G protein-coupled receptor kinase 2(GRK2)participates in the phosphorylation and desensitization of G protein-coupled receptor(GPCR),impacting various biological processes such as inflammation and cell proliferation.D...G protein-coupled receptor kinase 2(GRK2)participates in the phosphorylation and desensitization of G protein-coupled receptor(GPCR),impacting various biological processes such as inflammation and cell proliferation.Dysregulated expression and activity of GRK2 have been reported in multiple cells in rheumatoid arthritis(RA).However,whether and how GRK2 regulates synovial hyperplasia and fibroblast-like synoviocytes(FLSs)proliferation is poorly understood.In this study,we investigated the regulation of GRK2 and its biological function in RA.We found that GRK2 transmembrane activity was increased in FLSs of RA patients and collagen-induced arthritis(CIA)rats.Additionally,we noted a positive correlation between high GRK2 expression on the cell membrane and serological markers associated with RA and CIA.Immunoprecipitationemass spectrometry and pulldown analyses revealed tumor necrosis factor receptor-associated factor 2(TRAF2)as a novel substrate of GRK2.Furthermore,surface plasmon resonance(SPR)and molecular docking assays determined that the C-terminus of GRK2 binds to the C-terminus of TRAF2 at the Gln340 residue.GRK2 knockdown and the GRK2 inhibitor CP-25 attenuated synovial hyperplasia and FLS proliferation in CIA both in vitro and in vivo by decreasing GRK2 membrane expression and activity.Mechanistically,increased GRK2 transmembrane activity contributed to the recruitment of TRAF2 on the cell membrane,promoting GRK2-RAF2 interactions that facilitate the recruitment of the E3 ubiquitin ligase TRIM47 to TRAF2.This enhanced TRAF2 Lys63 polyubiquitylation and induced nuclear factor(NF)-kB activation,leading to synovial hyperplasia and abnormal proliferation of FLSs.Our study provides a mechanistic and preclinical rationale for further evaluation of GRK2 as a therapeutic target for RA.展开更多
基金supported by National Natural Science Foundation of China(Nos.82104187,82173824,and 82430114)Natural Science Foundation of Anhui Province(No.2108085QH382,China)+2 种基金Project funded by China Postdoctoral Science Foundation(Nos.2021T140002 and 2021M700184)the Postdoctoral Science Foundation of Anhui Province(No.2020B430,China)Postgraduate Innovation Research and Practice Program of Anhui Medical University(No.YJS20230162,China).
文摘G protein-coupled receptor kinase 2(GRK2)participates in the phosphorylation and desensitization of G protein-coupled receptor(GPCR),impacting various biological processes such as inflammation and cell proliferation.Dysregulated expression and activity of GRK2 have been reported in multiple cells in rheumatoid arthritis(RA).However,whether and how GRK2 regulates synovial hyperplasia and fibroblast-like synoviocytes(FLSs)proliferation is poorly understood.In this study,we investigated the regulation of GRK2 and its biological function in RA.We found that GRK2 transmembrane activity was increased in FLSs of RA patients and collagen-induced arthritis(CIA)rats.Additionally,we noted a positive correlation between high GRK2 expression on the cell membrane and serological markers associated with RA and CIA.Immunoprecipitationemass spectrometry and pulldown analyses revealed tumor necrosis factor receptor-associated factor 2(TRAF2)as a novel substrate of GRK2.Furthermore,surface plasmon resonance(SPR)and molecular docking assays determined that the C-terminus of GRK2 binds to the C-terminus of TRAF2 at the Gln340 residue.GRK2 knockdown and the GRK2 inhibitor CP-25 attenuated synovial hyperplasia and FLS proliferation in CIA both in vitro and in vivo by decreasing GRK2 membrane expression and activity.Mechanistically,increased GRK2 transmembrane activity contributed to the recruitment of TRAF2 on the cell membrane,promoting GRK2-RAF2 interactions that facilitate the recruitment of the E3 ubiquitin ligase TRIM47 to TRAF2.This enhanced TRAF2 Lys63 polyubiquitylation and induced nuclear factor(NF)-kB activation,leading to synovial hyperplasia and abnormal proliferation of FLSs.Our study provides a mechanistic and preclinical rationale for further evaluation of GRK2 as a therapeutic target for RA.
