Intercellular junctions mediate adhesion and communication between adjoining endothelial and epithelial cells. In the endothelium, junctional complexes comprise tight junctions, adherens junctions, and gap junctions. The expression and organization of these complexes depend on the type of vessels and the permeability requirements of perfused organs. Gap junctions are communication structures, which allow the passage of small molecular weight solutes between neighboring cells. Tight junctions serve the major functional purpose of providing a "barrier" and a "fence" within the membrane, by regulating paracellular permeability and maintaining cell polarity. Adherens junctions play an important role in contact inhibition of endothelial cell growth, paracellular permeability to circulating leukocytes and solutes. In addition, they are required for a correct organization of new vessels in angiogenesis. Extensive research in the past decade has identified several molecular components of the tight and adherens junctions, including integral membrane and intracellular proteins. These proteins interact both among themselves and with other molecules. Here, we review the individual molecules of junctions and their complex network of interactions. We also emphasize how the molecular architectures and interactions may represent a mechanistic basis for the function and regulation of junctions, focusing on junction assembly and permeability regulation. Finally, we analyze in vivo studies and highlight information that specifically relates to the role of junctions in vascular endothelial cells.

Junctional adhesion molecule (JAM) is an integral membrane protein that has been reported to colocalize with the tight junction molecules occludin, ZO-1, and cingulin. However, evidence for the association of JAM with these molecules is missing. Transfection of Chinese hamster ovary cells with JAM (either alone or in combination with occludin) resulted in enhanced junctional localization of both endogenous ZO-1 and cotransfected occludin. Additionally, JAM was coprecipitated with ZO-1 in the detergent-insoluble fraction of Caco-2 epithelial cells. A putative PDZ-binding motif at the cytoplasmic carboxyl terminus of JAM was required for mediating the interaction of JAM with ZO-1, as assessed by in vitro binding and coprecipitation experiments. JAM was also coprecipitated with cingulin, another cytoplasmic component of tight junctions, and this association required the amino-terminal globular head of cingulin. Taken together, these data indicate that JAM is a component of the multiprotein complex of tight junctions, which may facilitate junction assembly.

The monoclonal antibody 9EG7 has been previously found to recognize an epitope induced by manganese on the integrin ␤ 1 chain (Lenter, M., Uhlig, H., Hamann, A., Jeno, P., Imhof, B., and Vestweber, D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9051-9055). Here we show that treatment of ␤ 1 integrins with manganese or soluble integrin ligands (e.g. fibronectin and RGD peptide) induced the 9EG7 epitope. This epitope was also induced upon EGTA treatment to remove calcium, and the addition of calcium inhibited 9EG7 epitope induction by manganese or by ligand. Further emphasizing the importance of the 9EG7 epitope, the 9EG7 antibody itself stimulated adhesion mediated by multiple ␤ 1 integrins, and conversely, ligands for ␣ 2 ␤ 1 , ␣ 3 ␤ 1 , ␣ 4 ␤ 1 , and ␣ 5 ␤ 1 all stimulated 9EG7 expression. Together these results support a model whereby (i) calcium inhibits ␤ 1 integrin function because it prevents the appearance of a conformation favorable to ligand binding and (ii) manganese enhances ␤ 1 integrin function because it induces the same favorable conformation that is induced by adding ligand, or removing calcium. Notably, other ␤ 1 -stimulating agents (magnesium and mAb TS2/16) did not induce 9EG7 expression unless ligand was also present. Thus, although 9EG7 may reliably detect the ligand-bound conformation of ␤ 1 integrins, its expression does not always correlate with integrin "activation." Finally, mouse/chicken ␤ 1 chimeric molecules were used to map the 9EG7 epitope to ␤ 1 residues 495-602 within the cysteine-rich region, and antibody cross-blocking studies showed that the 9EG7 epitope is distinct from all previously defined human ␤ 1 epitopes.