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First published online September 2, 2003
doi: 10.1242/10.1242/jcs.00704

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The junctional adhesion molecule (JAM) family members JAM-2 and JAM-3 associate with the cell polarity protein PAR-3: a possible role for JAMs in endothelial cell polarity

¹ Institute of Cell Biology, ZMBE, University of Münster, Germany
² Department of Pathology, Centre Medical Universitaire, CH-1211 Geneva, Switzerland
³ Department of Molecular Biology, Yokohama City University School of Medicine, Yokohama 236-004, Japan
⁴ Max-Planck-Institute of Vascular Biology, D-48149 Münster, Germany

View larger version (30K): [in a new window]   Fig. 1. PAR-3 associates directly with JAM-2 and JAM-3. (A) Schematic view of PAR-3 and PAR-3 expression constructs used in this study. The three conserved regions (CR) are indicated by brackets. The aPKC-binding region (aa 712-936) is illustrated as grey bar encompassing CR3. The three PDZ domains are indicated. The expression constructs used in this study are schematically illustrated. (B) Full-length PAR-3 (PAR-3/1-1337) and a PAR-3 fragment comprising the three PDZ domains (PAR-3/PDZ1-3) were generated by in vitro transcription/translation in the presence of [35S]-methionine and incubated with GST-fusion proteins containing the cytoplasmic domains of JAM-2, JAM-3 and JAM-1. To analyse the requirement of the PDZ domain binding motif, the C-terminal five aa residues of JAM-2 and JAM-3 were deleted (JAM-25, JAM-35). As control for unspecific binding GST alone was used (GST-). In the lane marked with `lysate', 7% of the transcription/translation reaction was loaded. PAR-3 binds to both JAM-2 and JAM-3 in a PDZ domain-dependent manner.

View larger version (34K): [in a new window]   Fig. 2. PAR-3 associates with JAM-2 and JAM-3 through its first PDZ domain. Constructs comprising all three PDZ domains (PDZ 1-3) or individual PDZ domains of PAR-3 (PDZ 1, PDZ 2, PDZ 3) were generated by vitro transcription/translation and incubated with immobilized GST-fusion proteins as described in the legend to Fig. 1. From the three individual PDZ domains only PDZ 1 strongly bound to both JAM-2 and JAM-3; a weak association was observed with PDZ 3. As indicated in the lower panel all PDZ domains were generated with the same efficiencies.

View larger version (20K): [in a new window]   Fig. 3. PAR-3 generated in COS-7 cells associates with JAM-2 and JAM-3. Three T7 epitope-tagged PAR-3 constructs comprising either full-length PAR-3 (PAR-3/1-1337), or aa residues 583-1337 with the PDZ 3 and the aPKC binding domain (PAR-3/583-1337), or aa residues 258-936 with PDZ domains 1 to 3 and the aPKC binding domain (PAR-3/258-936) were transiently transfected into COS-7 cells. The lysates of transfected cells were incubated with immobilized GST-JAM fusion proteins and the resulting protein complexes were analysed by immunoblotting with antibodies against the T7 epitope. Arrowheads indicate the positions of recombinant PAR-3 molecules; the small arrow in the top right panel indicates PAR-3 degradation products. The two PAR-3 constructs containing PDZ domain 1 were efficiently affinity-isolated with both GST-JAM-2 and GST-JAM-3, whereas the construct lacking PDZ domains 1 and 2 did not bind to GST-JAM fusion proteins.

View larger version (50K): [in a new window]   Fig. 4. PAR-3 associates exclusively with JAMs 1 to 3. GST fusion proteins with the C-terminal cytoplasmic domains of JAM-2, JAM-3, JAM-1, ESAM, CAR, claudin-1 and claudin-5 were incubated with [35S]-methionine labelled PAR-3 constructs comprising PDZ domains 1 to 3 (PAR-3/PDZ1-3) or full length PAR-3 (PAR-3/1-1337) as described in the legend to Fig. 1. Both PAR-3 constructs efficiently associated only with JAMs 1 to 3.

View larger version (29K): [in a new window]   Fig. 5. JAM-2 and JAM-3 associate with ZO-1. (A) A ZO-1 construct comprising PDZ domains 1 to 3 of ZO-1 (ZO-1/PDZ1-3) was generated in vitro and incubated with immobilized GST-JAM fusion (JAM-1, JAM-2, JAM-3) proteins as described in the legend to Fig. 1. GST-fusion proteins lacking the C-terminal PDZ domain binding motifs were used as controls to analyse the PDZ domain-dependence of the association (JAM-19, JAM-25, JAM-35). All three JAMs bind to ZO-1 in a PDZ domain-dependent manner. (B) Lysates derived from CMT epithelial cells were incubated with immobilized JAM fusion proteins. The resulting protein complexes were subjected to SDS-PAGE and analysed by immunoblotting with antibodies directed against ZO-1; the lane marked with `lysate' contains an aliquot of CMT lysates directly immunoblotted with ZO-1 antibodies. All three JAM molecules isolate ZO-1 from CMT lysates. (C) GST-fusion proteins containing the C-terminal cytoplasmic domains of JAM-2, JAM-3, JAM-1, ESAM, CAR, claudin-1 and claudin-5 were incubated with [35S]-methionine-labelled ZO-1 constructs comprising PDZ domains 1 to 3 (ZO-1/PDZ1-3) or aa residues 6-1256 (ZO-1/6-1256) as described in the legend to Fig. 4. Besides JAMs 1 to 3, ZO-1 associates with claudin-1 and claudin-5; in addition, ZO-1 associates with CAR, probably in a PDZ-domain-independent manner.

View larger version (120K): [in a new window]   Fig. 6. PAR-3 localizes at cell-cell contacts of endothelial cells. (A) Human umbilical vein endothelial cells (HUVEC) were stained with antibodies against PAR-3, ZO-1 and AF-6. Bound antibodies were visualized with biotinylated donkey anti-rabbit IgG and Cy3-conjugated streptavidin. PAR-3 localizes at cell-cell contacts of HUVECs in a similar way to ZO-1 and AF-6. Bar, 20 µm. (B) Double-label immunofluorescence staining of HUVEC with antibodies against PAR-3 and JAM-2. Rat anti-JAM-2 antibodies were visualized with goat anti-rat FITC before further processing for incubations with rabbit anti-PAR-3 and goat anti-rabbit Texas Red in the presence of 0.2% of normal rat serum. PAR-3 colocalizes with JAM-2 at cell contacts of HUVECs. Bar, 25 µm.

View larger version (108K): [in a new window]   Fig. 7. PAR-3 is expressed by endothelial cells in various tissues. Cryostat sections of tongue, heart endocardium and a heart artery, as well as of mesenterial lymph node, were incubated with a polyclonal antibody against PAR-3. Bound antibodies were visualized by peroxidase-conjugated secondary antibodies. Antibodies against PECAM-1, von Willebrand factor (vWF) and the MECA-79 epitope were used as endothelial-specific markers. In negative control samples (neg. ctrl) the staining procedures were performed without primary antibodies. In the bottom panels, high endothelial venules appear as regions with lower cell densities. Note that PAR-3 is completely absent from high endothelial venule endothelial cells. Bars, 50 µm.

View larger version (97K): [in a new window]   Fig. 8. JAM-2 recruits PAR-3 and ZO-1 in CHO cells. (A) CHO cells stably transfected with JAM-2 (JAM-2, left panel), the S281A mutant of JAM-2 (J-2 S281A, middle panel) or the S281D mutant of JAM-2 (J-2 S281D, right panel) were stained with a mAb against JAM-2. Wild-type JAM-2 is barely detectable at cell-cell junctions and appears as discrete punctate staining (small inset in left panel). By contrast, the S281A mutant of JAM-2 is predominantly clustered at intercellular contacts. The S281D mutant of JAM-2 behaves like wt JAM-2 and is rarely localized at cell-cell contacts. All three cell lines showed a comparable surface expression of the transfected constructs as analysed by FACS analysis (not shown). Bar, 100 µm. (B) CHO cells stably transfected with the S281A mutant of JAM-2 were simultaneously stained with antibodies against JAM-2 and either PAR-3, ZO-1 or HSP-90, followed by Cy-3-conjugated secondary antibodies to detect JAM-2 or Cy-2-conjugated secondary antibodies to detect PAR-3, ZO-1 or HSP-90. Both PAR-3 and ZO-1 were recruited by JAM-2 to sites of cell-cell contacts. Bar, 5 µm.

View larger version (93K): [in a new window]   Fig. 9. JAM-2 is phosphorylated at serine residue S281 in CHO cells. (A) Phosphoamino acid analysis of JAM-2. CHO cells stably transfected with the S281A mutant of JAM-2 (JAM-2 S281A, left panel) or wild-type JAM-2 (JAM-2 wt, right panel) were metabolically labelled with [32P]-orthophosphate. Immunoprecipitated JAM-2 was hydrolyzed and the resulting amino acids were subjected to two-dimensional electrophoresis. The broken circles indicate the positions of comigrating cold phosphoamino acids. The inset illustrates the relative positions of free phosphate residues (Pi), phospho-serine (P-Ser), phosphothreonine (P-Thr) and phospho-tyrosine (P-Tyr). JAM-2 is phosphorylated exclusively on serine residues in both cell lines. (B) Two-dimensional phosphotryptic peptide maps of [32P]-labelled JAM-2 S281A and JAM-2 wt. Immunoprecipitated JAM-2 was subjected to trypsin digestion and the resulting peptides were subjected to electrophoresis and thin layer chromatography as indicated by the arrows. The origins of sample application are indicated by encircled black dots; the position of a marker dye for thin layer chromagtography is indicated by an encircled `M'. The positions of phosphopeptides are indicated by arrowheads. From two phosphopeptides that are identified in wt JAM-2, one is missing in JAM-2 S281A indicating that JAM-2 is phosphorylated at the S281 residue.

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