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2 Department of Geriatric Medicine, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan
3 KAN Research Institute, Inc., Chudoji, Kyoto 600-8317, Japan
Address correspondence to Shoichiro Tsukita, Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan. Tel.: 81-75-753-4372. Fax: 81-75-753-4660. E-mail: htsukita{at}mfour.med.kyoto-u.ac.jp
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Abstract |
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Key Words: JAM; PAR-3; claudin; ZO-1; tight junction
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Introduction |
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Occludin and claudins (claudin-124) are now known as constituents of TJ strands (Furuse et al., 1993, 1998a). Both occludin and claudins bear four transmembrane domains, but did not show any sequence similarity with each other. When claudins were overexpressed in mouse L fibroblasts, claudin molecules were polymerized within plasma membranes to reconstitute TJ strands (Furuse et al., 1998b). Another type of integral membrane protein, the junctional adhesion molecule (JAM), was also reported to be localized at TJs (Martin-Padura et al., 1998). JAM belongs to the immunoglobulin superfamily: it has a single transmembrane domain, and its extracellular portion is thought to be folded into two immunoglobulin-like domains. JAM was shown to be involved in cellcell adhesion/junctional assembly of epithelial/endothelial cells (Martin-Padura et al., 1998; Bazzoni et al., 2000a; Liu et al., 2000; Palmeri et al., 2000), as well as in the extravasation of monocytes through endothelial cells (Martin-Padura et al., 1998), but our knowledge on its localization and function at TJs is still fragmentary.
Most claudin species, as well as JAM, end in Val at their COOH termini, suggesting that these COOH termini directly bind to PDZ domains. Indeed, three related PDZ-containing proteins, ZO-1, ZO-2, and ZO-3, are known to be concentrated at TJs. ZO-1 (220 kD) was first identified as an antigen for a mAb raised against the junction-enriched fraction from the liver (Stevenson et al., 1986). Then, ZO-2 (
160 kD) was identified as a protein that was coimmunoprecipitated with ZO-1 (Gumbiner et al., 1991). A phosphorylated 130-kD protein was also found in the ZO-1 immunoprecipitate (Balda et al., 1993) and is now called ZO-3. Cloning and sequencing cDNAs encoding these molecules showed that all have three PDZ domains (PDZ13), one SH3 domain, and one GUK domain, in this order from their NH2 termini (Itoh et al., 1993; Willott et al., 1993; Jesaitis and Goodenough, 1994; Haskins et al., 1998). Among these three PDZ domains, PDZ1 domain was recently shown to bind directly to the COOH termini of claudins (Itoh et al., 1999).
Recently, another intriguing PDZ-containing protein, a mammalian homologue of PAR-3, was reported to be concentrated at TJs (Izumi et al., 1998). PAR-3, which contains three PDZ domains, was initially identified in C. elegans as a product of one of six partitioning-defective genes (par-16) that are essential for the first asymmetric divisions of early embryos (Kemphues et al., 1988; Guo and Kemphues, 1996). A mammalian homologue of PAR-3 was identified as a binding partner for atypical PKCs (ASIP) in epithelial cells (Izumi et al., 1998). As TJs are involved in the establishment of epithelial polarity, the molecular mechanism behind the recruitment of PAR-3 to TJs, as well as its physiological function at TJs, now attracts increasing interest.
Thus, for a better understanding of the molecular architecture of TJs, the most pressing questions concern the molecular mechanisms underlying the recruitment of JAM and PAR-3 (and their binding proteins) to TJs. In this study, we examined the detailed localization of JAM at TJs and the interactions between JAM and underlying PDZ-containing proteins including PAR-3. The results obtained led us to propose a new molecular architectural model for TJs that could explain how JAM and PAR-3 are recruited to TJs.
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Results and discussion |
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Direct association of JAM with PDZ3 domain of ZO-1
Recently, COOH termini of claudins were shown to directly bind to PDZ1 domain of ZO-1 (and also ZO-2/ZO-3) (Itoh et al., 1999). Considering that ZO-1 is a multidomain protein, it is tempting to speculate that JAM is associated with claudins through ZO-1. First, we examined the ability of JAM to recruit endogenous ZO-1 in L transfectants, JL cells (Fig. 3
a). As previously reported (Itoh et al., 1999), in C1L cells expressing claudin-1, endogenous ZO-1 was recruited to the claudin-based cellcell adhesion sites. Similarly, in JL cells, endogenous ZO-1 was concentrated precisely at JAM-based cellcell adhesion sites. In contrast, when the JAM mutant lacking its COOH-terminal -LV (JAMLV) was expressed in L fibroblasts (J
LVL cells), these JAM mutants were concentrated at cellcell borders, but ZO-1 was not recruited. These findings are consistent with a recent report on the association of JAM with ZO-1 (Bazzoni et al., 2000b) and suggest that some of the PDZ domains of ZO-1 interact with the COOH-terminal end of JAM.
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Taking into consideration that the COOH termini of claudins bind to PDZ1 of ZO-1 at a Kd of 1.3 x 10-7 M (Itoh et al., 1999), it can be speculated that ZO-1 tethers JAM to claudin-based strands at TJs in epithelial cells. As PDZ1 domains of ZO-2 and ZO-3 show affinity to claudins (Itoh et al., 1999), ZO-2 and ZO-3 would also be involved in the recruitment of JAM to TJ strands.
Recruitment of PAR-3 to JAM, not to claudin-1
During the course of this study, we noticed that, in JL cells, endogenous PAR-3 was also recruited to JAM-based cellcell adhesion sites: when JL cells were double stained with anti-JAM mAb and PAR-3/ASIP pAb, JAM and PAR-3 showed precise colocalization at cellcell adhesion sites (Fig. 4
a). JAMLV did not recruit PAR-3 to cellcell adhesion sites (Fig. 4 a). These findings suggested that, at least in the L fibroblast transfection system, JAM recruits PAR-3 to the plasma membrane at cellcell adhesion sites through the interaction between the COOH terminus of JAM and PDZ domains of PAR-3. In marked contrast, in C1L cells endogenous PAR-3 was not recruited to the claudin-1based cellcell adhesion sites (Fig. 4 a).
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A molecular architectural model of TJs
We were then led to the speculative model of the molecular architecture of TJs shown in Fig. 5
a, which could explain the recruitment of JAM as well as PAR-3 to TJs: it is expected that the cytoplasmic surface of individual TJ strands appears like a toothbrush consisting of densely packed numerous short COOH-terminal cytoplasmic tails of claudins. The cytoplasmic surface of strands would then strongly attract the PDZ1 domain of ZO-1 (and also ZO-2/ZO-3). JAM would be recruited and tethered to TJ strands through the direct binding of its COOH terminus to the PDZ3 domain of ZO-1. JAM molecules laterally aggregate to form oligomers, which would allow the recruitment of additional JAM molecules around TJ strands. Since these JAM molecules would be free of ZO-1, they could recruit PAR-3 and then its binding proteins, such as atypical PKC, PAR-6, and Cdc42, to TJs (Joberty et al., 2000; Lin et al., 2000; Qiu et al., 2000; Suzuki et al., 2001).
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In C. elegans germline cells, most PAR proteins were enriched at the cell periphery and localized to one or the other pole of cells undergoing asymmetric cell divisions (Guo and Kemphues, 1996; Kemphues, 2000). Detailed analyses of PAR mutants revealed that the asymmetric distribution of PAR proteins is important for their function as determinants of cell polarity in these cells. Therefore, there is a search for membrane proteins that recruit some PAR proteins to the plasma membrane and allow their asymmetric distribution. In this sense, JAM is the first protein shown to recruit PAR proteins to certain specified membrane domains. We showed here that JAM recruits PAR-3 to the cellcell adhesion sites in L transfectants and TJs in epithelial cells. This finding provides an important clue as to how PAR signaling determines cell polarity in general.
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Materials and methods |
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cDNA transfection
The cDNA fragments encoding full-length human JAM and a JAM mutant lacking its COOH-terminal residues LV were produced by PCR using human JAM cDNA (Ozaki et al., 1999) in the pcDNA vector as a template, and these fragments were subcloned into the mammalian expression vector pME18S. Mouse L cells were cotransfected with 2 µg of expression vector and 0.1 µg of pGKpuro and selected basically as described previously (Itoh et al., 1999).
Recombinant proteins and in vitro binding assay
The cDNA fragment encoding the cytoplasmic domain of JAM was produced by PCR, and the fragment was subcloned into the pGEX vector (Amersham Pharmacia Biotech). For production of maltose-binding protein fusion proteins with various ZO-1 mutants or with various PAR-3 mutants, the cDNAs were amplified by PCR and subcloned into the pMAL vector (New England Biolabs, Inc.) (Figs. 3 b and 4 b). These recombinant proteins were expressed in E. coli.
In vitro binding assay was performed basically as described previously (Itoh et al., 1999).
Immunofluorescence microscopy and immunoreplica electron microscopy
For immunofluorescence microscopy, cells plated on glass coverslips were rinsed in PBS, fixed with 1% formaldehyde in PBS for 15 min, and processed as described previously (Itoh et al., 1999). For immunoelectron microscopy for examining freeze fracture replicas, MDCK cells and L transfectants were fixed with 1% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3) for 5 min and then processed as described by Fujimoto (1995).
Immunoprecipitation
T84 cells cultured on 9-cm dishes were washed twice with PBS and lysed in 1-ml extraction buffer (0.1% nonidet P-40, 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, 5 mM MgCl2, 5 mM CaCl2) followed by sonication (5 times for 15 s). Cell lysates were clarified by centrifugation at 15,000 rpm for 20 min and incubated with 50 µl of protein GSepharose bead slurry (Zymed Laboratories) coupled with anti-JAM mAb (3D8), antiZO-1 pAb, or respective control IgG for 3 h. After washing five times with the extraction buffer, immunoprecipitates were eluted from beads with the SDS-PAGE sample buffer.
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Footnotes |
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Acknowledgments |
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This study was supported in part by a Grant-in-Aid for Cancer Research and a Grant-in-Aid for Scientific Research (A) from the Ministry of Education, Science and Culture of Japan to S. Tsukita, and by JSPS Research for the Future Program to M. Furuse.
Submitted: 12 March 2001
Revised: 8 June 2001
Accepted: 3 July 2001
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References |
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