The cytoplasmic domain of the myelin P0 protein influences the adhesive interactions of its extracellular domain

The extracellular domain of the myelin P0 protein is believed to engage in adhesive interactions and thus hold the myelin membrane compact. We have previously shown that P0 can behave as a homophilic adhesion molecule through interactions of its extracellular domains (Filbin, M. T., F. S. Walsh, B. D. Trapp, J. A. Pizzey, and G. I. Tennekoon. 1990. Nature (Lond.) 344:871-872). To determine if the cytoplasmic domain of P0 must be intact for the extracellular domains to adhere, we compared the adhesive capabilities of P0 proteins truncated at the COOH-terminal to the full-length P0 protein. P0 cDNAs lacking nucleotides coding for the last 52 or 59 amino acids were transfected into CHO cells, and surface expression of the truncated proteins was assessed by immunofluorescence, surface labeling followed by immunoprecipitation, and an ELISA. Cell lines were chosen that expressed at least equivalent amounts of the truncated P0 proteins at the surface as did a cell line expressing the full-length P0. The adhesive properties of these three cell lines were compared. It was found that when a suspension of single cells was allowed to aggregate for a period of 60 min, only the cells expressing the full-length P0 had formed large aggregates, while the cells expressing the truncated P0 molecules were still mostly single cells indistinguishable from the control cells. Furthermore, 25-30% of the full-length P0 was insoluble in NP40, indicative of an interaction with the cytoskeleton, whereas only 5-10% of P0 lacking 52 amino acids and none of P0 lacking 59 amino acids were insoluble. These results suggest that for the extracellular domain of P0 to behave as a homophilic adhesion molecule, its cytoplasmic domain must be intact, and most probably, it is interacting with the cytoskeleton.     

Dominant-negative effect on adhesion by myelin Po protein truncated in its cytoplasmic domain

The myelin Po protein is believed to hold myelin together via interactions of both its extracellular and cytoplasmic domains. We have already shown that the extracellular domains of Po can interact in a homophilic manner (Filbin, M.T., F.S. Walsh, B.D. Trapp, J.A. Pizzey, and G.I. Tennekoon. 1990. Nature (Lond.). 344:871-872). In addition, we have shown that for this homophilic adhesion to take place, the cytoplasmic domain of Po must be intact and most likely interacting with the cytoskeleton; Po proteins truncated in their cytoplasmic domains are not adhesive (Wong, M.H., and M.T. Filbin, 1994. J. Cell Biol. 126:1089-1097). To determine if the presence of these truncated forms of Po could have an effect on the functioning of the full-length Po, we coexpressed both molecules in CHO cells. The adhesiveness of CHO cells expressing both full-length Po and truncated Po was then compared to cells expressing only full-length Po. In these coexpressors, both the full-length and the truncated Po proteins were glycosylated. They reached the surface of the cell in approximately equal amounts as shown by an ELISA and surface labeling, followed by immunoprecipitation. Furthermore, the amount of full-length Po at the cell surface was equivalent to other cell lines expressing only full-length Po that we had already shown to be adhesive. Therefore, there should be sufficient levels of full-length Po at the surface of these coexpressors to measure adhesion of Po. However, as assessed by an aggregation assay, the coexpressors were not adhesive. By 60 min they had not formed large aggregates and were indistinguishable from the control transfected cells not expressing Po. In contrast, in the same time, the cells expressing only the full-length Po had formed large aggregates. This indicates that the truncated forms of Po have a dominant-negative effect on the adhesiveness of the full-length Po. Furthermore, from cross-linking studies, full-length Po, when expressed alone but not when coexpressed with truncated Po, appears to cluster in the membrane. We suggest that truncated Po exerts its dominant-negative effect by preventing clustering of full-length Po. We also show that colchicine, which disrupts microtubules, prevents adhesion of cells expressing only the full-length Po. This strengthens our suggestion that an interaction of Po with the cytoskeleton, either directly or indirectly, is required for adhesion to take place.     

Lateral dimerization is required for the homophilic binding activity of C-cadherin

Regulation of cadherin-mediated adhesion can occur rapidly at the cell surface. To understand the mechanism underlying cadherin regulation, it is essential to elucidate the homophilic binding mechanism that underlies all cadherin-mediated functions. Therefore, we have investigated the structural and functional properties of the extracellular segment of Xenopus C-cadherin using a purified, recombinant protein (CEC 1-5). CEC 1-5 supported adhesion of CHO cells expressing C-cadherin. The extracellular segment was also capable of mediating aggregation of microspheres. Chemical cross-linking and gel filtration revealed that CEC 1-5 formed dimers in the presence as well as absence of calcium. Analysis of the functional activity of purified dimers and monomers demonstrated that dimers retained substantially greater homophilic binding activity than monomers. These results demonstrate that lateral dimerization is necessary for homophilic binding between cadherin extracellular segments and suggest multiple potential mechanisms for the regulation of cadherin activity. Since the extracellular segment alone possessed significant homophilic binding activity, the adhesive activity of the extracellular segment in a cellular context was analyzed. The adhesion of CHO cells expressing a truncated version of C-cadherin lacking the cytoplasmic tail was compared to cells expressing the wild-type C-cadherin using a laminar flow assay on substrates coated with CEC 1-5. CHO cells expressing the truncated C-cadherin were able to attach to CEC 1-5 and to resist detachment by low shear forces, demonstrating that tailless C-cadherin can mediate basic, weak adhesion of CHO cells. However, cells expressing the truncated C-cadherin did not exhibit the complete adhesive activity of cells expressing wild-type C-cadherin. Cells expressing wild-type C-cadherin remained attached to CEC 1-5 at high shear forces, while cells expressing the tailless C-cadherin did not adhere well at high shear forces. These results suggest that there may be two states of cadherin-mediated adhesion. The first, relatively weak state can be mediated through interactions between the extracellular segments alone. The second strong adhesive state is critically dependent on the cytoplasmic tail.     

N-Cadherin signaling in synapse formation and neuronal physiology

Neural cadherin (N-cadherin) is an adhesion receptor that is localized in abundance at neuron-to-neuron synapses. N-cadherin contains an extracellular domain that binds to other cadherins on juxtaposed cell membranes, a single-pass transmembrane region, and a cytoplasmic tail that interacts with various proteins, including catenins, kinases, phosphatases, and presenilin 1. N-cadherin contributes to the structural and functional organization of the synaptic complex by ensuring the adhesion between synaptic membranes and organizing the underlying actin cytoskeleton. Additionally, recent findings have shown that N-cadherin may participate in synaptic physiology by regulating calcium influx through voltage-activated calcium currents. The diverse activities of N-cadherin stem from its ability to operate as both an adhesion molecule that links cytoskeletons across cell membranes and a ligand-activated homophilic receptor capable of initiating intracellular signaling. An important mechanism of cadherin signaling is the regulation of small Rho guanosine triphosphatase activity that affects cytoskeleton dynamics and calcium influx. Because both the regulation of cadherin adhesive activity and cadherin-mediated signaling are affected by the binding of molecules to the intracellular domain, changes in the composition of the N-cadherin complex are central to the regulation of cadherin-mediated functions. This article focuses on the roles that N-cadherin might play at the level of the synapse through its effect on adhesion and signaling in the proximity of the synaptic junction.     

Critical role of the fifth domain of E-cadherin for heterophilic adhesion with alpha E beta 7, but not for homophilic adhesion

The integrin alpha(E)beta(7) is expressed on intestinal intraepithelial T lymphocytes and CD8(+) T lymphocytes in inflammatory lesions near epithelial cells. Adhesion between alpha(E)beta(7)(+) T and epithelial cells is mediated by the adhesive interaction of alpha(E)beta(7) and E-cadherin; this interaction plays a key role in the damage of target epithelia. To explore the structure-function relationship of the heterophilic adhesive interaction between E-cadherin and alpha(E)beta(7), we performed cell aggregation assays using L cells transfected with an extracellular domain-deletion mutant of E-cadherin. In homophilic adhesion assays, L cells transfected with wild-type or a domain 5-deficient mutant formed aggregates, whereas transfectants with domain 1-, 2-, 3-, or 4-deficient mutants did not. These results indicate that not only domain 1, but domains 2, 3, and 4 are involved in homophilic adhesion. When alpha(E)beta(7)(+) K562 cells were incubated with L cells expressing the wild type, 23% of the resulting cell aggregates consisted of alpha(E)beta(7)(+) K562 cells. In contrast, the binding of alpha(E)beta(7)(+) K562 cells to L cells expressing a domain 5-deficient mutant was significantly decreased, with alpha(E)beta(7)(+) K562 cells accounting for only 4% of the cell aggregates, while homophilic adhesion was completely preserved. These results suggest that domain 5 is involved in heterophilic adhesion with alpha(E)beta(7), but not in homophilic adhesion, leading to the hypothesis that the fifth domain of E-cadherin may play a critical role in the regulation of heterophilic adhesion to alpha(E)beta(7) and may be a potential target for treatments altering the adhesion of alpha(E)beta(7)(+) T cells to epithelial cells in inflammatory epithelial diseases.     

Adhesive but not signaling activity of Drosophila N-cadherin is essential for target selection of photoreceptor afferents

Drosophila N-cadherin (CadN) is an evolutionarily conserved, atypical classical cadherin, which has a large complex extracellular domain and a catenin-binding cytoplasmic domain. We have previously shown that CadN regulates target selection of R7 photoreceptor axons. To determine the functional domains of CadN, we conducted a structure-function analysis focusing on its in vitro adhesive activity and in vivo function in R7 growth cones. We found that the cytoplasmic domain of CadN is largely dispensable for the targeting of R7 growth cones, and it is not essential for mediating homophilic interaction in cultured cells. Instead, the cytoplasmic domain of CadN is required for maintaining proper growth cone morphology. Domain swapping with the extracellular domain of CadN2, a related but non-adhesive cadherin, revealed that the CadN extracellular domain is required for both adhesive activity and R7 targeting. Using a target-mosaic system, we generated CadN mutant clones in the optic lobe and examined the target-selection of genetically wild-type R7 growth cones to CadN mutant target neurons. We found that CadN, but neither LAR nor Liprin-alpha, is required in the medulla neurons for R7 growth cones to select the correct medulla layer. Together, these data suggest that CadN mediates homophilic adhesive interactions between R7 growth cones and medulla neurons to regulate layer-specific target selection.     

Tools for studying the role of N-cadherin mediated extracellular interaction in neuronal development and function

N-cadherin is a homophilic cell adhesion molecule that plays important roles in many aspects of neuronal development. In order to better understand the function of N-cadherin mediated cell-cell contact in activity-dependent dendrite development, we generated a number of new tools. EC1, consisting of the first extracellular domain of N-cadherin, can specifically inhibit N-cadherin, but not E-cadherin, mediated cell-cell contact, both when overexpressed in neurons and added as a purified protein. Ncad-HA is an extracellularly epitope-tagged version of N-cadherin that, when overexpressed under the activity-independent pCS2-min promoter, can be used to assay surface N-cadherin level following various manipulations. These tools are likely to be very useful for studying the function of N-cadherin in multiple aspects of neural circuit development.Key words: N-cadherin, dendrite development, neuronal activity, homophilic interaction, cell-cell contact, activity-independent expression vector     

Multiple cadherin extracellular repeats mediate homophilic binding and adhesion.

The extracellular homophilic-binding domain of the cadherins consists of 5 cadherin repeats (EC1-EC5). Studies on cadherin specificity have implicated the NH(2)-terminal EC1 domain in the homophilic binding interaction, but the roles of the other extracellular cadherin (EC) domains have not been evaluated. We have undertaken a systematic analysis of the binding properties of the entire cadherin extracellular domain and the contributions of the other EC domains to homophilic binding. Lateral (cis) dimerization of the extracellular domain is thought to be required for adhesive function. Sedimentation analysis of the soluble extracellular segment of C-cadherin revealed that it exists in a monomer-dimer equilibrium with an affinity constant of approximately 64 microm. No higher order oligomers were detected, indicating that homophilic binding between cis-dimers is of significantly lower affinity. The homophilic binding properties of a series of deletion constructs, lacking successive or individual EC domains fused at the COOH terminus to an Fc domain, were analyzed using a bead aggregation assay and a cell attachment-based adhesion assay. A protein with only the first two NH(2)-terminal EC domains (CEC1-2Fc) exhibited very low activity compared with the entire extracellular domain (CEC1-5Fc), demonstrating that EC1 alone is not sufficient for effective homophilic binding. CEC1-3Fc exhibited high activity, but not as much as CEC1-4Fc or CEC1-5Fc. EC3 is not required for homophilic binding, however, since CEC1-2-4Fc and CEC1-2-4-5Fc exhibited high activity in both assays. These and experiments using additional EC combinations show that many, if not all, the EC domains contribute to the formation of the cadherin homophilic bond, and specific one-to-one interaction between particular EC domains may not be required. These conclusions are consistent with a previous study on direct molecular force measurements between cadherin ectodomains demonstrating multiple adhesive interactions (Sivasankar, S., W. Brieher, N. Lavrik, B. Gumbiner, and D. Leckband. 1999. PROC: Natl. Acad. Sci. USA. 96:11820-11824; Sivasankar, S., B. Gumbiner, and D. Leckband. 2001. Biophys J. 80:1758-68). We propose new models for how the cadherin extracellular repeats may contribute to adhesive specificity and function.     

Construction and expression of an enzymatically active form of PECAM-1 containing the phosphatase domain of the protein tyrosine phosphatase, SHP-2

Platelet endothelial cell adhesion molecule-1 (PECAM-1; CD31) is a 130-kDa transmembrane glycoprotein that is expressed on the surfaces of platelets, endothelial cells, and certain leukocyte subsets. The extracellular region of PECAM-1 contains six immunoglobulin homology domains, two of which (domains 1 and 2) mediate PECAM-1 homophilic interactions. Recent evidence suggests that a major function of the extracellular region of PECAM-1 is to determine its localization within the plane of the plasma membrane. The cytoplasmic domain of PECAM-1 contains an immunoreceptor tyrosine-based inhibitory motif that, upon tyrosine phosphorylation, supports recruitment of the Src homology 2 domain-containing protein tyrosine phosphatase, SHP-2. However, neither the targets of this PECAM-1/SHP-2 complex nor the significance of localizing SHP-2 to the borders of opposing PECAM-1-expressing cells is yet known. As a first step in addressing these issues, we designed a cDNA encoding a chimeric protein composed of the PECAM-1 extracellular domain fused to the phosphatase domain of SHP-2, which we call PECAM-1/PhD2. When immunopurified from stably transfected HEK293 cell lines expressing this recombinant protein, PECAM-1/PhD2 was found to possess constitutive enzymatic activity and appropriate border localization. This constitutively active chimeric protein will be useful in future studies designed to define the components of signal transduction pathways modulated by PECAM-1/SHP-2 signaling complexes.     

The receptor tyrosine kinase ARK mediates cell aggregation by homophilic binding.

The ARK (AXL, UFO) receptor is a member of a new family of receptor tyrosine kinases whose extracellular domain contains a combination of fibronectin type III and immunoglobulin motifs similar to those found in many cell adhesion molecules. ARK mRNA is expressed at high levels in the mouse brain, prevalently in the hippocampus and cerebellum, and this pattern of expression resembles that of adhesion molecules that are capable of promoting cell aggregation through homophilic or heterophilic binding. We report here the ability of the murine ARK receptor to mediate homophilic binding. Expression of the ARK protein in Drosophila S2 cells induces formation of cell aggregates consisting of ARK-expressing cells, and aggregation leads to receptor activation, with an increase in receptor phosphorylation. Homophilic binding does not require ARK tyrosine kinase activity, since S2 cells expressing a receptor in which the intracellular domain was deleted were able to undergo aggregation as well as cells expressing the wild-type ARK receptor. Similar results were obtained with NIH 3T3 and CHO cells expressing high levels of ARK, although in this case ARK expression appeared to be accompanied by constitutive activation. The purified recombinant extracellular domain of ARK can induce homotypic aggregation of coated fluorescent beads (Covaspheres), and this protein can also function as a substrate for adhesion by S2 and NIH 3T3 cells expressing ARK. These results suggest that ARK represents a new cell adhesion molecule that through its homophilic interaction may regulate cellular functions during cell recognition.     

Transmembrane control of cadherin-mediated cell adhesion: a 94 kDa protein functionally associated with a specific region of the cytoplasmic domain of E-cadherin.

Cadherins are a family of transmembrane glycoproteins which play a key role in Ca(2+)-dependent cell-cell adhesion. Cytoplasmic domains of these molecules are anchored to the cell cytoskeleton and are required for cadherin function. To elucidate how the function of cadherins is controlled through their cytoplasmic domains, we deleted five different regions in the cytoplasmic domain of E-cadherin. After transfecting L cells with cDNA encoding the mutant polypeptides, we assayed aggregating activity of these transfectants; all these mutant proteins were shown to have an extracellular domain with normal Ca(2+)-sensitivity and molecular weight. Two mutant polypeptides with deletions in the carboxy half of the cytoplasmic domain, however, did not promote cell-cell adhesion and had also lost the ability to bind to the cytoskeleton, whereas the mutant molecules with deletions of other regions retained the ability to promote cell adhesion and to anchor to the cytoskeleton. Thus, the cytoplasmic domain contains a subdomain which was involved in the cell adhesion and cytoskeleton-binding functions. When E-cadherin in F9 cells or in L cells transfected with wild-type or functional mutant cadherin polypeptides was solubilized with nonionic detergents and immunoprecipitated, two additional 94 and 102 kDa components were coprecipitated. The 94 kDa component, however, was not detected in the immunoprecipitates from cells expressing the mutant cadherins which had lost the adhesive function. These results suggest that the interaction of the carboxy half of the cytoplasmic domain with the 94 kDa component regulates the cell binding function of the extracellular domain of E-cadherin.     

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 α- and β-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.     

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.     

PECAM-1/CD31 trans-homophilic binding at the intercellular junctions is independent of its cytoplasmic domain; evidence for heterophilic interaction with integrin alphavbeta3 in Cis

PECAM-1/CD31 is a cell adhesion and signaling molecule that is enriched at the endothelial cell junctions. Alternative splicing generates multiple PECAM-1 splice variants, which differ in their cytoplasmic domains. It has been suggested that the extracellular ligand-binding property, homophilic versus heterophilic, of these isoforms is controlled by their cytoplasmic tails. To determine whether the cytoplasmic domains also regulate the cell surface distribution of PECAM-1 splice variants, we examined the distribution of CD31-EGFPs (PECAM-1 isoforms tagged with the enhanced green fluorescent protein) in living Chinese hamster ovary cells and in PECAM-1-deficient endothelial cells. Our results indicate that the extracellular, rather than the cytoplasmic domain, directs PECAM-1 to the cell-cell borders. Furthermore, coculturing PECAM-1 expressing and deficient cells along with transfection of CD31-EGFP cDNAs into PECAM-1 deficient cells reveal that this PECAM-1 localization is mediated by homophilic interactions. Although the integrin alphavbeta3 has been shown to interact with PECAM-1, this trans-heterophilic interaction was not detected at the borders of endothelial cells. However, based on cocapping experiments performed on proT cells, we provide evidence that the integrin alphavbeta3 associates with PECAM-1 on the same cell surface as in a cis manner.     

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.     

Deletions in the cytoplasmic domain of platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31) result in changes in ligand binding properties

Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a member of the immunoglobulin superfamily present on platelets, endothelial cells, and leukocytes that may function as a vascular cell adhesion molecule. The purpose of this study was to examine the role of the cytoplasmic domain in PECAM-1 function. To accomplish this, wild- type and mutated forms of PECAM-1 cDNA were transfected into murine fibroblasts and the functional characteristics of the cells analyzed. Wild-type PECAM-1 localized to the cell-cell borders of adjacently transfected cells and mediated heterophilic, calcium-dependent L-cell aggregation that was inhibitable by a polyclonal and two monoclonal anti-PECAM-1 antibodies. A mutant protein lacking the entire cytoplasmic domain did not support aggregation or move to cell-cell borders. In contrast, both forms of PECAM-1 with partially truncated cytoplasmic domains (missing either the COOH-terminal third or two thirds of the cytoplasmic domain) localized to cell-cell borders in 3T3 cells in a manner analogous to the distribution seen in cultured endothelial cells. L-cells expressing these mutants demonstrated homophilic, calcium-independent aggregation that was blocked by the polyclonal anti-PECAM-1 antibody, but not by the two bioactive monoclonal antibodies. Although changes in the cytoplasmic domain of other receptors have been shown to alter ligand-binding affinity, to our knowledge, PECAM-1 is the first example of a cell adhesion molecule where changes in the cytoplasmic domain result in a switch in the basic mechanism of adhesion leading to different ligand-binding specificity. Variations in the cytoplasmic domain could thus be a potential mechanism for regulating PECAM-1 activity in vivo.     

Signal transduction from N-cadherin increases Bcl-2. Regulation of the phosphatidylinositol 3-kinase/Akt pathway by homophilic adhesion and actin cytoskeletal organization

Associated with the metastatic progression of epithelial tumors is the dynamic regulation of cadherins. Whereas E-cadherin is expressed in most epithelium and carcinomas, recent studies suggest that the up-regulation of other cadherin subtypes in carcinomas, such as N-cadherin, may function in cancer progression. We demonstrate that a signal transduction cascade links the N-cadherin.catenin adhesion complex to up-regulation of the anti-apoptotic protein Bcl-2. In suspension, aggregates of DU-145 cells, an E-cadherin expressing human prostate carcinoma line, survive loss of integrin-dependent adhesion by a different anti-apoptotic signaling pathway than the N-cadherin expressing lines PC3 and PC3N. N-cadherin intercellular adhesion mediates a 3.5-fold increase in Bcl-2 protein expression, whereas the level of the proapoptotic protein Bax remains constant. Only N-cadherin ligation in PC3 cells, which express both N-cadherin and E-cadherin, is sufficient to induce activation of Akt/protein kinase B. N-cadherin homophilic ligation initiates phosphatidylinositol 3-kinase-dependent activation of Akt resulting in Akt phosphorylation of Bad on serine 136. Following N-cadherin homophilic adhesion phosphatidylinositol 3-kinase was identified in immunoprecipitates of the N-cadherin.catenin complex. The recruitment of phosphatidylinositol 3-kinase to the adhesion complex is dependent on ligation of N-cadherin and an organized actin cytoskeleton because cytochalasin D blocks the recruitment. We propose that N-cadherin homophilic adhesion can initiate anti-apoptotic signaling, which enhances the Akt cell survival pathway in metastatic cancer.     

Crystal Structure of the cis-Dimer of Nectin-1: implications for the architecture of cell-cell junctions

In multicellular organisms, cells are interconnected by cell adhesion molecules. Nectins are immunoglobulin (Ig)-like cell adhesion molecules that mediate homotypic and heterotypic cell-cell adhesion, playing key roles in tissue organization. To mediate cell-cell adhesion, nectin molecules dimerize in cis on the surface of the same cell, followed by trans-dimerization of the cis-dimers between the neighboring cells. Previous cell biological studies deduced that the first Ig-like domain of nectin and the second Ig-like domain are involved in trans-dimerization and cis-dimerization, respectively. However, to understand better the steps involved in nectin adhesion, the structural basis for the dimerization of nectin must be determined. In this study, we determined the first crystal structure of the entire extracellular region of nectin-1. In the crystal, nectin-1 formed a V-shaped homophilic dimer through the first Ig-like domain. Structure-based site-directed mutagenesis of the first Ig-like domain identified four essential residues that are involved in the homophilic dimerization. Upon mutating the four residues, nectin-1 significantly decreased cis-dimerization on the surface of cultured cells and abolished the homophilic and heterophilic adhesion activities. These results indicate that, in contrast with the previous notion, our structure represents a cis-dimer. Thus, our findings clearly reveal the structural basis for the cis-dimerization of nectins through the first Ig-like domains.     

Fast dissociation kinetics between individual E-cadherin fragments revealed by flow chamber analysis

E-cadherin is the predominant adhesion molecule of epithelia. The interaction between extracellular segments of E-cadherin in the membrane of opposing cells is homophilic and calcium dependent. Whereas it is widely accepted that the specificity of the adhesive interaction is localized to the N-terminal domain, the kinetics of the recognition process are unknown. We report the first quantitative data describing the dissociation kinetics of individual E-cadherin interactions. Aggregation assays indicate that the two outermost domains of E-cadherin (E/EC1-2) retain biological activity when chemically immobilized on glass beads. Cadherin fragment trans-interaction was analysed using a flow chamber technique. Transient tethers had first-order kinetics, suggesting a unimolecular interaction. The unstressed lifetime of individual E-cadherin interactions was as brief as 2 s. A fast off rate and the low tensile strength of the E-cadherin bond may be necessary to support the high selectivity and plasticity of epithelial cell interactions.