CP-25 Prevents PGE2-Induced FLS Proliferation of Patients with RA via Targeting the Kinase Domain of GRK2

Paeoniflorin-6'-O-benzene sulfonate (CP-25) has therapeutic potential for the treatment of AA and CIA models, anti-proliferation effect on FLS, inhibiting of release of inflammatory cytokines. Although our groups have revealed a role for CP-25 in improving FLS function by regulating GRK2 translocation, little is known as to how GRK2 translocation activity is suppressed. Here, we report that CP-25 inhibits FLS line of patients with rheumatoid arthritis (MH7A) proliferation by down-regulating GRK2 translocation to EP4 receptor. Binding affinity assay demonstrates that CP-25 and GRK2 have affinity. The docking of CP-25 and GRK2, site-directed mutagenesis and co-immunoprecipitation assay reveal that CP-25 regulated GRK2 translocation activity is related to G201, K220, K230, A321 and D335 in kinase domain of GRK2, especially A321, it may a key amino acid for CP-25 in inhibiting GRK2 translocation activity. Together, our study not only uncovers a molecular mechanism for the formation of a GRK2/EP4 complex that regulates MH7A abnormal proliferation but also suggests that CP-25 may be through forming hydrogen bonds with GRK2 to improve FLS abnormal proliferation. Funding: This research was supported by the Key Project of National Natural Science Foundation of China (No. 81330081), Surface Project of National Natural Science Foundation of China (No. 81673444) and Youth Science Fund Project of National Natural Science Foundation of China (No. 81502123). Declaration of Interest: The authors have no financial conflicts of interest. Ethical Approval: All patients gave their informed consent, and the study protocol was approved by Biomedical Ethic Committee of Anhui Medical University (No. 20131321).