Homophilic interaction of junctional adhesion molecule.
Junctional adhesion molecule (JAM) is an integral membrane protein that belongs to the immunoglobulin superfamily, localizes at tight junctions, and regulates both paracellular permeability and leukocyte transmigration. To investigate molecular determinants of JAM function, the extracellular domain of murine JAM was produced as a recombinant soluble protein (rsJAM) in insect cells. rsJAM consisted in large part of noncovalent homodimers, as assessed by analytical ultracentrifugation. JAM dimers were also detected at the surface of Chinese hamster ovary cells transfected with murine JAM, as evaluated by cross-linking and immunoprecipitation. Furthermore, fluid-phase rsJAM bound dose-dependently solid-phase rsJAM, and such homophilic binding was inhibited by anti-JAM Fab BV11, but not by Fab BV12. Interestingly, Fab BV11 exclusively bound rsJAM dimers (but not monomers) in solution, whereas Fab BV12 bound both dimers and monomers. Finally, we mapped the BV11 and BV12 epitopes to a largely overlapping sequence in proximity of the extracellular amino terminus of JAM. We hypothesize that rsJAM dimerization induces a BV11-positive conformation which in turn is critical for rsJAM homophilic interactions. Dimerization and homophilic binding may contribute to both adhesive function and junctional organization of JAM.
Laboratory of Vascular Biology, Istituto di Ricerche Farmacologiche Mario Negri, I-20157 Milano, Italy. bazzoni@irfmn.mnegri.it
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G
Bazzoni
O M
OM
Martinez-Estrada
F
F
Mueller
P
P
Nelboeck
G
G
Schmid
T
T
Bartfai
E
E
Dejana
M
M
Brockhaus
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D015815:Cell Adhesion Molecules / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004727:Endothelium / Q000737:chemistry; D018604:Epitope Mapping; D000939:Epitopes; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D062726:Junctional Adhesion Molecules; D007700:Kinetics; D007962:Leukocytes / Q000737:chemistry; D051379:Mice; D011233:Precipitin Tests; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014461:Ultracentrifugation
D015815:Cell Adhesion Molecules; D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D000939:Epitopes; D062726:Junctional Adhesion Molecules; D011994:Recombinant Proteins; D012965:Sodium Chloride