Secondary antibodies included goat antiCmouse, goat antiCmouse IgG1-isotypeCspecific and IgG2b-isotypeCspecific antibodies, and goat antiCrabbit. undergo clathrin-dependent endocytosis contain endocytosis signals that bind to adapter proteins, including the well-characterized tetramer AP2 and monomeric phosphotyrosine-binding domain proteins Dab2, Numb, and autosomal recessive hypercholesterolemia (ARH; Traub, 2003). Multiple low affinity interactions between adapter proteins and clathrin then cooperate to assemble a clathrin-coated pit that invaginates and pinches off to form an intracellular vesicle. Dab2 is an NPXY sequenceCspecific clathrin adapter that internalizes the low density lipoprotein receptor (LDLR) and related receptors (Keyel et al., 2006; Maurer and Cooper, 2006). It forms dynamic complexes with its cargoes and recruits clathrin (Morris and Cooper, 2001; Mishra et al., 2002; Keyel et al., 2006; Chetrit et al., 2009). Dab2 expression is strongly reduced in many different carcinomas, particularly ovarian and mammary tumors (Mok et al., 1994; Schwahn and Medina, 1998), and Dab2 loss allows carcinoma cells to resist anoikis (Sheng et al., 2000; Wang et al., 2001). Dab2 has also been reported to regulate the migration of various cell types (Hocevar et al., 2005; Orlandini et al., 2008). It is unclear whether the B-Raf IN 1 roles of Dab2 in cancer and migration stem from its function as an endocytic adapter or other mechanisms. Integrins are cell surface receptors for various ECM components, with different combinations of integrin and subunits conferring ECM ligand specificity (Hynes, 1992). Integrins act as bistable switches, toggling between an inactive, unbound state and an active conformation simultaneously able to bind the ECM and the cytoskeleton (Carman and Springer, 2003). Binding to the ECM and cytoskeleton induces the clustering of active integrins into structures known as focal complexes or adhesions from which signals are generated to regulate cellular B-Raf IN 1 responses. However, unbound integrins are inactive and diffuse rapidly in the plane of the membrane (Duband et al., 1988). Cell migration requires active focal adhesion disassembly and integrin recycling to allow new contacts to form near the front of the cell (Webb et al., 2004; Jones et al., 2006). After focal adhesion disassembly, integrins may diffuse or are actively recycled, via intracellular compartments, to sites of new adhesion assembly (Bretscher, 1996; Caswell and Norman, 2006). Intracellular integrin trafficking routes and their regulation are becoming understood (Lawson and Maxfield, 1995; Pierini et al., 2000; Laukaitis et al., 2001; Rappoport and Simon, 2003). Specifically, integrin recycling can occur through short loop, returning directly from early endosomes to the nearby cell surface, or long loop, passing via a perinuclear recycling compartment and then returning to the cell surface at distant sites, including the leading edge. In IL1B cancer and epithelial cells, the long-loop pathway is needed for migration on collagen-coated surfaces and invasion of the collagen matrix (Powelka et al., 2004; Roberts et al., 2004; Li et al., 2005; Jones B-Raf IN 1 et al., 2006). However, the molecules that internalize integrins and route them to the appropriate recycling pathway are less clear. Endocytosis of different integrins may be clathrin dependent or independent, depending on the cell type and environment (Altankov and Grinnell, 1993; Memmo and McKeown-Longo, 1998; Upla et al., 2004; Caswell and Norman, 2006). Importantly, dynamin-dependent integrin endocytosis may drive focal adhesion disassembly (Ezratty et al., 2005). However, cells B-Raf IN 1 in suspension also internalize integrins, suggesting that mechanisms for bulk turnover of inactive integrins exist (Bretscher, 1989). In an unbiased screen for Dab2-modulated receptors that may explain the role for Dab2 in cancer and cell migration, we found that depletion of Dab2 slows the endocytosis of several but not all integrins by HeLa cells. Measurements of specific integrins revealed that Dab2 regulates the bulk of constitutive endocytosis of inactive B-Raf IN 1 integrin 1 by HeLa cells and human foreskin fibroblasts (HFFs). Dab2 and integrin 1 colocalize in clathrin-coated pits at many sites dispersed over the cell surface, not specifically at adhesion sites, suggesting that Dab2 may trap freely diffusing integrins in coated pits. Dab2-dependent endocytosis maintains the intracellular pool of integrin 1. Dab2 also regulates cell migration depending on its endocytic function. Our data suggest that Dab2-mediated bulk integrin endocytosis is important to maintain an intracellular pool of integrin available for.