Mutation of Three Residues in the Third Intracellular Loop of the Dopamine D2 Receptor Creates an Internalization-defective Receptor
Mutation of Three Residues in the Third Intracellular Loop of the Dopamine D2 Receptor Creates an Internalization-defective Receptor
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DOI：
10.1074/jbc.m114.605378
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发表时间：
2014-11-28
影响因子：
4.8
通讯作者：
Neve, Kim A.
中科院分区：
文献类型：
--
作者：
Clayton, Cecilea C.;Donthamsetti, Prashant;Neve, Kim A.
抑制蛋白介导G蛋白偶联受体的脱敏和内化，并将受体信号传导导向异源三聚体G蛋白非依赖性信号传导途径。我们以前确定了多巴胺D2受体的四个残基的片段（残基212-215），这是必需的arrestin结合在体外异源表达系统，但也损害receptorexpression。我们现在描述在第三胞内环中抑制蛋白结合位点的额外突变的特征。将四个残基中的两个（残基214和215）或三个（残基213-215）突变为丙氨酸部分地减少激动剂诱导的arrestin 3的募集，而不改变aG蛋白的活化。抑制蛋白依赖性受体内化需要抑制蛋白与β 2-adaptin（网格蛋白相关衔接蛋白AP 2的β 2亚基）和网格蛋白结合，不成比例地受到三个残基突变的影响，即使在过度表达抑制蛋白或G蛋白偶联受体激酶2的存在下也没有观察到激动剂诱导的内化。arrestin招聘和内化之间的分离不能解释的受体-arrestin相互作用，G蛋白偶联受体激酶2的招聘，或受体诱导的arrestin和β 2-adaptin之间的相互作用的时间过程中的改变，这表明突变损害的内化复合物的属性尚未被确定。
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 receptorexpression. 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 aGprotein. Arrestin-dependent receptor internalization, which requires arrestin binding to beta 2-adaptin (the beta 2 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 over-expressed 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 beta 2-adaptin, suggesting that the mutation impairs a property of the internalization complex that has not yet been identified.