Junctional adhesion molecule interacts with the PDZ domain-containing proteins AF-6 and ZO-1

We have identified the PDZ domain protein AF-6 as an intracellular binding partner of the junctional adhesion molecule (JAM), an integral membrane protein located at cell contacts. Binding of AF-6 to JAM required the presence of the intact C terminus of JAM, which represents a classical type II PDZ domain-binding motif. Although JAM did not interact with the single PDZ domains of ZO-1 or of CASK, we found that a ZO-1 fragment containing PDZ domains 2 and 3 bound to JAM in vitro in a PDZ domain-dependent manner. AF-6 as well as ZO-1 could be coprecipitated with JAM from endothelial cell extracts, demonstrating the association of the endogenously expressed molecules in vivo. Targeting of JAM to sites of cell contacts could be affected by the loss of the PDZ domain-binding C terminus. Full-length mouse JAM co-distributed with endogenous AF-6 in human Caco-2 cells at sites of cell contact independent of whether adjacent cells expressed mouse JAM as an extracellular binding partner. In contrast, truncated JAM lacking the PDZ domain-binding C terminus did not co-distribute with endogenous AF-6, but was restricted to cell contacts between cells expressing mouse JAM. Our results suggest that JAM can be recruited to intercellular junctions by its interaction with the PDZ domain-containing proteins AF-6 and possibly ZO-1.     

Immunogold localization of tight junctional proteins in normal and osmotically-affected rat blood-brain barrier

The distribution of molecular components of interendothelial tight junctions (TJs) was studied in rat blood-brain barrier (BBB) microvessels, using immunogold cytochemistry applied to electron microscopy. Samples of rat brains, both normal (unaffected) and osmotically-affected (1, 5, and 30 min after intracarotid infusion of 1.8 M L(+)arabinose), were processed for immunocytochemical localization of TJ-specific integral membrane (occludin, JAM-1, claudin-5) and peripheral (ZO-1) protein molecules. In unaffected interendothelial junctions of control rats the immunosignals (represented by gold particles) for occludin and ZO-1 were of highest, whereas for claudin-5 and JAM-1 were of lower density. At 1 min after infusion, no discernible changes in distribution of junction-associated molecules were noted. At 5 min, however, changes were most conspicuous, and they consisted of segmental attenuation of the endothelial lining and dilatation (opening) of some junctional clefts accompanied by the diminution of the density of immunosignals for TJ-specific transmembrane and peripheral proteins. It was paralleled by disorganization of the spatial relation of these molecules to the junctional complexes. After 30 min, many interendothelial junctions appeared to be still open, whereas other junctions were partially or totally closed. In the opened interendothelial junctions the expression of TJ-associated molecules was weaker than in closed junctions. Our observations indicate that the localization and expression of TJ-specific proteins, especially occludin, and in lower degree claudin-5 and JAM-1, together with the peripheral ZO-1 molecules, are affected by osmotic shock. Presumably, some of these proteins (e.g., occludin, claudin-5 and ZO-1) could be considered sensitive indicators of normal and also of disturbed functional state of the BBB.     

JAM4, a junctional cell adhesion molecule interacting with a tight junction protein,MAGI-1

MAGI-1 is a membrane-associated guanylate kinase protein at tight junctions in epithelial cells. It interacts with various molecules and functions as a scaffold protein at cell junctions. We report here a novel MAGI-1-binding protein that we named junctional adhesion molecule 4 (JAM4). JAM4 belongs to an immunoglobulin protein family. JAM4 was colocalized with ZO-1 in kidney glomeruli and in intestinal epithelial cells. Biochemical in vitro studies revealed that JAM4 bound to MAGI-1 but not to ZO-1, whereas JAM1 did not bind to MAGI-1. JAM4 and MAGI-1 interacted with each other and formed clusters in COS-7 cells when coexpressed. JAM4 mediated calcium-independent homophilic adhesion and was accumulated at cell-cell contacts when expressed in L cells. MAGI-1, ZO-1, and occludin were recruited to JAM4-based cell contacts. JAM4 also reduced the permeability of CHO cell monolayers. MAGI-1 strengthened JAM4-mediated cell adhesion in L cells and sealing effects in CHO cells. These findings suggest that JAM4 together with MAGI-1 provides an adhesion machinery at tight junctions, which may regulate the permeability of kidney glomerulus and small intestinal epithelial cells.     

The junctional adhesion molecule-C promotes neutrophil transendothelial migration in vitro and in vivo

The third member of the family of junctional adhesion molecules (JAMs), JAM-3, also called JAM-C, was recently shown to be a novel counter-receptor on platelets for the leukocyte beta(2)-integrin Mac-1 (alphaMbeta(2), CD11b/CD18). Here, new functional aspects of the role of endothelial cell JAM-C were investigated. Endothelial cells express JAM-C, which is predominantly localized within junctions at interendothelial contacts, since it codistributes with a tight junction component, zonula occludens-1. Whereas JAM-C does not participate in neutrophil adhesion to endothelial cells, it mediates neutrophil transmigration in a Mac-1-dependent manner. In particular, inhibition of JAM-C significantly reduced neutrophil transendothelial migration, and the combination of JAM-C and platelet/endothelial cell adhesion molecule-1 blockade almost completely abolished neutrophil transendothelial migration in vitro. In vivo, inhibition of JAM-C with soluble mouse JAM-C resulted in a 50% reduction of neutrophil emigration in the mouse model of acute thioglycollate-induced peritonitis. Thus, JAM-C participates in neutrophil transmigration and thereby provides a novel molecular target for antagonizing interactions between vascular cells that promote inflammatory vascular pathologies.     

Molecular anatomy of interendothelial junctions in human blood-brain barrier microvessels

Immunogold cytochemical procedure was used to study the localization at the ultrastructural level of interendothelial junction-associated protein molecules in the human brain blood microvessels, representing the anatomic site of the blood-brain barrier (BBB). Ultrathin sections of Lowicryl K4M-embedded biopsy specimens of human cerebral cortex obtained during surgical procedures were exposed to specific antibodies, followed by colloidal gold-labeled secondary antibodies. All tight junction-specific integral membrane (transmembrane) proteins--occludin, junctional adhesion molecule (JAM-1), and claudin-5--as well as peripheral zonula occludens protein (ZO-1) were highly expressed. Immunoreactivity of the adherens junction-specific transmembrane protein VE-cadherin was of almost similar intensity. Immunolabeling of the adherens junction-associated peripheral proteins--alpha-catenin, beta-catenin, and p120 catenin--although positive, was evidently less intense. The expression of gamma-catenin (plakoglobin) was considered questionable because solitary immunosignals (gold particles) appeared in only a few microvascular profiles. Double labeling of some sections made possible to observe strict colocalization of the junctional molecules, such as occludin and ZO-1 or JAM-1 and VE-cadherin, in the interendothelial junctions. We found that in human brain microvessels, the interendothelial junctional complexes contain molecular components specific for both tight and adherens junctions. It is assumed that the data obtained can help us find the immunodetectable junctional molecules that can serve as sensitive markers of normal or abnormal function of the BBB.     

Junctional adhesion molecule 1 (JAM-1)

Junctional adhesion molecule 1 (JAM-1) was the first of a family of related proteins (JAM family) to be discovered. Two proteins with structural and sequence similarities to JAM-1, named JAM-2 and JAM-3, have been identified more recently. JAM-1 is specifically localized at the tight junctions of epithelial and endothelial cells and is involved in the regulation of junctional integrity and permeability. This function is attributed to its ability to interact in a homophilic manner. JAM-1 can also bind in a heterophilic manner as it serves as a ligand for integrin LFA-1 (CD11a/CD18), and plays a key role in the process of leukocyte transmigration. In addition, JAM-1 is also a receptor for reovirus, and is a platelet receptor involved in platelet adhesion and antibody-induced platelet aggregation. Further study of the mechanism of JAM-1 action within these diverse systems may demonstrate that JAM-1 is a key player in many different cellular functions.     

Extracellular domain of V-set and immunoglobulin domain containing 1 (VSIG1) interacts with sertoli cell membrane protein, while its PDZ-binding motif forms a complex with ZO-1

V-set and immunoglobulin domain containing 1 (VSIG1) is a newly discovered member of the junctional adhesion molecule (JAM) family; it is encoded by a gene located on human chromosome X and preferentially expressed in a variety of cancers in humans. Little is known about its physiological function. To determine the role(s) of VSIG1 in mammalian spermatogenesis, we first generated a specific antibody against mouse VSIG1 and examined the presence and localization of the protein in tissues. RTRCR and Western blot analysis of the mouse tissues indicated that VSIG1 was specifically expressed in the testis. Furthermore, the results of our trypsinization and biotinylation assays strongly support the assumption that VSIG1 is localized on the testicular germ cell surface. In order to determine whether VSIG1 is capable of participation in homotypic interactions, we performed a GST-pull down assay by using recombinant GST-fusion and Histagging proteins. The pull-down assay revealed that each GST-fusion Ig-like domain shows homotypic binding. We further show that mVSIG1 can adhere to the Sertoli cells through its first Ig-like domain. To identify the protein that interacted with cytoplasmic domain, we next performed co-immunoprecipitation analysis. This analysis showed that ZO-1, which is the central structural protein of the tight junction, is the binding partner of the cytoplasmic domain of mouse VSIG1. Our findings suggest that mouse VSIG1 interacts with Sertoli cells by heterophilic adhesion via its first Ig-like domain. In addition, its cytoplasmic domain is critical for binding to ZO-1.     

Immunogold study of interendothelial junction-associated and glucose transporter proteins during postnatal maturation of the mouse blood-brain barrier

The distribution of glucose transporter (GLUT-1) and of interendothelial junction—associated proteins—zonula occludens protein (ZO-1), occludin, and β-catenin—was studied using quantitative immunogold procedure. Lowicryl K4M-embedded samples of the cerebral cortex of 1-, 7-, and 14-day-, and 6-week-old (young-adult) mice were used. Ultrathin sections were exposed to specific rabbit polyclonal antibodies followed by colloidal gold-labelled secondary antibodies. We found that the density of immunosignals for GLUT-1 in both luminal and abluminal plasma membranes of the endothelial cells, and those closely related to the interendothelial junctions was low in blood microvessels from newborn mice, dropped slightly at the 7th day, and increased through the 14th day to the level of mature blood-brain barrier (BBB) observed in 6-week-old mice. The expression of ZO-1 was high in newborn mice and increased at the 7th day to the level similar to that found in 14-day- and 6-week-old mice. The expression of occludin was less intense than that of ZO-1 and increased from birth, reaching at the 14th day the level typical for mature BBB found in young-adult animals. The immunosignals for occludin were sparsely distributed inside the junctional clefts. Such a distribution indicates that the tight junctional characteristics are limited to a few short segments of the entire interendothelial cleft. The density of immunosignals for β-catenin was lowest, and it had the tendency to a gradual, although inconsiderable, drop in the time course of BBB maturation. These findings suggest that the relatively high concentration of GLUT-1 in the interendothelial junctions results from the participation of abluminal plasma membranes of adjacent endothelial cells in the formation of the junctional complexes. The interendothelial junctions of newborn mice are equipped already with the main components of the tight junctions, and the concentration of these components (ZO-1, occludin) reaches the level of the mature BBB at the 14th day of postnatal life.     

Association of junctional adhesion molecule with calcium/calmodulin-dependent serine protein kinase (CASK/LIN-2) in human epithelial caco-2 cells

We report here that junctional adhesion molecule (JAM) interacts with calcium/calmodulin-dependent serine protein kinase (CASK), a protein related to membrane-associated guanylate kinases. In Caco-2 cells, JAM and CASK were coprecipitated and found to colocalize at intercellular contacts along the lateral surface of the plasma membrane. Association of JAM with CASK requires the PSD95/dlg/ZO-1 (PDZ) domain of CASK and the putative PDZ-binding motif Phe-Leu-Val(COOH) in the cytoplasmic tail of JAM. Temporal dissociation in the junctional localization of the two proteins suggests that the association with CASK is not required for recruiting JAM to intercellular junctions. Compared with mature intercellular contacts, junction assembly was characterized by both enhanced solubility of CASK in Triton X-100 and reduced amounts of Triton-insoluble JAM-CASK complexes. We propose that JAM association with CASK is modulated during junction assembly, when CASK is partially released from its cytoskeletal associations.     

Dynamic assembly of tight junction-associated proteins ZO-1, ZO-2, ZO-3 and occludin during mouse tooth development

Tight junctions might play a role during tissue morphogenesis and cell differentiation. In order to address these questions, we have studied the distribution pattern of the tight junction-associated proteins ZO-1, ZO-2, ZO-3 and occludin in the developing mouse tooth as a model. A specific temporal and spatial distribution of tight junction-associated proteins during tooth development was observed. ZO-1 appeared discontinuously in the cell membrane of enamel organ and dental mesenchyme cells. However, endothelial cells of the dental mesenchyme capillaries displayed a continuous fluorescence at the cell membrane. Inner dental epithelium first showed an evident signal for ZO-1 at the basal pole of the cells at bud/cap stage, but ZO-1 was accumulated at the basal and apical pole of preameloblast/ameloblasts at late bell stage. Surprisingly, in the incisor ZO-1 decreased as the inner dental epithelium differentiated, and was re-expressed in secretory and mature ameloblasts. On the contrary, ZO-2 was confined to continuous cell-cell contacts of the enamel organ in both molars and incisors. The lateral cell membrane of inner dental epithelial cells was specifically ZO-2 labeled. However, ZO-3 was expressed in oral epithelium whereas dental embryo tissues were negative. In addition, occludin was hardly detected in dental tissues at the early stage of tooth development, but was distributed continuously at the cell membrane of endothelial cells of ED19.5 dental mesenchyme. In incisors, occludin was detected at the cell membrane of the secretory pole of ameloblasts. The occurrence and relation during tooth development of tight junction proteins ZO-1, ZO-2 and occludin, but not ZO-3, suggests a combinatory assembly in tooth morphogenesis and cell differentiation.     

Mapping the homotypic binding sites in CD31 and the role of CD31 adhesion in the formation of interendothelial cell contacts

CD31 is a member of the immunoglobulin superfamily consisting of six Ig- related domains. It is constitutively expressed by platelets, monocytes, and some lymphocytes, but at tenfold higher levels on vascular endothelial cells. CD31 has both homotypic and heterotypic adhesive properties. We have mapped the homotypic binding sites using a deletion series of CD31-Fc chimeras and a panel of anti-CD31 monoclonal antibodies. An extensive surface of CD31 is involved in homotypic binding with domains 2 and 3 and domains 5 and 6 playing key roles. A model consistent with the experimental data is that CD31 on one cell binds to CD31 on an apposing cell in an antiparallel interdigitating mode requiring full alignment of the six domains of each molecule. In addition to establishing intercellular homotypic contacts. CD31 binding leads to augmented adhesion via beta 1 integrins. The positive cooperation between CD31 and beta 1 integrins can occur in heterologous primate cells (COS cells). The interaction is specific to both CD31 and beta 1 integrins. Neither intercellular adhesion molecule-1 (ICAM- 1)/leukocyte function-associated antigen-1 (LCAM-1) nor neural cell adhesion molecule (NCAM)/NCAM adhesion leads to recruitment of beta 1 integrin adhesion pathways. Establishment of CD31 contacts have effects on the growth and morphology of endothelial cells. CD31(D1-D6)Fc inhibits the growth of endothelial cells in culture. In addition, papain fragments of anti-CD31 antibodies (Fab fragments) disrupt interendothelial contact formation and monolayer integrity when intercellular contacts are being formed. The same reagents are without effect once these contacts have been established, suggesting that CD31- CD31 interactions are critically important only in the initial phases of intercellular adhesion.     

Separation of luminal and abluminal membrane enriched domains from cultured bovine aortic endothelial cells: monoclonal antibodies specific for endothelial cell plasma membranes

Two kinds of membrane (luminal and abluminal membrane domains) fractions have been isolated from bovine aortic endothelial cells by fractionation of whole cell homogenate on discontinuous sucrose density gradients. The luminal membrane domain was enriched 12-16-fold for angiotensin-converting enzyme activity and 8-10-fold in alkaline phosphatase activity. The abluminal membrane domain displayed an enrichment of 8-fold in (Na+ + K+)-ATPase activity. Both of the membrane domains were minimally contaminated with mitochondria, microsomes and Golgi bodies, as assessed by their corresponding marker enzyme activities. 125I-labeling of endothelial cell monolayers by the Enzymo-Bead lactoperoxidase-catalyzed iodination procedure, followed by isolation of membranes, revealed that the radioactivity was predominantly associated with membranes enriched in angiotensin-converting enzyme activity, corresponding to the luminal membrane domain. However, when cells were radioiodinated in suspension culture, radioactivity was found equally associated in both the luminal and abluminal membrane fractions. Electron microscopy of freeze-fractured and sectioned material showed both luminal and abluminal membrane domains to be in the form of vesicles varying in size from 100 to 400 nm in diameter. To characterize the separation of endothelial cell membrane domains, we have attempted to prepare monoclonal antibodies specific for endothelial cells. Several clones were obtained, producing antibodies which bound to endothelial cells of arterial, venous and capillary origin. Two antibodies of these clones, XIVC6 and XVD2, were studied in more detail. In the ELISA assay, these antibodies reacted with bovine vascular endothelial cells, but not with human umbilical cord endothelial cells, nor with bovine corneal endothelial cells, smooth muscle cells or fibroblasts. Both of these antibodies are directed against an antigen of approximately 130 kDa, under reducing and non-reducing conditions, as assayed by the immunoprecipitation method. Western blot analysis of luminal and abluminal membrane fractions revealed that only MAb XVD2 reacted with an antigen, indicating that the antibody XIVC6 is directed against an epitope which is denatured by SDS. Moreover, MAb XVD2 preferentially reacted with the luminal membrane compared to the abluminal membrane domain of the endothelial cell. These monoclonal antibodies do not react with platelet membrane proteins, indicating that this 130 kDa membrane antigen is not common to both endothelial cells and platelets.(ABSTRACT TRUNCATED AT 400 WORDS)     

Accessory cell function of human endothelial cells. I. A subpopulation of Ia positive cells is required for antigen presentation

The expression of class I and class II HLA antigens on preparations of human endothelial cells, isolated from umbilical cord veins, was investigated by immunofluorescence. While virtually all endothelial cells expressed class I antigens, less than 1% were positive for class II antigens, as detected with a panel of 10 different monoclonal antibodies. Antigen specific T cell lines proliferated in response to mumps antigen in the presence of endothelial cells or blood monocytes from HLA-DR matched donors. However, these T cell lines failed to respond in the absence of accessory cells or when accessory cells from HLA-D-region mismatched cord donors were used. The ability of both monocytes and endothelial cells to present antigen was abolished by treatment of the cells with monoclonal antibodies specific for either class I or class II HLA antigens plus complement. Similar treatment with monoclonal antibodies specific for monocytes greatly reduced antigen presentation by endothelial cells. These results indicate that preparations of endothelial cells contain a subpopulation of Ia positive cells, distinct from monocytes, which are required for antigen presentation.     

Nepmucin/CLM-9, an Ig domain-containing sialomucin in vascular endothelial cells, promotes lymphocyte transendothelial migration in vitro

Nepmucin/CLM-9 is an Ig domain-containing sialomucin expressed in vascular endothelial cells. Here we show that, like CD31, nepmucin was localized to interendothelial contacts and to vesicle-like structures along the cell border and underwent intracellular recycling. Functional analyses showed that nepmucin mediated homotypic and heterotypic cell adhesion via its Ig domain. Nepmucin-expressing endothelial cells showed enhanced lymphocyte transendothelial migration (TEM), which was abrogated by anti-nepmucin mAbs that block either homophilic or heterophilic binding. Notably, the mAbs that inhibited homophilic binding blocked TEM without affecting lymphocyte adhesion. These results suggest that endothelial nepmucin promotes lymphocyte TEM using multiple adhesion pathways.     

Pilt, a novel peripheral membrane protein at tight junctions in epithelial cells

Tight junctions (TJs) serve as a barrier that prevents solutes and water from passing through the paracellular pathway, and as a fence between the apical and basolateral plasma membranes in epithelial cells. TJs consist of transmembrane proteins (claudin, occludin, and JAM) and many peripheral membrane proteins, including actin filament (F-actin)-binding scaffold proteins (ZO-1, -2, and -3), non-F-actin-binding scaffold proteins (MAGI-1), and cell polarity molecules (ASIP/PAR-3 and PAR-6). We identified here a novel peripheral membrane protein at TJs from a human cDNA library and named it Pilt (for protein incorporated later into TJs), because it was incorporated into TJs later after the claudin-based junctional strands were formed. Pilt consists of 547 amino acids with a calculated M(r) of 60,704. Pilt has a proline-rich domain. In cadherin-deficient L cells stably expressing claudin or JAM, Pilt was not recruited to claudin-based or JAM-based cell-cell contact sites, suggesting that Pilt does not directly interact with claudin or JAM. The present results indicate that Pilt is a novel component of TJs.     

JEAP, a novel component of tight junctions in exocrine cells.

Tight junctions (TJs) consist of transmembrane proteins and many peripheral membrane proteins. To further characterize the molecular organization of TJs, we attempted here to screen for novel TJ proteins by the fluorescence localization-based expression cloning method. We identified a novel peripheral membrane protein at TJs and named it junction-enriched and -associated protein (JEAP). JEAP consists of 882 amino acids with a calculated molecular weight of 98,444. JEAP contained a polyglutamic acid repeat at the N-terminal region, a coiled-coil domain at the middle region, and a consensus motif for binding to PDZ domains at the C-terminal region. Exogenously expressed JEAP co-localized with ZO-1 and occludin at TJs in polarized Madin-Darby canine kidney cells, but not with claudin-1, JAM, or ZO-1 in L cells. Endogenous JEAP localized at TJs of exocrine cells including pancreas, submandibular gland, lacrimal gland, parotid gland, and sublingual gland, but not at TJs of epithelial cells of small intestine or endothelial cells of blood vessels. The present results indicate that JEAP is a novel component of TJs, which is specifically expressed in exocrine cells.