Probing the phosphoinositide binding site of the clathrin assembly protein AP-2 with photoaffinity labels

The relative binding specificities of the subunits of bovine assembly protein AP-2 for the phosphatidylinositol polyphosphates (PtdInsP(n)) and inositol polyphosphates (InsP(n)) were determined by photoaffinity labeling. Three types of benzophenone-containing photoprobes were employed: (i) the water-soluble P-1- or P-2-tethered p-benzoyldihydrocinnamoyl-InsP(n) (BZDC- InsP(n)) analogs, (ii) P-1-linked phosphotriester PtdInsP(n) analogs that sampled the interface between the water and lipid phases, and (iii) sn-1-O- acyl-linked PtdInsP(n) analogs that interacted with proteins penetrating the bilayer. The InsP(n) and PtdInsP(n) probes 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 PtdlnsP(n) binding site of α-AP-2 prefers more highly phosphorylated species, and (iii) the protein-acyl chain interactions showed high capacity but low selectivity.