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

G 蛋白偶联受体的磷酸化和脱敏：G 蛋白 betaamma 亚基的调节。

• 批准号: 05454665
• 负责人: HAGA Tatsuya
• 金额: $ 4.16万
• 依托单位: UNIVERSITY OF TOKYO
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05454665/
• 关键词: muscarinic receptor; acetylcholine receptor; G protein; phosphorylation; desensitization; G protein-coupled receptor; G protein-coupled receptor kinase; sequestration; 受容体; レセプター; アセチルコリン; ムスカリン; 受容体キナーゼ; BARK; 神経伝達物質; キナーゼ

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.

毒蕈碱型乙酰胆碱受体m2亚型（m2受体）被G蛋白偶联受体激酶（GRK）以激动剂依赖性方式磷酸化。我们已经定位的磷酸化位点的中央部分的胞内环（I3）的m2受体。该区域的7 ~ 8个丝氨酸和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）融合蛋白的磷酸化作用被G蛋白β-γ亚基和乳霜菌素协同刺激。此外，I3-GST的磷酸化被发现刺激氨甲酰胆碱在存在的m2或I3-缺失的m2受体。这些结果表明，激动剂结合的m2受体既是GRK的底物，又是GRK的激活剂，在纯化蛋白的体外重组系统中，m2受体与G蛋白Gi或Go的相互作用不受GRK磷酸化m2受体或缺失m2受体上的I3的影响。在COS 7细胞中表达的M2受体在体内的激动剂依赖性磷酸化被共表达的GRK 2刺激，并被共表达的GRK 2的显性负性形式抑制。此外，发现GRK 2促进了M2受体的隔离，并通过GRK 2的显性负性形式减弱。

项目成果

Tsuga,H.: "Sequestration of muscarinic acetylcholine receptor m2 subtypes" J.Biol.Chem.269. 32522-32527 (1994)

Tsuga,H.：“毒蕈碱乙酰胆碱受体 m2 亚型的隔离”J.Biol.Chem.269。

芳賀和子: "Synergistic activation of a G protein-coupled receptor kinase by G protein beta gamma subunits and mastoparan-or-related peptides." J.Biol.Chem. 269. 12594-12599 (1994)

Kazuko Haga：“G 蛋白 β γ 亚基和 Mastoparan 或相关肽对 G 蛋白偶联受体激酶的协同激活。J.Biol.Chem。269。12594-12599 (1994)

亀山仁彦: "Activation of G protein and G protein-coupled receptor kinase(βARK1)by a muscarinic receptor m2 mutant lacking phosphorylation sites." Eun J.Biochem.226. 267-276 (1994)

Hitoshi Kameyama：“缺乏磷酸化位点的毒蕈碱受体 m2 突变体激活 G 蛋白和 G 蛋白偶联受体激酶 (βARK1)。”Eun J.Biochem.267-276 (1994)。

T.Haga: "G protein-coupled receptor kinases" Journal of Neuro chemistry. (in press).

T.Haga：“G 蛋白偶联受体激酶”神经化学杂志。

Kameyama,K: "Activation of G protein and G protein-coupled receptor kinase(βARK1)by a muscarinic receptor m2 mutant lacking phosphorylation sites." Eur.J.Biochem.226. 267-276 (1994)

Kameyama, K：“缺乏磷酸化位点的毒蕈碱受体 m2 突变体对 G 蛋白和 G 蛋白偶联受体激酶 (βARK1) 的激活。Eur.J.Biochem.226 (1994)。