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 α subunit (Gaidarov et al. 1996. J. Biol. Chem. 271:20922–20929). Here we used deletion and site- directed mutagenesis to determine that α 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 α 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.
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23 August 1999
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August 23 1999
Phosphoinositide–Ap-2 Interactions Required for Targeting to Plasma Membrane Clathrin-Coated Pits
Ibragim Gaidarov,
Ibragim Gaidarov
aKimmel Cancer Institute and the Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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James H. Keen
James H. Keen
aKimmel Cancer Institute and the Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Search for other works by this author on:
Ibragim Gaidarov
aKimmel Cancer Institute and the Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
James H. Keen
aKimmel Cancer Institute and the Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
1.used in this paper: AP, assembly or associated protein; CD, circular dichroism; MBP, maltose-binding protein; PH, pleckstrin homology; PPI, polyphosphoinositide or inositol polyphosphate
Online ISSN: 1540-8140
Print ISSN: 0021-9525
© 1999 The Rockefeller University Press
1999
The Rockefeller University Press
J Cell Biol (1999) 146 (4): 755–764.
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Ibragim Gaidarov, James H. Keen; Phosphoinositide–Ap-2 Interactions Required for Targeting to Plasma Membrane Clathrin-Coated Pits. J Cell Biol 23 August 1999; 146 (4): 755–764. doi: https://doi.org/10.1083/jcb.146.4.755
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