Phosphorylation of the δ-opioid receptor regulates its β-arrestins selectivity and subsequent receptor internalization and adenylyl cyclase desensitization
Phosphorylation of the δ-opioid receptor regulates its β-arrestins selectivity and subsequent receptor internalization and adenylyl cyclase desensitization
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DOI：
10.1074/jbc.m611258200
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发表时间：
2007-08-03
影响因子：
4.8
通讯作者：
Law, Ping-Yee
中科院分区：
文献类型：
--
作者：
Qiu, Yu;Loh, Horace H.;Law, Ping-Yee
在目前的研究中，我们研究了受体磷酸化和β-arrestins在δ-阿片受体（DOR）信号转导和运输中的作用，通过使用DOR突变体，其中C末端的所有Ser/Thr残基突变为Ala（Ala）。我们证明，DOR激动剂D-[Pen（2），Pen（5）]脑啡肽可诱导受体内化和腺苷酸环化酶（AC）脱敏，但与野生型DOR相比，动力学相对较慢。阻断显性负突变发动蛋白，发动蛋白K44 E的内化，不影响AC脱敏。然而，β-arrestins的耗尽几乎完全阻断了内化和AC脱敏。BRET测定表明DOR磷酸化促进β-抑制蛋白2的受体选择性超过β-抑制蛋白1。此外，在缺乏β-arrestin 1（β arr 1（-/-））或β-arrestin 2（β arr 2（-/-））的小鼠胚胎成纤维细胞（MEF）中，激动剂诱导的细胞脱敏和内化与野生型MEF中观察到的相似。相比之下，虽然DOR内化在β arr 1（-/-）MEFs和β arr 2（-/-）MEFs中均降低，但DPDE诱导的DOR脱敏在β arr 2（-/-）MEFs中显著降低，但在β arr 1（-/-）MEFs中没有。此外，BRET测定表明磷酸化的消耗不影响受体-β-抑制蛋白复合物的稳定性。与该观察结果一致，DOR在内化后不再循环，这与野生型DOR相似。总之，这些结果表明，受体磷酸化赋予β-抑制蛋白2的DOR选择性，而不影响受体-β-抑制蛋白复合物的稳定性和内化受体的命运。
In the current study, we investigated the role of receptor phosphorylation and beta-arrestins in delta-opioid receptor (DOR) signaling and trafficking by using a DOR mutant in which all Ser/Thr residues in the C terminus were mutated to Ala (DTS). We demonstrated that the DOR agonist D-[Pen(2),Pen(5)] enkephalin could induce receptor internalization and adenylyl cyclase (AC) desensitization of DTS, but with comparatively slower kinetics than those observed with wild type DOR. Blockade of the internalization of DTS by the dominant-negative mutant dynamin, dynamin K44E, did not affect AC desensitization. However, depletion of beta-arrestins almost totally blocked both internalization and AC desensitization of DTS. A BRET assay suggested that DOR phosphorylation promotes receptor selectivity for beta-arrestin 2 over beta-arrestin 1. Furthermore, in mouse embryonic fibroblast (MEF) cells lacking either beta-arrestin 1 (beta arr1(-/-)) or beta-arrestin 2 (beta arr2(-/-)), agonist-induced DTS desensitization and internalization were similar to that observed in wild type MEFs. In contrast, although DOR internalization decreased in both beta arr1(-/-) MEFs and beta arr2(-/-) MEFs, DPDPE-induced DOR desensitization was significantly reduced in beta arr2(-/-) MEFs, but not in beta arr1(-/-) MEFs. Additionally, the BRET assay suggested that depletion of phosphorylation did not influence the stability of the receptor-beta-arrestin complex. Consistent with this observation, DTS did not recycle after internalization, which is like wild type DOR. Taken together, these results indicate that receptor phosphorylation confers DOR selectivity for beta-arrestin 2 without affecting the stability of the receptor-beta-arrestin complex and the fate of the internalized receptor.