Localization of HIV RNA in mitochondria of infected cells: potential role in cytopathogenicity

A novel member of the cadherin family of cell adhesion molecules has been characterized by cloning from rat liver, sequencing of the corresponding cDNA, and functional analysis after heterologous expression in nonadhesive S2 cells. cDNA clones were isolated using a polyclonal antibody inhibiting Ca(2+)-dependent intercellular adhesion of hepatoma cells. As inferred from the deduced amino acid sequence, the novel molecule has homologies with E-, P-, and N-cadherins, but differs from these classical cadherins in four characteristics. Its extracellular domain is composed of five homologous repeated domains instead of four characteristic for the classical cadherins. Four of the five domains are characterized by the sequence motifs DXNDN and DXD or modifications thereof representing putative Ca(2+)-binding sites of classical cadherins. In its NH2-terminal region, this cadherin lacks both the precursor segment and the endogenous protease cleavage site RXKR found in classical cadherins. In the extracellular EC1 domain, the novel cadherin contains an AAL sequence in place of the HAV sequence motif representing the common cell adhesion recognition sequence of E-, P-, and N-cadherin. In contrast to the conserved cytoplasmic domain of classical cadherins with a length of 150-160 amino acid residues, that of the novel cadherin has only 18 amino acids. Examination of transfected S2 cells showed that despite these structural differences, this cadherin mediates intercellular adhesion in a Ca(2+)-dependent manner. The novel cadherin is solely expressed in liver and intestine and was, hence, assigned the name LI-cadherin. In these tissues, LI- cadherin is localized to the basolateral domain of hepatocytes and enterocytes. These results suggest that LI-cadherin represents a new cadherin subtype and may have a role in the morphological organization of liver and intestine.     

The cadherins: cell-cell adhesion molecules controlling animal morphogenesis

Cadherins are a family of glycoproteins involved in the Ca2+-dependent cell-cell adhesion mechanism which is detected in most kinds of tissues. Inhibition of the cadherin activity with antibodies induces dissociation of cell layers, indicating a fundamental importance of these molecules in maintaining the multicellular structure. Cadherins are divided into subclasses, including E-, N- and P-cadherins. While all subclasses are similar in molecular weight, Ca2+- and protease-sensitivity, each subclass is characterized by a unique tissue distribution pattern and immunological specificity. Analysis of amino acid sequences deduced from cDNA encoding these molecules showed that they are integral membrane proteins of 723-748 amino acids long and share common sequences; similarity in the sequences between subclasses is in a range of 50-60% when compared within a single animal species. L cells, with very little endogenous cadherin activity, transfected with the cadherin cDNA acquired high cadherin-mediated aggregating activity. Their colony morphology was altered by the ectopic expression of cadherins from the dispersed type to the compact type, providing direct evidence for a key role of cadherins in cell-cell adhesion. It has been suggested that cadherins bind cells by their homophilic interactions at the extracellular domain and are associated with actin bundles at the cytoplasmic domain. It appears that each cadherin subclass has binding specificity and this molecular family is involved in selective cell-cell adhesion. In development, the expression of each cadherin subclass is spatiotemporally regulated and associated with a variety of morphogenetic events; e.g. the termination or initiation of expression of a cadherin subclass in a given cell collective is correlated with its segregation from or connection with other cell collectives. Antibodies to cadherins were shown to perturb the morphogenesis of some embryonic organs in vitro. These observations suggest that cadherins play a crucial role in construction of tissues and the whole animal body.     

Structure and binding mechanism of vascular endothelial cadherin: a divergent classical cadherin

Vascular endothelial cadherin (VE-cadherin), a divergent member of the type II classical cadherin family of cell adhesion proteins, mediates homophilic adhesion in the vascular endothelium. Previous investigations with a bacterially produced protein suggested that VE-cadherin forms cell surface trimers that bind between apposed cells to form hexamers. Here we report studies of mammalian-produced VE-cadherin ectodomains suggesting that, like other classical cadherins, VE-cadherin forms adhesive trans dimers between monomers located on opposing cell surfaces. Trimerization of the bacterially produced protein appears to be an artifact that arises from a lack of glycosylation. We also present the 2.1-Å-resolution crystal structure of the VE-cadherin EC1-2 adhesive region, which reveals homodimerization via the strand-swap mechanism common to classical cadherins. In common with type II cadherins, strand-swap binding involves two tryptophan anchor residues, but the adhesive interface resembles type I cadherins in that VE-cadherin does not form a large nonswapped hydrophobic surface. Thus, VE-cadherin is an outlier among classical cadherins, with characteristics of both type I and type II subfamilies.     

Identification of a conserved region common to cadherins and influenza strain A hemagglutinins

Cadherins are a family of integral membrane glycoproteins that mediate homophilic, calcium-dependent cell adhesion in vertebrate species. The primary structures of six members of the cadherin family have recently been determined. The extracellular portion of these proteins is composed of five domains, the first of which is the most highly conserved among cadherins. Previous searches of protein sequence databases have revealed little or no sequence homology between cadherins and other proteins. Here we report that the first extracellular domain of cadherins exhibits substantial sequence homology with the amino termini of influenza strain A hemagglutinins. These regions of sequence homology have been shown to be functionally important in both cadherins and hemagglutinins. Our observations suggest that a functional domain of cadherins is conserved among other proteins.     

The cadherin-binding specificities of B-cadherin and LCAM

The cadherin family of calcium-dependent cell adhesion molecules plays an important part in the organization of cell adhesion and tissue segregation during development. The expression pattern and the binding specificity of each cadherin are of principal importance for its role in morphogenesis. B-Cadherin and LCAM, two chicken cadherins, have similar, but not identical, spatial and temporal patterns of expression. To examine the possibility that they might bind to one another in a heterophilic manner, we generated, by cDNA transfection, L- cell lines that express LCAM or B-cadherin. We then examined the abilities of these cells to coaggregate with each other and with other cadherin-expressing cells in short-term aggregation assays. The B- cadherin- and the LCAM-expressing cell lines segregate from P-, N-, or R-cadherin-expressing cells. B-cadherin- and LCAM-expressing cell lines, however, appear to be completely miscible, forming large mixed aggregates. Chick B-cadherin and murine E-cadherin also form mixed aggregates, indistinguishable from homophilic aggregates. Murine E- cadherin and chick LCAM coaggregate less completely, suggesting that the heterophilic interactions of these two cell lines are weak relative to homophilic interactions. These data suggest that heterophilic interactions between B-cadherin and LCAM are important during avian morphogenesis and help identify the amino acids in the binding domain that determine cadherin specificity.     

Phylogenetic analysis of the cadherin superfamily.

Cadherins are a multigene family of proteins which mediate homophilic calcium-dependent cell adhesion and are thought to play an important role in morphogenesis by mediating specific intercellular adhesion. Different lines of experimental evidence have recently indicated that the site responsible for mediating adhesive interactions is localized to the first extracellular domain of cadherin. Based upon an analysis of the sequence of this domain, I show that cadherins can be classified into three groups with distinct structural features. Furthermore, using this sequence information a phylogenetic tree relating the known cadherins was assembled. This is the first such tree to be published for the cadherins. One cadherin subtype, neural cadherin (N-cadherin), shows very little sequence divergence between species, whereas all other cadherin subtypes show more substantial divergence, suggesting that selective pressure upon this domain may be greater for N-cadherin than for other cadherins. Phylogenetic analysis also suggests that the gene duplications which established the main branches leading to the different cadherin subtypes occurred very early in their history. These duplications set the stage for the diversified superfamily we now observe.     

Structure of the neural (N-) cadherin prodomain reveals a cadherin extracellular domain-like fold without adhesive characteristics

Classical cadherins mediate cell-cell adhesion through calcium-dependent homophilic interactions and are activated through cleavage of a prosequence in the late Golgi. We present here the first three-dimensional structure of a classical cadherin prosequence, solved by NMR. The prototypic prosequence of N-cadherin consists of an Ig-like domain and an unstructured C-terminal region. The folded part of the prosequence-termed prodomain-has a striking structural resemblance to cadherin "adhesive" domains that could not have been predicted from the amino acid sequence due to low sequence similarities. Our detailed structural and evolutionary analysis revealed that prodomains are distant relatives of cadherin "adhesive" domains but lack all the features known to be important for cadherin-cadherin interactions. The presence of an additional "nonadhesive" domain seems to make it impossible to engage homophilic interactions between cadherins that are necessary to activate adhesion, thus explaining the inactive state of prodomain-bearing cadherins.     

Cadherins in neuronal morphogenesis and function

Classic cadherins represent a family of calcium-dependent homophilic cell–cell adhesion molecules. They confer strong adhesiveness to animal cells when they are anchored to the actin cytoskeleton via their cytoplasmic binding partners, catenins. The cadherin/catenin adhesion system plays key roles in the morphogenesis and function of the vertebrate and invertebrate nervous systems. In early vertebrate development, cadherins are involved in multiple events of brain morphogenesis including the formation and maintenance of the neuroepithelium, neurite extension and migration of neuronal cells. In the invertebrate nervous system, classic cadherin-mediated cell–cell interaction plays important roles in wiring among neurons. For synaptogenesis, the cadherin/catenin system not only stabilizes cell–cell contacts at excitatory synapses but also assembles synaptic molecules at synaptic sites. Furthermore, this system is involved in synaptic plasticity. Recent studies on the role of individual cadherin subtypes at synapses indicate that individual cadherin subtypes play their own unique role to regulate synaptic activities.     

Structure and Functions of Classical Cadherins

Cadherins are a family of membrane receptors that mediate calcium-dependent homophilic cell–cell adhesion. Cadherins play a key role in the regulation of organ and tissue development during embryogenesis. In adult organisms, these proteins are responsible for formation of stable cell–cell junctions and maintenance of normal tissue structure. Disruption in expression or function of cadherins may cause uncontrolled cell migration and proliferation during tumor development. This review focuses on the structure and physiological functions of classical cadherins.     

A complex of Protocadherin-19 and N-cadherin mediates a novel mechanism of cell adhesion

During embryonic morphogenesis, adhesion molecules are required for selective cell-cell interactions. The classical cadherins mediate homophilic calcium-dependent cell adhesion and are founding members of the large and diverse cadherin superfamily. The protocadherins are the largest subgroup within this superfamily, yet their participation in calcium-dependent cell adhesion is uncertain. In this paper, we demonstrate a novel mechanism of adhesion, mediated by a complex of Protocadherin-19 (Pcdh19) and N-cadherin (Ncad). Although Pcdh19 alone is only weakly adhesive, the Pcdh19-Ncad complex exhibited robust adhesion in bead aggregation assays, and Pcdh19 appeared to play the dominant role. Adhesion by the Pcdh19-Ncad complex was unaffected by mutations that disrupt Ncad homophilic binding but was inhibited by a mutation in Pcdh19. In addition, the complex exhibited homophilic specificity, as beads coated with Pcdh19-Ncad did not intermix with Ncad- or Pcdh17-Ncad-coated beads. We propose a model in which association of a protocadherin with Ncad acts as a switch, converting between distinct binding specificities.     

The Intracellular Domain of Cadherin-11 is not Required for the Induction of Cell Aggregation, Adhesion or Gap-Junction Formation

The cadherin family of cell adhesion molecules demonstrates calcium-dependent hemophilic binding, leading to cellular recognition and adhesion. The adhesion mediated by the classical type 1 cadherins is strengthened through catenin-mediated coupling of the cytoplasmic domain to the cytoskeleton. This cytoskeletal interaction may not be essential for the adhesion promoted by all cadherins, several of which lack cytosolic catenin-binding sequences. Cadherin-11, a classical cadherin, possesses a cytoplasmic domain that interacts with catenins, but may also occur as a variant form expressing a truncated cytoplasmic domain. To study the role of the cytoplasmic sequence in cadherin-11 mediated adhesion we have constructed and expressed a truncated cadherin-11 protein lacking the cytoplasmic domain and unable to bind β-catenin. Expression of the truncated cadherin-11 in MDA-MB-435S human mammary carcinoma cells reduced their motility and promoted calcium-dependent cell aggregation, frequent cell contacts, and functional gap-junctions. We conclude that the intracellular catenin-binding domain of cadherin-11, and by inference cytoskeletal interaction, is not required for the initiation and formation of cell adhesion.     

The Intracellular Domain of Cadherin-11 is not Required for the Induction of Cell Aggregation,Adhesion or Gap-Junction Formation

The cadherin family of cell adhesion molecules demonstrates calcium-dependent hemophilic binding, leading to cellular recognition and adhesion. The adhesion mediated by the classical type 1 cadherins is strengthened through catenin-mediated coupling of the cytoplasmic domain to the cytoskeleton. This cytoskeletal interaction may not be essential for the adhesion promoted by all cadherins, several of which lack cytosolic catenin-binding sequences. Cadherin-11, a classical cadherin, possesses a cytoplasmic domain that interacts with catenins, but may also occur as a variant form expressing a truncated cytoplasmic domain. To study the role of the cytoplasmic sequence in cadherin-11 mediated adhesion we have constructed and expressed a truncated cadherin-11 protein lacking the cytoplasmic domain and unable to bind β-catenin. Expression of the truncated cadherin-11 in MDA-MB-435S human mammary carcinoma cells reduced their motility and promoted calcium-dependent cell aggregation, frequent cell contacts, and functional gap-junctions. We conclude that the intracellular catenin-binding domain of cadherin-11, and by inference cytoskeletal interaction, is not required for the initiation and formation of cell adhesion.     

Cadherins: actin with the cytoskeleton to form synapses

Classic cadherins are calcium-dependent homophilic cell adhesion molecules that are enriched at synapses and thought to function in target recognition and adhesion at synaptic junctions. This brief review highlights evidence that cadherins and their associated catenins play a role in directing the development of pre- and postsynaptic specializations. In particular, the question of whether cadherin regulation of the actin cytoskeleton at discrete contact sites translates into the assembly of synaptic compartments will be explored.     

Structural and functional diversity of cadherin superfamily: Are new members of cadherin superfamily involved in signal transduction pathway?

A large number of cadherins and cadherin-related proteins are expressed in different tissues of a variety of multicellular organisms. These proteins share one property: their extracellular domains consist of multiple repeats of a cadherin-specific motif. A recent structure study has shown that the cadherin repeats roughly corresponding to the folding unit of the extracellular domains. The members of the cadherin superfamily are roughly classified into two groups, classical type cadherins proteins and protocadherin type according to their structural properties. These proteins appear to be derived from a common ancestor that might have cadherin repeats similar to those of the current protocadherins, and to have common functional properties. Among various cadherins, E-cadherin was the first to be identified as a Ca²⁺-dependent homophilic adhesion protein. Recent knockout mice experiments have proven its biological role, but there are still several puzzling unsolved properties of the cell adhesion activity. Other members of cadherin superfamily show divergent properties and many lack some of the expected properties of cell adhesion protein. Since recent studies of various adhesion proteins reveal that they are involved in different signal transduction pathways, the idea that the new members of cadherin superfamily may participate in more general cell-cell interaction processes including signal transduction is an intriguing hypothesis. The cadherin superfamily is structurally divergent and possibly functionally divergent as well. © 1996 Wiley-Liss, Inc.     

In the first extracellular domain of E-cadherin,heterophilic interactions,but not the conserved His-Ala-Val motif,are required for adhesion

The classical cadherins, definitive proteins of the cadherin superfamily, are characterized functionally by their ability to mediate calcium-dependent cell aggregation in vitro. To test hypothetical mechanisms of adhesion, we have constructed two mutants of the chicken E-cadherin protein, one with the highly conserved His-Ala-Val (HAV) sequence motif reversed to Val-Ala-His (VAH), the other lacking the first extracellular domain (EC1). The inversion of HAV to VAH has no effect on the capacity of E-cadherin to mediate adhesion. Deletion of EC1 completely eliminates the ability of E-cadherin to mediate homophilic adhesion, but the deletion mutant is capable of adhering heterophilically to both unmutated E-cadherin and to the HAV/VAH mutant. These results demonstrate that the conserved HAV sequence motif is not involved in cadherin-mediated adhesion as has been suggested previously and supports the idea that in the context of the cell surface, cadherin-mediated cell-cell adhesion involves an interaction of EC1 with other domains of the cadherin extracellular moiety and not the "linear zipper" model, which posits trans interactions only between EC1 on apposing cell surfaces.     

Cadherin-directed actin assembly: E-cadherin physically associates with the Arp2/3 complex to direct actin assembly in nascent adhesive contacts

Cadherin cell adhesion molecules are major determinants of tissue patterning which function in cooperation with the actin cytoskeleton. In the context of stable adhesion, cadherin/catenin complexes are often envisaged to passively scaffold onto cortical actin filaments. However, cadherins also form dynamic adhesive contacts during wound healing and morphogenesis. Here actin polymerization has been proposed to drive cell surfaces together, although F-actin reorganization also occurs as cell contacts mature. The interaction between cadherins and actin is therefore likely to depend on the functional state of adhesion. We sought to analyze the relationship between cadherin homophilic binding and cytoskeletal activity during early cadherin adhesive contacts. Dissecting the specific effect of cadherin ligation alone on actin regulation is difficult in native cell-cell contacts, due to the range of juxtacrine signals that can arise when two cell surfaces adhere. We therefore activated homophilic ligation using a specific functional recombinant protein. We report the first evidence that E-cadherin associates with the Arp2/3 complex actin nucleator and demonstrate that cadherin binding can exert an active, instructive influence on cells to mark sites for actin assembly at the cell surface.     

Expression of T-cadherin in the rat carotid artery wall after balloon injury and in different rat organs

T-cadherin is an unusual glycosilphosphatidylinositol (GPI)-anchored member of the cadherin family of cell adhesion proteins. In contrast to classical cadherins, tissue distribution of T-cadherin so far remained unknown. We examined tissue distribution of T-cadherin in rats using Western blotting and immunohistochemical method. Our results show that T-cadherin is expressed in all types of muscles (cardiac, striated, and smooth muscles), in brain neurons, and spinal cord, in the vessel endothelium, at the apical pole of intestinal villar epithelium, in the basal layer of skin, and eosophagal epithelium. Blood-derived and lymphoid cells as well as connective tissue were T-cadherin-negative. The highest level of T-cadherin expression was revealed in the cardiovascular system. Although T-cadherin was detected in smooth muscle cells, its role in the intimal thickening and restenosis is not known. We examined T-cadherin expression within 1-28 days after balloon injury of rat left carotid arteries. T-cadherin expression was valued immunohistochemically with semiquantitative method. In uninjured arteries, T-cadherin was expressed in endothelial (vWF-positive) cells, and smooth muscle (alpha-actin-positive) cells (SMCs). After denudation of arterial wall, T-cadherin was present both in the media and neointima. We revealed dynamics of T-cadherin expression in the media of injured artery: an essential increase being registered at the stage of cell migration and proliferation in the media and neointima (1-7 days), followed by its decrease to the baseline level (10-28 days). The high upregulation of T-cadherin expression in the media and neointima during migration and proliferation of vascular cells after vessel injury enables us to suggest the involvement of T-cadherin in vessel remodeling after balloon catheter injury.     

Antiadhesive molecule T-cadherin is an atypical low-density lipoprotein receptor in vascular cells

Elevated serum LDL level, which results in cholesterol accumulation in vascular wall, is widely accepted as a risk factor in atherosclerosis development. Additionally to metabolic effects, LDL can produce hormone-like effects in a number of cells: activate second messenger systems, regulate gene expression and activate platelets and stimulate cell proliferation. The responses elicited by LDL are rapid, dose-dependent and capable of being saturated, indicating the involvement of specific receptor/binding sites in LDI-stimulated signal transduction. This LDL-binding protein was isolated from human aorta media and identified as T-cadherin. Cadherins are a superfamily of adhesion molecules that mediate Ca2+ -dependent cell-cell adhesion in embryogenesis and in adult organism's solid tissues. Intercellular junctions are formed as a result of interactions between extracellular domains of the neighboring cells' cadherins. Binding of the intercellular domain to the acting cytoskeleton ensures stability of cadherin-mediated adhesive junctions. T-cadherin is a unique member of calcium-dependent adherent proteins; in contrast to classical cadherins T-cadherin is anchored to the cell surface membranes via a glycosyl phosphatidyl inositol (GPI) moiety. Subcellular distribution of T-cadherin is restricted to lipid rafts on the cell membranes where it co-localizes with signal-transducing molecules. The function of T-cadherin has not yet been revealed. It was originally cloned from chicken embryo brain where the spatial-temporally restricted pattern of T-cadherin suggests its role as a negative guidance cue in tegulating the segmental organization of trunk neural crest migration and motor axon projections. Comparative study of the T-cadherin expression in human organs and tissues revealed that T-cadherin content was maximal in cardiovascular system. Its expression in VSMC depends on the cell phenotype and proliferate activity and increases in atherosclerotic lesion and restenosis. T-cadherin seems to play a key role in the regulation of the vascular cell phenotype, migration and growth. We hypothesize that T-cadherin is an anti-adhesive molecule which participates in intercellular interactions informing cells about their environment and regulating migration and proliferation of cells in vascular wall, while LDL interfere with the normal function of T-cadherin.     

Disruption of epithelial cell-cell adhesion by exogenous expression of a mutated nonfunctional N-cadherin.

Cadherins, a family of transmembrane cell-cell adhesion receptors, require interactions with the cytoskeleton for normal function. To assess the mechanisms of these interactions, we studied the effect of exogenous expression of a mutant N-cadherin, cN390 delta; on epithelial cell-cell adhesion. The intracellular domain of cN390 delta was intact but its extracellular domain was largely deleted so that this molecule was not functional for cell adhesion. cDNA of cN390 delta was attached to the metallothionein promoter, and introduced into the keratinocyte line PAM212 expressing endogenous E- and P-cadherin. When the expression of cN390 delta was induced by Zn2+, cadherin-dependent adhesion of the transfected cells was inhibited, resulting in the dispersion of cell colonies, although their contacts were maintained under high cell density conditions. In these cultures, cN390 delta was expressed not only on the free surfaces of the cells but also at cell-cell junctions. The endogenous cadherins were concentrated at cell-cell junctions under normal conditions. As a result of cN390 delta expression, however, the endogenous cadherins localizing at the cell-cell junctions were largely diminished, suggesting that these molecules were replaced by the mutant molecules at these sites. As a control, we transfected the same cell line with cDNA of a truncated form of N-cadherin cadherin whose intracellular C terminus had been deleted leaving the extracellular domain intact. This molecule had no effect on cell-cell adhesion, nor did it localize to cell-cell contact sites. We also found that the association of the endogenous cadherins with alpha- and beta-catenins and plakoglobin was not affected by the expression of cN390 delta, which also formed a complex with these molecules, suggesting that no competition occurred between the endogenous and exogenous cadherins for these cytoplasmic proteins. These and other additional results suggest that the nonfunctional cadherins whose intracellular domain is intact occupy the sites where the endogenous cadherins should localize, through interactions with the cytoskeleton, and inhibit the cadherin adhesion system.