Figure 4

R-Ras regulates integrin localization in the plasma membrane. (A) Immunofluorescence of fixed Cos7 cells using antibodies against R-Ras and β₁-integrin reveal colocalization of endogenous R-Ras and β₁-integrin at ruffles. Scale bar = 20 μm. (B) R-Ras activation enhances integrin localization to low-density lipid fractions. Lysates from T47D cells stably expressing R-Ras(38V), R-Ras(41A) or control (vector only) were fractionated on an Optiprep™ gradient, and analyzed by subsequent SDS-PAGE and immunoblotting. Activated R-Ras increased the amount of β₁-integrin (determined by antibody to the α₂ subunit) at the plasma membrane, specifically in the low-density microdomain that contains Src, which itself was not increased in this membrane fraction by activation of R-Ras. Integrin was not found in the bulk plasma membrane, identified by Rack1. (C) Cos7 cell adhesion on increasing fibronectin (FN) concentrations was reduced following the transfection of 20 nM siRNA directed against R-Ras.