The State Key Laboratory of Medical Neurobiology and Pharmacology Research Center, Shanghai Medical College and Institutes of Brain Science, Fudan University, Shanghai, China.
PLoS One. 2012;7(8):e43997. doi: 10.1371/journal.pone.0043997. Epub 2012 Aug 27.
The G protein-coupled receptor kinases (GRKs) phosphorylate agonist occupied G protein-coupled receptors (GPCRs) and desensitize GPCR-mediated signaling. Recent studies indicate they also function non-catalytically via interaction with other proteins. In this study, a proteomic approach was used to screen interacting proteins of GRK5 in MDA-MB-231 cells and HUVEC cells. Mass spectrometry analysis reveals several proteins in the GRK5 immunocomplex including damaged DNA-binding protein 1 (DDB1), an adaptor subunit of the CUL4-ROC1 E3 ubiquitin ligase complex. Co-immunoprecipitation experiments confirmed the association of GRK5 with DDB1-CUL4 complex, and reveal that DDB1 acts as an adapter to link GRK5 to CUL4 to form the complex. Overexpression of DDB1 promoted, whereas knockdown of DDB1 inhibited the ubiquitination of GRK5, and the degradation of GRK5 was reduced in cells deficient of DDB1. Furthermore, the depletion of DDB1 decreased Hsp90 inhibitor-induced GRK5 destabilization and UV irradiation-induced GRK5 degradation. Thus, our study identified potential GRK5 interacting proteins, and reveals the association of GRK5 with DDB1 in cell and the regulation of GRK5 level by DDB1-CUL4 ubiquitin ligase complex-dependent proteolysis pathway.
G 蛋白偶联受体激酶（GRKs）磷酸化激动剂占据的 G 蛋白偶联受体（GPCR），使 GPCR 介导的信号转导脱敏。最近的研究表明，它们还通过与其他蛋白质相互作用发挥非催化功能。在这项研究中，采用蛋白质组学方法筛选 MDA-MB-231 细胞和 HUVEC 细胞中 GRK5 的相互作用蛋白。质谱分析显示，GRK5 免疫复合物中的几种蛋白质包括受损的 DNA 结合蛋白 1（DDB1），CUL4-ROC1 E3 泛素连接酶复合物的衔接子亚基。共免疫沉淀实验证实了 GRK5 与 DDB1-CUL4 复合物的关联，并揭示了 DDB1 作为接头将 GRK5 与 CUL4 连接形成复合物。DDB1 的过表达促进了 GRK5 的泛素化和降解，而 DDB1 敲低则抑制了 GRK5 的泛素化，并且 DDB1 缺陷细胞中 GRK5 的降解减少。此外，DDB1 的耗竭减少了 HSP90 抑制剂诱导的 GRK5 不稳定和 UV 照射诱导的 GRK5 降解。因此，我们的研究鉴定了潜在的 GRK5 相互作用蛋白，并揭示了 GRK5 在细胞中的 DDB1 关联以及 DDB1-CUL4 泛素连接酶复合物依赖性蛋白水解途径对 GRK5 水平的调节。
