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J. Biol. Chem., Vol. 282, Issue 13, 9789-9796, March 30, 2007

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The Gap Junction Protein Connexin32 Interacts with the Src Homology 3/Hook Domain of Discs Large Homolog 1^*

Heather S. Duffy¹, Ionela Iacobas, Kylie Hotchkiss, Bethany J. Hirst-Jensen^¶, Alejandra Bosco, Nadine Dandachi, Rolf Dermietzel^||, Paul L. Sorgen^¶, and David C. Spray

From the Department of Pharmacology, Columbia University, New York, New York 10032, Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461, ^¶Department of Biochemistry and Molecular Biology, University of Nebraska, Omaha, Nebraska 68918, and ^||Department of Neuroscience and Molecular Brain Research, University of Bochum, D-44780 Bochum, Germany

Scaffolding of membrane proteins is a common strategy for forming complexes of proteins, including some connexins, within membrane microdomains. Here we describe studies indicating that Cx32 interacts with a PDZ-containing scaffolding protein, Dlgh1 (Discs Large homolog 1). Initial screens of liver lysates using antibody arrays indicated an interaction between Cx32 and Dlgh1 that was confirmed using coimmunoprecipitation studies. Yeast two-hybrid complementation determined that the Cx32 bound via interaction with the SH3/Hook domain of Dlgh1. Confocal microscopy of liver sections revealed that Cx32 and Dlgh1 could colocalize in hepatocyte membranes in wild type mice. Examination of levels and localization of Dlgh1 in livers from Cx32 null mice indicate that, in the absence of Cx32, Dlgh1 was decreased, and the remainder was translocated from the hepatocyte membrane to the nucleus with some remaining in cytoplasmic compartments. This translocation was confirmed by Western blots comparing Dlgh1 levels in nuclear extracts from wild type and Cx32 null murine livers. Using SKHep cells stably transfected with Cx32 under the control of a tet-off promoter, we found that acute removal of Cx32 led to a decrease of membrane-localized Dlgh1 and an increase in the nuclear localization of this tumor suppressor protein. Together, these results suggest that loss of Cx32 alters the levels, localization, and interactions of the tumor suppressor protein Dlgh1, events known in other systems to alter cell cycle and increase tumorigenicity.

Received for publication, June 1, 2006 , and in revised form, February 5, 2007.

^* This work was funded, in part, by grants from National Institutes of Health (DK41918, to D. C. S.), the Marion Bessin Liver Center at the Albert Einstein College of Medicine (to H. S. D.), the United States Public Health Service Grant M072631, as well as the American Heart Association Grant 0560050Z (to P. L. S.) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Pharmacology, College of Physicians and Surgeons, Columbia University, 630 W. 168th St., New York, NY 10032. Tel.: 212-305-3411; Fax: 212-305-8780; E-mail: hsd2102{at}columbia.edu.

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