G12 regulates protein interactions within the MDCK cell tight junction and inhibits tight-junction assembly

The polarized functions of epithelia require an intact tight junction (TJ) to restrict paracellular movement and to separate membrane proteins into specific domains. TJs contain scaffolding, integral membrane and signaling proteins, but the mechanisms that regulate TJs and their assembly are not well defined. G12 (GNA12) binds the TJ protein ZO-1 (TJP1), and G12 activates Src to increase paracellular permeability via unknown mechanisms. Herein, we identify Src as a component of the TJ and find that recruitment of Hsp90 to activated G12 is necessary for signaling. TJ integrity is disrupted by G12-stimulated Src phosphorylation of ZO-1 and ZO-2 (TJP2); this phosphorylation leads to dissociation of occludin and claudin 1 from the ZO-1 protein complex. Inhibiting Hsp90 with geldanamycin blocks G12-stimulated Src activation and phosphorylation, but does not affect protein levels or the G12-ZO-1 interaction. Using the calcium-switch model of TJ assembly and GST-TPR (GST-fused TPR domain of PP5) pull-downs of activated G12, we demonstrate that switching to normal calcium medium activates endogenous G12 during TJ assembly. Thrombin increases permeability and delays TJ assembly by activating G12, but not G13, signaling pathways. These findings reveal an important role for G12, Src and Hsp90 in regulating the TJ in established epithelia and during TJ assembly.