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Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands
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Expanding role of G proteins in tight junction regulation: Galpha(s) stimulates TJ assembly

C Saha1, S K Nigam, B M Denker

  • 1Renal Division, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

Biochemical and Biophysical Research Communications
|July 11, 2001
PubMed

Multiple signaling mechanisms regulate epithelial cell tight junction (TJ) assembly and maintenance. Several G proteins are likely to regulate these processes, but only G(i/o) have been specifically tested. Treatment of MDCK cells with cholera toxin, a Galpha(s) activator, accelerated TJ development in the calcium switch as measured by the time to half-maximal [T(50) (H)] transepithelial resistance (TER). Galpha(s) was predominantly localized in the lateral membrane, but a fraction colocalizes with ZO-1 in the TJ. MDCK cell lines expressing epitope-tagged Galpha(s) and constitutively active (R201Calpha(s)) showed a similar localization. TJ assembly was significantly faster in R201Calpha(s)-MDCK cell lines (T(50) (H) of 1.7 versus 3.3 h for controls) without detectable differences in cAMP levels. Confocal studies showed R201Calpha(s)-MDCK cells more rapidly localized ZO-1 and occludin into the developing TJ without affecting E-cadherin or Na(+)/K(+) ATPase localization. Endogenous Galpha(s) and R201Calpha(s) were immunoprecipitated with ZO-1 at baseline and during TJ assembly. The data supports a model of multiple Galpha subunits interacting with TJ proteins to regulate the assembly and maintenance of the TJ.

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