Xiao Kunhong, McClatchy Daniel B, Shukla Arun K, Zhao Yang, Chen Minyong, Shenoy Sudha K, Yates John R, Lefkowitz Robert J
Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.
Proc Natl Acad Sci U S A. 2007 Jul 17;104(29):12011-6. doi: 10.1073/pnas.0704849104. Epub 2007 Jul 9.
Beta-arrestins are cytosolic proteins that form complexes with seven-transmembrane receptors after agonist stimulation and phosphorylation by the G protein-coupled receptor kinases. They play an essential role in receptor desensitization and endocytosis, and they also serve as receptor-regulated signaling scaffolds and adaptors. Moreover, in the past decade, a growing list of protein-protein interactions of beta-arrestins pertinent to these functions has been documented. The discovery of several novel functions of beta-arrestins stimulated us to perform a global proteomics analysis of beta-arrestin-interacting proteins (interactome) as modulated by a model seven-transmembrane receptor, the angiotensin II type 1a receptor, in an attempt to assess the full range of functions of these versatile molecules. As determined by LC tandem MS, 71 proteins interacted with beta-arrestin 1, 164 interacted with beta-arrestin 2, and 102 interacted with both beta-arrestins. Some proteins bound only after agonist stimulation, whereas others dissociated. Bioinformatics analysis of the data indicates that proteins involved in cellular signaling, organization, and nucleic acid binding are the most highly represented in the beta-arrestin interactome. Surprisingly, both S-arrestin (visual arrestin) and X-arrestin (cone arrestin) were also found in heteromeric complex with beta-arrestins. The beta-arrestin interactors distribute not only in the cytoplasm, but also in the nucleus as well as other subcellular compartments. The binding of 16 randomly selected newly identified beta-arrestin partners was validated by coimmunoprecipitation assays in HEK293 cells. This study provides a comprehensive analysis of proteins that bind beta-arrestin isoforms and underscores their potentially broad regulatory roles in mammalian cellular physiology.
β-抑制蛋白是一种胞质蛋白，在激动剂刺激以及被G蛋白偶联受体激酶磷酸化后，会与七次跨膜受体形成复合物。它们在受体脱敏和内吞作用中发挥着重要作用，并且还充当受体调节的信号支架和衔接蛋白。此外，在过去十年中，与这些功能相关的β-抑制蛋白的蛋白质-蛋白质相互作用的清单越来越长。β-抑制蛋白的几种新功能的发现促使我们对由模型七次跨膜受体——血管紧张素II 1a型受体调节的β-抑制蛋白相互作用蛋白（相互作用组）进行全局蛋白质组学分析，以试图评估这些多功能分子的全部功能范围。通过液相色谱串联质谱法测定，有71种蛋白质与β-抑制蛋白1相互作用，164种与β-抑制蛋白2相互作用，102种与两种β-抑制蛋白都相互作用。一些蛋白质仅在激动剂刺激后才结合，而其他蛋白质则会解离。对数据的生物信息学分析表明，参与细胞信号传导、组织和核酸结合的蛋白质在β-抑制蛋白相互作用组中占比最高。令人惊讶的是，还发现S-抑制蛋白（视抑制蛋白）和X-抑制蛋白（视锥抑制蛋白）也与β-抑制蛋白形成异源复合物。β-抑制蛋白相互作用分子不仅分布在细胞质中，还分布在细胞核以及其他亚细胞区室中。通过在HEK293细胞中进行免疫共沉淀试验，验证了16种随机选择的新鉴定的β-抑制蛋白伙伴的结合。这项研究对与β-抑制蛋白亚型结合的蛋白质进行了全面分析，并强调了它们在哺乳动物细胞生理学中潜在的广泛调节作用。
