Probing the Phosphoinositide Binding Site of the Clathrin Assembly Protein AP-2 with Photoaffinity Labels

The relative binding specificities of the subunitsof bovine assembly protein AP-2 for the phosphatidylinositol polyphosphates (PtdInsPn) and inositol polyphosphates (InsPn) were determined by photoaffinitylabeling. Three types of benzophenone-containing photoprobes were employed: (i) the water-solubleP-1- or P-2-tetheredp-benzoyldihydrocinnamoyl-InsPn(BZDC-InsPn) analogs, (ii) P-1-linked phosphotriester PtdInsPnanalogs that sampled the interface between the water and lipid phases, and (iii)sn-1-O-acyl-linked PtdInsPnanalogs that interacted with proteins penetrating the bilayer. The InsPnand PtdInsPnprobes bind with highest selectivity and affinity to the two α subunit isoforms, with certain probes and conditions resulting in strong labeling of the 50-kDa μ subunit. Three main conclusions were reached: (i) head group recognition predominated over acyl chain recognition, (ii) the PtdInsPnbinding site of α-AP-2 prefers more highly phosphorylated species, and (iii) the protein–acyl chain interactions showed high capacity but low selectivity.