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Zonula Occludens-1 Is a Scaffolding Protein for Signaling Molecules

2002; Elsevier BV; Volume: 277; Issue: 28 Linguagem: Inglês

10.1074/jbc.c200240200

1083-351X

Tobias Meyer, Catherine Schwesinger, Bradley M. Denker,

Cancer Cells and Metastasis

Zonula occludens proteins are multidomain proteins usually localized at sites of intercellular junctions, yet little is known about their role in regulating junctional properties. Multiple signaling proteins regulate the junctional complex, and several (including G proteins) have been co-localized with zonula occludens-1 (ZO-1) in the tight junction of epithelial cells. However, evidence for direct interactions between signaling proteins and tight junction proteins has been lacking. In these studies, we constructed Gα-glutathione S -transferase (GST) fusion proteins and tested for interactions with [ 35 S]methionine-labeled in vitro translated ZO-1 and ZO-2. Only Gα 12 directly interacted with in vitro translated ZO-1 and ZO-2. Using a series of ZO-1 domains expressed as GST fusion proteins and in vitro translated [ 35 S]methionine-labeled Gα 12 , we found that Gα 12 and constitutively active (Q229L) α 12 (QLα 12 ) bind to the Src homology 3 (SH3) domain of ZO-1. This binding was not detected with SH3 domains from other proteins. Inducible expression of wild-type α 12 and QLα 12 in Madin-Darby canine kidney (MDCK) cells was established using the Tet-Off system. In Gα 12 -expressing cells, we found that ZO-1 and Gα 12 co-localize by confocal microscopy and co-immunoprecipitate. Gα 12 from MDCK cell lysates bound to the GST-ZO-1-SH3 domain, and expression of QLα 12 in MDCK cells reversibly increased paracellular permeability. These studies indicated that ZO-1 directly interacts with Gα 12 and that Gα 12 regulates barrier function of MDCK cells.