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Originally published In Press as doi:10.1074/jbc.C200240200 on May 21, 2002

J. Biol. Chem., Vol. 277, Issue 28, 24855-24858, July 12, 2002
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ACCELERATED PUBLICATION
Zonula Occludens-1 Is a Scaffolding Protein for Signaling Molecules
Galpha 12 DIRECTLY BINDS TO THE Src HOMOLOGY 3 DOMAIN AND REGULATES PARACELLULAR PERMEABILITY IN EPITHELIAL CELLS*

Tobias N. MeyerDagger §, Catherine Schwesinger||, and Bradley M. DenkerDagger **

From the Dagger  Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115 and the  Division of Surgery, Children's Hospital, Boston, Massachusetts 02115

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 Galpha -glutathione S-transferase (GST) fusion proteins and tested for interactions with [35S]methionine-labeled in vitro translated ZO-1 and ZO-2. Only Galpha 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 [35S]methionine-labeled Galpha 12, we found that Galpha 12 and constitutively active (Q229L) alpha 12 (QLalpha 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 alpha 12 and QLalpha 12 in Madin-Darby canine kidney (MDCK) cells was established using the Tet-Off system. In Galpha 12-expressing cells, we found that ZO-1 and Galpha 12 co-localize by confocal microscopy and co-immunoprecipitate. Galpha 12 from MDCK cell lysates bound to the GST-ZO-1-SH3 domain, and expression of QLalpha 12 in MDCK cells reversibly increased paracellular permeability. These studies indicated that ZO-1 directly interacts with Galpha 12 and that Galpha 12 regulates barrier function of MDCK cells.

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by Grant Me 1760/1-1 from the Deutsche-Forschungs-gemeinschaft, Bonn, Germany.

|| Supported by the Daimler-Benz Foundation, Ladenburg, Germany.

** Supported by National Institutes of Health Grant GM55223 and a clinical scientist award from the National Kidney Foundation. To whom correspondence should be addressed: Renal Division, Brigham and Women's Hospital, 77 Ave. Louis Pasteur, Boston, MA 02115. Tel.: 617-525-5809; Fax: 617-525-5830; E-mail: bdenker@rics.bwh.harvard.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.


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