Phosphorylation and desensitization of G protein-coupled receptors : Regulation by G protein betagamma subunits.

毒蕈碱乙酰胆碱受体m2亚型（m2受体）被G蛋白偶联受体激酶（GRK）以激动剂依赖的方式磷酸化。我们已经在m2受体的细胞内环（I3）的中心部分定位了磷酸化位点。该区域的7 ~ 8个丝氨酸残基和3 ~ 4个苏氨酸残基被磷酸化。发现GRK对I3-GST（谷胱甘肽s -转移酶）融合蛋白的磷酸化受到G蛋白β - γ亚基和乳突蛋白的协同刺激。此外，发现在m2或i3缺失的m2受体存在时，氨甲酰胆碱会刺激I3-GST的磷酸化。这些结果表明，激动剂结合的m2受体既是GRK的底物，也是激活剂。在体外纯化蛋白重建系统中，m2受体与G蛋白、Gi或Go的相互作用不受GRK磷酸化m2受体或m2受体I3缺失的影响。在COS7细胞中表达的m2受体的体内激动剂依赖性磷酸化被共表达的GRK2刺激，并被共表达的显性GRK2阴性形式抑制。此外，发现GRK2促进了m2受体的隔离，而GRK2的显性阴性形式则减弱了m2受体的隔离。

Muscarinic acetylcholine receptor m2 subtypes (m2 receptors) are phosphorylated by G protein-coupled receptor kinase (GRK) in an agonist-dependent manner. We have located the phosphorylation sites in the central part of intracellular loop (I3) of m2 receptors. Seven to eight serine and three to four threonine residues in this region were phosphorylated. The phosphorylation of I3-GST (glutathione S-transferase) fusion protein by GRK was found to be synergistically stimulated by G protein betagamma subunits and mastoparan. In addition, the phosphorylation of I3-GST was found to be stimulated by carbamylcholine in the presence of m2 or I3-deleted m2 receptors. These results indicate that the agonist-bound m2 receptors serve both as substrates and activators of GRK.The interaction of m2 receptors with G proteins, Gi or Go, in in vitro reconstitution system of purified proteins was not affected by phosphorylation of m2 receptors by GRK or by deletion of I3 from m2 receptors. Agonist-dependent phosphorylation in vivo of m2 receptors expressed in COS7 cells was stimulated by coexpressed GRK2 and was inhibited by coexpressed dominant negative form of GRK2. In addition, the sequestration of m2 receptors was found to be facilitated by GRK2 and attenuated by dominant negative form of GRK2.