Fig. 5: Kp-10/GPR54 upregulated the expression of Dusp18 and recruited both active Src and its phosphatase Dusp18. | Nature Communications

Fig. 5: Kp-10/GPR54 upregulated the expression of Dusp18 and recruited both active Src and its phosphatase Dusp18.

From: Kisspeptin-10 binding to Gpr54 in osteoclasts prevents bone loss by activating Dusp18-mediated dephosphorylation of Src

Fig. 5

a, b IB analysis of lysates from RAW264.7 with indicated dose of Kp-10 treatment for 1 h (a) and quantification results (b). c Quantitative PCR of Dusp18 mRNA in RAW264.7 cells treated with inhibitors of PLC (U73122, 10 µM), PKC (Staurosporine, 0.25 µM), ERK (LY3214996, 20 µM), and P38 (SB203580, 10 µM) for 1 h, and then incubated with indicated dose of Kp-10 for 30 min. d–i Anti-Flag IP of lysates derived from 293 T cells transfected with indicated constructs and treated with PLC inhibitor (U73122, 10 µM), PKC inhibitor (Staurosporine, 0.25 µM), ERK inhibitor (LY3214996, 10 µM), Ca2+/CaMKII inhibitor (KN-93, 10 µM), and Src inhibitor (Saracatinib, 10 µM) for 1 h and then stimulated with 10 nM Kp-10 for 20 min. Anti-Flag IP of lysates derived from 293 T cells transfected with GPR54-flag and Src-HA constructs (d) and quantification results (e). Anti-Flag IP of lysates derived from 293 T cells transfected with GPR54-flag and DUSP18-HA constructs (f) and quantification results (g). Anti-Flag IP of lysates derived from 293 T cells transfected with Src-HA, and DUSP18-flag constructs (h) and quantification results (i). j, k IB analysis of lysates derived from WT BMMs and Gq/11 KO BMMs with or without Kp-10 treatment for 1 h (j) and quantification of results (k). l Working model of Kp-10 /Gpr54 mediated Src dephosphorylation. Low dose of Kp-10 (0.1, 1 nM) induced the expression of Dusp18 obviously but not by 10 nM Kp-10, which is dependent on Gq/11 signaling. However, both active Src and DUSP18 were recruited by GPR54 through the PR motif in GPR54 CT along with the increase of Kp-10 dose (1, 10 nM). Therefore, phosphorylation of Src was dose dependently suppressed when GPR54 was activated by Kp-10. Data represent means ± SEM. P values were determined by one-way ANOVA analysis (b, c, e, g, i, k). Representative results were obtained from at least three independent experiments. Source data are provided as a Source Data file.

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