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JAM-L-mediated leukocyte adhesion to endothelial cells is regulated in cis by alpha4beta1 integrin activation.

Luissint AC, Lutz PG, Calderwood DA, Couraud PO, Bourdoulous S - J. Cell Biol. (2008)

Bottom Line: Cis dimerization of JAM-L is required to engage in heterophilic interactions with its cognate counter-receptor CAR (coxsackie and adenovirus receptor).Interestingly, JAM-L expressed on neutrophils binds CAR independently of integrin activation.However, on resting monocytes and T lymphocytes, which express the integrin VLA-4, JAM-L molecules engage in complexes with VLA-4 and mainly accumulate in their monomeric form.

View Article: PubMed Central - PubMed

Affiliation: Institut Cochin, Université Paris Descartes, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8104, Paris F-75014, France.

ABSTRACT
Junctional adhesion molecules (JAMs) are endothelial and epithelial adhesion molecules involved in the recruitment of circulating leukocytes to inflammatory sites. We show here that JAM-L, a protein related to the JAM family, is restricted to leukocytes and promotes their adhesion to endothelial cells. Cis dimerization of JAM-L is required to engage in heterophilic interactions with its cognate counter-receptor CAR (coxsackie and adenovirus receptor). Interestingly, JAM-L expressed on neutrophils binds CAR independently of integrin activation. However, on resting monocytes and T lymphocytes, which express the integrin VLA-4, JAM-L molecules engage in complexes with VLA-4 and mainly accumulate in their monomeric form. Integrin activation is required for the dissociation of JAM-L-VLA-4 complexes and the accumulation of functional JAM-L dimers, which indicates that the leukocyte integrin VLA-4 controls JAM-L function in cis by controlling its dimerization state. This provides a mechanism through which VLA-4 and JAM-L functions are coordinately regulated, allowing JAM-L to strengthen integrin-dependent adhesion of leukocytes to endothelial cells.

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VLA-4 integrin controls the dimerization state of JAM-L. (A–C) K562 cells were transfected with JAM-L–HA or double transfected with JAM-L–HA and the human α4 subunit. Cells were then untreated (−) or treated with SDF-1α (100 ng/ml for 5 min) before lysis. (A) Whole cell extracts were analyzed by immunoblotting with anti-HA and anti-GFP to show that similar amounts of JAM-L and α4 were expressed in these different conditions. Arrows indicate the electrophoretic mobility of α4-GFP and of monomers and dimers of JAM-L–HA. Lysates were immunoprecipitated (IP; B) with anti-HA or with anti-GFP (C), and immune complexes were separated by SDS-PAGE and immunoblotted (IB) with anti-HA or anti-GFP, as indicated. (D) K562 cells were transfected with JAM-L–HA or double transfected with JAM-L–HA and the human α4 subunit. Cells were then untreated (−) or treated with SDF-1α (100 ng/ml for 5 min), then treated or not with BS3 before cell lysis and Western blot analysis. Numbers next to gel blots indicate molecular mass markers in kD. (E) The proportion of dimeric and monomeric forms of JAM-L in D was calculated using ImageJ.
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fig7: VLA-4 integrin controls the dimerization state of JAM-L. (A–C) K562 cells were transfected with JAM-L–HA or double transfected with JAM-L–HA and the human α4 subunit. Cells were then untreated (−) or treated with SDF-1α (100 ng/ml for 5 min) before lysis. (A) Whole cell extracts were analyzed by immunoblotting with anti-HA and anti-GFP to show that similar amounts of JAM-L and α4 were expressed in these different conditions. Arrows indicate the electrophoretic mobility of α4-GFP and of monomers and dimers of JAM-L–HA. Lysates were immunoprecipitated (IP; B) with anti-HA or with anti-GFP (C), and immune complexes were separated by SDS-PAGE and immunoblotted (IB) with anti-HA or anti-GFP, as indicated. (D) K562 cells were transfected with JAM-L–HA or double transfected with JAM-L–HA and the human α4 subunit. Cells were then untreated (−) or treated with SDF-1α (100 ng/ml for 5 min), then treated or not with BS3 before cell lysis and Western blot analysis. Numbers next to gel blots indicate molecular mass markers in kD. (E) The proportion of dimeric and monomeric forms of JAM-L in D was calculated using ImageJ.

Mentions: Because JAM-L dimerization is required to engage in heterophilic interactions with CAR (Fig. 2), we then investigated whether the interaction between JAM-L and VLA-4 controlled the dimerization state of JAM-L using K562 cells that we transfected with JAM-L–HA alone or together with α4 (Fig. 7 A). We observed that in K562 cells expressing JAM-L-HA alone, SDS-resistant dimers of JAM-L are constitutively present, even in the absence of SDF-1α treatment (Fig. 7 B, middle), which is consistent with our observation that JAM-L expression can promote the adhesion of K562 cells independently of integrin activation. However, when cotransfected with α4-GFP, a constitutive interaction between α4 and the monomeric form of JAM-L–HA was detected, as previously observed in U937 cells (Fig. 5 A). Indeed, α4-GFP was detected in JAM-L–HA immunoprecipitates (Fig. 7 B, top), and, conversely, JAM-L–HA was detected in α4-GFP immunoprecipitates (Fig. 7 C, top). In addition, this interaction was accompanied by a dramatic decrease in the amount of dimeric JAM-L (Fig. 7 B, middle), which is consistent with our observation that coexpression of both JAM-L and VLA-4 prevents JAM-L–mediated adhesion of K562 cells to CAR (Fig. 6 D).


JAM-L-mediated leukocyte adhesion to endothelial cells is regulated in cis by alpha4beta1 integrin activation.

Luissint AC, Lutz PG, Calderwood DA, Couraud PO, Bourdoulous S - J. Cell Biol. (2008)

VLA-4 integrin controls the dimerization state of JAM-L. (A–C) K562 cells were transfected with JAM-L–HA or double transfected with JAM-L–HA and the human α4 subunit. Cells were then untreated (−) or treated with SDF-1α (100 ng/ml for 5 min) before lysis. (A) Whole cell extracts were analyzed by immunoblotting with anti-HA and anti-GFP to show that similar amounts of JAM-L and α4 were expressed in these different conditions. Arrows indicate the electrophoretic mobility of α4-GFP and of monomers and dimers of JAM-L–HA. Lysates were immunoprecipitated (IP; B) with anti-HA or with anti-GFP (C), and immune complexes were separated by SDS-PAGE and immunoblotted (IB) with anti-HA or anti-GFP, as indicated. (D) K562 cells were transfected with JAM-L–HA or double transfected with JAM-L–HA and the human α4 subunit. Cells were then untreated (−) or treated with SDF-1α (100 ng/ml for 5 min), then treated or not with BS3 before cell lysis and Western blot analysis. Numbers next to gel blots indicate molecular mass markers in kD. (E) The proportion of dimeric and monomeric forms of JAM-L in D was calculated using ImageJ.
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fig7: VLA-4 integrin controls the dimerization state of JAM-L. (A–C) K562 cells were transfected with JAM-L–HA or double transfected with JAM-L–HA and the human α4 subunit. Cells were then untreated (−) or treated with SDF-1α (100 ng/ml for 5 min) before lysis. (A) Whole cell extracts were analyzed by immunoblotting with anti-HA and anti-GFP to show that similar amounts of JAM-L and α4 were expressed in these different conditions. Arrows indicate the electrophoretic mobility of α4-GFP and of monomers and dimers of JAM-L–HA. Lysates were immunoprecipitated (IP; B) with anti-HA or with anti-GFP (C), and immune complexes were separated by SDS-PAGE and immunoblotted (IB) with anti-HA or anti-GFP, as indicated. (D) K562 cells were transfected with JAM-L–HA or double transfected with JAM-L–HA and the human α4 subunit. Cells were then untreated (−) or treated with SDF-1α (100 ng/ml for 5 min), then treated or not with BS3 before cell lysis and Western blot analysis. Numbers next to gel blots indicate molecular mass markers in kD. (E) The proportion of dimeric and monomeric forms of JAM-L in D was calculated using ImageJ.
Mentions: Because JAM-L dimerization is required to engage in heterophilic interactions with CAR (Fig. 2), we then investigated whether the interaction between JAM-L and VLA-4 controlled the dimerization state of JAM-L using K562 cells that we transfected with JAM-L–HA alone or together with α4 (Fig. 7 A). We observed that in K562 cells expressing JAM-L-HA alone, SDS-resistant dimers of JAM-L are constitutively present, even in the absence of SDF-1α treatment (Fig. 7 B, middle), which is consistent with our observation that JAM-L expression can promote the adhesion of K562 cells independently of integrin activation. However, when cotransfected with α4-GFP, a constitutive interaction between α4 and the monomeric form of JAM-L–HA was detected, as previously observed in U937 cells (Fig. 5 A). Indeed, α4-GFP was detected in JAM-L–HA immunoprecipitates (Fig. 7 B, top), and, conversely, JAM-L–HA was detected in α4-GFP immunoprecipitates (Fig. 7 C, top). In addition, this interaction was accompanied by a dramatic decrease in the amount of dimeric JAM-L (Fig. 7 B, middle), which is consistent with our observation that coexpression of both JAM-L and VLA-4 prevents JAM-L–mediated adhesion of K562 cells to CAR (Fig. 6 D).

Bottom Line: Cis dimerization of JAM-L is required to engage in heterophilic interactions with its cognate counter-receptor CAR (coxsackie and adenovirus receptor).Interestingly, JAM-L expressed on neutrophils binds CAR independently of integrin activation.However, on resting monocytes and T lymphocytes, which express the integrin VLA-4, JAM-L molecules engage in complexes with VLA-4 and mainly accumulate in their monomeric form.

View Article: PubMed Central - PubMed

Affiliation: Institut Cochin, Université Paris Descartes, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8104, Paris F-75014, France.

ABSTRACT
Junctional adhesion molecules (JAMs) are endothelial and epithelial adhesion molecules involved in the recruitment of circulating leukocytes to inflammatory sites. We show here that JAM-L, a protein related to the JAM family, is restricted to leukocytes and promotes their adhesion to endothelial cells. Cis dimerization of JAM-L is required to engage in heterophilic interactions with its cognate counter-receptor CAR (coxsackie and adenovirus receptor). Interestingly, JAM-L expressed on neutrophils binds CAR independently of integrin activation. However, on resting monocytes and T lymphocytes, which express the integrin VLA-4, JAM-L molecules engage in complexes with VLA-4 and mainly accumulate in their monomeric form. Integrin activation is required for the dissociation of JAM-L-VLA-4 complexes and the accumulation of functional JAM-L dimers, which indicates that the leukocyte integrin VLA-4 controls JAM-L function in cis by controlling its dimerization state. This provides a mechanism through which VLA-4 and JAM-L functions are coordinately regulated, allowing JAM-L to strengthen integrin-dependent adhesion of leukocytes to endothelial cells.

Show MeSH
Related in: MedlinePlus