Cho Dongim, Zheng Mei, Min Chengchun, Ma Lan, Kurose Hitoshi, Park Jae H, Kim Kyeong-Man
Department of Pharmacology, College of Pharmacy, Chonnam National University, Gwang-Ju 500-757, Korea.
Mol Endocrinol. 2010 Mar;24(3):574-86. doi: 10.1210/me.2009-0369. Epub 2010 Feb 16.
The regulatory mechanisms and functional roles of agonist-induced internalization of G protein-coupled receptors (GPCRs) were analyzed using mutant dopamine D(2) receptors (D(2)Rs) in which all possible GPCR kinase (GRK) phosphorylation sites were mutated or the affinity for beta-arrestins was altered. Agonist-induced internalization of D(2)Rs involved a phosphorylation-dependent component, which was mediated by serine/threonine (S/T) residues in the second loop and T225 in the third loop, and a phosphorylation-independent component. GRK2-mediated enhancement of the internalization and inhibition of D(2)R signaling did not involve receptor phosphorylation, and only the former required the enzymatic activity of GRK2. The phosphorylation-deficient mutant (D(2)R-intracellular loop 2/3) recycled more slowly and showed more agonist-induced desensitization than did the wild-type D(2)R, suggesting that receptor phosphorylation mediates the recycling of the internalized receptors and enhances receptor resensitization. Blockade of the agonist-induced internalization of D(2)R-intracellular loop 2/3 provoked desensitization as in wild-type D(2)R, suggesting that certain cellular processes other than receptor dephosphorylation occurring within the endocytic vesicle are responsible for the resensitization of D(2)R. When dissociation between D(2)R and beta-arrestin was inhibited or when the expression of cellular beta-arrestins was decreased, agonist-induced desensitization of D(2)R did not occur, suggesting that dissociation from beta-arrestin is the main cellular process required for resensitization of D(2)R and is achieved through agonist-induced internalization. These results indicate that, in the regulation of some GPCRs, phosphorylation-independent association with beta-arrestin plays a major role in agonist-induced desensitization.
利用突变型多巴胺D(2)受体（D(2)Rs）分析了激动剂诱导的G蛋白偶联受体（GPCRs）内化的调控机制和功能作用，其中所有可能的GPCR激酶（GRK）磷酸化位点均发生突变，或对β-抑制蛋白的亲和力发生改变。激动剂诱导的D(2)Rs内化涉及一个磷酸化依赖性成分，该成分由第二个环中的丝氨酸/苏氨酸（S/T）残基和第三个环中的T225介导，以及一个磷酸化非依赖性成分。GRK2介导的内化增强和D(2)R信号抑制不涉及受体磷酸化，只有前者需要GRK2的酶活性。与野生型D(2)R相比，磷酸化缺陷型突变体（D(2)R-细胞内环2/3）的再循环更慢，且表现出更多激动剂诱导的脱敏，这表明受体磷酸化介导内化受体的再循环并增强受体再敏化。阻断激动剂诱导的D(2)R-细胞内环2/3内化会引发与野生型D(2)R相同的脱敏，这表明除了内吞小泡内发生的受体去磷酸化之外，某些细胞过程负责D(2)R的再敏化。当D(2)R与β-抑制蛋白之间的解离受到抑制或细胞β-抑制蛋白的表达降低时，激动剂诱导的D(2)R脱敏不会发生，这表明与β-抑制蛋白的解离是D(2)R再敏化所需的主要细胞过程，并且是通过激动剂诱导的内化实现的。这些结果表明，在某些GPCR的调控中，与β-抑制蛋白的磷酸化非依赖性结合在激动剂诱导的脱敏中起主要作用。
