Involvement of a heterotrimeric G protein alpha subunit in tight junction biogenesis

The tight junction (TJ) of polarized epithelial cells is critical for maintaining an impermeant barrier and epithelial cell polarity. The signaling events important for TJ assembly require regulated calcium stores and protein kinase C (PKC), but the earliest signaling events in the cascade have not been well defined. We now show that G alpha(i2) in Madin Darby canine kidney (MDCK) cells localizes to a region overlapping with the TJ. To further analyze the localization of G alpha subunits in epithelial cells, rat G alpha(o), Q205L alpha(o) (G alpha(o) ''activated'' by point mutation) and plasmid without insert (PC) were transfected into MDCK cells and localized by immunofluorescence and confocal microscopy, Similar to endogenous G alpha(i2), G alpha(o)-MDCK cells localize G alpha(o), (84% similar to G alpha(i2)) in the subapical region overlapping with ZO-1 (zona occludens-I), a key component of the TJ, PC-MDCK cells have no detectable G alpha(o). In Ga alpha(o)-MDCK cells, a physical association of G alpha(o) with components of the TJ was detectable by immunoprecipitation of ZO-1. Immunoprecipitates of ZO-1 from G alpha(o)-MDCK cells consistently coprecipitated G alpha(o). Constitutively active Q205LG alpha(o) localized to the subapical lateral membrane similar to wild-type G alpha(o). To determine if constitutively activated G alpha subunits can affect TJ biogenesis, the formation of tight junctions in PC, G alpha(o), and Q205L alpha(o)-MDCK cells was followed by measurement of transepithelial resistance (TER) during the Ca2+ switch, a model widely used to study mechanisms of junctional assembly, Baseline and post Ca2+ switch TER values did not differ among the cell lines, However, constitutively activated Q205L alpha(o)-MDCK, cells developed TER significantly faster than PC and G alpha(o) cells in the early phase (0-4 h) (54 +/- 4 versus 23 +/- 3 (PC); 12 +/- 1 (G alpha(o)) Ohm . m(2)/h) and late phase (4-h peak) (117 +/- 10 versus 45 +/- 5 (PC); 66 +/- 7 (G alpha(o)) Ohm . cm(2)/h) after Ca2+ switch, Peak TER values were significantly higher in Q205L alpha(o)-MDCK cells (1168 +/- 107 versus 437 +/- 37 (PC); 548 +/- 54 (G alpha(o)) Ohm . cm(2)). These results indicate that G alpha(o) and Q205L alpha(o) expressed in MDCK cells are localized near the junctional complex, associate with at least one TJ protein, and that activated G alpha(o) accelerates TJ biogenesis without significantly affecting the maintenance of the TJ, Together, these results suggest an important role for heterotrimeric G proteins in TJ assembly.