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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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Cis-dimerization of JAM-L is required for its interaction with CAR. (A) K562 cells were double transfected with vectors encoding JAM-L–HA and JAM-L–GFP or with JAM-L–HA and ICAM-1–GFP. After lysis, immunoprecipitations (IP) were performed with anti-HA or anti-GFP, as indicated. Immune complexes were subjected to immunoblotting (IB) with anti-HA or anti-GFP. Arrows indicate the electrophoretic mobility of JAM-L–HA, JAM-L–GFP, or ICAM-1–GFP. The bracket indicates an IgG heavy chain. (B) K562 cells were transfected with vectors encoding wild-type (JAM-L–WT, gray) or mutated (JAM-L–K54D, black) JAM-L coupled to HA, or empty vector (white) as control. Cells were then loaded with CMFDA, and adhesion assays were performed on immobilized CAR-Fc or CD25-Fc proteins in RPMI medium. Cells were incubated for 40 min at 37°C, adherent cells were lysed, and fluorescence intensity was measured. The basal adhesion of K562 empty vector cells on CD25-Fc protein was 8% of the total number of incubated cells. Results are means ± SD (n = 4) of a representative three independent experiments (***, P < 0.001). (C) K562 cells expressing JAM-L–WT–HA or JAM-L–K54D–HA were labeled using anti–JAM-L, fixed, and analyzed by flow cytometry (bold black lines). Gray areas represent staining with the secondary antibody alone. (D) K562 cells expressing JAM-L–WT–HA or JAM-L–K54D–HA were lysed, then immunoprecipitations and immunoblots were performed with anti-HA. Two exposure times are shown for each blot. Arrows indicate the electrophoretic mobility of monomers and dimers of JAM-L–HA. Black lines indicate that intervening lanes have been spliced out. Numbers next to gel blots indicate molecular mass markers in kD. (E) K562 cells were transfected with JAM-L–WT–HA or JAM-L–K54D–HA and treated or not treated with BS3 before cell lysis and Western blot analysis with anti-HA. (F) The proportion of dimeric and monomeric forms of JAM-L–WT–HA (gray) or JAM-L–K54D–HA (black) in E was calculated using ImageJ.
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fig2: Cis-dimerization of JAM-L is required for its interaction with CAR. (A) K562 cells were double transfected with vectors encoding JAM-L–HA and JAM-L–GFP or with JAM-L–HA and ICAM-1–GFP. After lysis, immunoprecipitations (IP) were performed with anti-HA or anti-GFP, as indicated. Immune complexes were subjected to immunoblotting (IB) with anti-HA or anti-GFP. Arrows indicate the electrophoretic mobility of JAM-L–HA, JAM-L–GFP, or ICAM-1–GFP. The bracket indicates an IgG heavy chain. (B) K562 cells were transfected with vectors encoding wild-type (JAM-L–WT, gray) or mutated (JAM-L–K54D, black) JAM-L coupled to HA, or empty vector (white) as control. Cells were then loaded with CMFDA, and adhesion assays were performed on immobilized CAR-Fc or CD25-Fc proteins in RPMI medium. Cells were incubated for 40 min at 37°C, adherent cells were lysed, and fluorescence intensity was measured. The basal adhesion of K562 empty vector cells on CD25-Fc protein was 8% of the total number of incubated cells. Results are means ± SD (n = 4) of a representative three independent experiments (***, P < 0.001). (C) K562 cells expressing JAM-L–WT–HA or JAM-L–K54D–HA were labeled using anti–JAM-L, fixed, and analyzed by flow cytometry (bold black lines). Gray areas represent staining with the secondary antibody alone. (D) K562 cells expressing JAM-L–WT–HA or JAM-L–K54D–HA were lysed, then immunoprecipitations and immunoblots were performed with anti-HA. Two exposure times are shown for each blot. Arrows indicate the electrophoretic mobility of monomers and dimers of JAM-L–HA. Black lines indicate that intervening lanes have been spliced out. Numbers next to gel blots indicate molecular mass markers in kD. (E) K562 cells were transfected with JAM-L–WT–HA or JAM-L–K54D–HA and treated or not treated with BS3 before cell lysis and Western blot analysis with anti-HA. (F) The proportion of dimeric and monomeric forms of JAM-L–WT–HA (gray) or JAM-L–K54D–HA (black) in E was calculated using ImageJ.

Mentions: Crystal structures have revealed that JAM-A molecules form homodimers in cis via a dimerization motif in the membrane distal Ig-like domain (Kostrewa et al., 2001; Prota et al., 2003). Moreover, Cis dimerization of JAM-A was shown to be required for homophilic interaction and function in intercellular junction assembly (Bazzoni et al., 2000; Liu et al., 2000; Mandell et al., 2004). As this dimerization motif is conserved between JAM members and JAM-L, we questioned whether JAM-L forms homodimers in cis. For this purpose, K562 cells were cotransfected with two constructs of JAM-L bearing different tags, JAM-L-GFP and JAM-L-HA, or cotransfected with JAM-L-HA and ICAM-1–GFP, as a control. As shown in Fig. 2 A, JAM-L–GFP was coimmunoprecipitated with JAM-L–HA, and vice versa. However, no coimmunoprecipitation between JAM-L–HA and ICAM-1–GFP was observed, therefore specifically demonstrating that JAM-L is able to homodimerize.


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)

Cis-dimerization of JAM-L is required for its interaction with CAR. (A) K562 cells were double transfected with vectors encoding JAM-L–HA and JAM-L–GFP or with JAM-L–HA and ICAM-1–GFP. After lysis, immunoprecipitations (IP) were performed with anti-HA or anti-GFP, as indicated. Immune complexes were subjected to immunoblotting (IB) with anti-HA or anti-GFP. Arrows indicate the electrophoretic mobility of JAM-L–HA, JAM-L–GFP, or ICAM-1–GFP. The bracket indicates an IgG heavy chain. (B) K562 cells were transfected with vectors encoding wild-type (JAM-L–WT, gray) or mutated (JAM-L–K54D, black) JAM-L coupled to HA, or empty vector (white) as control. Cells were then loaded with CMFDA, and adhesion assays were performed on immobilized CAR-Fc or CD25-Fc proteins in RPMI medium. Cells were incubated for 40 min at 37°C, adherent cells were lysed, and fluorescence intensity was measured. The basal adhesion of K562 empty vector cells on CD25-Fc protein was 8% of the total number of incubated cells. Results are means ± SD (n = 4) of a representative three independent experiments (***, P < 0.001). (C) K562 cells expressing JAM-L–WT–HA or JAM-L–K54D–HA were labeled using anti–JAM-L, fixed, and analyzed by flow cytometry (bold black lines). Gray areas represent staining with the secondary antibody alone. (D) K562 cells expressing JAM-L–WT–HA or JAM-L–K54D–HA were lysed, then immunoprecipitations and immunoblots were performed with anti-HA. Two exposure times are shown for each blot. Arrows indicate the electrophoretic mobility of monomers and dimers of JAM-L–HA. Black lines indicate that intervening lanes have been spliced out. Numbers next to gel blots indicate molecular mass markers in kD. (E) K562 cells were transfected with JAM-L–WT–HA or JAM-L–K54D–HA and treated or not treated with BS3 before cell lysis and Western blot analysis with anti-HA. (F) The proportion of dimeric and monomeric forms of JAM-L–WT–HA (gray) or JAM-L–K54D–HA (black) in E was calculated using ImageJ.
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fig2: Cis-dimerization of JAM-L is required for its interaction with CAR. (A) K562 cells were double transfected with vectors encoding JAM-L–HA and JAM-L–GFP or with JAM-L–HA and ICAM-1–GFP. After lysis, immunoprecipitations (IP) were performed with anti-HA or anti-GFP, as indicated. Immune complexes were subjected to immunoblotting (IB) with anti-HA or anti-GFP. Arrows indicate the electrophoretic mobility of JAM-L–HA, JAM-L–GFP, or ICAM-1–GFP. The bracket indicates an IgG heavy chain. (B) K562 cells were transfected with vectors encoding wild-type (JAM-L–WT, gray) or mutated (JAM-L–K54D, black) JAM-L coupled to HA, or empty vector (white) as control. Cells were then loaded with CMFDA, and adhesion assays were performed on immobilized CAR-Fc or CD25-Fc proteins in RPMI medium. Cells were incubated for 40 min at 37°C, adherent cells were lysed, and fluorescence intensity was measured. The basal adhesion of K562 empty vector cells on CD25-Fc protein was 8% of the total number of incubated cells. Results are means ± SD (n = 4) of a representative three independent experiments (***, P < 0.001). (C) K562 cells expressing JAM-L–WT–HA or JAM-L–K54D–HA were labeled using anti–JAM-L, fixed, and analyzed by flow cytometry (bold black lines). Gray areas represent staining with the secondary antibody alone. (D) K562 cells expressing JAM-L–WT–HA or JAM-L–K54D–HA were lysed, then immunoprecipitations and immunoblots were performed with anti-HA. Two exposure times are shown for each blot. Arrows indicate the electrophoretic mobility of monomers and dimers of JAM-L–HA. Black lines indicate that intervening lanes have been spliced out. Numbers next to gel blots indicate molecular mass markers in kD. (E) K562 cells were transfected with JAM-L–WT–HA or JAM-L–K54D–HA and treated or not treated with BS3 before cell lysis and Western blot analysis with anti-HA. (F) The proportion of dimeric and monomeric forms of JAM-L–WT–HA (gray) or JAM-L–K54D–HA (black) in E was calculated using ImageJ.
Mentions: Crystal structures have revealed that JAM-A molecules form homodimers in cis via a dimerization motif in the membrane distal Ig-like domain (Kostrewa et al., 2001; Prota et al., 2003). Moreover, Cis dimerization of JAM-A was shown to be required for homophilic interaction and function in intercellular junction assembly (Bazzoni et al., 2000; Liu et al., 2000; Mandell et al., 2004). As this dimerization motif is conserved between JAM members and JAM-L, we questioned whether JAM-L forms homodimers in cis. For this purpose, K562 cells were cotransfected with two constructs of JAM-L bearing different tags, JAM-L-GFP and JAM-L-HA, or cotransfected with JAM-L-HA and ICAM-1–GFP, as a control. As shown in Fig. 2 A, JAM-L–GFP was coimmunoprecipitated with JAM-L–HA, and vice versa. However, no coimmunoprecipitation between JAM-L–HA and ICAM-1–GFP was observed, therefore specifically demonstrating that JAM-L is able to homodimerize.

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