Junctional adhesion molecule (JAM) binds to PAR-3

Abstract

At tight junctions (TJs), claudins with four transmembrane domains are incorporated into TJ strands. Junctional adhesion molecule (JAM), which belongs to the immunoglobulin superfamily, is also localized at TJs, but it remains unclear how JAM is integrated into TJs. Immunoreplica electron microscopy revealed that JAM showed an intimate spatial relationship with TJ strands in epithelial cells. In L fibroblasts expressing exogenous JAM, JAM was concentrated at cell–cell adhesion sites, where there were no strand-like structures, but rather characteristic membrane domains free of intramembranous particles were detected. These domains were specifically labeled with anti-JAM polyclonal antibody, suggesting that JAM forms planar aggregates through their lateral self-association. Immunofluorescence microscopy and in vitro binding assays revealed that ZO-1 directly binds to the COOH termini of claudins and JAM at its PDZ1 and PDZ3 domains, respectively. Furthermore, another PDZ-containing polarity-related protein, PAR-3, was directly bound to the COOH terminus of JAM, but not to that of claudins. These findings led to a molecular architectural model for TJs: small aggregates of JAM are tethered to claudin-based strands through ZO-1, and these JAM aggregates recruit PAR-3 to TJs. We also discuss the importance of this model from the perspective of the general molecular mechanisms behind the recruitment of PAR proteins to plasma membranes.

Multi-PDZ Domain Protein 1 (MUPP1) Is Concentrated at Tight Junctions through Its Possible Interaction with Claudin-1 and Junctional Adhesion Molecule

“…One of these pAbs (B4) was used after affinity purification with the GST fusion protein. Mouse anti-ZO-1 mAb (T8-754) and rabbit anti-JAM pAb (C4) were generated and characterized previously (23,31). Rat anti-E-cadherin mAb (ECCD2) was a generous gift from Dr. M. Takeichi (Kyoto University, Kyoto, Japan).…”

“…Rabbit anti-ZO-1 pAb, mouse anti-Myc tag mAb, and rabbit anti-maltose-binding protein (MBP) pAb were purchased from Zymed Laboratories Inc. (San Francisco, CA), MBL (Nagoya, Japan), and New England BioLabs Inc. (Beverly, MA), respectively. MDCK cells, F9 cells, Eph4 cells, L cells, and JL cells (23) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum.…”

“…It was recently shown that heterogeneous claudin species (and also occludin) are co-polymerized to form individual TJ strands as heteropolymers and that between adjacent cells claudin molecules adhere with each other in both homotypic and heterotypic manners except in some combinations (20,21). In addition to claudins and occludin, another type of integral membrane protein, JAM (junctional adhesion molecule) belonging to the immunoglobulin superfamily, was also reported to be concentrated at TJs (22), but this molecule did not appear to constitute TJ strands per se but to laterally associate with strands (23).…”

“…This suggests that these COOH termini bind directly to PDZ domains (24,25). As the cytoplasmic surface of individual TJ strands is expected to appear as a toothbrush consisting of numerous densely packed short COOH-terminal cytoplasmic tails of claudins, TJ strands including laterally associated JAM molecules may strongly attract and recruit many PDZ domain-containing proteins (5,23). Indeed, three structurally related PDZ domain-containing proteins, ZO-1, ZO-2, and ZO-3, have been shown to be concentrated at the cytoplasmic surface of TJs (26 -30).…”

“…In addition, several PDZ domain-containing proteins such as MAGI (membrane-associated guanylate kinase inverted)-1/-2/-3 (34 -37) and mammalian homologues of Caenorhabditis elegans PAR (partitioning)-3/-6 (38 -42) were also reported to be concentrated at TJs. Recently PDZ domains of PAR-3 were shown to be associated with JAM (and not with claudins) (23,43), but it remains unknown how the other PDZ domain-containing proteins are recruited to TJs.…”