Ferguson S S, Zhang J, Barak L S, Caron M G
John P. Robarts Research Institute and Department of Physiology, University of Western Ontario, London.
Life Sci. 1998;62(17-18):1561-5. doi: 10.1016/s0024-3205(98)00107-6.
Beta-arrestin proteins play a dual role in regulating G protein-coupled receptor (GPCR) responsiveness by contributing to both receptor desensitization and internalization. Recently, beta-arrestins were also shown to be critical determinants for beta2-adrenergic receptor (beta2AR) resensitization. This was demonstrated by overexpressing wild-type beta-arrestins to rescue the resensitization-defect of a beta2AR (Y326A) mutant (gain of function) and overexpressing a dominant-negative beta-arrestin inhibitor of beta2AR sequestration to impair beta2AR dephosphorylation and resensitization (loss of function). Moreover, the ability of the beta2AR to resensitize in different cell types was shown to be dependent upon beta-arrestin expression levels. To further study the mechanisms underlying beta-arrestin function, green fluorescent protein was coupled to beta-arrestin2 (beta arr2GFP), thus allowing the real-time visualization of the agonist-dependent trafficking of beta-arrestin in living cells. Beta arr2GFP translocation from the cytoplasm to the plasma membrane proceeded with a time course, sensitivity and specificity that was indistinguishable from the most sensitive second messenger readout systems. Beta arr2GFP translocation was GRK-dependent and was demonstrated for 16 different ligand-activated GPCRs. Because beta-arrestin binding is a common divergent step in GPCR signalling, this assay represents a universal methodology for screening orphan receptors, GRK inhibitors and novel GPCR ligands. Moreover, beta arr2GFP provides a valuable new tool to dissect the biological function and regulation of beta-arrestin proteins.
β-抑制蛋白在调节G蛋白偶联受体（GPCR）反应性方面发挥双重作用，既参与受体脱敏又参与内化过程。最近，β-抑制蛋白还被证明是β2肾上腺素能受体（β2AR）再敏化的关键决定因素。这一点通过过表达野生型β-抑制蛋白来挽救β2AR（Y326A）突变体（功能获得）的再敏化缺陷，以及过表达一种抑制β2AR隔离的显性负性β-抑制蛋白以损害β2AR去磷酸化和再敏化（功能丧失）得以证明。此外，β2AR在不同细胞类型中再敏化的能力被证明取决于β-抑制蛋白的表达水平。为了进一步研究β-抑制蛋白功能的潜在机制，绿色荧光蛋白与β-抑制蛋白2（βarr2GFP）偶联，从而能够实时观察活细胞中激动剂依赖的β-抑制蛋白转运。βarr2GFP从细胞质向质膜的转位具有时间进程、敏感性和特异性，与最敏感的第二信使读出系统难以区分。βarr2GFP转位依赖于GRK，并且在16种不同的配体激活的GPCR中得到证实。由于β-抑制蛋白结合是GPCR信号传导中一个常见的分歧步骤，该检测方法代表了一种筛选孤儿受体、GRK抑制剂和新型GPCR配体的通用方法。此外，βarr2GFP为剖析β-抑制蛋白的生物学功能和调节提供了一种有价值的新工具。
