We are interested in determining the molecular mechanism by which Rac affects the expression of the polarized phenotype in FRT thyroid epithelial cells. To this aim an inducible, constitutively-active form of Rac, ER-Rac(QL), and the inducible, dominant-negative ER-Rac(N17) were stably expressed in FRT cells. By immunofluorescence analysis and cell fractionation we determined that upon tamoxifen treatment of FRT clones expressing ER-Rac(QL), the protein moves from the cytosol to the plasma membrane. The same is true for the dominant-negative ER-Rac(N17) after tamoxifen treatment. The bulk of endogenous Rac is also localized on the plasma membrane of wild-type FRT cells. Treatment with the specific Rac inhibitor NSC23766, removes endogenous Rac from the plasma membrane. Strikingly, E-cadherin is correspondingly removed from the membrane, likely by endocytosis. Chelation of calcium in the culture medium, in a Ca++ switch assay, also causes internalization of E-cadherin from the plasma membrane and the partial removal of Rac. Intracellular E-cadherin and Rac do not colocalize. The coordinate regulation of the association of both proteins to the plasma membrane is under investigation
