title: The gap junction protein connexin32 interacts with the Src homology 3/hook domain of discs large homolog 1.
abstract: 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.
authors.affiliation: Department of Pharmacology, Columbia University, New York, New York 10032. Electronic address: hsd2102@columbia.edu.
authors.forename: Heather S
authors.initials: HS
authors.lastname: Duffy
authors.affiliation: Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461.
authors.forename: Ionela
authors.initials: I
authors.lastname: Iacobas
authors.affiliation: Department of Pharmacology, Columbia University, New York, New York 10032.
authors.forename: Kylie
authors.initials: K
authors.lastname: Hotchkiss
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nebraska, Omaha, Nebraska 68918.
authors.forename: Bethany J
authors.initials: BJ
authors.lastname: Hirst-Jensen
authors.affiliation: Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Bosco
authors.affiliation: Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461.
authors.forename: Nadine
authors.initials: N
authors.lastname: Dandachi
authors.affiliation: Department of Neuroscience and Molecular Brain Research, University of Bochum, D-44780 Bochum, Germany.
authors.forename: Rolf
authors.initials: R
authors.lastname: Dermietzel
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nebraska, Omaha, Nebraska 68918.
authors.forename: Paul L
authors.initials: PL
authors.lastname: Sorgen
authors.affiliation: Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461.
authors.forename: David C
authors.initials: DC
authors.lastname: Spray
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D045744:Cell Line, Tumor; D017630:Connexins / Q000172:deficiency / Q000378:metabolism / Q000502:physiology*; D000074288:Discs Large Homolog 1 Protein; D017629:Gap Junctions / Q000235:genetics / Q000502:physiology*; D051528:Guanylate Kinases; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D021381:Protein Transport / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000378:metabolism / Q000502:physiology; D018909:src Homology Domains / Q000235:genetics / Q000502:physiology*; D000097002:Gap Junction beta-1 Protein
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D017630:Connexins; D000074288:Discs Large Homolog 1 Protein; D051528:Guanylate Kinases; D008565:Membrane Proteins; D025521:Tumor Suppressor Proteins; D000097002:Gap Junction beta-1 Protein; C000614189:Dlg1 protein, mouse