Involvement of Gα(i2) in the maintenance and biogenesis of epithelial cell tight junctions

Abstract

Polarized epithelial cells have highly developed tight junctions (TJ) to maintain an impermeant barrier and segregate plasma membrane functions, but the mechanisms that promote TJ formation and maintain its integrity are only partially defined. Treatment of confluent monolayers of Madin-Darby canine kidney (MDCK) cells with AlF4/- (activator of heterotrimeric G protein α subunits) results in a 3-4-fold increase in transepithelial resistances (TER), a reliable indicator of TJ integrity. MOCK cells transfected with activated Gα0 (Q205L) have acclerated TJ formation (Denker, B. M., Saha, C., Khawaja, S., and Nigam, S. J. (1996) J. Biol. Chem. 271, 25750-25753). Gα(i2) has been localized within the tight junction, and a role for Gα(i2) in the formation and/or maintenance of the tight junction was studied by transfection of MDCK cells with vector without insert (PC), wild type Gα(i2), or a GTPase-deficient mutant (constitutively activated), Q205Lα(i2). Tryptic conformational analysis confirmed expression of a constitutively active Gα(i2) in Q205Lα(i2)-MDCK cells, and confocal microscopy showed a similar pattern of Gα(i2) localization in the three cell lines. Q205Lα(i2)-MDCK cells had significantly higher base-line TER values than wild type Gα(i2)- or PC-MDCK cells (1187 ± 150 versus 576 ± 89 (Gα(i2)); 377 ± 52 Ω · cm2 (PC)), and both Gα(i2)- and Q205Lα(i2)- transfected cell lines more rapidly develop TER in the Ca2+ switch, a model widely used to study the mechanisms of junctional assembly. Treatment of cells with AlF4/- during the Ca2+ switch had little effect on the kinetics of TER development in Gα(i2)- or Q205Lα(i2)-MDCK cells, but PC cells reached half-maximal TER significantly sooner in the presence of AlF4/- (similar times to Gα(i2)-transfected cells). Base-line TER values obtained after the switch were significantly higher for all three cell lines in the presence of AlF4/-. These findings indicate that Gα(i2) is important for both the maintenance and development of the TJ, although additional Gα subunits are likely to play a role.