AP-3 adaptor functions in targeting P-selectin to secretory granules in endothelial cells

P-selectin, a cell adhesion protein participating in the early stages of in flammation, contains multiple sorting signals that regulate its cell surfac e expression. Targeting to secretory granules regulates delivery of P-selec tin to the cell surface. Internalization followed by sorting from early to late endosomes mediates rapid removal of P-selectin from the surface. We sh ow here that the P-selectin cytoplasmic domain bound AP-2 and AP-3 adaptor complexes in vitro. The amino acid substitution L768A, which abolishes endo somal sorting and impairs granule targeting of P-selectin, reduced binding of AP-3 adaptors but not AP-2 adaptors. Turnover of P-selectin was 2.4-fold faster than turnover of transferrin receptor in AP-3-deficient mocha fibro blasts, similar to turnover of these two proteins in AP-3-competent cells, demonstrating that AP-3 function is not required for endosomal sorting. How ever, sorting P-selectin to secretory granules was defective in endothelial cells from AP-3-deficient pearl mice, demonstrating a role for AP-3 adapto rs in granule assembly in enclothelial cells. P-selectin sorting to platele t alpha -granules was normal in pearl mice, consistent with earlier evidenc e that granule targeting of P-selectin is mechanistically distinct in endot helial cells and platelets. These observations establish that AP-3 adaptor functions in assembly of conventional secretory granules, in addition to ly sosomes and the 'lysosome-like' secretory granules of platelets and melanoc ytes.