Cis-dimerization mediates function of junctional adhesion molecule A

Abstract

Junctional adhesion molecule-A (JAM-A) is a transmembrane component of tight junctions that has been proposed to play a role in regulating epithelial cell adhesion and migration, yet mechanistic structure-function studies are lacking. Although biochemical and structural studies indicate that JAM-A forms cis-homodimers, the functional significance of dimerization is unclear. Here, we report the effects of cis-dimerization-defective JAM-A mutants on epithelial cell migration and adhesion. Overexpression of dimerization-defective JAM-A mutants in 293T cells inhibited cell spreading and migration across permeable filters. Similar inhibition was observed with using dimerization-blocking antibodies. Analyses of cells expressing the JAM-A dimerization-defective mutant proteins revealed diminished beta1 integrin protein but not mRNA levels. Further analyses of beta1 protein localization and expression after disruption of JAM-A dimerization suggested that internalization of beta1 integrin precedes degradation. A functional link between JAM-A and beta1 integrin was confirmed by restoration of cell migration to control levels after overexpression of beta1 integrin in JAM-A dimerization-defective cells. Last, we show that the functional effects of JAM dimerization require its carboxy-terminal postsynaptic density 95/disc-large/zonula occludins-1 binding motif. These results suggest that dimerization of JAM-A regulates cell migration and adhesion through indirect mechanisms involving posttranscriptional control of beta1 integrin levels.

Figure 1.

Stable expression of JAM-A mutants in 293T cells. (A) Structure for endogenous JAM-A is shown and contains two Ig-like loops, a transmembrane domain, and a cytoplasmic tail with a carboxy-terminal PDZ binding domain. The star in the 6163 mutant highlights the region of mutations at amino acids 61 and 63 that forms a salt-bridge between two JAM-A molecules in cis. The DL1 mutant completely lacks the distal most Ig-like domain that mediates cis-dimerization. (B) Western blots demonstrating overexpression of wild-type and mutant proteins in 293T stable transfectants. Tubulin is shown as a protein loading control. A 10-s film exposure (B1) and a 2-min exposure (B2) are shown to demonstrate the presence of JAM-A overexpression in the stable cell lines and to highlight the presence of endogenous JAM-A, respectively. (C) Immunofluorescence labeling of 293T cells expressing mutant constructs for JAM-A protein demonstrating similar localization of endogenous JAM-A, exogenous JAM-A, and mutant JAM-A to cell–cell contacts.

Figure 2.

JAM-A dimerization-defective mutant effects cell migration. (A) Schematic for cell migration assays. As detailed in the methods, 293T cells suspended in DMEM without serum were added to the upper chamber of transwell filters coated with 10 μg/ml fibronectin on the bottom of the filters. To assess the extent of cell migration, cells were stained with phallodin and confocal images were taken of the underside of the transwell filter. (B) Cell migration assays revealed that overexpression of DL1 and 6163, but not wild-type JAM-A resulted in decreased cell migration. Bar, 200 μm. (C) The number of cells that migrated per square millimeter over 3 h was determined from three separate filters after assessing two photomicrographs of each filter and counting the number of cells by using MetaMorph software. Average counts ± SEM are shown. As can be seen, overexpression of 6163 and DL1 mutants significantly decreased cell migration (*p < 0.05).

Figure 3.

JAM-A mutants alter 293T cell spreading. (A) Cell spreading assays reveal that dimerization-defective mutants 6163 and DL1 decrease cell spreading. Serum-starved 293T cells were added to 10 μg/ml fibronectin-coated coverslips. After 1 h at 37°C, phase contrast images were taken (2 images each for 3 coverslips/sample). Black arrows point to spreading cells, and white arrows point to rounded cells. (B) Spreading versus rounded cells were counted manually for each image, and average ± SEM was plotted. As can be seen, expression of dimerization-defective JAM-A significantly decreases 293T cell spreading (*p < 0.05).

Figure 4.

JAM-A dimerization defective-mutants decrease the length of 293T cell protrusions. (A) Confocal microscopy revealed a decrease in both the length and number of cellular protrusions (white arrows) in 293T cells overexpressing the 6163 and DL1 mutants. Bar, 50 μM. (B) All cell protrusions in eight images were measured using the program ImageJ. Bars are the average length in micrometers + SEM. As can be seen, cellular protrusions in JAM-A dimerization-defective cells are significantly shorter than those in cells expressing wild-type JAM-A (*p < 0.05).

Figure 5.

The number of PY118-paxillin focal concentrations is decreased by overexpression of 6163 and DL1 mutants in 293T cells. (A) Staining with phallodin, anti-PY118 paxillin, and Topro revealed that overexpression of both the 6163 and DL1 mutants significantly decreased the number and density of focal contacts as determined by PY-118 paxillin staining. Bar, 20 μm. (B) PY118-paxillin containing rod-shaped structures were counted with MetaMorph software based on staining with anti-PY118 paxillin. Five slides were counted per sample, and average + SEM/mm² was reported (*p < 0.05).

Figure 6.

The β1 integrin protein expression, but not mRNA levels is decreased in the 293T JAM-A dimerization-defective cell lines. (A) Immunoblotting revealed that 293T cells expressing 6163 or DL1 had dramatically decreased amounts of β1 integrin protein levels, with no change in β4 integrin or tubulin, which was used as a loading control. (B) Real-time PCR analysis from control and transfected cell lines demonstrating no significant change in β1 Integrin mRNA for any of the overexpressing 293T cell lines.

Figure 7.

Antibodies blocking JAM-A dimerization inhibits 293T cell migration and alters β1 integrin localization. (A) JAM-A antibody J10.4 (inhibits JAM-A dimerization) inhibits 293T cell migration. Serum-starved 293T cells were treated with 10 μg/ml mouse IgG, 1H2A9 (does not inhibit JAM-A dimerization), or J10.4 before migration assays. Confocal analyses revealed that J10.4 significantly inhibited 293T cell migration. Bar, 200 μm. (B) Blocking of JAM-A dimerization (J10.4) significantly reduced 293T cell migration compared with isotype (mouse IgG) and antigen-binding (1H2A9) antibody controls (*p < 0.05). (C and D) 293T cells were Ca⁺⁺ depleted for 5 min to expose intercellular junctions followed by treatment with J10.4 (JAM-A dimerization blocking antibody), 1H2A9 (nondimerization blocking JAM-A antibody), or mouse IgG at 10 μg/ml for 3 h in DMEM. Western blots of cell lysates were then probed for total levels of β1 integrin (C), demonstrating no change in levels of β1 integrin. However, immunofluorescence photomicrographs of such cells stained for β1 integrin by using a rat anti-β1 integrin antibody (D) reveal loss of lateral cell border staining after treatment with J10.4 but not 1H2A9 or mouse IgG. Note the appearance of β1 integrin labeled cytoplasmic vesicles (white arrow) in J10.4-treated cells, suggesting internalization. (E) Concurrent treatment with J10.4 and Cyclohexamide results in decreased B1 integrin.

Figure 8.

β1 Integrin is the putative effector for JAM-A–mediated regulation of 293T cell migration. (A) Western blot demonstrating that transfection of 293T cells with siRNA specific for β1 integrin results in significantly decreased β1 integrin protein levels compared with transfection with siRNA targeting cyclophilin (CB). JAM-A protein levels were not changed. (B and C) Transfection of 293T cells with siRNA specific for β1 integrin revealed significantly reduced 293T cell migration compared with controls treated with siRNA specific for cyclophilin B. Bar, 200 μM. Bar graph is average number of cells per field + SEM (*p < 0.05). (D) Western blot demonstrating increased β1 integrin protein levels in JAM-A dimerization-defective cell lines transfected with a plasmid encoding β1 integrin. JAM-A protein levels were not changed. (E and F) Migration assays with mutant cell lines overexpressing β1 Integrin. As can be seen, overexpression of β1 integrin in the 6163 and DL1 mutant cell lines restores cell migration to that of control 293T cells. Transient transfection of a control plasmid had no effect on cell migration. Bar, 200 μm (*p < 0.05 for β1 integrin-transfected cells vs. control).

Figure 9.

The PDZ binding domain of JAM-A is critical for dominant-negative effects on cell migration and β1 integrin expression by dimerization-defective mutants. (A) Expression of JAM-A mutant proteins and β1 integrin after transient transfection. As can be seen in the Western blot, full-length (wild type), and the 6163 mutant of JAM-A have an M_r of 37 kDa, whereas constructs lacking the membrane-distal loop (DL1 and DL1-dFLV) have an M_r of ∼25 kDa. Also shown are immunoblots for β1 integrin after transient transfection with JAM-A constructs, demonstrating decreased expression with 6163, DL1, and dFLV, respectively. However, transfection with the DL1-dFLV double mutant results in no change in β1 integrin protein expression. (B) Transient expression of DL1 or dFLV resulted in decreased cell migration, as measured by confocal analysis of Topro-3 nuclear staining, whereas expression of the DL1-dFLV construct had no effect. These findings suggest that the PDZ binding motif is required for regulation of β1 integrin protein levels and cell migration by the DL1 mutant. (C) Average cells migrated ± SEM are shown. As can be seen, overexpression of 6163 and DL1 mutants significantly decreased cell migration, whereas expression of the double mutant DL1-dFLV had no effect (*p < 0.05).

Figure 10.

Model for effect of JAM-A dimerization on β1 integrin levels. In this model, JAM-A dimers are required for close apposition of cytoplasmic tails bound to scaffolding complexes containing signaling elements. Under this scenario, disruption of cis-dimerization by either mutagenesis or a blocking antibody would result in loss of close apposition of these cytoplasmic tail complexes, inhibition of signaling, and subsequent internalization/degradation of β1 integrin through an as of yet undetermined mechanism. Diminished cell surface expression of β1 Integrin would then result in decreased adhesion to matrix and affect cell migration.