3762

R-Ras is a member of the Ras superfamily of small GTPases, which act as molecular on/off switches for intracellular signaling pathways. R-Ras shares ≈55% identity with classical Ras proteins H-, N-, and K-Ras (1), and has a unique 26-amino acid N-terminal extension (2). Activation of R-Ras disrupts ductal morphogenesis of mammary epithelial cells (MECs) in 3D collagen gels and enhances cell migration (3,4). We show here that constitutive activation of R-Ras causes a decrease in adherens junction (AJ) formation and corresponding increase in focal adhesions (FAs). R-Ras inhibits AJ formation to a greater extent on collagen than on fibronectin, suggesting that R-Ras disrupts AJs by affecting a collagen receptor. It has been shown previously that R-Ras enhances adhesion through the α2β1 integrin, a collagen and laminin receptor (4). The α2β1 integrin not only mediates MEC polarization and tubulogenesis in 3D collagen matrices, it also mediates haptotactic migration on collagen (5,6,7). Because the α2β1 integrin can mediate these two opposing phenotypes, regulation of α2β1 integrin is likely important in epithelial vs. mesenchymal phenotype choice made by MECs in response to the extracellular matrix (ECM). We find that inhibition of the α2β1 integrin through addition of a blocking antibody, P1E6, causes a partial rescue of AJ formation on collagen but not fibronectin. In addition, E-cadherin co-immunoprecipitates with the α2β1 integrin in control cells, but not in cells overexpressing activated R-Ras. These data suggest that R-Ras disrupts adherens junctions in mammary epithelial cells through its effects on the α2β1 integrin, likely through increasing cell-substratum adhesion. (1) Self, A.J., H.F. Paterson, and A. Hall. Oncogene, 1993. 8(3):655-61. (2) Lowe, D.G., et al. Cell, 1987. 48(1):137-46. (3) Keely, P.J., et al. Journal of Cell Biology, 1999. 145(5):1077-88. (4) Kwong, L., et al. Molecular and Cellular Biology, 2003. 23(3):933-49. (5) Berdichevsky, F., et al. Journal of Cell Science, 1992. 102(Pt 3):437-46. (6) Chen, J., et al. American Journal of Pathology, 2002. 161(1):337-44. (7) Zutter, M.M., et al. Molecular Biology of the Cell, 2001. 12(S):8-9a.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]