Gα12 regulates protein interactions within the MDCK cell tight junction and inhibits tight-junction assembly

Abstract

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. Gα12 (GNA12) binds the TJ protein ZO-1 (TJP1), and Gα12 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 Gα12 is necessary for signaling. TJ integrity is disrupted by Gα12-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 Gα12-stimulated Src activation and phosphorylation, but does not affect protein levels or the Gα12–ZO-1 interaction. Using the calcium-switch model of TJ assembly and GST-TPR (GST-fused TPR domain of PP5) pull-downs of activated Gα12, we demonstrate that switching to normal calcium medium activates endogenous Gα12 during TJ assembly. Thrombin increases permeability and delays TJ assembly by activating Gα12, but not Gα13, signaling pathways. These findings reveal an important role for Gα12, Src and Hsp90 in regulating the TJ in established epithelia and during TJ assembly.

Membrane-Initiated Estradiol Signaling of Epithelial-Mesenchymal Transition-Associated Mechanisms Through Regulation of Tight Junctions in Human Breast Cancer Cells

“…This observation was in agreement with previous reports showing that formation of the c-Src/ZO-1 complex was related to increased paracellular permeability in MDCK cells. This mechanism involves disrupting protein-protein interactions within the multiple protein TJ complexes, including the displacement of ZO-1 and occludin from this complex [32].…”

Polycystin-1 Protein Level Determines Activity of the Gα12/JNK Apoptosis Pathway

“…23), and maintained in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum, 100 g/ml G418, and 100 units/ml penicillin/streptomycin. MDCK cells with Tet-Off inducible G␣ 12 expression (G␣ 12 -MDCK cells) (17,24,25) were maintained in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum, 100 g/ml G418, 100 g/ml hygromycin, and 40 ng/ml doxycycline. G␣ 12 expression was induced by removal of doxycycline, and cells were analyzed at 48 h.…”

“…Glutathioneagarose beads (Amersham Biosciences) were added for 2 h, and samples were centrifuged, washed, and eluted in SDS sample buffer, followed by SDS-PAGE and autoradiography. For thrombin-stimulated G␣ 12 binding to the PC1 C terminus, TetOff G␣ 12 -MDCK cells (described previously in detail (17,24,25)) were treated with thrombin (2 units/ml) for the indicated times after overnight serum starvation. Cells were lysed on ice and incubated with GST fusion proteins (GST alone or with the PC1 C terminus) immobilized on glutathione-Sepharose beads at 4°C for 2 h. Beads were centrifuged and washed five times with 1 ml of ice-cold PBS containing 0.5% Triton X-100, and samples were eluted with SDS sample buffer and analyzed by SDS-PAGE and Western blotting with anti-rabbit G␣ 12 antibody (1:500 dilution; Santa Cruz Biotechnology).…”

“…To examine whether the PC1 C terminus can distinguish between the two family members G␣ 12 and G␣ 13 (67% identical), we used Tet-Off inducible MDCK cells expressing G␣ 12 and QL␣ 12 , that have been characterized previously (17,24,25). Endogenous G␣ 12 is below the detection limit in most cell types, whereas G␣ 13 is readily detectable.…”

The Biology of Epithelial Cell Tight Junctions in the Kidney

“…G␣ 12 activates during TJ assembly in the calcium switch model of TJ biogenesis, suggesting an important role for this G protein in regulating both baseline TJ permeability and its assembly or disassembly. 24 Rho GTPases are also major regulators of the TJ and the actin cytoskeleton. The inhibition of Rho leads to disassembly of TJ in various epithelial cell lines, and basal Rho activity is required for normal TJ function.…”