Characterization of ZO-2 as a MAGUK Family Member Associated with Tight as well as Adherens Junctions with a Binding Affinity to Occludin and α Catenin

doi:10.1074/jbc.274.9.5981

Characterization of ZO-2 as a MAGUK Family Member Associated with Tight as well as Adherens Junctions with a Binding Affinity to Occludin and α Catenin*

From the ^‡Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan and the ^§Department of Dermatology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan

Abstract

ZO-2, a member of the MAGUK family, was thought to be specific for tight junctions (TJs) in contrast to ZO-1, another MAGUK family member, which is localized at TJs and adherens junctions (AJs) in epithelial and nonepithelial cells, respectively. Mouse ZO-2 cDNA was isolated, and a specific polyclonal antibody was generated using corresponding synthetic peptides as antigens. Immunofluorescence microscopy with this polyclonal antibody revealed that, similarly to ZO-1, in addition to TJs in epithelial cells, ZO-2 was also concentrated at AJs in nonepithelial cells such as fibroblasts and cardiac muscle cells lacking TJs. When NH₂-terminal dlg-like and COOH-terminal non-dlg-like domains of ZO-2 (N-ZO-2 and C-ZO-2, respectively) were separately introduced into cultured cells, N-ZO-2 was colocalized with endogenous ZO-1/ZO-2, i.e. at TJs in epithelial cells and at AJs in non-epithelial cells, whereas C-ZO-2 was distributed along actin filaments. Consistently, occludin as well as α catenin directly bound to N-ZO-2 as well as the NH₂-terminal dlg-like portion of ZO-1 (N-ZO-1) in vitro. Furthermore, immunoprecipitation experiments revealed that the second PDZ domain of ZO-2 was directly associated with N-ZO-1. These findings indicated that ZO-2 forms a complex with ZO-1/occludin or ZO-1/α catenin to establish TJ or AJ domains, respectively.

Footnotes

↵* This study was supported in part by a Grant-in-Aid for Cancer Research and a Grant-in-Aid for Scientific Research (A) from the Ministry of Education, Science and Culture of Japan (to S. T.).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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AF113005.
↵¶ To whom correspondence should be addressed: Dept. of Cell Biology, Kyoto University Faculty of Medicine, Konoe-Yoshida, Sakyo-ku, Kyoto 606, Japan. Tel.: 81-75-753-4372; Fax: 81-75-753-4660; E-mail:htsukita{at}mfour.med.kyoto-u.ac.jp.
Abbreviations:

TJ

tight junction

AJ

adherens junction

N-ZO-2

NH₂-terminal dlg-like domain of ZO-2

C-ZO-2

COOH-terminal non-dlg-like domain of ZO-2

F-ZO-2

full-length ZO-2

mAb

monoclonal antibody

pAb

polyclonal antibody

RT-PCR

reverse transcriptase polymerase chain reaction

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline

GST

glutathione S-transferase

HA

hemagglutinin

MDCK

Madin-Darby canine kidney
- Received October 20, 1998.
- Revision received November 23, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.