Gα12 Inhibits α2β1 Integrin–mediated Madin-Darby Canine Kidney Cell Attachment and Migration on Collagen-I and Blocks Tubulogenesis

Abstract

Regulation of epithelial cell attachment and migration are essential for normal development and maintenance of numerous tissues. G proteins and integrins are critical signaling proteins regulating these processes, yet in polarized cells little is known about the interaction of these pathways. Herein, we demonstrate that Gα12 inhibits interaction of MDCK cells with collagen-I, the major ligand for α2β1 integrin. Activating Gα12 (QL point mutation or stimulating endogenous Gα12 with thrombin) inhibited focal adhesions and lamellipodia formation and led to impaired cell migration. Consistent with Gα12-regulated attachment to collagen-I, Gα12-silenced MDCK cells revealed a more adherent phenotype. Inhibiting Rho kinase completely restored normal attachment in Gα12-activated cells, and there was partial recovery with inhibition of Src and protein phosphatase pathways. Gα12 activation led to decreased phosphorylation of focal adhesion kinase and paxillin with displacement of α2 integrin from the focal adhesion protein complex. Using the MDCK cell 3D-tubulogenesis assay, activated Gα12 inhibited tubulogenesis and led to the formation of cyst-like structures. Furthermore, Gα12-silenced MDCK cells were resistant to thrombin-stimulated cyst development. Taken together, these studies provide direct evidence for Gα12–integrin regulation of epithelial cell spreading and migration necessary for normal tubulogenesis.