Regulation of AP-3 function by inositides. Identification of phosphatidylinositol 3,4,5-trisphosphate as a potent ligand.

As part of the growing effort to understand the role inositol phosphates and inositol lipids play in the regulation of vesicle traffic within nerve terminals, we determined whether or not the synapse-specific clathrin assembly protein AP-3 can interact with inositol lipids. We found that soluble dioctanoyl-phosphatidylinositol 3,4,5-trisphosphate (DiC8PtdIns(3,4, 5)P3) was only 7.5-fold weaker a ligand than D-myo-inositol hexakisphosphate in assays that measured the displacement of D-myo-[3H]inositol hexakisphosphate. In functional assays we found that both of these ligands inhibited clathrin assembly, but DiC8-PtdIns(3,4,5)P3 was more potent and exhibited a larger maximal effect. We also examined the structural features of DiC8-PtdIns(3,4, 5)P3 that establish specificity. Dioctanoyl-phosphatidylinositol 3, 4-bisphosphate, which does not have a 5-phosphate, and 4, 5-O-bisphosphoryl-D-myo-inosityl 1-O-(1, 2-O-diundecyl)-sn-3-glycerylphosphate, which does not have a 3-phosphate, were, respectively, 2-fold and 4-fold less potent than DiC8-PtdIns(3,4,5)P3 as inhibitors of clathrin assembly. Deacylation of DiC8-PtdIns(3,4,5)P3 reduced its affinity for AP-3 almost 20-fold, and also dramatically lowered its ability to inhibit clathrin assembly. The deacylated products of the soluble derivatives of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 4, 5-bisphosphate were both not significant inhibitors of clathrin assembly. It therefore appears that the interactions of inositides with AP-3 should not be considered simply in terms of electrostatic effects of the highly charged phosphate groups. Ligand specificity appears also to be mediated by hydrophobic interactions with the fatty-acyl chains of the inositol lipids.