Activated leukocyte cell adhesion molecule (CD166/ALCAM): developmental and mechanistic aspects of cell clustering and cell migration

Activated leukocyte cell adhesion molecule (ALCAM/CD166) is a member of the immunoglobulin superfamily and belongs to a recent subgroup with five extracellular immunoglobulin-like domains (VVC2C2C2). ALCAM mediates both heterophilic (ALCAM-CD6) and homophilic (ALCAM-ALCAM) cell-cell interactions. While expressed in a wide variety of tissues, ALCAM is usually restricted to subsets of cells involved in dynamic growth and/or migration, including neural development, branching organ development, hematopoiesis, immune response and tumor progression. Recent structure-function analyses of ALCAM hint at how its cytoskeletal anchoring and the integrity of the extracellular immunoglobulin-like domains may regulate complex cellular properties in regard to cell adhesion, growth and migration. Accumulating evidence suggests that ALCAM expression may reflect the onset of a cellular program for homeostatic control of growth saturation, which induces either growth arrest or cell migration when the upper limits are exceeded.     

Dual knockdown of Galectin-8 and its glycosylated ligand,the activated leukocyte cell adhesion molecule (ALCAM/CD166), synergistically delays in vivo breast cancer growth

Galectin-8 (Gal-8), a ‘tandem-repeat’-type galectin, has been described as a modulator of cellular functions including adhesion, spreading, growth arrest, apoptosis, pathogen recognition, autophagy, and immunomodulation. We have previously shown that activated leukocyte cell adhesion molecule (ALCAM), also known as CD166, serves as a receptor for endogenous Gal-8. ALCAM is a member of the immunoglobulin superfamily involved in cell-cell adhesion through homophilic (ALCAM-ALCAM) and heterophilic (i.e. ALCAM-CD6) interactions in different tissues. Here we investigated the physiologic relevance of ALCAM-Gal-8 association and glycosylation-dependent mechanisms governing these interactions. We found that silencing of ALCAM in MDA-MB-231 triple negative breast cancer cells decreases cell adhesion and migration onto Gal-8-coated surfaces in a glycan-dependent fashion. Remarkably, either Gal-8 or ALCAM silencing also disrupted cell-cell adhesion, and led to reduced tumor growth in a murine model of triple negative breast cancer. Moreover, structural characterization of endogenous ALCAM N-glycosylation showed abundant permissive structures for Gal-8 binding. Importantly, we also found that cell sialylation controls Gal-8-mediated cell adhesion. Altogether, these findings demonstrate a central role of either ALCAM or Gal-8 (or both) in controlling triple negative breast cancer.     

CD6 recognizes the neural adhesion molecule BEN.

CD6 and its ligand activated leukocyte cell adhesion molecule (ALCAM, CD166) have been detected on various immune cells and in the brain. CD6-ligand interactions have been implicated in the regulation of T cell function. ALCAM shares the same extracellular domain organization and significant sequence homology with the chicken neural adhesion molecule BEN. Although ALCAM's CD6 binding site is only partially conserved in BEN, CD6 specifically binds BEN, albeit with approximately 10-fold lower avidity than ALCAM. Differences in binding avidity are not detected when ALCAM and BEN fusion proteins containing the full-length extracellular regions are tested. Homotypic interactions between full-length forms are likely to account for these observations. The identified cross-species interaction between CD6 and BEN suggests that CD6-ligand interactions are highly conserved.     

Dynamic regulation of activated leukocyte cell adhesion molecule-mediated homotypic cell adhesion through the actin cytoskeleton

Restricted expression of activated leukocyte cell adhesion molecule (ALCAM) by hematopoietic cells suggests an important role in the immune system and hematopoiesis. To get insight into the mechanisms that control ALCAM-mediated adhesion we have investigated homotypic ALCAM-ALCAM interactions. Here, we demonstrate that the cytoskeleton regulates ALCAM-mediated cell adhesion because inhibition of actin polymerization by cytochalasin D (CytD) strongly induces homotypic ALCAM-ALCAM interactions. This induction of cell adhesion is likely due to clustering of ALCAM at the cell surface, which is observed after CytD treatment. Single-particle tracking demonstrated that the lateral mobility of ALCAM in the cell membrane is increased 30-fold after CytD treatment. In contrast, both surface distribution and adhesion of a glycosylphosphatidylinositol (GPI)-anchored ALCAM mutant are insensitive to CytD, despite the increase in lateral mobility of GPI-ALCAM upon CytD treatment. This demonstrates that clustering of ALCAM is essential for cell adhesion, whereas enhanced diffusion of ALCAM alone is not sufficient for cluster formation. In addition, upon ligand binding, both free diffusion and the freely dragged distance of wild-type ALCAM, but not of GPI-ALCAM, are reduced over time, suggesting strengthening of the cytoskeleton linkage. From these findings we conclude that activation of ALCAM-mediated adhesion is dynamically regulated through actin cytoskeleton-dependent clustering.     

Platelet endothelial cell adhesion molecule 1 (PECAM-1) and its interactions with glycosaminoglycans: 1. Molecular modeling studies

Platelet endothelial cell adhesion molecule 1 (PECAM-1) has many functions, including its roles in leukocyte extravasation as part of the inflammatory response and in the maintenance of vascular integrity through its contribution to endothelial cell-cell adhesion. PECAM-1 has been shown to mediate cell-cell adhesion through homophilic binding events that involve interactions between domain 1 of PECAM-1 molecules on adjacent cells. However, various heterophilic ligands of PECAM-1 have also been proposed. The possible interaction of PECAM-1 with glycosaminoglycans (GAGs) is the focus of this study. The three-dimensional structure of the extracellular immunoglobulin (Ig) domains of PECAM-1 were constructed using homology modeling and threading methods. Potential heparin/heparan sulfate-binding sites were predicted on the basis of their amino acid consensus sequences and a comparison with known structures of sulfate-binding proteins. Heparin and other GAG fragments have been docked to investigate the structural determinants of their protein-binding specificity and selectivity. The modeling has predicted two regions in PECAM-1 that appear to bind heparin oligosaccharides. A high-affinity binding site was located in Ig domains 2 and 3, and evidence for a low-affinity site in Ig domains 5 and 6 was obtained. These GAG-binding regions were distinct from regions involved in PECAM-1 homophilic interactions.     

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.     

Three-Dimensional Analysis of CD6 Site-Directed Mutagenesis and Monoclonal Antibody Binding Studies Using the X-ray Structure of Mac-2 Binding Protein and a Molecular Model of the CD6 Ligand Binding Domain

The extracellular region of CD6 consists of three scavenger receptor cysteine-rich (SRCR) domains and binds activated leukocyte cell adhesion molecule (ALCAM), a member of the immunoglobulin superfamily (IgSF). Residues important for the CD6-ALCAM interaction have previously been identified by mutagenesis. A total of 22 CD6 residues were classified according to their importance for anti-CD6 monoclonal antibody (mAb) and/or ALCAM binding. The three-dimensional structure of the SRCR domain of Mac-2 binding protein has recently been determined, providing a structural prototype for the SRCR protein superfamily. This has made a thorough three-dimensional analysis of CD6 mutagenesis and mAb binding experiments possible. Mutation of buried residues compromised both mAb and ALCAM binding, consistent with the presence of structural perturbations. However, several residues whose mutation affected both mAb and ALCAM binding or, alternatively, only ligand binding were found to map to the surface in the same region of the domain. This suggests that the CD6 ligand binding site and epitopes of tested mAbs overlap and provides an explanation for the finding that these mAbs effectively block ALCAM binding. An approximate molecular model of CD6 was used to delineate the ALCAM binding site.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s0089490050263Abbreviations ALCAM activated leukocyte cell adhesion molecule - CD6D3 third (membrane-proxi-mal) extracellular domain of CD6 - IgSF immunoglobulin superfamily - mAb monoclonal antibody - M2BP Mac-2 binding protein - SRCR scavenger receptor cysteine-rich domain - SRCRSF scavenger receptor cysteine-rich protein superfamily     

Neural cell adhesion molecule (N-CAM) homophilic binding mediated by the two N-terminal Ig domains is influenced by intramolecular domain-domain interactions

The mechanism by which the neural cell adhesion molecule, N-CAM, mediates homophilic interactions between cells has been variously attributed to an isologous interaction of the third immunoglobulin (Ig) domain, to reciprocal binding of the two N-terminal Ig domains, or to reciprocal interactions of all five Ig domains. Here, we have used a panel of recombinant proteins in a bead binding assay, as well as transfected and primary cells, to clarify the molecular mechanism of N-CAM homophilic binding. The entire extracellular region of N-CAM mediated bead aggregation in a concentration- and temperature-dependent manner. Interactions of the N-terminal Ig domains, Ig1 and Ig2, were essential for bead binding, based on deletion and mutation experiments and on antibody inhibition studies. These findings were largely in accord with aggregation experiments using transfected L cells or primary chick brain cells. Additionally, maximal binding was dependent on the integrity of the intramolecular domain-domain interactions throughout the extracellular region. We propose that these interactions maintain the relative orientation of each domain in an optimal configuration for binding. Our results suggest that the role of Ig3 in homophilic binding is largely structural. Several Ig3-specific reagents failed to affect N-CAM binding on beads or on cells, while an inhibitory effect of an Ig3-specific monoclonal antibody is probably due to perturbations at the Ig2-Ig3 boundary. Thus, it appears that reciprocal interactions between Ig1 and Ig2 are necessary and sufficient for N-CAM homophilic binding, but that maximal binding requires the quaternary structure of the extracellular region defined by intramolecular domain-domain interactions.     

Cancer-Derived Mutations in the Fibronectin III Repeats of PTPRT/PTPρ Inhibit Cell-Cell Aggregation

Abstract

The receptor protein tyrosine phosphatase T PTPρ is the most frequently mutated tyrosine phosphatase in human cancer. PTPρ mediates homophilic cell-cell aggregation. In its extracellular region, PTPρ has cell adhesion molecule–like motifs, including a MAM domain, an immunoglobulin domain, and four fibronectin type III (FNIII) repeats. Tumor-derived mutations have been identified in all of these extracellular domains. Previously, the authors determined that tumor-derived mutations in the MAM and immunoglobulin domains of PTPρ reduce homophilic cell-cell aggregation. In this paper, the authors describe experiments in which the contribution of the FNIII repeats to PTPρ-mediated cell-cell adhesion was evaluated. The results demonstrate that deletion of the FNIII repeats of PTPρ result in defective cell-cell aggregation. Furthermore, all of the tumor-derived mutations in the FNIII repeats of PTPρ also disrupt cell-cell aggregation. These results further support the hypothesis that mutational inactivation of PTPρ may lead to cancer progression by disrupting cell-cell adhesion.     

Molecular model of the N-terminal receptor-binding domain of the human CD6 ligand ALCAM.

CD6-ligand interactions have been implicated in the regulation of T-cell adhesion and activation. CD6 is a member of the scavenger receptor family, whereas its human ligand (ALCAM) belongs to the immunoglobulin superfamily. The extracellular region of ALCAM includes five immunoglobulin-like domains. As a fusion protein, the N-terminal extracellular domain of ALCAM (ALCAMD1) binds specifically to CD6. We report the construction, assessment, and analysis of a molecular model of ALCAMD1. The model defines the CDR-analogous loops, the location of N-linked glycosylation sites, and residues that form the beta-sheet faces of the immunoglobulin-like domain. Predicted structural characteristics of the A'GFCC'C" face of the model are consistent with the presence of monomeric and dimeric forms of ALCAMD1, which has implications for the receptor-ligand interactions.     

Identification of a peptide sequence involved in homophilic binding in the neural cell adhesion molecule NCAM

The neural cell adhesion molecule NCAM is capable of mediating cell-cell adhesion via homophilic interactions. In this study, three strategies have been combined to identify regions of NCAM that participate directly in NCAM-NCAM binding: analysis of domain deletion mutations, mapping of epitopes of monoclonal antibodies, and use of synthetic peptides to inhibit NCAM activity. Studies on L cells transfected with NCAM mutant cDNAs using cell aggregation and NCAM-covasphere binding assays indicate that the third immunoglobulin-like domain is involved in homophilic binding. The epitopes of four monoclonal antibodies that have been previously shown to affect cell-cell adhesion mediated by NCAM were also mapped to domain 3. Overlapping hexapeptides were synthesized on plastic pins and assayed for binding with these monoclonal antibodies. One of them (PP) reacted specifically with the sequence KYSFNY. Synthetic oligopeptides containing the PP epitope were potent and specific inhibitors of NCAM binding activity. A substratum containing immobilized peptide conjugates also exhibited adhesiveness for neural retinal cells. Cell attachment was specifically inhibited by peptides that contained the PP-epitope and by anti-NCAM univalent antibodies. The shortest active peptide has the sequence KYSFNYDGSE, suggesting that this site is directly involved in NCAM homophilic interaction.     

Homophilic adhesion of E-cadherin occurs by a co-operative two-step interaction of N-terminal domains.

Cluster formation of E-cadherin on the cell surface is believed to be of major importance for cell-cell adhesion. To mimic this process the extracellular part of mouse E-cadherin (ECAD) was recombinantly fused to the assembly domain of rat cartilage oligomeric matrix protein (COMP), resulting in the chimeric protein ECAD-COMP. The COMP domain formed a five-stranded alpha-helical coiled-coil. This enabled the formation of a pentameric ECAD with bundled C-termini and free N-termini. The pentameric protein construct ECAD-COMP and the monomeric ECAD were expressed in human embryonal kidney 293 cells. Electron microscopy, analytical ultracentrifugation, solid phase binding and cell attachment assays revealed that pentamers showed strong self-association and cell attachment, whereas monomers exhibited no activity. At the high internal concentration in the pentamer the N-terminal EC1 domains of two E-cadherin arms interact to form a ring-like structure. Then the paired domains interact with a corresponding pair from another pentamer. None of the four other extracellular domains of E-cadherin is involved in this interaction. Based on these results, an in vivo mechanism is proposed whereby two N-terminal domains of neighbouring E-cadherins at the cell surface first form a pair, which binds with high affinity to a similar complex on another cell. The strong dependence of homophilic interactions on C-terminal clustering points towards a regulation of E-cadherin mediated cell-cell adhesion via lateral association.     

The conserved immunoglobulin domain controls the subcellular localization of the homophilic adhesion receptor protein-tyrosine phosphatase mu

The receptor protein-tyrosine phosphatase mu (PTPmu) is a homophilic adhesion protein thought to regulate cell-cell adhesion in the vascular endothelium through dephosphorylation of cell junction proteins. In subconfluent cell cultures, PTPmu resides in an intracellular membrane pool; however, as culture density increases and cell contacts form, the phosphatase localizes to sites of cell-cell contact, and its expression level increases. These characteristics of PTPmu, which are consistent with a role in cell-cell adhesion, suggest that control of subcellular localization is an important mechanism to regulate the function of this phosphatase. To gain a better understanding of how PTPmu is regulated, we examined the importance of the conserved immunoglobulin domain, containing the homophilic binding site, in control of the localization of the enzyme. Deletion of the immunoglobulin domain impaired localization of PTPmu to the cell-cell contacts in endothelial and epithelial cells. In addition, deletion of the immunoglobulin domain affected the distribution of PTPmu in subconfluent endothelial cells when homophilic binding to another PTPmu molecule on an apposing cell was not possible, resulting in an accumulation of the mutant phosphatase at the cell surface with a concentration at the cell periphery in the region occupied by focal adhesions. This aberrant localization correlated with reduced survival and alterations in normal focal adhesion and cytoskeleton morphology. This study therefore illustrates the critical role of the immunoglobulin domain in regulation of the localization of PTPmu and the importance of such control for the maintenance of normal cell physiology.     

Homophilic binding of PTP mu, a receptor-type protein tyrosine phosphatase, can mediate cell-cell aggregation

The receptor-like protein tyrosine phosphatase, PTPmu, displays structural similarity to cell-cell adhesion molecules of the immunoglobulin superfamily. We have investigated the ability of human PTPmu to function in such a capacity. Expression of PTPmu, with or without the PTPase domains, by recombinant baculovirus infection of Sf9 cells induced their aggregation. However, neither a chimeric form of PTPmu, containing the extracellular and transmembrane segments of the EGF receptor and the intracellular segment of PTPmu, nor the intracellular segment of PTPmu expressed as a soluble protein induced aggregation. PTPmu mediates aggregation via a homophilic mechanism, as judged by lack of incorporation of uninfected Sf9 cells into aggregates of PTPmu-expressing cells. Homophilic binding has been demonstrated between PTPmu-coated fluorescent beads (Covaspheres) and endogenously expressed PTPmu on MvLu cells. Additionally the PTPmu-coated beads specifically bound to a bacterially expressed glutathione-S-transferase fusion protein containing the extracellular segment of PTPmu (GST/PTPmu) adsorbed to petri dishes. Covaspheres coated with the GST/PTPmu fusion protein aggregated in vitro and also bound to PTPmu expressed endogenously on MvLu cells. These results suggest that the ligand for this transmembrane PTPase is another PTPmu molecule on an adjacent cell. Thus homophilic binding interactions may be an important component of the function of PTPmu in vivo.     

Structural basis of cell-cell adhesion by NCAM

The neural cell adhesion molecule NCAM, a member of the immunoglobulin superfamily, mediates cell-cell recognition and adhesion via a homophilic interaction. NCAM plays a key role during development and regeneration of the nervous system and is involved in synaptic plasticity associated with memory and learning. The 1.85 A crystal structure of the two N-terminal extracellular domains of NCAM reported here provides a structural basis for the homophilic interaction. The molecular packing of the two-domain structure reveals a cross shaped antiparallel dimer, and provides fundamental insight into trans-cellular recognition mediated by NCAM.     

Primary structure of ICAM-1 demonstrates interaction between members of the immunoglobulin and integrin supergene families

Intercellular adhesion molecule 1 (ICAM-1) is a 90 kd inducible surface glycoprotein that promotes adhesion in immunological and inflammatory reactions. ICAM-1 is a ligand of lymphocyte function-associated antigen-1 (LFA-1), an alpha beta complex that is a member of the integrin family of cell-cell and cell-matrix receptors. ICAM-1 is encoded by an inducible 3.3 kb mRNA. The amino acid sequence specifies an integral membrane protein with an extracellular domain of 453 residues containing five immunoglobulin-like domains. Highest homology is found with neural cell adhesion molecule (NCAM) and myelin-associated glycoprotein (MAG), which also contain five Ig-like domains. NCAM and MAG are nervous system adhesion molecules, but unlike ICAM-1, NCAM is homophilic. The ICAM-1 and LFA-1 interaction is heterophilic and unusual in that it is between members of the immunoglobulin and intergrin families. Unlike other integrin ligands, ICAM-1 does not contain an RGD sequence.     

Cloning of an immunoglobulin family adhesion molecule selectively expressed by endothelial cells

To gain fundamental information regarding the molecular basis of endothelial cell adhesive interactions during vascular formation, we have cloned and characterized a unique cell adhesion molecule. This molecule, named endothelial cell-selective adhesion molecule (ESAM), is a new member of the immunoglobulin superfamily. The conceptual protein encoded by cDNA clones consists of V-type and C2-type immunoglobulin domains as well as a hydrophobic signal sequence, a single transmembrane region, and a cytoplasmic domain. Northern blot analysis showed ESAM to be selectively expressed in cultured human and murine vascular endothelial cells and revealed high level expression in lung and heart and low level expression in kidney and skin. In situ hybridization analysis indicated that ESAM is primarily expressed in the developing vasculature of the embryo in an endothelial cell-restricted pattern. Epitope-tagged ESAM was shown to co-localize with cadherins and catenins in cell-cell junctions. In aggregation assays employing ESAM-expressing Chinese hamster ovary cells, this novel molecule was shown to mediate cell-cell adhesion through homophilic interactions. The endothelial cell-selective expression of this immunoglobulin-like adhesion molecule coupled with its in vitro functional profile strongly suggests a role in cell-cell interactions that is critical for vascular development or function.     

Glycosylation-dependent binding of galectin-8 to activated leukocyte cell adhesion molecule (ALCAM/CD166) promotes its surface segregation on breast cancer cells

BackgroundWe previously demonstrated that the activated leukocyte cell adhesion molecule (ALCAM/CD166) can interact with galectin-8 (Gal-8) in endothelial cells. ALCAM is a member of the immunoglobulin superfamily that promotes homophilic and heterophilic cell–cell interactions. Gal-8 is a “tandem-repeat”-type galectin, known as a matricellular protein involved in cell adhesion. Here, we analyzed the physical interaction between both molecules in breast cancer cells and the functional relevance of this phenomenon.MethodsWe performed binding assays by surface plasmon resonance to study the interaction between Gal-8 and the recombinant glycosylated ALCAM ectodomain or endogenous ALCAM from MDA-MB-231 breast cancer cells. We also analyzed the binding of ALCAM-silenced or control breast cancer cells to immobilized Gal-8 by SPR. In internalization assays, we evaluated the influence of Gal-8 on ALCAM surface localization.ResultsWe showed that recombinant glycosylated ALCAM and endogenous ALCAM from breast carcinoma cells physically interacted with Gal-8 in a glycosylation-dependent fashion displaying a differential behavior compared to non-glycosylated ALCAM. Moreover, ALCAM-silenced breast cancer cells exhibited reduced binding to Gal-8 relative to control cells. Importantly, exogenously added Gal-8 provoked ALCAM segregation, probably trapping this adhesion molecule at the surface of breast cancer cells.ConclusionsOur data indicate that Gal-8 interacts with ALCAM at the surface of breast cancer cells through glycosylation-dependent mechanisms.General significanceA novel heterophilic interaction between ALCAM and Gal-8 is demonstrated here, suggesting its physiologic relevance in the biology of breast cancer cells.     

Tumor-derived extracellular mutations of PTPRT /PTPrho are defective in cell adhesion

Receptor protein tyrosine phosphatase T (PTPRT/PTPrho) is frequently mutated in human cancers including colon, lung, gastric, and skin cancers. More than half of the identified tumor-derived mutations are located in the extracellular part of PTPrho. However, the functional significance of those extracellular domain mutations remains to be defined. Here we report that the extracellular domain of PTPrho mediates homophilic cell-cell aggregation. This homophilic interaction is very specific because PTPrho does not interact with its closest homologue, PTPmu, in a cell aggregation assay. We further showed that all five tumor-derived mutations located in the NH(2)-terminal MAM and immunoglobulin domains impair, to varying extents, their ability to form cell aggregates, indicating that those mutations are loss-of-function mutations. Our results suggest that PTPrho may play an important role in cell-cell adhesion and that mutational inactivation of this phosphatase could promote tumor migration and metastasis.