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J. Cell Biol., Volume 146, Number 4, August 23, 1999 755-764
Copyright © 1999 by The Rockefeller University Press.

Phosphoinositide–AP-2 Interactions Required for Targeting to Plasma Membrane Clathrin-coated Pits

Ibragim Gaidarova and James H. Keena
a Kimmel Cancer Institute and the Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Correspondence to: James H. Keen, Kimmel Cancer Institute and the Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA 19107. Tel:(215) 503-4624 Fax:(215) 503-0622 E-mail:jim.keen{at}mail.tju.edu.

The clathrin-associated AP-2 adaptor protein is a major polyphosphoinositide-binding protein in mammalian cells. A high affinity binding site has previously been localized to the NH2-terminal region of the AP-2 {alpha} subunit (Gaidarov et al. 1996 Down. J. Biol. Chem. 271:20922–20929). Here we used deletion and site- directed mutagenesis to determine that {alpha} residues 21–80 comprise a discrete folding and inositide-binding domain. Further, positively charged residues located within this region are involved in binding, with a lysine triad at positions 55–57 particularly critical. Mutant peptides and protein in which these residues were changed to glutamine retained wild-type structural and functional characteristics by several criteria including circular dichroism spectra, resistance to limited proteolysis, and clathrin binding activity. When expressed in intact cells, mutated {alpha} subunit showed defective localization to clathrin-coated pits; at high expression levels, the appearance of endogenous AP-2 in coated pits was also blocked consistent with a dominant-negative phenotype. These results, together with recent work indicating that phosphoinositides are also critical to ligand-dependent recruitment of arrestin-receptor complexes to coated pits (Gaidarov et al. 1999 Down. EMBO (Eur. Mol. Biol. Organ.) J. 18:871–881), suggest that phosphoinositides play a critical and general role in adaptor incorporation into plasma membrane clathrin-coated pits.

Key Words: clathrin, adaptor, phosphatidylinositols, endocytosis, adaptins


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