Regulatory interactions in the recognition of endocytic sorting signals by AP-2 complexes

Abstract

1995; Nesterov et al., 1995a;Ohno et al., 1995;Heilker et al., 1996). The α chain of the AP-2 heterotetramer can Many plasma membrane proteins destined for endobind clathrin (Goodman and Keen, 1995), synaptotagmin cytosis are concentrated into clathrin-coated pits (Zhang et al., 1994), Eps15 (Benmerah et al., 1995 Tebar through the recognition of a tyrosine-based motif ), Grb2 (Okabayashi et al., 1996 and small their cytosolic domains by an adaptor (AP-2) complex.phosphorylated molecules like inositol phosphates and The μ2 subunit of isolated AP-2 complexes binds phosphoinositides (Beck and Keen, 1991a; Timerman specifically, but rather weakly, to proteins bearing et al., 1992;Voglmaier et al., 1992; Gaidarov et al., 1996). the tyrosine-based signal. We now demonstrate, usingThe physiological significance of these interactions is peptides with a photoreactive probe, that this binding under investigation. The β chain contacts clathrin and is strengthened significantly when the AP-2 complex drives coat assembly (Ahle and Ungewickell, 1989; is present in clathrin coats, indicating that there is Schroder and Ungewickell, 1991; Gallusser and cooperativity between receptor-AP-2 interactions and Shih et al., 1995). The μ2 chain coat formation. Phosphoinositides with a phosphate at recognizes the tyrosine-based endocytic motif (Ohno et al., the D-3 position of the inositol ring, but not other 1995; Boll et al., 1996), a sequence of four amino isomers, also increase the affinity of the AP-2 complex acids of the form tyrosine-polar-polar-large hydrophobic for the tyrosine-based motif. AP-2 is the first protein (YppØ), which is used for sorting proteins from the plasma known (in any context) to interact with phosphatidylmembrane to the endosome (Trowbridge et al., 1993; inositol 3-phosphate. Our findings indicate that recep- Thomas and Roth, 1994). tor recruitment can be coupled to clathrin coatThe interactions just described do not by themselves assembly and suggest a mechanism for regulation of explain how cargo recruitment and coat formation are membrane traffic by lipid products of phosphoinositide coupled and how concentration of cargo into coated 3-kinases.structures is achieved. Previous work has shown that the Keywords: adaptors/clathrin/coated pits/membrane isolated AP-2 complex, or even its isolated μ2 subunit, traffic/protein sorting can recognize the tyrosine-based motif (Ohno et al., 1995;Boll et al., 1996). This interaction reflects the specificity seen in vivo, but it is rather weak (dissociation constant Introduction~1 0 μM). Are there regulatory mechanisms that enhance

Phosphoinositides in Constitutive Membrane Traffic

“…Previously, it was observed that PtdIns(3)P and, to a lesser extent, PtdIns(3,4)P 2 enhanced the binding of AP2 adaptors to peptides containing internalization signals. Clathrin binding enhanced the affinity of AP2 for internalization signals to a similar extent, but the effect of clathrin and PtdIns(3)P were not additive (286). However, when fluorescent proteins that bind PtdIns(3)P are expressed in cells, they label endosomes and not the plasma membrane (32,92,318), suggesting that there is little free PtdIns(3)P at the cell surface.…”

“…Mutation of the PIP binding site on the AP2␣ subunit inhibits membrane binding, and mutation of the binding site on the AP2 subunit prevents binding to cargo, demonstrating the importance of the interaction with PIPs (111,291). AP2 can bind multiple PIPs, and in vitro those with D-3 phosphate increase the affinity of AP2 complexes for peptides that have internalization signals (286). This may be relevant to the observations that PtdIns3KIIC2␣ localizes in clathrin-coated pits (112) and that PtdIns(3,4,5)P 3 is important for recruitment of AP2 to activated ␤-adrenergic receptors (248).…”

Molecular Structure, Function, and Dynamics of Clathrin-Mediated Membrane Traffic

“…The heterotetramers of AP2 and AP1 have at least two distinct states-a closed or "locked" state and one or more open states (Rapoport et al 1997;Collins et al 2002;Jackson et al 2010). In the closed state, two well-studied cargo-binding sites are buried (Fig.…”

Direct interaction of Cbl with pTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR to lysosomes for degradation

“…A clathrin box in the hinge domain and an additional domain near the ear domain of the ß2-subunit facilitates direct binding to clathrin (Ahle and Ungewickell, 1989;Kirchhausen et al, 1989;Mousavi et al, 2004) and facilitates clathrin assembly. The ß2-subunit may also be involved in cargo recognition (Rapoport et al, 1998). Cargo is recruited through interactions between sorting motifs in protein cargo and the AP-2 complex.…”