G-protein-coupled receptor kinase specificity for β-arrestin recruitment to the β2-adrenergic receptor revealed by fluorescence resonance energy transfer
G-protein-coupled receptor kinase specificity for β-arrestin recruitment to the β2-adrenergic receptor revealed by fluorescence resonance energy transfer
复制标题
DOI：
10.1074/jbc.m513605200
复制
发表时间：
2006-07-21
影响因子：
4.8
通讯作者：
Lefkowitz, Robert J.
中科院分区：
文献类型：
--
作者：
Violin, Jonathan D.;Ren, Xiu-Rong;Lefkowitz, Robert J.
g蛋白偶联受体激酶（GRKs）小家族通过磷酸化七螺旋受体来调节细胞信号传导，从而促进受体与β -抑制素的相互作用。这将受体从g蛋白激活转换为g蛋白脱敏、受体内化和β -抑制蛋白依赖的信号激活。然而，GRKs募集β -抑制素到特定受体的特异性尚未被阐明。在这里，我们使用β(2)-肾上腺素能受体（β (2)AR），原型非视觉七螺旋受体，作为模型来测试功能性GRK特异性。我们通过测量β (2)AR和β -抑制素之间相互作用的动力学，在活细胞中使用荧光共振能量转移法监测内源性GRK活性。我们发现β (2)AR磷酸化是高亲和力β -抑制蛋白结合所必需的，我们使用小干扰RNA沉默来证明HEK-293和U2-OS细胞使用其表达的GRK的不同亚群来促进β -抑制蛋白募集，在两种细胞类型中都有明显的GRK冗余。令人惊讶的是，GRK对β -抑制蛋白募集的特异性与大量受体磷酸化的特异性并不相关，这表明β -抑制蛋白募集对特定位点的受体磷酸化的子集是特异性的。此外，GRK家族的多个成员能够磷酸化β (2)AR并诱导β -阻滞蛋白募集，它们的相对贡献在很大程度上取决于它们的相对表达水平。由于GRK同种异构体的调控不同，这个部分冗余的系统确保了β -抑制素的募集，同时为组织特异性调节β -抑制素募集率提供了机会。
The small family of G-protein-coupled receptor kinases (GRKs) regulate cell signaling by phosphorylating heptahelical receptors, thereby promoting receptor interaction with beta-arrestins. This switches a receptor from G-protein activation to G-protein desensitization, receptor internalization, and beta-arrestin-dependent signal activation. However, the specificity of GRKs for recruiting beta-arrestins to specific receptors has not been elucidated. Here we use the beta(2)-adrenergic receptor (beta(2)AR), the archetypal nonvisual heptahelical receptor, as a model to test functional GRK specificity. We monitor endogenous GRK activity with a fluorescence resonance energy transfer assay in live cells by measuring kinetics of the interaction between the beta(2)AR and beta-arrestins. We show that beta(2)AR phosphorylation is required for high affinity beta-arrestin binding, and we use small interfering RNA silencing to show that HEK-293 and U2-OS cells use different subsets of their expressed GRKs to promote beta-arrestin recruitment, with significant GRK redundancy evident in both cell types. Surprisingly, the GRK specificity for beta-arrestin recruitment does not correlate with that for bulk receptor phosphorylation, indicating that beta-arrestin recruitment is specific for a subset of receptor phosphorylations on specific sites. Moreover, multiple members of the GRK family are able to phosphorylate the beta(2)AR and induce beta-arrestin recruitment, with their relative contributions largely determined by their relative expression levels. Because GRK isoforms vary in their regulation, this partially redundant system ensures beta-arrestin recruitment while providing the opportunity for tissue-specific regulation of the rate of beta-arrestin recruitment.