Involvement of the junctional adhesion molecule-1 (JAM1) homodimer interface in regulation of epithelial barrier function.
Junctional adhesion molecule-1 (JAM1) is a tight junction-associated immunoglobulin superfamily protein implicated in the regulation of tight junctions and leukocyte transmigration. The structural basis for the function of JAM1 has yet to be determined. Here we provide evidence that JAM1 homodimer formation is important for its function in epithelial cells. Experiments were conducted to determine the effects of a panel of JAM1 monoclonal antibodies on epithelial barrier recovery after transient disruption by calcium switch. Two monoclonal antibodies were observed to inhibit barrier recovery in contrast to another monoclonal antibody that had no effect. Epitope mapping by phage display revealed that both inhibitory antibodies bind to a region of JAM1 located within the N-terminal Ig-like loop (residues 111-123). Competition experiments with synthetic peptides and site-directed mutagenesis confirmed the location of this epitope. Analysis of the crystal structure of JAM1 revealed that this epitope includes residues within the putative homodimer interface, and one of the two inhibitory antibodies was then shown to block JAM1 homodimer formation in vitro. Finally, mutations within the homodimer interface were shown to prevent enrichment of JAM1 at points of cell contact, presumably by interference with homophilic interactions. These findings suggest that homodimer formation may be important for localization of JAM1 at tight junctions and for regulation of epithelial barrier function.
Mandell
Kenneth J
KJ
Epithelial Pathobiology Research Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA. kjmande@emory.edu
McCall
Ingrid C
IC
Parkos
Charles A
CA
D000818:Animals; D019556:COS Cells; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000502:physiology*; D019281:Dimerization; D004847:Epithelial Cells / Q000502:physiology*; D006801:Humans; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000502:physiology*; D019108:Tight Junctions / Q000502:physiology
D015815:Cell Adhesion Molecules; C407243:F11R protein, human; D011956:Receptors, Cell Surface