The juxtamembrane region of the cadherin cytoplasmic tail supports lateral clustering, adhesive strengthening, and interaction with p120ctn

Cadherin cell-cell adhesion molecules form membrane-spanning molecular complexes that couple homophilic binding by the cadherin ectodomain to the actin cytoskeleton. A fundamental issue in cadherin biology is how this complex converts the weak intrinsic binding activity of the ectodomain into strong adhesion. Recently we demonstrated that cellular cadherins cluster in a ligand-dependent fashion when cells attached to substrata coated with the adhesive ectodomain of Xenopus C-cadherin (CEC1-5). Moreover, forced clustering of the ectodomain alone significantly strengthened adhesiveness (Yap, A.S., W.M. Brieher, M. Pruschy, and B.M. Gumbiner. Curr. Biol. 7:308-315). In this study we sought to identify the determinants of the cadherin cytoplasmic tail responsible for clustering activity. A deletion mutant of C-cadherin (CT669) that retained the juxtamembrane 94-amino acid region of the cytoplasmic tail, but not the beta-catenin-binding domain, clustered upon attachment to substrata coated with CEC1-5. Like wild-type C-cadherin, this clustering was ligand dependent. In contrast, mutant molecules lacking either the complete cytoplasmic tail or just the juxtamembrane region did not cluster. The juxtamembrane region was itself sufficient to induce clustering when fused to a heterologous membrane-anchored protein, albeit in a ligand-independent fashion. The CT669 cadherin mutant also displayed significant adhesive activity when tested in laminar flow detachment assays and aggregation assays. Purification of proteins binding to the juxtamembrane region revealed that the major associated protein is p120(ctn). These findings identify the juxtamembrane region of the cadherin cytoplasmic tail as a functionally active region supporting cadherin clustering and adhesive strength and raise the possibility that p120(ctn) is involved in clustering and cell adhesion.     

Contribution of cadherins to directional cell migration and histogenesis in Xenopus embryos

Perturbation of adhesion mediated by cadherins was achieved by over-expressing truncated forms of E- and EP-cadherins (in which the extracellular domain was deleted) in different blastomeres of stage 6 Xenopus laevis embryos. Injections of mRNA encoding truncated E- and EP-cadherins into A1A2 blastomeres resulted in inhibition of cell adhesion and, at later stages, in morphogenetic defects in the anterior neural tissues to which they mainly contribute. In addition, truncated EP-cadherin mRNA produced a duplication of the dorso-posterior axis in a significant number of cases. The expression of truncated E- and EP-cadherins in blastomeres involved in gastrulation and neural induction (B1B2 and C1), led to the duplication of the dorso-posterior axis as well as to defects in anterior structures. Morphogenetic defects obtained with truncated EP-cadherin were more severe than those induced with truncated E-cadherin. Cells derived from blastomeres injected with truncated EP-cadherin mRNA, dispersed more readily at the blastula and gastrula stages than the cells derived from the blastomeres expressing truncated E-cadherin. Presumptive mesodermal cells expressing truncated cadherins did not engage in coherent directional migration. The alteration of cadherin-mediated cell adhesion led directly to the perturbation of the convergent-extension movements during gastrulation as shown in the animal cap assays and indirectly to perturbation of neural induction. Although the cytoplasmic domains of type I cadherins share a high degree of sequence identity, the over-expression of their cytoplasmic domains induces a distinct pattern of perturbations, strongly suggesting that in vivo, each cadherin may transduce a specific adhesive signal. These graded perturbations may in part result from the relative ability of each cadherin cytoplasmic domain to titer the beta-catenin.     

Apoptosis of endothelial cells is associated with paracrine induction of adhesion molecules: evidence for an interleukin-1beta-dependent paracrine loop

Cadherin-mediated adhesion depends on the association of its cytoplasmic domain with the actin-containing cytoskeleton. This interaction is mediated by a group of cytoplasmic proteins: alpha-and beta- or gamma- catenin. Phosphorylation of beta-catenin on tyrosine residues plays a role in controlling this association and, therefore, cadherin function. Previous work from our laboratory suggested that a nonreceptor protein tyrosine phosphatase, bound to the cytoplasmic domain of N-cadherin, is responsible for removing tyrosine-bound phosphate residues from beta-catenin, thus maintaining the cadherin-actin connection (). Here we report the molecular cloning of the cadherin-associated tyrosine phosphatase and identify it as PTP1B. To definitively establish a causal relationship between the function of cadherin-bound PTP1B and cadherin-mediated adhesion, we tested the effect of expressing a catalytically inactive form of PTP1B in L cells constitutively expressing N-cadherin. We find that expression of the catalytically inactive PTP1B results in reduced cadherin-mediated adhesion. Furthermore, cadherin is uncoupled from its association with actin, and beta-catenin shows increased phosphorylation on tyrosine residues when compared with parental cells or cells transfected with the wild-type PTP1B. Both the transfected wild-type and the mutant PTP1B are found associated with N-cadherin, and recombinant mutant PTP1B binds to N-cadherin in vitro, indicating that the catalytically inactive form acts as a dominant negative, displacing endogenous PTP1B, and rendering cadherin nonfunctional. Our results demonstrate a role for PTP1B in regulating cadherin-mediated cell adhesion.     

Structural basis of calcium-induced E-cadherin rigidification and dimerization

The cadherins mediate cell adhesion and play a fundamental role in normal development. They participate in the maintenance of proper cell-cell contacts: for example, reduced levels of epithelial cadherin (E-cadherin) correlate with increased invasiveness in many human tumour cell types. The cadherins typically consist of five tandemly repeated extracellular domains, a single membrane-spanning segment and a cytoplasmic region. The N-terminal extracellular domains mediate cell-cell contact while the cytoplasmic region interacts with the cytoskeleton through the catenins. Cadherins depend on calcium for their function: removal of calcium abolishes adhesive activity, renders cadherins vulnerable to proteases (reviewed in ref. 4) and, in E-cadherin, induces a dramatic reversible conformational change in the entire extracellular region. We report here the X-ray crystal structure at 2.0 A resolution of the two N-terminal extracellular domains of E-cadherin in the presence of calcium. The structure reveals a two-fold symmetric dimer, each molecule of which binds a contiguous array of three bridged calcium ions. Not only do the bound calcium ions linearize and rigidify the molecule, they promote dimerization. Although the N-terminal domain of each molecule in the dimer is aligned in a parallel orientation, the interactions between them differ significantly from those found in the neural cadherin (N-cadherin) N-terminal domain (NCD1) structure. The E-cadherin dual-domain structure reported here defines the role played by calcium in the cadherin-mediated formation and maintenance of solid tissues.     

Epithelial cell adhesion molecule (Ep-CAM) modulates cell-cell interactions mediated by classic cadherins

The contribution of noncadherin-type, Ca2+-independent cell-cell adhesion molecules to the organization of epithelial tissues is, as yet, unclear. A homophilic, epithelial Ca2+-independent adhesion molecule (Ep-CAM) is expressed in most epithelia, benign or malignant proliferative lesions, or during embryogenesis. Here we demonstrate that ectopic Ep-CAM, when expressed in cells interconnected by classic cadherins (E- or N-cadherin), induces segregation of the transfectants from the parental cell type in coaggregation assays and in cultured mixed aggregates, respectively. In the latter assay, Ep-CAM-positive transfectants behave like cells with a decreased strength of cell-cell adhesion as compared to the parental cells. Using transfectants with an inducible Ep-CAM-cDNA construct, we demonstrate that increasing expression of Ep-CAM in cadherin-positive cells leads to the gradual abrogation of adherens junctions. Overexpression of Ep-CAM has no influence on the total amount of cellular cadherin, but affects the interaction of cadherins with the cytoskeleton since a substantial decrease in the detergent-insoluble fraction of cadherin molecules was observed. Similarly, the detergent-insoluble fractions of alpha- and beta-catenins decreased in cells overexpressing Ep-CAM. While the total beta-catenin content remains unchanged, a reduction in total cellular alpha-catenin is observed as Ep-CAM expression increases. As the cadherin-mediated cell-cell adhesions diminish, Ep-CAM-mediated intercellular connections become predominant. An adhesion-defective mutant of Ep-CAM lacking the cytoplasmic domain has no effect on the cadherin-mediated cell-cell adhesions. The ability of Ep-CAM to modulate the cadherin-mediated cell-cell interactions, as demonstrated in the present study, suggests a role for this molecule in development of the proliferative, and probably malignant, phenotype of epithelial cells, since an increase of Ep-CAM expression was observed in vivo in association with hyperplastic and malignant proliferation of epithelial cells.     

Elevated neointimal endothelin-1 in transplantation-associated arteriosclerosis of renal allograft recipients

The cell adhesion molecule L1 plays an important role in neural development, and mutations in human L1 have been implicated in X-linked hydrocephalus and related neurological diseases. We have previously demonstrated that recombinant proteins containing the second immunoglobulin-like domain (Ig2) of L1 contain both homophilic binding and neuritogenic activities. In this report, the involvement of L1 Ig2 in cell-cell adhesion and neuritogenesis was further evaluated in cell transfection studies. Transfectants expressing intact L1 were capable of undergoing L1-dependent self-aggregation and promoting neurite outgrowth from neural retinal cells. However, both activities were abolished in transfectants expressing L1delta2, a mutant L1 with Ig2 deleted. In competition experiments, the wild-type Ig2 fusion protein inhibited L1-dependent cell aggregation, whereas an Ig2 fusion protein containing the hydrocephalus mutation R184Q did not. Oligopeptides flanking Arg184 were therefore synthesized and assayed for their effects on L1-mediated cell-cell binding and neuritogenesis. The peptide L1-A, spanning the residues His178 and Gly191, inhibited both L1- and Ig2 fusion protein-mediated homophilic binding. When neural retinal cells were cultured on substrate-coated Ig2 fusion protein, peptide L1-A also abolished L1-dependent neurite outgrowth. Substitutions of several charged residues and hydrophobic residues with alanine in peptide analogues led to the loss of inhibitory effects, suggesting that multiple amino acids might be involved in L1-L1 binding. Taken together, these results identify an L1 homophilic binding site within the sequence HIKQDERVTMGQNG of Ig2 and demonstrate the requirement of L1 homophilic binding in the promotion of neurite outgrowth.     

Interactions of the cytoplasmic domain of P-selectin with clathrin-coated pits enhance leukocyte adhesion under flow

Flowing leukocytes tether to and roll on P-selectin, a receptor on endothelial cells that is rapidly internalized in clathrin-coated pits. We asked whether the association of P-selectin with clathrin-coated pits contributes to its adhesive function. Under flow, rolling neutrophils accumulated efficiently on CHO cells expressing wild-type P-selectin or a P-selectin construct with a substitution in the cytoplasmic domain that caused even faster internalization than that of the wild-type protein. By contrast, far fewer rolling neutrophils accumulated on CHO cells expressing P-selectin constructs with a deletion or a substitution in the cytoplasmic domain that impaired internalization. Neutrophils rolled on the internalization-competent constructs with greater adhesive strength, slower velocity, and more uniform motion. Flowing neutrophils tethered equivalently to internalization-competent or internalization-defective P-selectin, but after tethering, they rolled further on internalization-competent P-selectin. Confocal microscopy demonstrated colocalization of alpha-adaptin, a component of clathrin-coated pits, with wild-type P-selectin, but not with P-selectin lacking the cytoplasmic domain. Treatment of CHO cells or endothelial cells with hypertonic medium reversibly impaired the clathrin-mediated internalization of P-selectin and its ability to support neutrophil rolling. Interactions of the cytoplasmic domain of P-selectin with clathrin-coated pits provide a novel mechanism to enhance leukocyte adhesion under flow.     

Differential localization of VE- and N-cadherins in human endothelial cells: VE-cadherin competes with N-cadherin for junctional localization

The two major cadherins of endothelial cells are neural (N)-cadherin and vascular endothelial (VE)- cadherin. Despite similar level of protein expression only VE-cadherin is located at cell-cell contacts, whereas N-cadherin is distributed over the whole cell membrane. Cotransfection of VE-cadherin and N-cadherin in CHO cells resulted in the same distribution as that observed in endothelial cells indicating that the behavior of the two cadherins was not cell specific but related to their structural characteristics. Similar amounts of alpha- and beta-catenins and plakoglobin were associated to VE- and N-cadherins, whereas p120 was higher in the VE-cadherin complex. The presence of VE-cadherin did not affect N-cadherin homotypic adhesive properties or its capacity to localize at junctions when cotransfectants were cocultured with cells transfected with N-cadherin only. To define the molecular domain responsible for the VE-cadherin-dominant activity we prepared a chimeric construct formed by VE-cadherin extracellular region linked to N-cadherin intracellular domain. The chimera lost the capacity to exclude N-cadherin from junctions indicating that the extracellular domain of VE-cadherin alone is not sufficient for the preferential localization of the molecule at the junctions. A truncated mutant of VE-cadherin retaining the full extracellular domain and a short cytoplasmic tail (Arg621-Pro702) lacking the catenin-binding region was able to exclude N-cadherin from junctions. This indicates that the Arg621-Pro702 sequence in the VE-cadherin cytoplasmic tail is required for N-cadherin exclusion from junctions. Competition between cadherins for their clustering at intercellular junctions in the same cell has never been described before. We speculate that, in the endothelium, VE- and N-cadherin play different roles; whereas VE-cadherin mostly promotes the homotypic interaction between endothelial cells, N-cadherin may be responsible for the anchorage of the endothelium to other surrounding cell types expressing N-cadherin such as vascular smooth muscle cells or pericytes.     

The role of LFA-1 (CD11a/CD18) cytoplasmic domains in binding to intercellular adhesion molecule-1 (CD54) and in postreceptor cell spreading

We have investigated the role of the cytoplasmic domains of LFA-1 in binding to ICAM-1 and in postadhesion events. Various truncated and chimeric forms of LFA-1 alpha (CD11a) and beta (CD18) chains were generated and transfected into murine fibroblast TNR-2 cells. Transfected fibroblasts expressing wild-type LFA-1 adhered only weakly to ICAM-1 immobilized on plastic, and phorbol ester pretreatment enhanced this adhesion significantly. In contrast, transfected cells expressing LFA-1 lacking both the alpha and the beta cytoplasmic domains, the beta cytoplasmic domain alone, or GPI-anchored LFA-1 adhered to immobilized ICAM-1 without prior activation. Truncation of the alpha cytoplasmic domain alone resulted in much reduced cell adhesion which could be only weakly upregulated by PMA. The presence of manganese dramatically enhanced the binding to ICAM-1 of LFA-1 lacking the alpha cytoplasmic domain or both cytoplasmic domains, whereas it had relatively little effect on wild-type LFA-1 or the mutant lacking the beta cytoplasmic domain. Soluble LFA-1, generated by phosphatidylinositol-specific phospholipase-C treatment of GPI-anchored LFA-1, was capable of binding ICAM-1+ cells. Although doubly truncated or GPI-anchored LFA-1 mediated cell adhesion to immobilized ICAM-1, cells expressing these mutants, as well as those expressing individual alpha and beta chain truncations, failed to spread out following this adhesion, whereas the wild-type transfectants did so readily. Manganese had no effect on cell spreading. Fluorescent staining of these cells indicated no significant variation in the distribution of LFA-1 on the cell surface. From these results we conclude that (1) cells expressing LFA-1 lacking both the alpha and the beta cytoplasmic domains adhere to ICAM-1 without prior stimulation, indicating the importance of LFA-1 cytoplasmic domains in inside-out signaling, (2) truncation of the alpha cytoplasmic domain alone inhibits cell adhesion by making LFA-1 nonresponsive to inside-out signaling, and (3) both cytoplasmic domains are required for cell spreading following adhesion to immobilized ICAM-1.     

The protein tyrosine kinase p56lck regulates cell adhesion mediated by CD4 and major histocompatibility complex class II proteins

The CD4 protein is expressed on a subset of human T lymphocytes that recognize antigen in the context of major histocompatibility complex (MHC) class II molecules. Using Chinese hamster ovary (CHO) cells expressing human CD4, we have previously demonstrated that the CD4 protein can mediate cell adhesion by direct interaction with MHC class II molecules. In T lymphocytes, CD4 can also function as a signaling molecule, presumably through its intracellular association with p56lck, a member of the src family of protein tyrosine kinases. In the present report, we show that p56lck can affect cell adhesion mediated by CD4 and MHC class II molecules. The expression of wild-type p56lck in CHO-CD4 cells augments the binding of MHC class II+ B cells, whereas the expression of a mutant p56lck protein with elevated tyrosine kinase activity results in decreased binding of MHC class II+ B cells. Using site-specific mutants of p56lck, we demonstrate that the both the enzymatic activity of p56lck and its association with CD4 are required for this effect on CD4/MHC class II adhesion. Further, the binding of MHC class II+ B cells induces CD4 at the cell surface to become organized into structures resembling adhesions-type junctions. Both wild-type and mutant forms of p56lck influence CD4-mediated adhesion by regulating the formation of these structures. The wild-type lck protein enhances CD4/MHC class II adhesion by augmenting the formation of CD4-associated adherens junctions whereas the elevated tyrosine kinase activity of the mutant p56lck decreases CD4-mediated cell adhesion by preventing the formation of these structures.     

Microbial glycoconjugates

Cadherin-mediated adhesion regulates transitions from initial cell-cell recognition to loosely adherent cell clusters and ultimately, to strongly compacted groups of cells in colonies. Recent studies have described distinct roles for intermolecular clustering of cadherins as well as interactions of cadherin with the actin cytoskeleton in establishing cell-cell adhesion. Integrating cytomechanical roles of cadherin-mediated adhesion will lead to a greater understanding of how cadherins regulate tissue morphogenesis.     

On the role of myosin-II in cytokinesis: division of Dictyostelium cells under adhesive and nonadhesive conditions

We have investigated the role of myosin in cytokinesis in Dictyostelium cells by examining cells under both adhesive and nonadhesive conditions. On an adhesive surface, both wild-type and myosin-null cells undergo the normal processes of mitotic rounding, cell elongation, polar ruffling, furrow ingression, and separation of daughter cells. When cells are denied adhesion through culturing in suspension or on a hydrophobic surface, wild-type cells undergo these same processes. However, cells lacking myosin round up and polar ruffle, but fail to elongate, furrow, or divide. These differences show that cell division can be driven by two mechanisms that we term Cytokinesis A, which requires myosin, and Cytokinesis B, which is cell adhesion dependent. We have used these approaches to examine cells expressing a myosin whose two light chain-binding sites were deleted (DeltaBLCBS-myosin). Although this myosin is a slower motor than wild-type myosin and has constitutively high activity due to the abolition of regulation by light-chain phosphorylation, cells expressing DeltaBLCBS-myosin were previously shown to divide in suspension (Uyeda et al., 1996). However, we suspected their behavior during cytokinesis to be different from wild-type cells given the large alteration in their myosin. Surprisingly, DeltaBLCBS-myosin undergoes relatively normal spatial and temporal changes in localization during mitosis. Furthermore, the rate of furrow progression in cells expressing a DeltaBLCBS-myosin is similar to that in wild-type cells.     

The membrane-proximal region of the E-cadherin cytoplasmic domain prevents dimerization and negatively regulates adhesion activity

Cadherins are transmembrane glycoproteins involved in Ca2+-dependent cell-cell adhesion. Deletion of the COOH-terminal residues of the E-cadherin cytoplasmic domain has been shown to abolish its cell adhesive activity, which has been ascribed to the failure of the deletion mutants to associate with catenins. Based on our present results, this concept needs revision. As was reported previously, leukemia cells (K562) expressing E-cadherin with COOH-terminal deletion of 37 or 71 amino acid residues showed almost no aggregation. Cells expressing E-cadherin with further deletion of 144 or 151 amino acid residues, which eliminates the membrane-proximal region of the cytoplasmic domain, showed E-cadherin-dependent aggregation. Thus, deletion of the membrane-proximal region results in activation of the nonfunctional E-cadherin polypeptides. However, these cells did not show compaction. Chemical cross-linking revealed that the activated E-cadherin polypeptides can be cross-linked to a dimer on the surface of cells, whereas the inactive polypeptides, as well as the wild-type E-cadherin polypeptide containing the membrane-proximal region, can not. Therefore, the membrane-proximal region participates in regulation of the adhesive activity by preventing lateral dimerization of the extracellular domain.     

Catenin-dependent and -independent functions of vascular endothelial cadherin

Vascular endothelial cadherin (VE-cadherin, cadherin-5, or 7B4) is an endothelial specific cadherin that regulates cell to cell junction organization in this cell type. Cadherin linkage to intracellular catenins was found to be required for their adhesive properties and for localization at cell to cell junctions. We constructed a mutant form of VE-cadherin lacking the last 82 amino acids of the cytoplasmic domain. Surprisingly, despite any detectable association of this truncated VE-cadherin to catenin-cytoskeletal complex, the molecule was able to cluster at cell-cell contacts in a manner similar to wild type VE-cadherin. Truncated VE-cadherin was also able to promote calcium-dependent cell to cell aggregation and to partially inhibit cell detachment and migration from a confluent monolayer. In contrast, intercellular junction permeability to high molecular weight molecules was severely impaired by truncation of VE-cadherin cytoplasmic domain. These results suggest that the VE-cadherin extracellular domain is enough for early steps of cell adhesion and recognition. However, interaction of VE-cadherin with the cytoskeleton is necessary to provide strength and cohesion to the junction. The data also suggest that cadherin functional regulation might not be identical among the members of the family.     

The human REC2/RAD51B gene acts as a DNA damage sensor by inducing G1 delay and hypersensitivity to ultraviolet irradiation

HsRec2/Rad51B is a 350-amino acid protein with a molecular mass of 38,300 Da that appears to be involved in cell cycle regulation and UV-induced apoptosis. The mouse and human genes were isolated based on their homology to a recombinational repair gene from Ustilago maydis and contain functional domains to hRAD51 and hLIM 15 (M. C. Rice et al., Proc. Natl. Acad. Sci. USA, 94: 7417-7422, 1997). Here, we report the results of studies on the behavior of CHO cells containing a plasmid encoding a wild-type hsRec2/Rad51B, a full-length protein with a single mutation at residue 163, which lies in the putative src site, and a truncated version of hsRec2/Rad51B, containing only the first 100 amino acids at the NH2 terminus. Using fluorescence-activated cell sorting analysis to follow the progression of cells through the cell cycle, we find that stable transfectants constitutively overexpressing the wild-type human Rec2/Rad51B protein exhibit a G1 delay. In addition, when irradiated with UV at a dose of 15 J/m2, CHO cells transfected with the various hREC2/RAD51B vectors exhibited different responses. Cells expressing the wild-type human Rec2/Rad51B underwent apoptosis, with the greatest cell death occurring 24 h after irradiation. The control cells, which contained an empty vector, and the cells expressing truncated hsRec2/Rad51B or the full-length Rec2 with a mutation at residue 163 did not. In summary, these findings of cell cycle slowing and UV-induced apoptosis in CHO cells constitutively expressing the human Rec2/Rad51B protein suggest that hsRec2/Rad51B plays a role in a DNA damage surveillance pathway.     

N-cadherin redistribution during synaptogenesis in hippocampal neurons

Cadherins are homophilic adhesion molecules that, together with their intracellular binding partners the catenins, mediate adhesion and signaling at a variety of intercellular junctions. This study shows that neural (N)-cadherin and beta-catenin, an intracellular binding partner for the classic cadherins, are present in axons and dendrites before synapse formation and then cluster at developing synapses between hippocampal neurons. N-cadherin is expressed initially at all synaptic sites but rapidly becomes restricted to a subpopulation of excitatory synaptic sites. Sites of GABAergic, inhibitory synapses in mature cultures therefore lack N-cadherin but are associated with clusters of beta-catenin, implying that they contain a different classic cadherin. These findings indicate that N-cadherin adhesion may stabilize early synapses that can then be remodeled to express a different cadherin and that cadherins systematically differentiate between functionally (excitatory and inhibitory) and spatially distinct synaptic sites on single neurons. These results suggest that differential cadherin expression may orchestrate the point-to-point specificity displayed by developing synapses.     

Neurite fasciculation mediated by complexes of axonin-1 and Ng cell adhesion molecule

Neural cell adhesion molecules composed of immunoglobulin and fibronectin type III-like domains have been implicated in cell adhesion, neurite outgrowth, and fasciculation. Axonin-1 and Ng cell adhesion molecule (NgCAM), two molecules with predominantly axonal expression exhibit homophilic interactions across the extracellular space (axonin- 1/axonin-1 and NgCAM/NgCAM) and a heterophilic interaction (axonin-1-NgCAM) that occurs exclusively in the plane of the same membrane (cis-interaction). Using domain deletion mutants we localized the NgCAM homophilic binding in the Ig domains 1-4 whereas heterophilic binding to axonin-1 was localized in the Ig domains 2-4 and the third FnIII domain. The NgCAM-NgCAM interaction could be established simultaneously with the axonin-1-NgCAM interaction. In contrast, the axonin-1-NgCAM interaction excluded axonin-1/axonin-1 binding. These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-12/NgCAM2 tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM. The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-12/NgCAM2 complexes and, thus, neurite fasciculation in DRG neurons.     

Molecular cloning and characterization of a novel human classic cadherin homologous with mouse muscle cadherin

We used a novel cDNA cloning method based on the cadherin-beta-catenin protein interaction and identified a new human classic-type cadherin, which we named cadherin-15, from adult brain and skeletal muscle cDNA libraries. Sequence analysis revealed that this cadherin was closely related to mouse muscle cadherin and seemed to be its human counterpart. However, its deduced amino acid sequence differed from that of mouse muscle cadherin in that it had an extra 31-amino acid sequence at its C terminus that has been found neither in mouse muscle cadherin nor in any other known classic cadherin. Analysis of cadherin-15 protein expressed in L fibroblasts showed that it was cleaved proteolytically, expressed on the cell surfaces as a mature form of about 124-kDa, and functioned as a cell-cell adhesion molecule in a homophilic and specific manner, but Ca2+ did not protect it against degradation by trypsin. Our findings also suggest that cadherin-15 mediates cell-cell adhesion with a binding strength comparable to that of E-cadherin.     

Enhancing clinical efficacy of oral rehydration therapy: is low osmolality the key?

The cadherins are a family of homophilic adhesion molecules that play a vital role in the formation of cellular junctions and in tissue morphogenesis. Members of the integrin family are also involved in cell to cell adhesion, but bind heterophilically to immunoglobulin superfamily molecules such as intracellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, or mucosal addressin cell adhesion molecule (MadCAM)-1. Recently, an interaction between epithelial (E-) cadherin and the mucosal lymphocyte integrin, alphaEbeta7, has been proposed. Here, we demonstrate that a human E-cadherin-Fc fusion protein binds directly to soluble recombinant alphaEbeta7, and to alphaEbeta7 solubilized from intraepithelial T lymphocytes. Furthermore, intraepithelial lymphocytes or transfected JY' cells expressing the alphaEbeta7 integrin adhere strongly to purified E-cadherin-Fc coated on plastic, and the adhesion can be inhibited by antibodies to alphaEbeta7 or E-cadherin. The binding of alphaEbeta7 integrin to cadherins is selective since cell adhesion to P-cadherin-Fc through alphaEbeta7 requires >100-fold more fusion protein than to E-cadherin-Fc. Although the structure of the alphaE-chain is unique among integrins, the avidity of alphaEbeta7 for E-cadherin can be regulated by divalent cations or phorbol myristate acetate. Cross-linking of the T cell receptor complex on intraepithelial lymphocytes increases the avidity of alphaEbeta7 for E-cadherin, and may provide a mechanism for the adherence and activation of lymphocytes within the epithelium in the presence of specific foreign antigen. Thus, despite its dissimilarity to known integrin ligands, the specific molecular interaction demonstrated here indicates that E-cadherin is a direct counter receptor for the alphaEbeta7 integrin.     

HIV service provision and training needs in outpatient mental health settings

The erythropoietin receptor (EpoR) has been previously shown to contain a cytoplasmic C-terminal negative regulatory domain, experimental deletion or mutation of which leads to increased sensitivity of expressing cells to the effects erythropoietin (Epo). We have studied a naturally occurring C-terminal truncation mutant of the human EpoR by stably transfecting the growth factor-dependent hematopoietic tissue culture cell line 32D with expression plasmids containing either the wildtype or mutant human EpoR cDNA, thus rendering the cells dependent on Epo for viability and proliferation. In Epo dose-response assays, cells expressing the mutant EpoR displayed hyperresponsiveness to Epo compared with cells expressing comparable numbers of the wild-type EpoR cultured in the presence of fetal bovine serum. We investigated whether enhanced Epo sensitivity of cells expressing the truncated EpoR is associated with alteration in Epo receptor-mediated activation of Stat5, which could have a role in Epo-induced proliferation. Although maximal Stat5 activation in response to a given concentration of Epo was comparable in 32D cells expressing the wild-type or truncated EpoRs, the time course of Epo-induced Stat5 activation was very different. Gel-mobility shift studies revealed the presence of Stat5 DNA-binding activity in nuclear and cytoplasmic extracts of cells expressing the truncated EpoR for a significantly longer time than that observed in similar extracts of cells expressing the wild-type EpoR consistent with decreased rate of inactivation of Stat5 in cells expressing the mutant EpoR. Epo-dependent tyrosine phosphorylation of both Stat5 and Jak2 was also substantially prolonged in cells expressing the truncated EpoR. These results suggest a role for Stat5 in regulation of Epo-mediated cell growth and implicate altered kinetics of Epo-induced Jak2 and Stat5 activation in the pathogenesis of familial erythrocytosis associated with this naturally occurring EpoR gene mutation.