Fig. 2: GPR54 recruited Src via the PR motif in GPR54 C-Terminus.
a Sequence analysis of the PR motif in multiple species of GPR54 CT. b SH3 domain protein-binding array using the TranSignal™ SH3 Domain Array kit. The principal Src binding region is circled in red evident (1 Src; 2 the dilution of Src in half). c SPR binding-affinity measurement of Src and GPR54 CT. Src and GPR54 CT binding affinity is 2.6 nM (kinetic analysis, RUmax = 36.31, Chi = 1.08). d Crystallization structure analysis of the SH2-SH3 domain of Src and the human GPR54 336–356 piptide including PR motif. GPR54 CT bound with the SH3 domain of Src mainly via the key residues including R336, P339 and P342. e–h IB analysis of total samples and GST pull-downs using GST proteins including GST, GST-GPR54 CT, GST-GPR54 CT m1 (R336A&P339A), GST-GPR54 CT m2 (delta 339–344) incubated separately with WCL of 293 T cells transfected with HA-Src (e) and quantification of protein levels (f), IB analysis of WCL and anti-GPR54 IP assays derived from RAW264.7 cells induced by Kp-10 for 20 min (g) and quantification of protein levels (h). i, j IF staining was carried out using pre-osteoclasts differentiated from BMMs with M-CSF (10 ng/ml) and RANKL (50 ng/ml) stimulation for 2 days, then treated with 10 nM Kp-10 for 20 min. Representative images of endogenous Gpr54 and Src were shown by total internal reflection fluorescence (TIRF) microscopy (i), and the Mander’s overlap coefficient (MOC) as a measure of colocalization in cells from multiple images taken randomly (j). Scale bar, 50 μm. Data represent means ± SEM. P values were determined by one-way ANOVA analysis (f, h) or two-tailed Student’s t-test (j). Representative results were obtained from at least three independent experiments. Source data are provided as a Source Data file.
