Connexin45 directly binds to ZO‐1 and localizes to the tight junction region in epithelial MDCK cells

Abstract

Zonula occludens protein 1 (ZO‐1) is a cytosolic tight junction protein that tethers transmembrane proteins such as occludin, claudin and junctional adhesion molecule to the actin cytoskeleton. The interaction between ZO‐1 and claudin or junctional adhesion molecule occurs via the amino‐terminal PSD95/Dlg/ZO‐1 (PDZ) domains in ZO‐1. A yeast two‐hybrid screen to search for proteins that interact with the PDZ domains of ZO‐1 identified connexin (Cx) 45. Cx45 interacts with the PDZ domains of ZO‐1 and ZO‐3, but not ZO‐2, via a short C‐terminal PDZ binding motif (SVWI). In transfected epithelial Madin–Darby canine kidney cells, Cx45 co‐localizes with endogenous ZO‐1 at or near tight junctions and co‐precipitation experiments show that Cx45 and ZO‐1 directly interact. Inactivating the C‐terminal PDZ‐binding motif in Cx45 affects its co‐precipitation and co‐localization with ZO‐1. The growing number of connexins (i.e. Cx43 and Cx45) that can associate with ZO proteins indicate that ZO proteins may play a more general role in organizing gap junctions and/or in recruiting signaling molecules that regulate intercellular communication.