A zyxin–nectin interaction facilitates zyxin localization to cell–cell adhesions

► Zyxin localizes to cell–cell adhesion via amino acids 230–280 of the central hinge region. ► Zyxin directly binds nectin cell–cell adhesion receptors. ► Zyxin localization to cadherin-based cell–cell adhesions requires nectin expression. Cell–cell junction remodeling is associated with dramatic actin reorganizations. Several actin regulatory systems have been implicated in actin remodeling events as cell–cell contacts are assembled and disassembled, including zyxin/LPP–VASP complexes. These complexes facilitate strong cell–cell adhesion by maintaining actin-membrane connections. It has been proposed that zyxin and LPP localize to cell–cell junctions via a well-defined interaction with alpha-actinin. This was recently confirmed for LPP, but zyxin localization at cell–cell contacts occurs independently of alpha-actinin binding. Here we seek to map the zyxin sequence responsible for localization to cell–cell contacts and identify the protein that docks zyxin at this cellular location. Previous results have shown that a zyxin fragment excluding the alpha-actin binding site and the LIM domains (amino acids 51–392) can independently localize to cell–cell contacts. Here, expression of smaller zyxin fragments show that zyxin localization requires amino acids 230–280. A yeast-two-hybrid screen, using the central region of zyxin as bait, resulted in the identification of the cell–cell adhesion receptor nectin-4 as a zyxin binding partner. Further demonstrating zyxin–nectin interactions, zyxin binds the intracellular domain of nectin-2 in vitro. Depletion of nectin-2 from L cells expressing E-cadherin results in a loss of zyxin localization to cell–cell contacts, demonstrating that the zyxin–nectin interaction plays a critical role in zyxin targeting to these sites.