Oakley R H, Laporte S A, Holt J A, Barak L S, Caron M G
Howard Hughes Medical Institute Laboratories, Departments of Cell Biology and Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 2001 Jun 1;276(22):19452-60. doi: 10.1074/jbc.M101450200. Epub 2001 Mar 9.
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 beta2-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.
β抑制蛋白结合激动剂激活的G蛋白偶联受体（GPCR），并介导其脱敏和内化。尽管β抑制蛋白在质膜上与某些受体解离，如β2肾上腺素能受体，但它们仍与其他GPCR结合并与之一起内化到内吞小泡中。在细胞内持续存在的稳定受体 - β抑制蛋白复合物的形成会阻碍受体的再敏化，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（巴拉克，L.S.，奥克利，R.H.，拉波特，S.A.，和卡隆，M.G.（2001年）美国国家科学院院刊98，93 - 98）。在这里，我们研究负责持续受体/β抑制蛋白相互作用的分子决定因素。我们在实时和活的人胚肾（HEK - 293）细胞中表明，β抑制蛋白 - 2 - 绿色荧光蛋白缀合物与激动剂激活的神经降压素 - 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小泡中。使用受体诱变，我们证明β抑制蛋白与这些受体保持结合的能力是由位于受体羧基末端尾巴中的特定丝氨酸和苏氨酸残基簇介导的。这些簇在羧基末端结构域内的位置非常保守，并作为激动剂依赖性受体磷酸化的主要位点。此外，我们鉴定出一种对激动剂激活的β2肾上腺素能受体具有增强亲和力 的β抑制蛋白突变体，该突变体与缺乏丝氨酸/苏氨酸簇且通常在质膜上与野生型β抑制蛋白解离的受体一起转运到内吞小泡中。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。
