FUNCTIONAL SPECIALIZATION OF BETA-ARRESTIN INTERACTIONS REVEALED BY PROTEOMICS

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 β-抑制蛋白是在激动剂刺激和被G蛋白偶联受体激酶磷酸化后与七跨膜受体形成复合物的胞质蛋白。它们在受体脱敏和内吞作用中起重要作用，并且它们还充当受体调节的信号传导支架和衔接子。此外，在过去的十年中，越来越多的β-arrestins相关的这些功能的蛋白质-蛋白质相互作用的列表已被记录。β-arrestins的几个新功能的发现刺激我们进行一个全球性的β-arrestin相互作用蛋白质（interactome）的蛋白质组学分析的模型七跨膜受体，血管紧张素II 1a型受体，在试图评估这些多功能分子的功能的全方位。如通过LC串联MS所确定的，71种蛋白质与β-抑制蛋白1相互作用，164种蛋白质与β-抑制蛋白2相互作用，并且102种蛋白质与两种β-抑制蛋白相互作用。一些蛋白质仅在激动剂刺激后结合，而另一些蛋白质解离。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最具代表性。令人惊讶的是，S-抑制蛋白（视觉抑制蛋白）和X-抑制蛋白（视锥抑制蛋白）也发现在异聚复合物与β-抑制蛋白。β-arrestin相互作用物不仅分布在细胞质中，而且还分布在细胞核和其他亚细胞区室中。在HEK 293细胞中通过共免疫沉淀试验验证了16个随机选择的新鉴定的β-抑制蛋白伴侣的结合。这项研究提供了一个全面的分析蛋白结合β-抑制蛋白亚型，并强调其潜在的广泛的调节作用，在哺乳动物细胞生理学。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.