Molecular determinants underlying the formation of stable intracellular G protein-coupled receptor-β-arrestin complexes after receptor endocytosis
Molecular determinants underlying the formation of stable intracellular G protein-coupled receptor-β-arrestin complexes after receptor endocytosis
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DOI：
10.1074/jbc.m101450200
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发表时间：
2001-06-01
影响因子：
4.8
通讯作者：
Caron, MG
中科院分区：
文献类型：
--
作者：
Oakley, RH;Laporte, SA;Caron, MG
β-抑制蛋白结合激动剂活化的G蛋白偶联受体（GPCR）并介导其脱敏和内化。尽管β-抑制蛋白与质膜上的某些受体（如β 2肾上腺素能受体）解离，但它们仍与其它GPCR结合，并与它们一起内化到内吞囊泡中.持续存在于细胞内的稳定受体-β-抑制蛋白复合物的形成阻碍受体再敏化，并且这些复合物的异常形成可能在基于GPCR的疾病中起作用（Barak，L，S.，奥克利，R，H，，Laporte，S.一、和Caron，M. G.等人（2001）Proc.美国科学院98，93-98），在这里，我们研究了负责持续的受体/β-抑制蛋白相互作用的分子决定因素。我们在真实的时间和活的人胚肾（HEK-293）细胞中显示，β-抑制蛋白-2-绿色荧光蛋白缀合物与激动剂活化的神经降压素-1受体、催产素受体、血管紧张素II 1A型受体和P物质受体一起内化到内吞囊泡中。使用受体诱变，我们证明了β-抑制蛋白保持与这些受体相关的能力是由位于受体羧基末端尾部的丝氨酸和苏氨酸残基的特定簇介导的。这些簇在羧基末端结构域中的位置非常保守，并作为激动剂依赖性受体磷酸化的主要位点。此外，我们鉴定了对激动剂活化的β-肾上腺素能受体具有增强的亲和力的β-抑制蛋白突变体，所述β-抑制蛋白突变体运输到具有缺乏丝氨酸/苏氨酸簇的受体的内吞囊泡中，并且通常在质膜处与野生型β-抑制蛋白解离。通过鉴定对于形成稳定的受体-β-抑制蛋白复合物至关重要的受体和β-抑制蛋白残基，这些研究为调节GPCR反应性和治疗由异常GPCR/β-抑制蛋白相互作用引起的疾病提供了新的靶点。
beta -Arrestins bind agonist-activated G protein-coupled receptors (GPCRs) and mediate their desensitization and internalization. Although beta -arrestins dissociate from some receptors at the plasma membrane, such as the beta2 adrenergic receptor, they remain associated with other GPCRs and internalize with them into endocytic vesicles. Formation of stable receptor-beta -arrestin complexes that persist inside the cell impedes receptor resensitization, and the aberrant formation of these complexes may play a role in GPCR-based diseases (Barak, L, S., Oakley, R, H,, Laporte, S. A., and Caron, M. G. (2001) Proc. Natl. Acad, Sci, U, S, A. 98, 93-98), Here, we investigate the molecular determinants responsible for sustained receptor/beta -arrestin interactions. We show in real time and in live human embryonic kidney (HEK-293) cells that a beta -arrestin-2-green fluorescent protein conjugate internalizes into endocytic vesicles with agonist-activated neurotensin-1 receptor, oxytocin receptor, angiotensin II type 1A receptor, and substance P receptor. Using receptor mutagenesis, we demonstrate that the ability of beta -arrestin to remain associated with these receptors is mediated by specific clusters of serine and threonine residues located in the receptor carboxyl-terminal tail. These clusters are remarkably conserved in their position within the carboxyl-terminal domain and serve as primary sites of agonist-dependent receptor phosphorylation. In addition, we identify a beta -arrestin mutant with enhanced affinity for the agonist-activated beta -adrenergic receptor that traffics into endocytic vesicles with receptors that lack serine/threonine clusters and normally dissociate from wild-type beta -arrestin at the plasma membrane, By identifying receptor and beta -arrestin residues critical for the formation of stable receptor-beta -arrestin complexes, these studies provide novel targets for regulating GPCR responsiveness and treating diseases resulting from abnormal GPCR/beta -arrestin interactions.