Phosphoinositide-AP-2 interactions required for targeting to plasma membrane clathrin-coated pits

The clathrin-associated AP-2 adaptor protein is a major polyphosphoinositid e-binding protein in mammalian cells. A high affinity binding site has prev iously been localized to the NH2-terminal region of the AP-2 alpha subunit (Gaidarov et al. 1996, J. Biol, Chem. 271:20922-20929), Here we used deleti on and site-directed mutagenesis to determine that ct residues 21-80 compri se a discrete folding and inositide-binding domain. Further, positively cha rged residues located within this region are involved in binding, with a ly sine triad at positions 55-57 particularly critical. Mutant peptides and pr otein in which these residues were changed to glutamine retained wild-type structural and functional characteristics by several criteria including cir cular dichroism spectra, resistance to limited proteolysis, and clathrin bi nding activity. When expressed in intact cells. mutated alpha subunit showe d defective localization to clathrin-coated pits; at high expression levels , the appearance of endogenous AP-2 in coated pits was also blocked consist ent with a dominant-negative phenotype, These results, together with recent work indicating that phosphoinositides are also critical to ligand-depende nt recruitment of arrestin-receptor complexes to coated pits (Gaidarov et a l. 1999. EMBO (Eur. Mol, Biol. Orgnn.) J, 18:871-881), suggest that phospho inositides play a critical and general role in adaptor incorporation into p lasma membrane clathrin-coated pits.