Membrane association of Rac and E-cadherin in FRT thyroid epithelial cells.

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.