Dimeric versions of two short N-cadherin binding motifs (HAVDI and INPISG) function as N-cadherin agonists.

N-cadherin is a member of the classical cadherin family of homophilic binding molecules. Peptide competition studies have identified the HAVDI and INPISGQ sequences as functional binding motifs in extracellular domain 1 (ECD1) of N-cadherin. Whereas monomeric versions of these motifs function as specific N-cadherin antagonists, we now show that cyclic peptides containing a tandem repeat of the individual motifs function as N-cadherin agonists. In this context, when presented to neurons as soluble molecules, the dimeric versions of the motifs stimulate neurite outgrowth in a similar manner to native N-cadherin. The response to the dimeric agonist peptides was inhibited by monomeric versions of the same motif and also by recombinant N-cadherin ECD1 protein. The responses were also inhibited by antibodies to a fibroblast growth factor receptor (FGFR) binding motif in ECD4 of N-cadherin and by a specific FGFR antagonist (PD17304). These data suggest that the peptides function by binding to and clustering N-cadherin in neurons and thereby activating an N-cadherin/FGFR signaling cascade. The novel agonists will be invaluable for dissecting out those cadherin functions that rely on signaling as opposed to adhesion and clearly have the potential to be developed as therapeutic agents for the promotion of cell survival and axonal regeneration.     

A novel family of cyclic peptide antagonists suggests that N-cadherin specificity is determined by amino acids that flank the HAV motif

The classical cadherins (e.g. N-, E-, and P- cadherin) are well established homophilic adhesion molecules; however, the mechanism that governs cadherin specificity remains contentious. The classical cadherins contain an evolutionarily conserved His-Ala-Val (HAV) sequence, and linear peptides harboring this motif are capable of inhibiting a variety of cadherin-dependent processes. We now demonstrate that short cyclic HAV peptides can inhibit N-cadherin function. Interestingly, the nature of the amino acids that flank the HAV motif determine both the activity and specificity of the peptides. For example, when the HAV motif is flanked by a single aspartic acid, which mimics the natural HAVD sequence of N-cadherin, the peptide becomes a much more effective inhibitor of N-cadherin function. In contrast, when the HAV motif is flanked by a single serine, which mimics the natural HAVS sequence of E-cadherin, it loses its ability to inhibit the N-cadherin response. Our results demonstrate that subtle changes in the amino acids that flank the HAV motif can account for cadherin specificity and that small cyclic peptides can inhibit cadherin function. An emerging role for cadherins in a number of pathological processes suggests that the cyclic peptides reported in this study might be developed as therapeutic agents.     

Soluble N-cadherin stimulates fibroblast growth factor receptor dependent neurite outgrowth and N-cadherin and the fibroblast growth factor receptor co-cluster in cells

A chimeric molecule consisting of the extracellular domain of the adhesion molecule, N-cadherin, fused to the Fc region of human IgG (NCAD-Fc) supports calcium-dependent cell adhesion and promotes neurite outgrowth following affinity-capture to a tissue culture substrate. When presented to cerebellar neurons as a soluble molecule, the NCAD-Fc stimulated neurite outgrowth in a manner equivalent to that seen for N-cadherin expressed as a cell surface glycoprotein. Neurons expressing a dominant-negative version of the fibroblast growth factor (FGF) receptor did not respond to soluble NCAD-Fc. In cells transfected with full-length N-cadherin and the FGF receptor, antibody-clustering of N-cadherin resulted in a co-clustering of the FGF receptor to discrete patches in the cell membrane. The data demonstrate that the ability of N-cadherin to stimulate neurite outgrowth can be dissociated from its ability to function as a substrate associated adhesion molecule. The N-cadherin and the FGF receptor co-clustering in cells provides a basis for the neurite outgrowth response stimulated by N-cadherin being dependent on FGF receptor function.     

Identification of a murine ICAM-1-specific peptide by subtractive phage library selection on cells

The ICAM-1 adhesion molecule is expressed selectively at low levels on endothelial cells but is strongly upregulated in dysfunctional endothelial cells associated with inflammation, cancer, and atherogenesis. Using COS-7 cells transfected with murine ICAM-1 (mICAM-1) as a target receptor, a phage display library was screened. Clones were selected by elution with a mAb specific for a functional epitope of ICAM-1 and a novel peptide sequence binding to the extracellular domain of mICAM-1 was identified that can potentially be used as a targeting vector aimed at dysfunctional endothelium. We further showed that the targeting specificity of the peptide was retained following its incorporation at the N terminal end of a large chimeric protein. Moreover, this chimeric protein containing the mICAM-1-specific sequence was found to inhibit ICAM-1-mediated intercellular adhesion during antigen presentation. Taken together, these results demonstrate the potential for improving the cell-selectivity and properties of therapeutical agents toward targeting adhesion molecules involved in cell-cell interactions.     

Discovery and development of N-cadherin antagonists

This article describes over 20 years of research on antagonists of the cell adhesion molecule, N-cadherin. Four types of antagonists are discussed: synthetic linear peptides, synthetic cyclic peptides, non-peptidyl peptidomimetics of the disulfide linked cyclic peptide N-Ac-CHAVC-NH(2) and monoclonal antibodies directed against the N-cadherin ectodomain. The biological activities of these antagonists are also discussed. In particular, the ability of N-cadherin antagonists to act as anti-cancer drugs is considered.     

N-cadherin function is required for differentiation-dependent cytoskeletal reorganization in lens cells in vitro

Members of the cadherin family of cell adhesion molecules participate in calcium-dependent cell-cell adhesions that are necessary for the cell sorting events that regulate early developmental processes. Although individual cadherin molecules have been shown to participate in tissue histogenesis, the regulation of function of these receptors in cell differentiation has been more difficult to identify. We have determined that N-cadherin linkage to the cytoskeleton is correlated with lens cell differentiation in vivo. Through the use of a chick embryo lens culture system that mimics differentiation in vivo, we have determined that N-cadherin linkage to the cytoskeleton is altered and lens differentiation is blocked by function-blocking antibodies to N-cadherin. In the presence of the N-cadherin function-blocking antibody, NCD-2, both N-cadherin and filamentous actin are prevented from organizing at the cortical membranes. This correlates with an inhibition of lens morphogenesis and differentiation. These results are paralleled by changes in the expression of the molecular components of the cadherin-catenin complex and their linkage to the actin cytoskeleton. In the presence of NCD-2, expression of N-cadherin, alpha-catenin, and beta-catenin is inhibited and their association with the cytoskeleton blocked. Overall cadherin expression, however, remains unchanged as demonstrated by studies with a pan-cadherin antibody. This is accompanied by an increase in expression of the cadherin cytoskeletal protein plakoglobin. Although the cells have tried to compensate for the loss of N-cadherin by up-regulation of another cadherin(s) and plakoglobin, this is unable to compensate for N-cadherin function. The data strongly suggest that N-cadherin and its associated cytoskeleton play an important role in the differentiation process that leads to the formation of the crystalline lens.     

Identification of mammalian and invertebrate analogues of the avian calcium-dependent cell adhesion protein N-cadherin with synthetic-peptide directed antibodies against a conserved cytoplasmic domain

N-cadherin, a 130kD transmembrane adhesive glycoprotein, is a mediator of specific cellular interactions during development. Analysis of N-cadherin at the protein level, to date, has been largely dependent upon monoclonal antibody NCD-2 which recognizes only avian N-cadherin. We produced a monospecific polyclonal antiserum, C-NCAD(838-856), to a synthetic peptide corresponding to a portion of the highly conserved c-terminal cytoplasmic domain of chick N-cadherin. Using polyacrylamide gel electrophoresis and immunoblotting to map tissue distribution we show that the antiserum detects chick N-cadherin with a similar tissue distribution as NCD-2. Unlike NCD-2, however, anti-C-NCAD(838-856) recognizes N-cadherin analogues in a wide variety of species, including mouse, human, fish and drosophila. The results of comparative immunoblot studies demonstrate similar tissue-specific patterns and apparent molecular weight variation in the chick, mouse and human. This indicates that N-cadherin structure and expression, and most likely function as well, have been highly conserved in evolution. The antiserum recognizes an epitope unique to N-cadherin which is conserved among N-cadherins from a variety of species but is absent from other members of the cadherin gene family, as no immunoreactivity was detected with tissues bearing these other cadherins. The antiserum is thus a useful tool for the phylogenetic and biochemical investigation of N-cadherin from a variety of tissue sources.     

Chimeric synthetic peptides from the envelope (gp46) and the transmembrane (gp21) glycoproteins for the detection of antibodies to human T-cell leukemia virus type II.

Two chimeric synthetic peptides incorporating immunodominant sequences from HTLV-II virus were synthesized. Monomeric peptides P2 and P3 represent sequences from transmembrane protein (gp21) and envelope protein (gp46) of the virus. The peptide P2 is a gp21 (370-396) sequence and the peptide P3 is a gp46 (178-205) sequence. Those peptides were arranged in a way that permits one to obtain different combinations of chimeric peptides (P2-GG-P3 and P3-GG-P2), separated by two glycine residues as spacer arms. The antigenic activity of these peptides was evaluated by UltramicroEnzyme-linked immunosorbent assay (UMELISA) by using panels anti-HTLV-II-positive sera (n = 11), anti-HTLV-I/II-positive sera (n = 2), HTLV-positive (untypeable) serum samples (n = 2), and anti-HTLV-I-positive sera (n = 22), while specificity was evaluated with anti-HIV-positive samples (n = 19) and samples from healthy blood donors (n = 30). The efficacy of the chimeric peptides in solid-phase immunoassays was compared with the monomeric peptides and a mixture of the monomeric peptides. Higher sensitivity was observed for chimeric peptide Q5 assay. Those results may be related to a higher peptide adsorption capacity to the solid surface and for epitope accessibility to the antibodies. This chimeric peptide would be very useful for HTLV-II diagnostic.     

Cloning and expression of cDNA encoding a neural calcium-dependent cell adhesion molecule: its identity in the cadherin gene family

The neural cadherin (N-cadherin) is a Ca2+-dependent cell-cell adhesion molecule detected in neural tissues as well as in non-neural tissues. We report here the nucleotide sequence of the chicken N-cadherin cDNA and the deduced amino acid sequence. The sequence data suggest that N-cadherin has one transmembrane domain which divides the molecule into an extracellular and a cytoplasmic domain; the extracellular domain contains internal repeats of characteristic sequences. When the N-cadherin cDNA connected with virus promoters was transfected into L cells which have no endogenous N-cadherin, the transformants acquired the N-cadherin-mediated aggregating property, indicating that the cloned cDNA contained all information necessary for the cell-cell binding action of this molecule. We then compared the primary structure of N-cadherin with that of other molecules defined as cadherin subclasses. The results showed that these molecules contain common amino acid sequences throughout their entire length, which confirms our hypothesis that cadherins make a gene family.     

Chimeric synthetic peptides containing two immunodominant epitopes from the envelope gp46 and the transmembrane gp21 glycoproteins of HTLV-I virus.

Two chimeric synthetic peptides incorporating immunodominant sequences from HTLV-I virus were synthesized. Monomeric peptides P7 and P8 represent sequences from transmembrane protein (gp21) and envelope protein (gp46) of the virus. The peptide P7 is a gp21 (374-400) sequence and the peptide P8 is a gp46 (190-207) sequence. Those peptides were arranged in a way that permits one to obtain different combinations of chimeric peptides (P7-GG-P8 and P8-GG-P7), separated by two glycine residues as spacer arms. The antigenic activity of these peptides were evaluated by UltramicroEnzyme-linked immunosorbent assay (UMELISA) by using panels of anti-HTLV-I-positive sera (n = 22), anti-HTLV-I/II-positive sera (n = 2), HTLV-positive (untypeable) serum samples (n = 2), and anti-HTLV-II-positive sera (n = 11), while specificity was evaluated with anti-HIV-positive samples (n = 19) and samples from healthy blood donors (n = 30). The efficacy of the chimeric peptides in solid-phase immunoassays was compared with the monomeric peptides and monomeric peptides together. The chimeric peptide P7-GG-P8 proved to be the most reactive with anti-HTLV-I-positive sera. These results may be related to a higher peptide adsorption capacity to the solid surface and for epitope accessibility to the antibodies. This chimeric peptide would be very useful for HTLV-I diagnostics.     

A synthetic peptide from the COOH-terminal heparin-binding domain of fibronectin promotes focal adhesion formation.

Cell adhesion to extracellular matrix molecules such as fibronectin involves complex transmembrane signaling processes. Attachment and spreading of primary fibroblasts can be promoted by interactions of cell surface integrins with RGD-containing fragments of fibronectin, but the further process of focal adhesion and stress fiber formation requires additional interactions. Heparin-binding fragments of fibronectin can provide this signal. The COOH-terminal heparin-binding domain of fibronectin contains five separate heparin-binding amino acid sequences. We show here that all five sequences, as synthetic peptides coupled to ovalbumin, can support cell attachment. Only three of these sequences can promote focal adhesion formation when presented as multicopy complexes, and only one of these (WQPPRARI) retains this activity as free peptide. The major activity of this peptide resides in the sequence PRARI. The biological response to this peptide and to the COOH-terminal fragment may be mediated through cell surface heparan sulfate proteoglycans because treatment of cells with heparinase II and III, or competition with heparin, reduces the response. Treatment with chondroitinase ABC or competition with chondroitin sulfate does not.     

N-cadherin regulates cytoskeletally associated IQGAP1/ERK signaling and memory formation

Cadherin-mediated interactions are integral to synapse formation and potentiation. Here we show that N-cadherin is required for memory formation and regulation of a subset of underlying biochemical processes. N-cadherin antagonistic peptide containing the His-Ala-Val motif (HAV-N) transiently disrupted hippocampal N-cadherin dimerization and impaired the formation of long-term contextual fear memory while sparing short-term memory, retrieval, and extinction. HAV-N impaired the learning-induced phosphorylation of a distinctive, cytoskeletally associated fraction of hippocampal Erk-1/2 and altered the distribution of IQGAP1, a scaffold protein linking cadherin-mediated cell adhesion to the cytoskeleton. This effect was accompanied by reduction of N-cadherin/IQGAP1/Erk-2 interactions. Similarly, in primary neuronal cultures, HAV-N prevented NMDA-induced dendritic Erk-1/2 phosphorylation and caused relocation of IQGAP1 from dendritic spines into the shafts. The data suggest that the newly identified role of hippocampal N-cadherin in memory consolidation may be mediated, at least in part, by cytoskeletal IQGAP1/Erk signaling.     

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.     

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     

Inhibiting N-cadherin-mediated adhesion affects gap junction communication in isolated rat hearts

Cadherin-mediated adherens junctions is impaired concomitant with a decrease in connexin 43 (Cx43) in diseases or pathological processes. We have investigated the acute effects of adherens junction impairment in isolated rat hearts by introducing Ala-His-Ala-Val-Asp-NH₂ (AHAVD, a synthetic peptide) as a specific inhibitor of N-cadherin. Effect of AHAVD on N-cadherin mediated adhension was analyzed by Cardiomy-ocyte aggregation assay. Laser confocal microscopy showed disrupted cell-cell contacts in cultured neonatal cardiomyocytes co-incubated with 0.2 mM AHAVD. In isolated adult rat hearts, Cx43 was redistributed along the bilateral of cardiomyocytes from the intercalated discs and significant dephosphorylation of Cx43 on serine368 occurred concomitantly with decreased gap junction (GJ) function in dose dependent manner after 1 h perfusion with AHAVD. These results indicate that impairing cad-herin-mediated adhesion by AHAVD rapidly results in Cx43 redistribution and dephosphorylation of serine368, thereby impairing GJ communication function.     

Evidence that tyrosine phosphorylation regulates N-cadherin turnover during retinal development

N-cadherin, a member of the cadherin family of calcium-dependent cell adhesion molecules, mediates adhesive and signaling interactions between cells during development. N-Cadherin undergoes dynamic spatiotemporal changes in expression which correlate with morphogenetic movements of cells during organogenesis and histogenesis. We have previously shown that N-cadherin expression during development is regulated by several mechanisms, including mRNA expression, cytokine modulation, and proteolytically mediated turnover, yielding the NCAD90 protein. The present study was directed at determining the extent to which N-cadherin in primary embryonic cells is the target of endogenous kinases and phosphatases, as well as the effects of modulation of these enzymes on NCAD90 expression. The results of phosphoamino acid analyses, peptide mapping, and measurements of N-cadherin and NCAD90 expression in embryonic tissues indicate that N-cadherin is indeed the target of endogenous kinase and phosphatase action, and that modulation of different classes of these enzymes can result in either stimulation or inhibition of NCAD90 production. These results provide a mechanistic explanation for observations that cadherin function is downregulated following expression of exogenously introduced viral tyrosine kinases and provide a function for the tyrosine phosphatases recently found in association with cadherins. The results indicate that N-cadherin expression during retinal development is possibly regulated in part by modulation of its phosphorylation state, the balance of which may determine whether N-cadherin remains stably expressed or is targeted for proteolytically mediated turnover to produce NCAD90. Dev. Genet. 20:224–234, 1997. © 1997 Wiley-Liss, Inc.     

The "linker" region (amino acids 38-47) of the disintegrin elegantin is a novel inhibitory domain of integrin alpha5beta1-dependent cell adhesion on fibronectin: evidence for the negative regulation of fibronectin synergy site biological activity

Disintegrins are a family of potent inhibitors of cell-cell and cell-matrix adhesion. In this study we have identified a region of the disintegrin elegantin, termed the "linker domain" (amino acids 38-47), with inhibitory activity toward alpha(5)beta(1)-mediated cell adhesion on fibronectin (Fn). Using a chimeric structure-function approach in which sequences of the functionally distinct disintegrin kistrin were introduced into the elegantin template at targeted sites, a loss of inhibitory function toward alpha(5)beta(1)-mediated adhesion on Fn was observed when the elegantin linker domain was substituted. Subsequent analysis comparing the inhibitory efficacies of the panel of elegantin-kistrin chimeras toward CHO alpha(5) cell adhesion on recombinant Fn III(6-10) fragments showed that the loss of inhibitory activity associated with the disruption of the elegantin linker domain was dependent upon the presence of a functional Fn III(9) synergy site within the Fn III(6-10) substrate. This suggested that the elegantin linker domain inhibits primarily the activity of the Fn synergy domain in promoting alpha(5)beta(1) integrin-mediated cell adhesion. Construction of a cyclic peptide corresponding to the entire region of the elegantin linker domain showed that this domain has intrinsic alpha(5)beta(1) inhibitory activity comparable with the activity of the RGDS peptide. These data demonstrate a novel biological function for a disintegrin domain that antagonizes integrin-mediated cell adhesion.     

Evidence for endogenous proteases, mRNA level and insulin as multiple mechanisms of N-cadherin down-regulation during retinal development.

Our previous studies of the role of cell adhesion in retinal development have focused on the expression and function of N-cadherin, the predominant calcium-dependent intercellular adhesion protein of neural tissues. During the course of retinal development, N-cadherin expression undergoes significant qualitative and quantitative changes in its pattern of expression, most prominently a sharp down-regulation of expression throughout most of the retina. The present studies were directed at investigating the epigenetic mechanisms that could mediate this loss of N-cadherin from the retina. Using an in vitro intact retinal organ culture system, results were obtained which suggest that insulin enhances the down-regulation of N-cadherin expression in a protein-synthesis-dependent fashion. Furthermore, the metalloprotease inhibitor 1,10-phenanthroline inhibits the loss of N-cadherin from the retina. While N-cadherin is down-regulated in organ culture, other cell adhesion molecules, which are not down-regulated in vivo, are also not down-regulated in organ culture. The defined organ culture medium conditioned by the retina accumulates both a soluble 90 x 10(3) M(r) N-terminal fragment of N-cadherin as well as a number of secreted proteases. Both of these components are also shown to be present in vivo in the vitreous humor. Northern blot analysis indicates a single mRNA encoding N-cadherin in the retina and no evidence for a second message that could encode the 90 x 10(3) M(r) fragment. However, the amount of N-cadherin mRNA detectable on northern blots decreases during development. The results reported here suggest that the down-regulation of N-cadherin that occurs during retinal development is possibly mediated by multiple mechanisms, which include turnover at the cell surface mediated by endogenous proteolysis, reduced levels of N-cadherin mRNA and modulation by growth factors.     

PTPmu regulates N-cadherin-dependent neurite outgrowth

Cell adhesion is critical to the establishment of proper connections in the nervous system. Some receptor-type protein tyrosine phosphatases (RPTPs) have adhesion molecule-like extracellular segments with intracellular tyrosine phosphatase domains that may transduce signals in response to adhesion. PTPmu is a RPTP that mediates cell aggregation and is expressed at high levels in the nervous system. In this study, we demonstrate that PTPmu promotes neurite outgrowth of retinal ganglion cells when used as a culture substrate. In addition, PTPmu was found in a complex with N-cadherin in retinal cells. To determine the physiological significance of the association between PTPmu and N-cadherin, the expression level and enzymatic activity of PTPmu were perturbed in retinal explant cultures. Downregulation of PTPmu expression through antisense techniques resulted in a significant decrease in neurite outgrowth on an N-cadherin substrate, whereas there was no effect on laminin or L1-dependent neurite outgrowth. The overexpression of a catalytically inactive form of PTPmu significantly decreased neurite outgrowth on N-cadherin. These data indicate that PTPmu specifically regulates signals required for neurites to extend on an N-cadherin substrate, implicating reversible tyrosine phosphorylation in the control of N-cadherin function. Together, these results suggest that PTPmu plays a dual role in the regulation of neurite outgrowth.