nucleotide exchange factor for Gα.
磷酸脂酶 Cβ（PLCβ）酶通过 G 蛋白偶联受体被 G 蛋白三聚体中的 G 蛋白偶联受体催化的鸟嘌呤核苷酸交换激活，G 蛋白三聚体含有 Gq 家族 G 蛋白。在这里，我们报告了 M3 毒蕈碱受体（M3R）和 PLCβ3 之间直接相互作用的证据。表达和内源性 M3R 在免疫沉淀实验中与 PLCβ相互作用。用卡巴胆碱刺激 M3R 显著增加了这种关联。在 CHO 细胞中表达 M3R 促进了 YFP-PLCβ3 的质膜定位。PLCβ3 的 C 端缺失或 PLCβ3 的 PDZ 配体缺失抑制了与 M3R 的共免疫沉淀和 M3R 依赖性 PLCβ3 质膜定位。纯化的 PLCβ3 直接与谷胱甘肽 S-转移酶（GST）融合的 M3R 细胞内环 2 和 3（M3Ri2 和 M3Ri3）以及 M3R C 末端（M3R/H8-CT）结合。当 PDZ 配体被去除时，PLCβ3 与 M3Ri3 的结合被抑制。在体外使用重组纯化成分的测定中，M3Ri2、M3Ri3 和 M3R/H8-CT 增强了 Gαq 依赖性但不依赖于 Gβγ 的 PLCβ3 激活。M3Ri3N 中的关键残基和 PLCβ3 中的 PDZ 配体的破坏抑制了 M3Ri3 介导的增强作用。我们提出，M3 毒蕈碱受体使 PLCβ3 信号转导的效率最大化，超出了其作为 Gα 的鸟嘌呤核苷酸交换因子的典型作用。
