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© The Rockefeller University Press, 0021-9525/1999//755 $5.00
The Journal of Cell Biology, Volume 146, Number 4, , 1999 755-764

Phosphoinositide–Ap-2 Interactions Required for Targeting to Plasma Membrane Clathrin-Coated Pits

^a Kimmel Cancer Institute and the Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Kimmel Cancer Institute and the Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA 19107.(215) 503-0622(215) 503-4624

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 subunit (Gaidarov et al. 1996. J. Biol. Chem. 271:20922–20929). Here we used deletion and site- directed mutagenesis to determine that 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 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. 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

© 1999 The Rockefeller University Press

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