title: Targeted ubiquitination and degradation of G-protein-coupled receptor kinase 5 by the DDB1-CUL4 ubiquitin ligase complex.
abstract: 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.
authors.affiliation: The State Key Laboratory of Medical Neurobiology and Pharmacology Research Center, Shanghai Medical College and Institutes of Brain Science, Fudan University, Shanghai, China.
authors.forename: Ziyan
authors.initials: Z
authors.lastname: Wu
authors.forename: Yuejun
authors.initials: Y
authors.lastname: Chen
authors.forename: Tong
authors.initials: T
authors.lastname: Yang
authors.forename: Qinqin
authors.initials: Q
authors.lastname: Gao
authors.forename: Man
authors.initials: M
authors.lastname: Yuan
authors.forename: Lan
authors.initials: L
authors.lastname: Ma
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002417:Cattle; D044842:Cullin Proteins / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D054774:G-Protein-Coupled Receptor Kinase 5 / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry; D059748:Proteolysis / Q000528:radiation effects; D040901:Proteomics; D054875:Ubiquitination / Q000528:radiation effects; D014466:Ultraviolet Rays
chemical_list: C114234:CUL4A protein, human; D002352:Carrier Proteins; D044842:Cullin Proteins; C091435:DDB1 protein, human; D004268:DNA-Binding Proteins; C479529:RBX1 protein, human; D054774:G-Protein-Coupled Receptor Kinase 5