Clayton Cecilea C, Donthamsetti Prashant, Lambert Nevin A, Javitch Jonathan A, Neve Kim A
the Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, Oregon 97239.
the Departments of Psychiatry and Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York 10032, the Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York 10032, and.
J Biol Chem. 2014 Nov 28;289(48):33663-75. doi: 10.1074/jbc.M114.605378. Epub 2014 Oct 21.
Arrestins mediate desensitization and internalization of G protein-coupled receptors and also direct receptor signaling toward heterotrimeric G protein-independent signaling pathways. We previously identified a four-residue segment (residues 212-215) of the dopamine D2 receptor that is necessary for arrestin binding in an in vitro heterologous expression system but that also impairs receptor expression. We now describe the characterization of additional mutations at that arrestin binding site in the third intracellular loop. Mutating two (residues 214 and 215) or three (residues 213-215) of the four residues to alanine partially decreased agonist-induced recruitment of arrestin3 without altering activation of a G protein. Arrestin-dependent receptor internalization, which requires arrestin binding to β2-adaptin (the β2 subunit of the clathrin-associated adaptor protein AP2) and clathrin, was disproportionately affected by the three-residue mutation, with no agonist-induced internalization observed even in the presence of overexpressed arrestin or G protein-coupled receptor kinase 2. The disjunction between arrestin recruitment and internalization could not be explained by alterations in the time course of the receptor-arrestin interaction, the recruitment of G protein-coupled receptor kinase 2, or the receptor-induced interaction between arrestin and β2-adaptin, suggesting that the mutation impairs a property of the internalization complex that has not yet been identified.
抑制蛋白介导G蛋白偶联受体的脱敏和内化，还将受体信号导向异源三聚体G蛋白非依赖性信号通路。我们之前在体外异源表达系统中鉴定出多巴胺D2受体的一个四残基片段（第212 - 215位残基），它是抑制蛋白结合所必需的，但也会损害受体表达。我们现在描述了第三细胞内环中该抑制蛋白结合位点其他突变的特征。将这四个残基中的两个（第214和215位残基）或三个（第213 - 215位残基）突变为丙氨酸，部分降低了激动剂诱导的抑制蛋白3募集，而不改变G蛋白的激活。依赖抑制蛋白的受体内化需要抑制蛋白与β2 -衔接蛋白（网格蛋白相关衔接蛋白AP2的β2亚基）和网格蛋白结合，三残基突变对其影响尤为显著，即使在过表达抑制蛋白或G蛋白偶联受体激酶2的情况下，也未观察到激动剂诱导的内化。抑制蛋白募集与内化之间的脱节无法通过受体 - 抑制蛋白相互作用的时间进程、G蛋白偶联受体激酶2的募集或抑制蛋白与β2 -衔接蛋白之间的受体诱导相互作用的改变来解释，这表明该突变损害了尚未确定的内化复合物的一种特性。
