【摘 要】
：
Rheumatoid arthritis(RA)is an autoimmune disease and is mainly characterized by abnormal proliferation of fibroblast-like synoviocytes(FLS).The up-regulated cellular membrane expres-sion of G protein coupled receptor kinase 2(GRK2)of FLS plays a critical
【机 构】
：
Institute of Clinical Pharmacology,Anhui Medical University,Key Laboratory of Anti-Inflammatory and
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Rheumatoid arthritis(RA)is an autoimmune disease and is mainly characterized by abnormal proliferation of fibroblast-like synoviocytes(FLS).The up-regulated cellular membrane expres-sion of G protein coupled receptor kinase 2(GRK2)of FLS plays a critical role in RA progression,the increase of GRK2 translocation activity promotes dysfunctional prostaglandin E4 receptor(EP4)signaling and FLS abnormal proliferation.Recently,although our group found that paeoniflorin-6\'-O-ben-zene sulfonate(CP-25),a novel compound,could reverse FLS dysfunction via GRK2,little is known as to how GRK2 translocation activity is suppressed.Our findings revealed that GRK2 expression up-regulated and EP4 expression down-regulated in synovial tissues of RA patients and collagen-induced arthritis(CIA)rats,and prostaglandin E2(PGE2)level increased in arthritis.CP-25 could down-regulate GRK2 expression,up-regulate EP4 expression,and improve synovitis of CIA rats.CP-25 and GRK2 in-hibitors(paroxetine or GSK180736A)inhibited the abnormal proliferation of FLS in RA patients and CIA rats by down-regulating GRK2 translocation to EP4 receptor.The results of microscale thermophor-esis(MST),cellular thermal shift assay,and inhibition of kinase activity assay indicated that CP-25 could directly target GRK2,increase the protein stability of GRK2 in cells,and inhibit GRK2 kinase activity.The docking of CP-25 and GRK2 suggested that the kinase domain of GRK2 might be an important active pocket for CP-25.G201,K220,K230,A321,and D335 in kinase domain of GRK2 might form hydrogen bonds with CP-25.Site-directed mutagenesis and co-immunoprecipitation assay further re-vealed that CP-25 down-regulated the interaction of GRK2 and EP4 via controlling the key amino acid residue of Ala321 of GRK2.Our data demonstrate that FLS proliferation is regulated by GRK2 translo-cation to EP4.Targeted inhibition of GRK2 kinase domain by CP-25 improves FLS function and repre-sents an innovative drug for the treatment of RA by targeting GRK2.
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