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Junctional adhesion molecule (JAM) binds to PAR-3: a possible mechanism for the recruitment of PAR-3 to tight junctions.

Itoh M, Sasaki H, Furuse M, Ozaki H, Kita T, Tsukita S - J. Cell Biol. (2001)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1Department of Cell Biology, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan.

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.

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Interaction between JAM and PAR-3. (a) Recruitment of endogenous PAR-3 to cell–cell adhesion sites in L transfectants. C1L cells, JL cells, or JΔLVL cells were double stained. Claudin-1, JAM, and JAMΔLV were all concentrated at cell–cell adhesion sites. JAM, but not claudin-1–JAMΔLV, recruited PAR-3 to cell–cell contact sites (arrowheads). (b) Six distinct portions of PAR-3 were produced as recombinant fusion proteins with MBP in E. coli, and then the same in vitro binding analysis as described in the legend to Fig. 3 b was performed. Among six types of MBP fusion proteins, only MBP–PDZ1-PDZ3 was bound to GST-JAMcyt. (c) Quantitative analysis of the binding between MBP–PDZ1-PDZ3 of PAR-3 and GST-JAMcyt. Kd value was determined to be 7.5 × 10−8 M.
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fig4: Interaction between JAM and PAR-3. (a) Recruitment of endogenous PAR-3 to cell–cell adhesion sites in L transfectants. C1L cells, JL cells, or JΔLVL cells were double stained. Claudin-1, JAM, and JAMΔLV were all concentrated at cell–cell adhesion sites. JAM, but not claudin-1–JAMΔLV, recruited PAR-3 to cell–cell contact sites (arrowheads). (b) Six distinct portions of PAR-3 were produced as recombinant fusion proteins with MBP in E. coli, and then the same in vitro binding analysis as described in the legend to Fig. 3 b was performed. Among six types of MBP fusion proteins, only MBP–PDZ1-PDZ3 was bound to GST-JAMcyt. (c) Quantitative analysis of the binding between MBP–PDZ1-PDZ3 of PAR-3 and GST-JAMcyt. Kd value was determined to be 7.5 × 10−8 M.

Mentions: During the course of this study, we noticed that, in JL cells, endogenous PAR-3 was also recruited to JAM-based cell–cell 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 cell–cell adhesion sites (Fig. 4 a). JAMΔLV did not recruit PAR-3 to cell–cell 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 cell–cell 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-1–based cell–cell adhesion sites (Fig. 4 a).


Junctional adhesion molecule (JAM) binds to PAR-3: a possible mechanism for the recruitment of PAR-3 to tight junctions.

Itoh M, Sasaki H, Furuse M, Ozaki H, Kita T, Tsukita S - J. Cell Biol. (2001)

Interaction between JAM and PAR-3. (a) Recruitment of endogenous PAR-3 to cell–cell adhesion sites in L transfectants. C1L cells, JL cells, or JΔLVL cells were double stained. Claudin-1, JAM, and JAMΔLV were all concentrated at cell–cell adhesion sites. JAM, but not claudin-1–JAMΔLV, recruited PAR-3 to cell–cell contact sites (arrowheads). (b) Six distinct portions of PAR-3 were produced as recombinant fusion proteins with MBP in E. coli, and then the same in vitro binding analysis as described in the legend to Fig. 3 b was performed. Among six types of MBP fusion proteins, only MBP–PDZ1-PDZ3 was bound to GST-JAMcyt. (c) Quantitative analysis of the binding between MBP–PDZ1-PDZ3 of PAR-3 and GST-JAMcyt. Kd value was determined to be 7.5 × 10−8 M.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196413&req=5

fig4: Interaction between JAM and PAR-3. (a) Recruitment of endogenous PAR-3 to cell–cell adhesion sites in L transfectants. C1L cells, JL cells, or JΔLVL cells were double stained. Claudin-1, JAM, and JAMΔLV were all concentrated at cell–cell adhesion sites. JAM, but not claudin-1–JAMΔLV, recruited PAR-3 to cell–cell contact sites (arrowheads). (b) Six distinct portions of PAR-3 were produced as recombinant fusion proteins with MBP in E. coli, and then the same in vitro binding analysis as described in the legend to Fig. 3 b was performed. Among six types of MBP fusion proteins, only MBP–PDZ1-PDZ3 was bound to GST-JAMcyt. (c) Quantitative analysis of the binding between MBP–PDZ1-PDZ3 of PAR-3 and GST-JAMcyt. Kd value was determined to be 7.5 × 10−8 M.
Mentions: During the course of this study, we noticed that, in JL cells, endogenous PAR-3 was also recruited to JAM-based cell–cell 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 cell–cell adhesion sites (Fig. 4 a). JAMΔLV did not recruit PAR-3 to cell–cell 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 cell–cell 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-1–based cell–cell adhesion sites (Fig. 4 a).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1Department of Cell Biology, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan.

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.

Show MeSH
Related in: MedlinePlus