Role of Receptor-attached Phosphates in Binding of Visual and Non-visual Arrestins to G Protein-coupled Receptors
Role of Receptor-attached Phosphates in Binding of Visual and Non-visual Arrestins to G Protein-coupled Receptors
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DOI：
10.1074/jbc.m111.311803
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发表时间：
2012-03-16
影响因子：
4.8
通讯作者：
Gurevich, Vsevolod V.
中科院分区：
文献类型：
--
作者：
Gimenez, Luis E.;Kook, Seunghyi;Gurevich, Vsevolod V.
抑制蛋白是调节G蛋白偶联受体（GPCR）的蛋白质的小家族。抑制蛋白特异性结合磷酸化的活性受体，终止G蛋白偶联，靶向受体的内吞囊泡，并启动G蛋白非依赖性信号。视紫红质连接的磷酸盐与β链I中的Lys-14和Lys-15的相互作用被证明破坏了视觉抑制蛋白-1的α-螺旋I、β链I和C-尾的相互作用，促进其转变为活性受体结合状态。在这里，我们测试了保守的赖氨酸在非视觉抑制蛋白的同源位置的作用，通过产生K2 A突变体，其中两个赖氨酸被替换为丙氨酸。抑制蛋白-1、-2和-3中的K2 A突变在体外显著降低了它们与活性磷紫质的结合。通过基于生物发光共振能量转移（BRET）的测定来监测抑制蛋白与完整细胞中的几种GPCR的相互作用。BRET数据证实了Lys-14和Lys-15在arrestin-1与非同源受体结合中的作用。然而，对于非视觉抑制蛋白，情况并非如此，其中K2 A突变对M2毒蕈碱乙酰胆碱（M2 R）、β（2）-肾上腺素能（β（2）AR）或D2多巴胺受体的BRETmax净值几乎没有影响。此外，M2 R的磷酸化缺陷突变体与野生型非视觉抑制蛋白正常相互作用，而磷酸化缺陷β（2）AR突变体结合抑制蛋白的水平为野生型β（2）AR的20-50%。因此，受体连接的磷酸盐对抑制蛋白结合的贡献取决于受体-抑制蛋白对。尽管arrestin-1总是依赖于受体磷酸化，但在β（2）AR的情况下，其在arrestin-2和-3的募集中的作用比M2 R和D2多巴胺受体大得多。
Arrestins are a small family of proteins that regulate G protein-coupled receptors (GPCRs). Arrestins specifically bind to phosphorylated active receptors, terminating G protein coupling, targeting receptors to endocytic vesicles, and initiating G protein-independent signaling. The interaction of rhodopsin-attached phosphates with Lys-14 and Lys-15 in beta-strand I was shown to disrupt the interaction of alpha-helix I, beta-strand I, and the C-tail of visual arrestin-1, facilitating its transition into an active receptor-binding state. Here we tested the role of conserved lysines in homologous positions of non-visual arrestins by generating K2A mutants in which both lysines were replaced with alanines. K2A mutations in arrestin-1, -2, and -3 significantly reduced their binding to active phosphorhodopsin in vitro. The interaction of arrestins with several GPCRs in intact cells was monitored by a bioluminescence resonance energy transfer (BRET)-based assay. BRET data confirmed the role of Lys-14 and Lys-15 in arrestin-1 binding to non-cognate receptors. However, this was not the case for non-visual arrestins in which the K2A mutations had little effect on net BRETmax values for the M2 muscarinic acetylcholine (M2R), beta(2)-adrenergic (beta(2)AR), or D2 dopamine receptors. Moreover, a phosphorylation-deficient mutant of M2R interacted with wild type non-visual arrestins normally, whereas phosphorylation-deficient beta(2)AR mutants bound arrestins at 20-50% of the level of wild type beta(2)AR. Thus, the contribution of receptor-attached phosphates to arrestin binding varies depending on the receptor-arrestin pair. Although arrestin-1 always depends on receptor phosphorylation, its role in the recruitment of arrestin-2 and -3 is much greater in the case of beta(2)AR than M2R and D2 dopamine receptor.