doi: 10.1128/MCB.23.12.4267-4282.2003.
JAM4, a junctional cell adhesion molecule interacting with a tight junction protein, MAGI-1
Affiliations
- PMID: 12773569
- PMCID: PMC156145
- DOI: 10.1128/MCB.23.12.4267-4282.2003
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JAM4, a junctional cell adhesion molecule interacting with a tight junction protein, MAGI-1
Mol Cell Biol.
2003 Jun.
doi: 10.1128/MCB.23.12.4267-4282.2003.
- PMID: 12773569
- PMCID: PMC156145
- DOI: 10.1128/MCB.23.12.4267-4282.2003
Item in Clipboard
Abstract
MAGI-1 is a membrane-associated guanylate kinase protein at tight junctions in epithelial cells. It interacts with various molecules and functions as a scaffold protein at cell junctions. We report here a novel MAGI-1-binding protein that we named junctional adhesion molecule 4 (JAM4). JAM4 belongs to an immunoglobulin protein family. JAM4 was colocalized with ZO-1 in kidney glomeruli and in intestinal epithelial cells. Biochemical in vitro studies revealed that JAM4 bound to MAGI-1 but not to ZO-1, whereas JAM1 did not bind to MAGI-1. JAM4 and MAGI-1 interacted with each other and formed clusters in COS-7 cells when coexpressed. JAM4 mediated calcium-independent homophilic adhesion and was accumulated at cell-cell contacts when expressed in L cells. MAGI-1, ZO-1, and occludin were recruited to JAM4-based cell contacts. JAM4 also reduced the permeability of CHO cell monolayers. MAGI-1 strengthened JAM4-mediated cell adhesion in L cells and sealing effects in CHO cells. These findings suggest that JAM4 together with MAGI-1 provides an adhesion machinery at tight junctions, which may regulate the permeability of kidney glomerulus and small intestinal epithelial cells.
Figures
FIG. 1.
Mouse JAM4 amino acid sequence.…
FIG. 1.
Mouse JAM4 amino acid sequence. (A) Alignment of the amino acid sequences of…
Mouse JAM4 amino acid sequence. (A) Alignment of the amino acid sequences of pPrey 10427 and mouse BC004806. Amino acid residues are shown by single-letter codes. pPrey 10427 has 76 amino acids. Among them, 61 amino acids are identical to the C-terminal region of BC004806. (B) Structural model of JAM4 and mouse BC004806. The exrtacellular region of JAM4 contains two domains with intramolecular disulfide bonds typical of Ig-like loops of the V type. BC004806 has only the first loop. JAM4 and BC004806 have the C-terminal PDZ-binding motif. (C) Phylogenetic tree indicating the relationship between JAM4 and other Ig protein family members. Sequences were obtained from GenBank. The alignment was analyzed with Phylotree (http://cbrg.inf.ethz.ch/Server/AllAll.html ). Accession numbers are as follows: for Nectin-1α, AAF76195; for Nectin-2, P32507; for Nectin-3α, AAF63685; for Nectin-4, AAL79833; for coxsackievirus and adenovirus receptor (CAR), AAH16457; for intestinal A33 antigen, AAH08528; for endothelial cell-selective adhesion molecule (ESAM), AAK51504; for Nephrin, AAK38483; for receptor for advanced glycation end product (RAGE), AAB82007; for JAM1, AAC32982; for JAM2, NP076333; and for JAM3, NP075766. (D) Amino acid sequence of mouse JAM4 and alignment with mouse JAM1, JAM2, and JAM3. The putative hydrophobic signal peptide (indicated with double solid underlines) and transmembrane (dashed single underline) sequences of mouse JAM4 are marked. Cysteines likely to form disulfide bonds are indicated by asterisks. Putative N-linked glycosylation sites are indicated by boldface dots. Numbers indicate the numbers of amino acid residues of each protein. Amino acid residues conserved among more than three proteins are shown on a black background. (E) Constructs of JAM4 used in this study. SP, signal peptide; FLAG, FLAG tag. Solid ovals correspond to the C-terminal 9 amino acids containing the PDZ-binding motif.
FIG. 2.
(A) Northern blot analysis of…
FIG. 2.
(A) Northern blot analysis of JAM4. A uniformly labeled probe corresponding to the…
(A) Northern blot analysis of JAM4. A uniformly labeled probe corresponding to the full length of JAM4 was prepared. Blots with 20 μg of total RNA from each mouse tissue were hybridized with the probe (3,600,000 cpm) and exposed for 8 days. The mobilities of molecular mass standards (in kilobases) are indicated at the left. (B) Western blot with the anti-JAM4 antibody of rat kidney, intestine, spleen, and MTD-1A cells. Homogenates (total protein, 20 μg) from rat kidney, small intestine, spleen, and MTD-1A cells were resolved by SDS-PAGE and immunoblotted with the affinity-purified antibody against JAM4. Lane 1, spleen cells; lanes 2 and 4, kidney cells; lanes 3 and 5, small intestine cells; lanes 6 and 7, MTD-1A cells. For lanes 4, 5, and 7, the antibody was preincubated with 5 μM immunogen. The mobilities of molecular mass standards (in kilodaltons) are indicated at left. (C) FLAG-JAM4 in various cells. (a) Homogenates (total protein, 20 μg) from various stable transformants expressing FLAG-JAM4 were resolved by SDS-PAGE and immunoblotted with the anti-FLAG antibody. (b) Homogenates of stable transformants were treated with N-glycosidase F and charged onto SDS-PAGE gel with the in vitro transcription and translation product. The lane for the in vitro transcription and translation product was separately analyzed with the image analyzer. The remaining lanes were immunoblotted with the anti-FLAG antibody. The mobilities of molecular mass standards (in kilodaltons) are indicated at the left.
FIG. 3.
Immunohistological analysis of JAM4. (A)…
FIG. 3.
Immunohistological analysis of JAM4. (A) JAM4 in kidney cells. The sections of rat…
Immunohistological analysis of JAM4. (A) JAM4 in kidney cells. The sections of rat kidney were stained with the affinity-purified antibody in the absence (a) or the presence (b) of 5 μM immunogen. Signals were detected in glomeruli (arrow) and on apical membranes of proximal tubules (arrowheads). (a2) The demarcated area shown in panel a1 at higher magnification. Bars, 100 μm (a1 and b) and 20 μm (a2). (B) Colocalization of JAM4 and MAGI-1 with ZO-1 in kidney glomeruli. Sections of rat kidney were double stained with various antibodies. (a) JAM4 and ZO-1; (b) MAGI-1 and anti-ZO-1. Bar, 20 μm. (C) Electron microscopy localization of JAM4 in podocytes. (a) JAM4; (b) MAGI-1. Bar, 0.1 μm. (D) Colocalization of JAM4 with ZO-1 in small intestine epithelial cells. (a) JAM4 and ZO-1; (b) JAM4 and β-catenin. Bar, 50 μm.
FIG. 4.
Immunofluorescence analysis of JAM4 in…
FIG. 4.
Immunofluorescence analysis of JAM4 in MTD-1A and MDCK cells. (A) MTD-1A cells were…
Immunofluorescence analysis of JAM4 in MTD-1A and MDCK cells. (A) MTD-1A cells were fixed and immunostained with anti-JAM4, anti-MAGI-1, anti-β-catenin, and anti-ZO-1 antibodies. JAM4 and MAGI-1 were detected by anti-rabbit antibodies. β-Catenin and ZO-1 were detected by anti-mouse monoclonal and anti-rat monoclonal antibodies, respectively. (a) JAM4, β-catenin, and ZO-1 in MTD-1A cells; (b) MAGI-1, β-catenin, and ZO-1 in MTD-1A cells. Bar, 20 μm. (B) MDCK-FLAG-JAM4 cells were fixed and immunostained with anti-FLAG, anti-MAGI-1, anti-ERBIN, and anti-ZO-1 antibodies. MAGI-1 and ERBIN were detected by anti-rabbit antibodies. FLAG and ZO-1 were detected by anti-mouse monoclonal and anti-rat monoclonal antibodies, respectively. (a) FLAG-JAM4, MAGI-1, and ZO-1 in MDCK cells; (b) FLAG-JAM4, ERBIN, and ZO-1 in MDCK cells. Bar, 20 μm.
FIG. 5.
Interaction of JAM4 with MAGI-1.…
FIG. 5.
Interaction of JAM4 with MAGI-1. (A) Coimmunoprecipitation of JAM4 and MAGI-1. Myc-MAGI-1 with…
Interaction of JAM4 with MAGI-1. (A) Coimmunoprecipitation of JAM4 and MAGI-1. Myc-MAGI-1 with either FLAG-JAM4 or FLAG-JAM4ΔC was coexpressed in COS-7 cells. Anti-MAGI-1 antibody coprecipitated FLAG-JAM4 but not FLAG-JAM4ΔC with Myc-MAGI-1. The mobilities of molecular mass standards (in kilodaltons) are indicated at the left. Input, cell lysates; PIS IP, immunoprecipitate with the preimmune serum; MAGI IP, immunoprecipitate with the anti-MAGI-1 serum. (B) GST pulldown assay (using GST fusion proteins containing various PDZ domains of MAGI-1) of FLAG-JAM4. Lysates of COS cells expressing FLAG-JAM4 were incubated with GST alone or GST-MAGI-1-PDZ1, -PDZ2, -PDZ3, -PDZ4, or -PDZ5 immobilized on glutathione-Sepharose beads. The resulting complexes were analyzed by immunoblotting with the anti-FLAG antibody. The mobilities of molecular mass standards (in kilodaltons) are indicated at the left. (C) Specificity of the interaction of MAGI-1 with JAM4. Myc-MAGI-1 was coexpressed with either FLAG-JAM4 or FLAG-JAM1 in COS-7 cells. Lysates were incubated with either preimmune serum or anti-MAGI-1 serum immobilized on protein G-Sepharose beads. The resulting complexes were analyzed with either the anti-Myc or anti-FLAG antibody. MAGI-1 did not bind JAM1. The mobilities of molecular mass standards (in kilodaltons) are indicated at the left. (D) Specificity of the interaction of JAM4 with MAGI-1. Lysates of COS-7 cells expressing Myc-MAGI-1, Myc-ZO-1-1, Myc-CASK, or Myc-Lin-7 were incubated with GST alone or GST-JAM4-C immobilized on glutathione-Sepharose beads. The resulting complexes were analyzed by immunoblotting with the anti-Myc antibody. JAM4 did not bind ZO-1, CASK, or Lin-7. The mobilities of molecular mass standards (in kilodaltons) are indicated at the left. (E) Interaction of JAM4 and MAGI-1 in COS-7 cells. Subcellular localization of Myc-MAGI-1 (a), FLAG-JAM4 (b), and FLAG-JAM4ΔC (c) in COS-7 cells is shown. (d) Myc-MAGI-1 and FLAG-JAM4 were colocalized and formed clusters in COS-7 cells when coexpressed. (e) Myc-MAGI-1 and FLAG-JAM4ΔC did not interact with each other in COS-7 cells. (f) The effect of latrunculin A on the clustering of FLAG-JAM4 and Myc-MAGI-1. COS-7 cells were transfected with pFLAG JAM4 and pClneo Myc MAGI-1 and were treated (before fixation) with 5 μM latrunculin A for 5 min.
FIG. 6.
Cell aggregation activity of JAM4.…
FIG. 6.
Cell aggregation activity of JAM4. (A) Cell aggregation assays were performed using L…
Cell aggregation activity of JAM4. (A) Cell aggregation assays were performed using L cells or stable transformants of L cells expressing JAM4 (L-JAM4) or JAM4ΔC (L-JAM4ΔC). The extent of cell aggregation is represented by the ratio of the total particle number at the indicated time point (Nt) to the initial particle number (N0). The Nt/N0 values are representative of the means ± standard errors of three independent experiments. (B) Expression of JAM4 and JAM4ΔC. Cell surface proteins were biotinylated, precipitated by avidin-agarose beads, and then immunoblotted with the anti-JAM4 antibody. Lane 1, wild-type L cells; lane 2, L-JAM4 cells; lane 3, L-JAM4ΔC cells. (C) Cell aggregation of L-JAM4 cells. Results for wild-type L cells (L-w.t.), L-JAM4 cells (L-JAM4), and L-JAM4ΔC cells (L-JAM4ΔC) at 60 min in a cell aggregation assay are shown. Bar, 200 μm.
FIG. 7.
Recruitment of Myc-MAGI-1, ZO-1, and…
FIG. 7.
Recruitment of Myc-MAGI-1, ZO-1, and occludin to JAM4-based cell contacts. (A) Stable transformants…
Recruitment of Myc-MAGI-1, ZO-1, and occludin to JAM4-based cell contacts. (A) Stable transformants of L cells expressing FLAG-JAM4 (L-FLAG-JAM4) (a) and FLAG-JAM4ΔC (L-FLAG-JAM4ΔC) (b) were grown in cultures to generate cell contacts. FLAG-JAM4 and FLAG-JAM4ΔC were accumulated at cell contacts (arrows). (c) L-Myc-MAGI-1 cells. Myc-MAGI-1 was diffusely distributed. (B) Myc-MAGI-1 in L-FLAG-JAM4/Myc-MAGI-1 (a) and L-FLAG-JAM4ΔC/Myc-MAGI-1 (b) cells. FLAG-JAM4 (arrows) and Myc-MAGI-1 (arrowheads) were accumulated at cell contacts in L-FLAG-JAM4/Myc-MAGI-1 cells. (C) (a) L-FLAG-JAM4 cells were double stained with anti-FLAG and anti-ZO-1 antibodies. (b) L-FLAG-JAM4/Myc-MAGI-1 cells; (c) L-FLAG-JAM4ΔC/Myc-MAGI-1 cells. L-FLAG-JAM4/Myc-MAGI-1 cells and L-FLAG-JAM4ΔC/Myc-MAGI-1 cells were triple stained with anti-FLAG, anti-Myc, and anti-ZO-1 antibodies. ZO-1 (arrowheads) was recruited with Myc-MAGI-1 (arrows) to cell contacts in L-FLAG-JAM4/Myc-MAGI-1 cells. (D) (a) L-FLAG-JAM4 cells were double stained with anti-FLAG and anti-occludin antibodies. (b) L-FLAG-JAM4/Myc-MAGI-1 cells were triple stained with anti-FLAG, anti-Myc, and anti-occludin antibodies. Occludin (arrowheads) was recruited with Myc-MAGI-1 (arrows) to cell contacts in L-FLAG-JAM4/Myc-MAGI-1 cells. Bar, 20 μm.
FIG. 8.
Effect of MAGI-1 on the…
FIG. 8.
Effect of MAGI-1 on the cell adhesive activity of JAM4. (A) Phase-contrast images…
Effect of MAGI-1 on the cell adhesive activity of JAM4. (A) Phase-contrast images of L-FLAG-JAM4 (a), L-FLAG-JAM4ΔC (b), L-FLAG-JAM4/Myc-MAGI-1 (c), and L-FLAG-JAM4ΔC/Myc-MAGI-1 (d) cells grown in cultures on plastic dishes. Bar, 50 μm. (B) Immunofluorescence of FLAG-JAM4 in L-FLAG-JAM4 (a) and L-FLAG-JAM4/Myc-MAGI-1 (b) cells. Bar, 10 μm. (C) Phase-contrast images of L-FLAG-JAM4 (a) and L-FLAG-JAM4/Myc-MAGI-1 (b) cells in collagen cultures. Bar, 100 μm. (D) Dissociation assay of L-FLAG-JAM4 and L-FLAG-JAM4/Myc-MAGI-1 cells. (a) L-FLAG-JAM4 cells after being pipetted five times; (b) L-FLAG-JAM4/Myc-MAGI-1 cells after being pipetted five times. Bar, 200 μm. (c) Western blot with the anti-FLAG antibody of L-FLAG-JAM4 and L-FLAG-JAM4/Myc-MAGI-1 cells. Lane 1, L-FLAG-JAM4 cells; lane 2, L-FLAG-JAM4/Myc-MAGI-1 cells. The mobilities of molecular mass standards (in kilodaltons) are indicated at the left.
FIG. 9.
Effect of disruption of the…
FIG. 9.
Effect of disruption of the cytoskeleton on JAM4/MAGI-1-mediated cell adhesion. (A) Subcellular fractionation…
Effect of disruption of the cytoskeleton on JAM4/MAGI-1-mediated cell adhesion. (A) Subcellular fractionation of L-Myc-MAGI-1, L-FLAG-JAM4, and L-FLAG-JAM4/Myc-MAGI-1 cells. Comparable amounts of the fractions from various L cells were immunoblotted with the anti-Myc or the anti-FLAG antibody. Insol., insoluble. (a) L-Myc-MAGI-1 cells; (b) L-FLAG-JAM4 cells; (c) L-FLAG-JAM4/Myc-MAGI-1 cells. (B) Dissociation assay of L-FLAG-JAM4/Myc-MAGI-1 cells after mock, nocodazole, and latrunculin A treatment. L-FLAG-JAM4/Myc-MAGI-1 cells were treated with DMSO (a), 1 μg of nocodazole/ml (b), or 5 μM latrunculin A (c) for 60 min before the dissociation assay. Upper photos, cells after the treatment and before scraping; lower photos, cells after being pipetted five times. Bar, 200 μm.
FIG. 10.
Effect of JAM4 and MAGI-1…
FIG. 10.
Effect of JAM4 and MAGI-1 on the permeability of CHO cell monolayers. (A)…
Effect of JAM4 and MAGI-1 on the permeability of CHO cell monolayers. (A) Wild-type CHO and CHO-FLAG-JAM4 cells were grown in cultures in Transwells. FITC-dextran was added to the upper chamber, and the permeability was measured. The permeability of wild-type CHO cells was taken as 100%. Data are indicated as the means ± standard errors of three independent experiments. (B) (a) Wild-type CHO and CHO-FLAG-JAM4 cells were infected by adenovirus to express either GFP or GFP-MAGI-1. At 2 h after the infection, these cells were plated on Transwells for the permeability assay. (b) Immunoblots (IB) of CHO and CHO-FLAG-JAM4 cells to confirm the protein expression. Left panel, immunoblot with the anti-GFP antibody; right panel, immunoblot with the anti-FLAG antibody. Lanes 1, wild-type CHO cells with GFP; lanes 2, wild-type CHO cells with GFP-MAGI-1; lanes 3, CHO-FLAG-JAM4 cells with GFP; lanes 4, CHO-FLAG-JAM4 cells with GFP-MAGI-1. The mobilities of molecular mass standards (in kilodaltons) are indicated at the left.
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