FIG. 5.
G171V mutation disrupts LRP5 trafficking. (A and B) Interaction of LRP5 with Mesd. HEK cells were transfected with expression plasmids as indicated. One day later, the cells were lysed and immunoprecipitation (IP) was carried out using an anti-Flag antibody. Mesd is Flag tagged, while all the LRP5 molecules are HA tagged. (C and D) Secretion of LRP5 mutants lacking the transmembrane domains. HEK cells were transfected with the Mesd plasmid and expression plasmids indicated in the figure. R1-4 and R1-4GV (GV) are AP fusion proteins. One day later, CM was collected and centrifuged at high speed. The supernatants were immunoprecipitated by an anti-HA antibody (C) or used for the AP assay (D). Cells were also lysed in the SDS sample buffer and analyzed by Western blotting (lower panels). (E) Evaluation of cell surface LRP5 levels. HEK cells were transfected with LacZ, wt HA-LRP5, or HA-LRP5G171V expression plasmid. The levels of cell surface LRP5 molecules were detected by Western analysis using streptavidin-horseradish peroxidase after the cell surfaces were biotinylated, and LRP5 molecules were precipitated with anti-HA antibody (upper panel). The levels of LRP5 in the immunocomplexes are shown in the lower panel.