Junctional Adhesion Molecule Interacts with the PDZ Domain-containing Proteins AF-6 and ZO-1

Abstract

We have identified the PDZ domain protein AF-6 as an intracellular binding partner of the junctional adhesion molecule (JAM), an integral membrane protein located at cell contacts. Binding of AF-6 to JAM required the presence of the intact C terminus of JAM, which represents a classical type II PDZ domain-binding motif. Although JAM did not interact with the single PDZ domains of ZO-1 or of CASK, we found that a ZO-1 fragment containing PDZ domains 2 and 3 bound to JAM in vitro in a PDZ domain-dependent manner. AF-6 as well as ZO-1 could be coprecipitated with JAM from endothelial cell extracts, demonstrating the association of the endogenously expressed molecules in vivo. Targeting of JAM to sites of cell contacts could be affected by the loss of the PDZ domain-binding C terminus. Full-length mouse JAM co-distributed with endogenous AF-6 in human Caco-2 cells at sites of cell contact independent of whether adjacent cells expressed mouse JAM as an extracellular binding partner. In contrast, truncated JAM lacking the PDZ domain-binding C terminus did not co-distribute with endogenous AF-6, but was restricted to cell contacts between cells expressing mouse JAM. Our results suggest that JAM can be recruited to intercellular junctions by its interaction with the PDZ domain-containing proteins AF-6 and possibly ZO-1.

Junctional Adhesion Molecule A Interacts with Afadin and PDZ-GEF2 to Activate Rap1A, Regulate β1 Integrin Levels, and Enhance Cell Migration

“…The PDZ binding motif of JAM-A has been reported to associate with the PDZ domain-containing proteins Afadin and ZO-1 in endothelial and epithelial cells (Yamamoto et al, 1997;Ebnet et al, 2000). However, the signaling events downstream of JAM-A dimerization that mediate these functional effects are not understood.…”

“…To identify scaffolding proteins that mediate JAM-A function, we focused on the candidate proteins Afadin and ZO-1, which have previously been reported to associate with JAM-A (Ebnet et al, 2000;Fukuhara et al, 2002). Specifically, we investigated whether these proteins are required for JAM-A regulation of cell migration.…”

Rap1: a key regulator in cell-cell junction formation

“…One protein heavily involved in this process is afadin/AF6, an adaptor protein that binds to various junctional proteins, such as nectins, ZO-1 and JAM-A (Boettner et al, 2000;Ebnet et al, 2000;Takahashi et al, 1999). Afadin/AF6 has two Ras-association (RA) domains, which can bind to both Ras and Rap1.…”

Neutrophil Migration across Tight Junctions Is Mediated by Adhesive Interactions between Epithelial Coxsackie and Adenovirus Receptor and a Junctional Adhesion Molecule-like Protein on Neutrophils

“…An important distinction between JAML and other JAM proteins is in the lack of a traditional PDZ-binding motif at its C-terminus. Because the PDZ-binding motif of related proteins such as JAM-A mediates affiliation with scaffolding proteins such as ZO-1 (Ebnet et al, 2000), PAR3 (Itoh et al, 2001), and AF6 (Ebnet et al, 2000) and appears to be critical for targeting to specific sites such as intercellular junctions, the absence of this motif in JAML raises the likelihood of differences in function(s) and targeting patterns from those of other CTX proteins.Identification of Epithelial CAR as a Cellular Ligand for JAML CAR, the receptor shared between coxsackie B and adenoviruses (Bergelson et al, 1997;Carson et al, 1997;Tomko et al, 1997), is a member of the CTX subfamily of IgSFs and is characterized by an extracellular domain containing one Vand one C-type Ig domain, a single membrane-spanning region, and an intracellular tail with a PDZ-binding motif After incubation with primary antiserum, PMN were fixed, labeled with FITC goat anti-mouse IgG, and viewed with a fluorescence microscope. Bar, 20 m. (C) Up-regulation of JAML on the PMN cell surface after fLMP stimulation.…”