I. Gaidarov et Jh. Keen, Phosphoinositide-AP-2 interactions required for targeting to plasma membrane clathrin-coated pits, J CELL BIOL, 146(4), 1999, pp. 755-764
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.