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. 1999 Aug 23;146(4):755-64.
doi: 10.1083/jcb.146.4.755.

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

I Gaidarov  1 , J H Keen
Affiliations

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

I Gaidarov et al. J Cell Biol. .

Abstract

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 NH(2)-terminal region of the AP-2 alpha subunit (Gaidarov et al. 1996. 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. 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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Figures

Figure 1
Figure 1
A PPI binding site in the NH2-terminal region of AP-2 α. Indicated segments of the AP-2α sequence coupled at their NH2 terminus to MBP were analyzed for [3H]IP6 binding and approximate binding affinity. A high affinity site is present within residues 21–80, while shorter segments do not retain specific binding activity.
Figure 2
Figure 2
Site-directed mutagenesis of the PPI binding domain of AP-2 α MBP fusion proteins containing the indicated residue alterations were expressed and tested for retention of [3H]IP6 binding. The lysine triad at positions 55–57 was most critical, and alteration to glutaminyl residues virtually abolished binding.
Figure 3
Figure 3
Secondary structure of the PPI binding domain of AP-2 α. (a) Circular dichroism spectrum in aqueous solution (solid line) of the isolated wild-type AP-2 α5-80 fragment cleaved from MBP reveals substantial secondary structure. In 50% trifluoroethanol (dashed line) it exhibits almost complete α-helical folding. (b) The circular dichroism spectra of the wild-type (solid line) and KKK/Q mutant (dashed line) AP-2 α5-80 polypeptides are virtually identical, consistent with retention of the secondary structure present in the native structure.
Figure 3
Figure 3
Secondary structure of the PPI binding domain of AP-2 α. (a) Circular dichroism spectrum in aqueous solution (solid line) of the isolated wild-type AP-2 α5-80 fragment cleaved from MBP reveals substantial secondary structure. In 50% trifluoroethanol (dashed line) it exhibits almost complete α-helical folding. (b) The circular dichroism spectra of the wild-type (solid line) and KKK/Q mutant (dashed line) AP-2 α5-80 polypeptides are virtually identical, consistent with retention of the secondary structure present in the native structure.
Figure 4
Figure 4
AP-2 α and the KKK/Q mutant lacking PPI binding have similar domain structure and clathrin binding activity. (a) In vitro translated wild-type AP-2 α subjected to limited proteolysis with 0, 50, 100, or 150 ng/ml trypsin (lanes 1–4, respectively) for 15 min at room temperature results in generation of fragments corresponding to the 60–66-kD core (C) and 40-kD appendage (A) domains, following preferential cleavage in the hinge region as previously described (Goodman and Keen 1995). The cleavage pattern of the mutant AP-2 α KKK/Q translation product is virtually identical in terms of both protease sensitivity and fragments generated, indicating the presence of similar folded domains. (b) Most of input (I) of in vitro–translated wild-type AP-2 α binds to clathrin cages (30 μg) and becomes sedimentable in their presence (+), while little sediments in their absence (−), as previously reported (Goodman and Keen 1995). The mutant AP-2 α KKK/Q translation product shows essentially identical behavior.
Figure 5
Figure 5
Wild-type and mutant KKK/Q AP-2 α polypeptides are associated with AP-2 subunits in intact cells. Equal amounts of lysates prepared under nondenaturing conditions from untransfected mouse MOP8 cells (mock) or cells transfected with either wild-type AP-2 αγα (wt) or mutant αγα (KKK/Q) cDNAs were immunoprecipitated (IP) with antibody 100/3, which recognizes only the bovine γ hinge insert present in the exogenous transiently expressed protein. Both the wild-type and PPI binding defective KKK/Q mutant AP-2 α polypeptides (revealed by blotting with anti-γ antibodies) are incorporated into AP-2 complexes as revealed by immunoblotting of the immunoprecipitates with anti-β, anti-μ2, and anti-σ2 antibodies.
Figure 7
Figure 7
Clathrin distribution is normal at low expression levels of AP-2PPI−, but is perturbed at higher levels. Bar, 10 μm.
Figure 6
Figure 6
AP-2WT, but not AP-2PPI−, is targeted normally to clathrin-coated pits at the plasma membrane. (a) AP-2WT expressed in BALB/c-3T3 cells exhibits a punctate pattern entirely coincident with that of endogenous AP-2 α and is localized to plasma membrane clathrin-coated pits. With increasing levels of αγα expression (right panels), the incorporation of the endogenous AP-2 α polypeptide into coated pits is diminished. (b) Expressed AP-2PPI− does not colocalize with endogenous AP-2 α in coated pits. With increasing expression of AP-2PPI−, correct localization of endogenous AP-2 to plasma membrane clathrin–coated pits is also impaired (right panels). Bar, 10 μm.
Figure 8
Figure 8
Basic residues involved in PPI binding in mammalian AP-2 α are highly conserved. Sequence alignments of mouse AP-2 α (Mouse α, accession number P17426) with Drosophila melanogaster (Dm α, accession number Y13092), Caenorhabditis elegans (Ce α, accession number U28742), Saccharomyces cerevisiae (Sc α, accession number P38065), Schizosaccharomyces pombe (Sp α, accession number AB004535), mouse γ adaptin (mouse γ, accession number X54424), human δ (accession number AF002163), and human ∈ (I.M.A.G.E. Clone ID 1031294; Dell'Angelica, E., personal communication). Sequence comparisons were performed with PILEUP, with numbering according to the mouse AP-2 α sequence. Residues identical to the mouse AP-2 α protein in four or more other sequences are highlighted on a black background; nonidentical but conserved residues in three or more sequences are shaded. Asterisks denote basic residues critical to PPI binding in the AP-2 α sequence.
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