Journal: Nature Communications

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

doi: 10.1038/s41467-024-44852-9

Figure Lengend Snippet: 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.

Article Snippet: Fig. 4 Src was dephosphorylated via DUSP18 following GPR54 activation by Kp-10. a The binding affinity of DUSP18 and Src was measured at 5.9 nM (RUmax = 130.6, Chi = 3.42) by SPR binding analysis. b – f IB and quantification of protein co-IP levels.

Techniques: Sequencing, Protein Binding, Binding Assay, Crystallization Assay, Incubation, Transfection, Derivative Assay, Staining, Fluorescence, Microscopy, Two Tailed Test

Journal: Nature Communications

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

doi: 10.1038/s41467-024-44852-9

Figure Lengend Snippet: a – d Anti-Gpr54 IP was performed on WCL derived from RAW264.7 cells treated with indicated dose of Kp-10 for 20 min. Beads were analyzed by phosphatase activity assay ( a ), mass spectrometry assay ( b ), IB analysis ( c ) and quantification of protein levels ( d ). e , f Protein co-localization was analyzed by IF staining in primary pre-osteoclast with 10 nM Kp-10 treatment for 20 min. Representative images of endogenous Gpr54 and Dusp18 were shown by TIRF microscopy ( e ), and MOC was used to quantify the degree of colocalization in cells from multiple images taken randomly ( f ). Scale bar, 50 µm. g , h binding affinity was measured by SPR binding analysis. The binding affinity of DUSP18 and human GPR54 CT was measured at 40 nM, RUmax = 68.22, Chi = 1.01 ( g ), DUSP18 and mouse Gpr54 336–342 was measured at 1.8 µM, RUmax = 154.6, Chi = 3.42 ( h ). i – l IB and quantification of protein levels 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 with WCL of 293 T cells transfected with DUSP18-HA ( i ) and quantification of protein levels ( j ), Total samples and GST pull-downs using His-SRC protein (0 or 0.1 μg) purified from Sf9 cells, His-DUSP18 (0, 0.1 or 0.2 μg), and GST proteins purified from E. coli ( k ) and quantification of protein levels ( l ). Data represent means ± SEM. P values were determined by one-way ANOVA analysis ( a , d , j , l ) or two-tailed Student’s t -test ( f ). Representative results were obtained from at least three independent experiments. Source data are provided as a Source Data file.

Article Snippet: Fig. 4 Src was dephosphorylated via DUSP18 following GPR54 activation by Kp-10. a The binding affinity of DUSP18 and Src was measured at 5.9 nM (RUmax = 130.6, Chi = 3.42) by SPR binding analysis. b – f IB and quantification of protein co-IP levels.

Techniques: Derivative Assay, Phosphatase Assay, Mass Spectrometry, Staining, Microscopy, Binding Assay, Incubation, Transfection, Purification, Two Tailed Test

Journal: Nature Communications

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

doi: 10.1038/s41467-024-44852-9

Figure Lengend Snippet: a The binding affinity of DUSP18 and Src was measured at 5.9 nM (RUmax = 130.6, Chi = 3.42) by SPR binding analysis. b – f IB and quantification of protein co-IP levels. Anti-Flag IP derived from 293 T cells transfected with HA-Src and either DUSP18-Flag or DUSP18 (C104S)-Flag constructs with or without Kp-10 treatment for 20 min were lysed and subjected to anti-FLAG IP ( b ). Phosphatase reaction products using His-DUSP18 and His-DUSP18 (C104S) proteins purified from E. coli and SRC proteins purified from Sf9 insect cells ( c ) and quantification of protein levels ( d ). Anti-Src IP derived from RAW264.7 cells treated with or without Kp-10 treatment for 20 min were lysed and subjected to anti-Src IP ( e ) and quantification of protein levels ( f ). g , h Src and Dusp18 co-localization was analyzed by IF staining in primary pre-osteoclasts treated with 10 nM Kp-10 treatment for 20 min. Representative images of endogenous Dusp18 and Src were shown by TIRF microscopy ( g ), and MOC was used to quantify the degree of colocalization in cells from multiple images taken randomly ( h ). Scale bar, 50 µm. i – m IB and quantification of protein levels analysis. Anti-Flag IP derived from 293 T cells transfected with HA-Src, GPR54-myc, and either DUSP18-Flag or DUSP18 (C104S)-Flag constructs after Kp-10 treatment for 20 min ( i ) and quantification of protein levels ( j , k ). WCL derived from WT and Dusp18 −/− BMMs treated with or without Kp-10 for 20 min ( l ) and quantification of protein levels ( m ). n , o WT and Dusp18 −/− BMMs were cultured in vitro on dentin slices, and after incubation with M-CSF (10 ng/ml) and RANKL (50 ng/ml) for 5–7 days, the pits formed by osteoclast absorption activity were scanned by confocal microscopy (XY and z sections). Scale bars, 125 µm ( n ), and pits depth were quantified by confocal microscopy ( o ). Data represent means ± SEM. P values were determined by one-way ANOVA analysis ( d , f , j , k , m , o ) or by two-tailed Student’s t -test ( h ). Representative results were obtained from at least three independent experiments. Source data are provided as a Source Data file.

Article Snippet: Fig. 4 Src was dephosphorylated via DUSP18 following GPR54 activation by Kp-10. a The binding affinity of DUSP18 and Src was measured at 5.9 nM (RUmax = 130.6, Chi = 3.42) by SPR binding analysis. b – f IB and quantification of protein co-IP levels.

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Derivative Assay, Transfection, Construct, Purification, Staining, Microscopy, Cell Culture, In Vitro, Incubation, Activity Assay, Confocal Microscopy, Two Tailed Test