The Carboxyl Terminus of Zona Occludens-3 Binds and Recruits a Mammalian Homologue of Discs Lost to Tight Junctions

Ben Margolis,Chia-Jen Liu,Michael H. Roh,Stephanie Laurinec

doi:10.1074/jbc.m201177200

Ben Margolis, Chia-Jen Liu + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m201177200

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Abstract

Mammalian homologues of the Drosophila polarity proteins Stardust, Discs Lost, and Crumbs have been identified as Pals1, Pals1-associated tight junction protein (PATJ), and human Crumbs homologue 1 (CRB1), respectively. We have previously demonstrated that PATJ, Pals1, and CRB1 can form a tripartite tight junction complex in epithelial cells and that PATJ recruits Pals1 to tight junctions. Here, we observed that the Pals1/PATJ interaction was not crucial for the ultimate targeting of PATJ itself to tight junctions. This prompted us to examine if any of the 10 post-synaptic density-95/Discs Large/zona occludens-1 (PDZ) domains of PATJ could bind to the carboxyl termini of known tight junction constituents. We found that the 6th and 8th PDZ domains of PATJ can interact with the carboxyl termini of zona occludens-3 (ZO-3) and claudin 1, respectively. PATJ missing the 6th PDZ domain was found to mislocalize away from cell contacts. Surprisingly, deleting the 8th PDZ domain had little effect on PATJ localization. Finally, reciprocal co-immunoprecipitation experiments revealed that full-length ZO-3 can associate with PATJ. Hence, the PATJ/ZO-3 interaction is likely important for recruiting PATJ and its associated proteins to tight junctions.

Highlights

Polarized epithelia primarily serve to separate and regulate the movement of ions and metabolites between physiologically distinct compartments [1, 2]
We have previously demonstrated that PATJ, Pals1, and Crumbs homologue 1 (CRB1) can form a tripartite tight junction complex in epithelial cells and that PATJ recruits Pals1 to tight junctions
We found that the 6th and 8th post-synaptic density-95/Discs Large/zona occludens-1 (PDZ) domains of PATJ can interact with the carboxyl termini of zona occludens-3 (ZO-3) and claudin 1, respectively

Summary

Introduction

Polarized epithelia primarily serve to separate and regulate the movement of ions and metabolites between physiologically distinct compartments [1, 2]. We wondered if the association between PATJ and Pals1 was important for localizing PATJ itself to TJs. To address this issue, we expressed an EYFP-PATJ mutant protein missing the MRE domain (EYFP-PATJ⌬MRE) in MDCK cells.

Results

Conclusion