A phosphatidylinositol (4,5)-bisphosphate binding site within μ2-adaptin regulates clathrin-mediated endocytosis

Abstract

The clathrin adaptor complex AP-2 serves to coordinate clathrin-coated pit assembly with the sorting of transmembrane cargo proteins at the plasmalemma. How precisely AP-2 assembly and cargo protein recognition at sites of endocytosis are regulated has remained unclear, but recent evidence implicates phosphoinositides, in particular phosphatidylinositol (4,5)-bisphosphate (PI[4,5]P2), in these processes. Here we have identified and functionally characterized a conserved binding site for PI(4,5)P2 within μ2-adaptin, the medium chain of the clathrin adaptor complex AP-2. Mutant μ2 lacking a cluster of conserved lysine residues fails to bind PI(4,5)P2 and to compete the recruitment of native clathrin/AP-2 to PI(4,5)P2-containing liposomes or to presynaptic membranes. Moreover, we show that expression of mutant μ2 inhibits receptor-mediated endocytosis in living cells. We suggest that PI(4,5)P2 binding to μ2-adaptin regulates clathrin-mediated endocytosis and thereby may contribute to structurally linking cargo recognition to coat formation.

Specific Interaction between SNAREs and Epsin N-terminal Homology (ENTH) Domains of Epsin-related Proteins in trans-Golgi Network to Endosome Transport

“…Liposomes were generated, and liposome binding was performed as described (17). 100 g of liposomes (70% phosphatidylcholine, 20% phosphatidylethanolamine, and 10% variable lipids) were incubated with 4 g of the Strep-tagged ENTH domain of Ent3p in 150 l of 25 mM Hepes, pH 8, and 50 mM NaCl at 4°C for 15 min.…”

Phosphoinositides in Constitutive Membrane Traffic

“…This change may be regulated by phosphorylation and the open conformation stabilized by binding to PIPs. 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).…”

Stimulation of phosphatidylinositol kinase type I-mediated phosphatidylinositol (4,5)-bisphosphate synthesis by AP-2μ–cargo complexes

“…Biochemical, genetic, and cell biological data suggest that PI(4,5)P 2 plays an essential role in clathrin-dependent endocytosis of plasma membrane proteins including nutrient (4) and growth factor receptors, postsynaptic ion channels, as well as synaptic vesicle proteins (9)(10)(11). Endocytic adaptor proteins like the heterotetrameric AP-2 complex (via its ␣ and 2 subunits) (12,13), AP180͞CALM, epsin (14), Dab2, and HIP1͞1R as well as the large GTPase dynamin (15) all bind directly to PI(4,5)P 2 . In addition to serving as a membrane attachment site for endocytic proteins, PI(4,5)P 2 also plays an active role by allosterically eliciting a conformational change within AP-2 that is required for its interaction with sorting signals of transmembrane proteins (16) and to stably associate with the plasmalemma (17).…”