Key points are not available for this paper at this time.
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 Galphai2 in Madin Darby canine kidney (MDCK) cells localizes to a region overlapping with the TJ. To further analyze the localization of Galpha subunits in epithelial cells, rat Galphao, Q205Lalphao (Galphao "activated" by point mutation) and plasmid without insert (PC) were transfected into MDCK cells and localized by immunofluorescence and confocal microscopy. Similar to endogenous Galphai2, Galphao-MDCK cells localize Galphao, (84% similar to Galphai2) in the subapical region overlapping with ZO-1 (zona occludens-I), a key component of the TJ. PC-MDCK cells have no detectable Galphao. In Galphao-MDCK cells, a physical association of Galphao with components of the TJ was detectable by immunoprecipitation of ZO-1. Immunoprecipitates of ZO-1 from Galphao-MDCK cells consistently coprecipitated Galphao. Constitutively active Q205LGalphao localized to the subapical lateral membrane similar to wild-type Galphao. To determine if constitutively activated Galpha subunits can affect TJ biogenesis, the formation of tight junctions in PC, Galphao, and Q205Lalphao-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 Q205Lalphao-MDCK cells developed TER significantly faster than PC and Galphao cells in the early phase (0-4 h) (54 +/- 4 versus 23 +/- 3 (PC); 12 +/- 1 (Galphao) Omega.m2/h) and late phase (4-h peak) (117 +/- 10 versus 45 +/- 5 (PC); 66 +/- 7 (Galphao) Omega.m2/h) after Ca2+ switch. Peak TER values were significantly higher in Q205Lalphao-MDCK cells (1168 +/- 107 versus 437 +/- 37 (PC); 548 +/- 54 (Galphao) Omega.cm2). These results indicate that Galphao and Q205Lalphao expressed in MDCK cells are localized near the junctional complex, associate with at least one TJ protein, and that activated Galphao 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.
Denker et al. (Tue,) studied this question.