Gα12 regulates epithelial cell junctions through Src tyrosine kinases

Regulation and assembly of the epithelial cell junctional complex involve multiple signaling mechanisms, including heterotrimeric G proteins. Recently, we demonstrated that Gα12 binds to the tight junction scaffolding protein ZO-1 through the SH3 domain and that activated Gα12 increases paracellular permeability in Madin-Darby canine kidney (MDCK) cells (Meyer et al. J Biol Chem 277: 24855-24858, 2002). In the present studies, we explore the effects of Gα12 expression on tight and adherens junction proteins and examine downstream signaling pathways. By confocal microscopy, we detect disrupted tight and adherens junction proteins with increased actin stress fibers in constitutively active Gα12 (QLα12)-expressing MDCK cells. The normal distribution of ZO-1 and Na-K-ATPase was altered in QLα12-expressing MDCK cells, consistent with loss of polarity. We found that the tyrosine kinase inhibitor genistein and the Src-specific inhibitor PP-2 reversibly abrogated the QLα12 phenotype on the junctional complex. Junctional protein localization was preserved in PP-2- or genistein-treated QLα12-expressing cells, and the increase in paracellular permeability as measured by transepithelial resistance and [3H]mannitol flux was prevented by the inhibitors. Src activity was increased in QLα12-expressing MDCK cells as assessed by Src autophosphorylation, and β-catenin tyrosine phosphorylation was also increased, although there was no detectable increase in Rho activity. Taken together, these results indicate that Gα12 regulates MDCK cell junctions, in part through Src tyrosine kinase pathways.