Phosphatidylinositol-(4,5)-bisphosphate regulates sorting signal recognition by the clathrin-associated adaptor complex AP2

The alpha,beta 2,mu 2,sigma 2 heterotetrameric AP2 complex is recruited exclusively to the phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P(2))-rich plasma membrane where, amongst other roles, it selects motif-containing cargo proteins for incorporation into clathrin-coated vesicles. Unphosphorylated and mu 2Thr156-monophosphorylated AP2 mutated in their alpha PtdIns4,5P2, mu 2PtdIns4,5P(2), and mu 2Yxx phi binding sites were produced, and their interactions with membranes of different phospholipid and cargo composition were measured by surface plasmon resonance. We demonstrate that recognition of Yxx phi and acidic dileucine motifs is dependent on corecognition with PtdIns4,5P2, explaining the selective recruitment of AP2 to the plasma membrane. The interaction of AP2 with PtdIns4,5P(2)/Yxx phi-containing membranes is two step: initial recruitment via the aPtdIns4,5P2 site and then stabilization through the binding of mu 2Yxx phi and mu 2PtdIns4,5P2 sites to their ligands. The second step is facilitated by a conformational change favored by mu 2Thr156 phosphorylation. The binding of AP2 to acidic-dileucine motifs occurs at a different site from Yxx phi binding and is not enhanced by mu 2Thr156 phosphorylation.