Fig. 5: LAPTM5 directly interacts with WWP2 and promotes BMPR1A lysosomal sorting.

a Schematic illustration of TMT-labeled LC-MS/MS analysis in 786O^LAPTM5 and Renca^Laptm5 cells treated or untreated with BMP4 (10 ng/mL) for 60 min. b Coomassie brilliant blue staining of 786O^LAPTM5 cells treated or untreated with BMP4 (10 ng/mL) for 60 min. The arrowheads indicate bands of WWP2 and LAPTM5, respectively. c Overlap of identified LAPTM5-binding proteins in 786O^LAPTM5 and Renca^Laptm5 cells. d Heatmap of the nine proteins identified binding to LAPTM5 by TMT-labeled LC-MS/MS analysis. Schematic illustration (e) and IB result (f) of serial immunoprecipitation (IP) analysis using anti-Myc (first round) and anti-HA (second round) in 293T cells transfected with expression vectors for Flag-LAPTM5, HA-WWP2, and Myc-BMPR1A. Arrowhead indicates the band of HA-WWP2. g IP and IB analyses of 293T cells transfected with expression vectors for Flag-LAPTM5, HA-WWP2, and Myc-BMPR1A. h Quantitation of the binding between HA-WWP2 and Myc-BMPR1A as treated in (g) (n = 3 per group). i IP and IB analyses of 786O^luci/eGFP Parental and LuM1a cells. j Schematic illustration of full-length (FL) WWP2 and its deletion mutants lacking functional domains (ranges indicate amino acids present in construct). C838A represents the mutation of cytosine to adenine in the 838th nucleotide. k IP and IB analyses of 786-O cells transfected with expression vector for Flag-LAPTM5, control (Vector) or Myc-tagged full-length WWP2 or various deletion mutants. l Schematic illustration of LAPTM5, WWP2 and BMPR1A interactions in cells. m Immunofluorescence (IF) analysis of control (upper panel) and LAPTM5-overexpressing (lower panel) 786-O cells treated with Mock (ddH₂O), or BMP4 (10 ng/mL) for 60 min or 120 min. Scale bar, 10 μm. n Quantification of BMPR1A punctate spots per 786-O cell treated as in (M) [n = biological replicates, 6 in Vector, 4 in LAPTM5 (Mock); n = 4 in Vector, 8 in LAPTM5 (60 min); n = 3 in Vector, 9 in LAPTM5 (120 min)]. Data are presented as whisker plots: midline, median; box, 25–75th percentile; whisker, minimum to maximum values. o Ratio of co-localized spots to total BMPR1A (red line) or LAPTM5 (green line) spots and quantification of co-localized spots per 786-O cell (yellow line) treated as in (m) (n = 4, 8, 9 biological replicates in group Mock, 60 min, 120 min, respectively). Immunoblots are representative of three biological replicates. Data are presented as mean ± SD in (h), and mean ± SEM in (o). Two-tailed Student’s unpaired t-test was used for statistical analysis in all panels. Source data are provided as a Source data file.