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

Abstract

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 Gα12 in Madin Darby canine kidney (MDCK) cells localizes to a region overlapping with the TJ. To further analyze the localization of Gα subunits in epithelial cells, rat Gα(o), Q205Lα(o) (Gα(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 Gα12, Gα(o)-MDCK cells localize Gα(o), (84% similar to Gα12) in the subapical region overlapping with ZO-1 (zona occludens-I), a key component of the TJ. PC-MDCK cells have no detectable Gα(o). In Gα(o)-MDCK cells, a physical association of Gα(o) with components of the TJ was detectable by immunoprecipitation of ZO-1. Immunoprecipitates of ZO-1 from Gα(o)-MDCK cells consistently coprecipitated Gα(o). Constitutively active Q205LGα(o) localized to the subapical lateral membrane similar to wild-type Gα(o). To determine if constitutively activated Gα subunits can affect TJ biogenesis, the formation of tight junctions in PC, Gα(o), and Q205Lα(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 Q205Lα(o)-MDCK cells developed TER significantly faster than PC and Gα(o) cells in the early phase (0-4 h) (54 ± 4 versus 23 ± 3 (PC); 12 ± 1 (Gα(o)) Ω·m2/h) and late phase (4-h peak) (117 ± 10 versus 45 ± 5 (PC); 66 ± 7 (Gα(o)) Ω·cm2/h) after Ca2+ switch. Peak TER values were significantly higher in Q205Lα(o)-MDCK cells (1168 ± 107 versus 437 ± 37 (PC); 548 ± 54 (Gα(o)) Ω·cm2). These results indicate that Gα(o) and Q205Lα(o) expressed in MDCK cells are localized near the junctional complex, associate with at least one TJ protein, and that activated Gα(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.