Getting AP2 to the Plasma Membrane The Role of Phosphoinositides

Overwhelming genetic, cell biological, and biochemical evidence implicates phos-phoinositides, specifically phosphatidylinositol 4,5-bisphosphate (PIP2), in clathrin-mediated endocytosis (Di Paolo and De Camilli 2006; Krauss and Haucke 2007). The AP-2 complex binds to PIP2 and perhaps also to phosphatidyl-inositol (3,4,5) trisphosphate (PIP3) (Beck and Keen 1991; Haucke 2005). PIP3 can be synthesized at sites of endocytosis from PIP2 by a clathrin activated PI3 kinase C2a (Gaidarov et al. 2001) or downstream of receptor activation (Lefkowitz and Shenoy 2005). The a-chain of AP-2 interacts with high affinity with PIP2 via a positively charged site, including R11, N39, K43, K57, Y58, and K61 (Collins et al. 2002; Honing et al. 2005). Mutations in the putative binding motif of the a chain lead to AP-2 mislocali-zation (Gaidarov and Keen 1999), suggesting that this site serves as the primary determinant for targeting AP-2 to sites of endocytosis. Consistent with this, masking or depleting PIP2 in living cells has been shown to result in AP-2 mislocalization and inhibition of endocytosis (Jost et al. 1998; Krauss et al. 2003; Varnai et al. 2006).

The |2-chain of the AP-2 complex was also found to interact with PIP2. The interaction of PIP2 with |2 chain is again mediated by a number of basic residues (Lys341, Lys343, Lys345, Lys354, and Lys356) (Collins et al. 2002). This lysine cluster is a surface-exposed positively charged patch within subdomain B of the cargo recognition domain of |2 (Owen and Evans 1998; Nesterov et al. 1999). Changing residues K345, K354, and K356 to glutamates reduced the |2 affinity to PIP2. Mutant |2 lacking this 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 (Rohde et al. 2002). Moreover, it was shown that expression of mutant |2 inhibits receptor-mediated endocytosis in living cells (Rohde et al. 2002). However, mutating residues K341, K343, and K345 lead to fully functional |2 following siRNA-mediated knockdown and rescue (Motley et al. 2006).

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