The clathrin adaptor Dab2 recruits EH domain scaffold proteins to regulate integrin ß1 endocytosis.

Endocytic adaptor proteins facilitate cargo recruitment and clathrin-coated pit nucleation. The prototypical clathrin adaptor AP2 mediates cargo recruitment, maturation, and scission of the pit by binding cargo, clathrin, and accessory proteins, including the Eps-homology (EH) domain proteins Eps15 and intersectin. However, clathrin-mediated endocytosis of some cargoes proceeds efficiently in AP2-depleted cells. We found that Dab2, another endocytic adaptor, also binds to Eps15 and intersectin. Depletion of EH domain proteins altered the number and size of clathrin structures and impaired the endocytosis of the Dab2- and AP2-dependent cargoes, integrin ß1 and transferrin receptor, respectively. To test the importance of Dab2 binding to EH domain proteins for endocytosis, we mutated the EH domain-binding sites. This mutant localized to clathrin structures with integrin ß1, AP2, and reduced amounts of Eps15. Of interest, although integrin ß1 endocytosis was impaired, transferrin receptor internalization was unaffected. Surprisingly, whereas clathrin structures contain both Dab2 and AP2, integrin ß1 and transferrin localize in separate pits. These data suggest that Dab2-mediated recruitment of EH domain proteins selectively drives the internalization of the Dab2 cargo, integrin ß1. We propose that adaptors may need to be bound to their cargo to regulate EH domain proteins and internalize efficiently.

Quantitative proteomics identifies a Dab2/integrin module regulating cell migration.

Clathrin-associated endocytic adapters recruit cargoes to coated pits as a first step in endocytosis. We developed an unbiased quantitative proteomics approach to identify and quantify glycoprotein cargoes for an endocytic adapter, Dab2. Surface levels of integrins beta1, alpha1, alpha2, and alpha3 but not alpha5 or alphav chains were specifically increased on Dab2-deficient HeLa cells. Dab2 colocalizes with integrin beta1 in coated pits that are dispersed over the cell surface, suggesting that it regulates bulk endocytosis of inactive integrins. Depletion of Dab2 inhibits cell migration and polarized movement of integrin beta1 and vinculin to the leading edge. By manipulating intracellular and surface integrin beta1 levels, we show that migration speed correlates with the intracellular integrin pool but not the surface level. Together, these results suggest that Dab2 internalizes integrins freely diffusing on the cell surface and that Dab2 regulates migration, perhaps by maintaining an internal pool of integrins that can be recycled to create new adhesions at the leading edge.

Expression of integrin-linked kinase in the murine lens is consistent with its role in epithelial-mesenchymal transition of lens epithelial cells in vitro.

PURPOSE: To evaluate the expression and location of integrin-linked kinase (ILK) within the mouse lens and to characterize the role of this protein during mouse lens epithelial cells (LEC) differentiation in vitro. METHODS: Transcription levels of ILK mRNA were determined by RT-PCR in cultured cells and lens tissue. ILK protein was detected by immunoblotting, immunocytochemistry, immunohistochemistry, and immunoprecipitation. A role for ILK in the outgrowth of LEC from dissected mouse lens explants was determined by the use of ILK short interfering RNA (siRNA). Affinity-purified polyclonal anti-recombinant human ILK IgG was prepared and characterized for these experiments. A comparison of several anti-ILK antibodies was performed by immunoblotting, immunoprecipitation, and ELISA. RESULTS: ILK was transcribed in LEC and lens fiber cells in vivo. ILK protein was expressed in the differentiating LEC at the equatorial region of the lens and, to a lesser extent, within the cortical and nuclear fiber cells. LEC in vitro produced copious ILK, which exhibited a filamentous pattern throughout the cytoplasm. The expression of ILK was increased during epithelial-mesenchymal-transition (EMT) of LEC from lens explants, whereas inhibition of ILK by siRNA delayed expression of the EMT markers smooth muscle alpha-actin and fibronectin. CONCLUSIONS: Analysis of ILK expression, localization, and activity in the mouse lens and cultured LEC is substantially facilitated by the generation of a multi-functional, polyclonal, affinity-purified anti-ILK antibody. Expressed in most tissues and cells lines, ILK is unexpectedly restricted to the equatorial LEC and differentiated fiber cells of the mouse lens. The occurrence of ILK expression with LEC differentiation is consistent with the positive regulatory function of ILK, which is revealed in a model of EMT in vitro. This is the first study to show the expression of ILK in the lens and its unique distribution pattern within cultured lens epithelia.