Homophilic Cell Adhesion Molecule Research Articles

Cadherins in tissue architecture and disease.

Cadherins are homophilic cell adhesion molecules that determine tissue architecture and control cell contact formation and dissociation in development and tissue homeostasis of all metazoans. These adhesion molecules mediate homophilic interactions between cells and are linked inside the cell via a complex set of cytosolic factors to the actin cytoskeleton. These interactions are key to the plasticity of intercellular junctions and to the various signaling functions of the cadherins. This forms the basis for cadherin-driven cell behavior, cell differentiation, and regeneration of tissue structures. Consequently, mutations in cadherins are the cause of various human pathologies, with cancer representing one of the most prominent examples.

Bead aggregation assays for the characterization of putative cell adhesion molecules.

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.

FLRT Structure: Balancing Repulsion and Cell Adhesion in Cortical and Vascular Development

SummaryFLRTs are broadly expressed proteins with the unique property of acting as homophilic cell adhesion molecules and as heterophilic repulsive ligands of Unc5/Netrin receptors. How these functions direct cell behavior and the molecular mechanisms involved remain largely unclear. Here we use X-ray crystallography to reveal the distinct structural bases for FLRT-mediated cell adhesion and repulsion in neurons. We apply this knowledge to elucidate FLRT functions during cortical development. We show that FLRTs regulate both the radial migration of pyramidal neurons, as well as their tangential spread. Mechanistically, radial migration is controlled by repulsive FLRT2-Unc5D interactions, while spatial organization in the tangential axis involves adhesive FLRT-FLRT interactions. Further, we show that the fundamental mechanisms of FLRT adhesion and repulsion are conserved between neurons and vascular endothelial cells. Our results reveal FLRTs as powerful guidance factors with structurally encoded repulsive and adhesive surfaces.

Abstract 66: p120 catenin: A novel regulator of PanIN epithelial cell delamination in preinvasive pancreatic cancer

Abstract Pancreatic cancer is among the deadliest human malignancies ranking 4th in the United States for cancer-related deaths among both men and women. Due to the invasive nature of pancreatic cancer, metastasis to the lymphatic system and distant organs is a major contributor to pancreatic cancer-related death. Genetic alterations in cell adhesion molecules contribute to human disease including developmentally related syndromes and cancer. In a sequencing study of 24 primary pancreatic tumor exomes published by the Hopkins pancreatic cancer team in 2008, 79% of tumors had at least one mutation in a homophilic cell adhesion molecule, and this class of molecules was named as one of the twelve core signaling pathways in pancreatic cancer. Among this class of cell adhesion molecules are adherens junctions. Mislocalization of the adherens protein p120 catenin has been identified in almost all of the major types of human carcinomas including pancreatic cancer. An accumulating body of evidence has identified p120 catenin as a prognostic marker in pancreatic cancer. Based on relevant literature, we hypothesize that misexpression and mislocalization of p120 catenin in pancreatic cancer is pathologic in the progression of this deadly disease. To test this hypothesis, we have ablated p120 catenin in the KCiMist1 mouse model of pancreatic cancer. The KCiMist1 mouse model activates oncogenic Kras in adult pancreatic acinar cells and displays the preinvasive PanIN 1 - PanIN 3 lesions in a manner that faithfully recapitulates the human disease. Homozygous deletion of p120 catenin in this model resulted in an almost complete replacement of acinar cells by acinar to ductal metaplastic lesions, accelerated PanIN formation, and stromal infiltration at 1 month. At 2 months, the stromal infiltrate persists, and we see considerable evidence of fibrosis and fatty deposition in the pancreas, which are pathologic features of pancreatitis. We used the KCiMist1Gp120f/f model to trace the lineage of acinar cells undergoing Kras activation and p120 catenin excision, and observed a marked increase in isolated GFP+ Ecadherin+ cells located in the extensive stroma at 1 and 2 months suggesting that p120 catenin normally restrains PanIN epithelial cell delamination. We have begun to study potential mechanisms for this delamination phenotype including epithelial-mesenchymal transition (EMT) and non-EMT based mechanisms. Research suggests that EMT is a contributing factor to the development of drug resistance, which makes EMT a promising target for the development of future therapies that reduce invasion and drug resistance leading to better prognosis for patients with pancreatic cancer. Our preliminary findings suggest that the KCiMist1Gp120f/f mouse model represents a valuable tool to study both EMT and non-EMT based mechanisms for PanIN epithelial cell delamination. Citation Format: Audrey M. Hendley, Jennifer M. Bailey, Janivette Alsina, Christine Iacobuzio-Donahue, Anirban Maitra, Albert Reynolds, Steven D. Leach. p120 catenin: A novel regulator of PanIN epithelial cell delamination in preinvasive pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 66. doi:10.1158/1538-7445.AM2014-66

Latrophilins Function as Heterophilic Cell-adhesion Molecules by Binding to Teneurins: REGULATION BY ALTERNATIVE SPLICING

Latrophilin-1, -2, and -3 are adhesion-type G protein-coupled receptors that are auxiliary α-latrotoxin receptors, suggesting that they may have a synaptic function. Using pulldowns, we here identify teneurins, type II transmembrane proteins that are also candidate synaptic cell-adhesion molecules, as interactors for the lectin-like domain of latrophilins. We show that teneurin binds to latrophilins with nanomolar affinity and that this binding mediates cell adhesion, consistent with a role of teneurin binding to latrophilins in trans-synaptic interactions. All latrophilins are subject to alternative splicing at an N-terminal site; in latrophilin-1, this alternative splicing modulates teneurin binding but has no effect on binding of latrophilin-1 to another ligand, FLRT3. Addition to cultured neurons of soluble teneurin-binding fragments of latrophilin-1 decreased synapse density, suggesting that latrophilin binding to teneurin may directly or indirectly influence synapse formation and/or maintenance. These observations are potentially intriguing in view of the proposed role for Drosophila teneurins in determining synapse specificity. However, teneurins in Drosophila were suggested to act as homophilic cell-adhesion molecules, whereas our findings suggest a heterophilic interaction mechanism. Thus, we tested whether mammalian teneurins also are homophilic cell-adhesion molecules, in addition to binding to latrophilins as heterophilic cell-adhesion molecules. Strikingly, we find that although teneurins bind to each other in solution, homophilic teneurin-teneurin binding is unable to support stable cell adhesion, different from heterophilic teneurin-latrophilin binding. Thus, mammalian teneurins act as heterophilic cell-adhesion molecules that may be involved in trans-neuronal interaction processes such as synapse formation or maintenance.

Cajal-Retzius Cells Instruct Neuronal Migration by Coincidence Signaling between Secreted and Contact-Dependent Guidance Cues

Cajal-Retzius (CR) cells are a transient cell population of the CNS that is critical for brain development. In the neocortex, CR cells secrete reelin to instruct the radial migration of projection neurons. It has remained unexplored, however, whether CR cells provide additional molecular cues important for brain development. Here, we show that CR cells express the immunoglobulin-like adhesion molecule nectin1, whereas neocortical projection neurons express its preferred binding partner, nectin3. We demonstrate that nectin1- and nectin3-mediated interactions between CR cells and migrating neurons are critical for radial migration. Furthermore, reelin signaling to Rap1 promotes neuronal Cdh2 function via nectin3 and afadin, thus directing the broadly expressed homophilic cell adhesion molecule Cdh2 toward mediating heterotypic cell-cell interactions between neurons and CR cells. Our findings identify nectins and afadin as components of the reelin signaling pathway and demonstrate that coincidence signaling between CR cell-derived secreted and short-range guidance cues direct neuronal migration.

Coupling of NF-protocadherin signaling to axon guidance by cue-induced translation

Cell adhesion molecules and diffusible cues both regulate axon pathfinding, yet how these two modes of signaling interact is poorly understood. The homophilic cell adhesion molecule NF-protocadherin (NFPC) is expressed in the mid-dorsal optic tract neuroepithelium and in the axons of developing retinal ganglion cells (RGC) in Xenopus laevis. Here we report that targeted disruption of NFPC function in RGC axons or the optic tract neuroepithelium results in unexpectedly localized pathfinding defects at the caudal turn in the mid-optic tract. Semaphorin 3A (Sema3A), which lies adjacent to this turn, stimulates rapid, protein synthesis-dependent increases in growth cone NFPC and its cofactor, TAF1, in vitro. In vivo, growth cones exhibit marked increases in NFPC translation reporter activity in this mid-optic tract region that are attenuated by blocking neuropilin-1 function. Our results suggest that translation-linked coupling between regionally localized diffusible cues and cell adhesion can help axons navigate discrete segments of the pathway.

Single-Molecule Force Spectroscopy of the Aplysia Cell Adhesion Molecule Reveals Two Homophilic Bonds

Aplysia californica neurons comprise a powerful model system for quantitative analysis of cellular and biophysical properties that are essential for neuronal development and function. The Aplysia cell adhesion molecule (apCAM), a member of the immunoglobulin superfamily of cell adhesion molecules, is present in the growth cone plasma membrane and involved in neurite growth, synapse formation, and synaptic plasticity. apCAM has been considered to be the Aplysia homolog of the vertebrate neural cell adhesion molecule (NCAM); however, whether apCAM exhibits similar binding properties and neuronal functions has not been fully established because of the lack of detailed binding data for the extracellular portion of apCAM. In this work, we used the atomic force microscope to perform single-molecule force spectroscopy of the extracellular region of apCAM and show for the first time (to our knowledge) that apCAM, like NCAM, is indeed a homophilic cell adhesion molecule. Furthermore, like NCAM, apCAM exhibits two distinct bonds in the trans configuration, although the kinetic and structural parameters of the apCAM bonds are quite different from those of NCAM. In summary, these single-molecule analyses further indicate that apCAM and NCAM are species homologs likely performing similar functions.

Determinants of the Drosophila Odorant Receptor Pattern

In most olfactory systems studied to date, neurons that express the same odorant receptor (Or) gene are scattered across sensory epithelia, intermingled with neurons that express different Or genes. In Drosophila, olfactory sensilla that express the same Or gene are dispersed on the antenna and the maxillary palp. Here we show that Or identity is specified in a spatially stereotyped pattern by the cell-autonomous activity of the transcriptional regulators Engrailed and Dachshund. Olfactory sensilla then become highly motile and disperse beneath the epidermis. Thus, positional information and cell motility underlie the dispersed patterns of Drosophila Or gene expression.

The cell adhesion molecule DdCAD-1 regulates morphogenesis through differential spatiotemporal expression inDictyostelium discoideum

During development of Dictyostelium, multiple cell types are formed and undergo a coordinated series of morphogenetic movements guided by their adhesive properties and other cellular factors. DdCAD-1 is a unique homophilic cell adhesion molecule encoded by the cadA gene. It is synthesized in the cytoplasm and transported to the plasma membrane by contractile vacuoles. In chimeras developed on soil plates, DdCAD-1-expressing cells showed greater propensity to develop into spores than did cadA-null cells. When development was performed on non-nutrient agar, wild-type cells sorted from the cadA-null cells and moved to the anterior zone. They differentiated mostly into stalk cells and eventually died, whereas the cadA-null cells survived as spores. To assess the role of DdCAD-1 in this novel behavior of wild-type and mutant cells, cadA-null cells were rescued by the ectopic expression of DdCAD-1-GFP. Morphological studies have revealed major spatiotemporal changes in the subcellular distribution of DdCAD-1 during development. Whereas DdCAD-1 became internalized in most cells in the post-aggregation stages, it was prominent in the contact regions of anterior cells. Cell sorting was also restored in cadA(-) slugs by exogenous recombinant DdCAD-1. Remarkably, DdCAD-1 remained on the surface of anterior cells, whereas it was internalized in the posterior cells. Additionally, DdCAD-1-expressing cells migrated slower than cadA(-) cells and sorted to the anterior region of chimeric slugs. These results show that DdCAD-1 influences the sorting behavior of cells in slugs by its differential distribution on the prestalk and prespore cells.

A Combinatorial Semaphorin Code Instructs the Initial Steps of Sensory Circuit Assembly in the Drosophila CNS

Longitudinal axon fascicles within the Drosophila embryonic CNS provide connections between body segments and are required for coordinated neural signaling along the anterior-posterior axis. We show here that establishment of select CNS longitudinal tracts and formation of precise mechanosensory afferent innervation to the same CNS region are coordinately regulated by the secreted semaphorins Sema-2a and Sema-2b. Both Sema-2a and Sema-2b utilize the same neuronal receptor, plexin B (PlexB), but serve distinct guidance functions. Localized Sema-2b attraction promotes the initial assembly of a subset of CNS longitudinal projections and subsequent targeting of chordotonal sensory afferent axons to these same longitudinal connectives, whereas broader Sema-2a repulsion serves to prevent aberrant innervation. In the absence of Sema-2b or PlexB, chordotonal afferent connectivity within the CNS is severely disrupted, resulting in specific larval behavioral deficits. These results reveal that distinct semaphorin-mediated guidance functions converge at PlexB and are critical for functional neural circuit assembly.

Asymmetric distribution of Echinoid defines the epidermal leading edge during Drosophila dorsal closure

During Drosophila melanogaster dorsal closure, lateral sheets of embryonic epidermis assemble an actomyosin cable at their leading edge and migrate dorsally over the amnioserosa, converging at the dorsal midline. We show that disappearance of the homophilic cell adhesion molecule Echinoid (Ed) from the amnioserosa just before dorsal closure eliminates homophilic interactions with the adjacent dorsal-most epidermal (DME) cells, which comprise the leading edge. The resulting planar polarized distribution of Ed in the DME cells is essential for the localized accumulation of actin regulators and for actomyosin cable formation at the leading edge and for the polarized localization of the scaffolding protein Bazooka/PAR-3. DME cells with uniform Ed fail to assemble a cable and protrude dorsally, suggesting that the cable restricts dorsal migration. The planar polarized distribution of Ed in the DME cells thus provides a spatial cue that polarizes the DME cell actin cytoskeleton, defining the epidermal leading edge and establishing its contractile properties.

Cancer Cells Cut Homophilic Cell Adhesion Molecules and Run

The term contact inhibition (CI) encompasses the cellular changes that result in cessation of cell migration and of proliferation due to signals transduced when one cell comes into physical contact with another cell. Cancer cells, however, do not contact inhibit. A molecular understanding of the loss of CI in cancer cells is important for understanding tumor progression. In this Perspective, we propose that the loss of CI observed in cancer cells is the result of extracellular proteolysis of transmembrane cell-cell cell adhesion molecules (CAM) in the tumor microenvironment. Proteolysis of homophilic cell-cell CAMs results in a shed extracellular fragment and released cytoplasmic fragment(s) that disrupts adhesion and induces signals that promote proliferation and/or migration. The importance of this observation in tumor progression is supported by the presence of the shed extracellular fragments of homophilic cell-cell CAMs in serum and tumor tissue of cancer patients suggesting that instead of acting as tumor suppressors, the shed CAM extracellular and cytoplasmic fragments actually function as oncogenes. The study of cell-cell CAM cleavage will provide important and novel means of diagnosing, imaging, and treating tumor progression.

Echinoid regulates tracheal morphology and fusion cell fate in Drosophila

Morphogenesis of the Drosophila embryonic trachea involves a stereotyped pattern of epithelial tube branching and fusion. Here, we report unexpected phenotypes resulting from maternal and zygotic (M/Z) loss of the homophilic cell adhesion molecule Echinoid (Ed), as well as the subcellular localization of Ed in the trachea. ed(M/Z) embryos have convoluted trachea reminiscent of septate junction (SJ) and luminal matrix mutants. However, Ed does not localize to SJs, and ed(M/Z) embryos have intact SJs and show normal luminal accumulation of the matrix-modifying protein Vermiform. Surprisingly, tracheal length is not increased in ed(M/Z) mutants, but a previously undescribed combination of reduced intersegmental spacing and deep epidermal grooves produces a convoluted tracheal phenotype. In addition, ed(M/Z) mutants have unique fusion defects involving supernumerary fusion cells, ectopic fusion events and atypical branch breaks. Tracheal-specific expression of Ed rescues these fusion defects, indicating that Ed acts in trachea to control fusion cell fate.

Androgen depletion up‐regulates cadherin‐11 expression in prostate cancer

Men with castration-resistant prostate cancer (PCa) frequently develop metastasis in bone. The reason for this association is unclear. We have previously shown that cadherin-11 (also known as OB-cadherin), a homophilic cell adhesion molecule that mediates osteoblast adhesion, plays a role in the metastasis of PCa to bone. Here, we report that androgen-deprivation therapy up-regulates cadherin-11 expression in PCa. In human PCa specimens, immunohistochemical staining showed that 22/26 (85%) primary PCa tumours from men with castration-resistant PCa expressed cadherin-11. In contrast, only 7/50 (14%) androgen-dependent PCa tumours expressed cadherin-11. In the MDA-PCa-2b xenograft animal model, cadherin-11 was expressed in the recurrent tumours following castration. In the PCa cell lines, there is an inverse correlation between expression of cadherin-11 and androgen receptor (AR), and cadherin-11 is expressed in very low levels or not expressed in AR-positive cell lines, including LNCaP, C4-2B4 and VCaP cells. We showed that AR likely regulates cadherin-11 expression in PCa through an indirect mechanism. Although re-expression of AR in the AR-negative PC3 cells led to the inhibition of cadherin-11 expression, depletion of androgen from the culture medium or down-regulation of AR by RNA interference in the C4-2B4 cells or VCaP cells only produced a modest increase of cadherin-11 expression. Promoter analysis indicated that cadherin-11 promoter does not contain a typical AR-binding element, and AR elicits a modest inhibition of cadherin-11 promoter activity, suggesting that AR does not regulate cadherin-11 expression directly. Together, these results suggest that androgen deprivation up-regulates cadherin-11 expression in prostate cancer, and this may contribute to the metastasis of PCa to bone. Our study suggests that therapeutic strategies that block cadherin-11 expression or function may be considered when applying androgen-ablation therapy.

PTPμ suppresses glioma cell migration and dispersal

The cell-surface receptor protein tyrosine phosphatase mu (PTPmu) is a homophilic cell adhesion molecule expressed in CNS neurons and glia. Glioblastomas (GBMs) are the highest grade of primary brain tumors with astrocytic similarity and are characterized by marked dispersal of tumor cells. PTPmu expression was examined in human GBM, low-grade astrocytoma, and normal brain tissue. These studies revealed a striking loss of PTPmu protein expression in highly dispersive GBMs compared to less dispersive low-grade astrocytomas and normal brain. We hypothesized that PTPmu contributes to contact inhibition of glial cell migration by transducing signals in response to cell adhesion. Therefore, loss of PTPmu may contribute to the extensive dispersal of GBMs. The migration of brain tumor cells was assessed in vitro using a scratch wound assay. Parental U-87 MG cells express PTPmu and exhibited limited migration. However, short-hairpin RNA (shRNA)-mediated knockdown of PTPmu induced a morphological change and increased migration. Next, a brain slice assay replicating the three-dimensional environment of the brain was used. To assess migration, labeled U-87 MG glioma cells were injected into adult rat brain slices, and their movement was followed over time. Parental U-87 MG cells demonstrated limited dispersal in this assay. However, PTPmu shRNA induced migration and dispersal of U-87 MG cells in the brain slice. Finally, in a mouse xenograft model of intracranially injected U-87 MG cells, PTPmu shRNA induced morphological heterogeneity in these xenografts. Together, these data suggest that loss of PTPmu in human GBMs contributes to tumor cell migration and dispersal, implicating loss of PTPmu in glioma progression.

Proteolytic Cleavage of Protein Tyrosine Phosphatase μ Regulates Glioblastoma Cell Migration

Glioblastoma multiforme (GBM), the most common malignant primary brain tumor, represents a significant disease burden. GBM tumor cells disperse extensively throughout the brain parenchyma, and the need for tumor-specific drug targets and pharmacologic agents to inhibit cell migration and dispersal is great. The receptor protein tyrosine phosphatase mu (PTPmu) is a homophilic cell adhesion molecule. The full-length form of PTPmu is down-regulated in human glioblastoma. In this article, overexpression of full-length PTPmu is shown to suppress migration and survival of glioblastoma cells. Additionally, proteolytic cleavage is shown to be the mechanism of PTPmu down-regulation in glioblastoma cells. Proteolysis of PTPmu generates a series of proteolytic fragments, including a soluble catalytic intracellular domain fragment that translocates to the nucleus. Only proteolyzed PTPmu fragments are detected in human glioblastomas. Short hairpin RNA-mediated down-regulation of PTPmu fragments decreases glioblastoma cell migration and survival. A peptide inhibitor of PTPmu function blocks fragment-induced glioblastoma cell migration, which may prove to be of therapeutic value in GBM treatment. These data suggest that loss of cell surface PTPmu by proteolysis generates catalytically active PTPmu fragments that contribute to migration and survival of glioblastoma cells.

The Drosophila cell adhesion molecule Klingon is required for long-term memory formation and is regulated by Notch

The ruslan (rus) mutant was previously identified in a behavioral screen for mutants defective in long-lasting memory, which consists of two consolidated memory types, anesthesia-resistant memory, and protein synthesis-dependent long-term memory (LTM). We demonstrate here that rus is a new allele of klingon (klg), which encodes a homophilic cell adhesion molecule. Klg is acutely required for LTM but not anesthesia-resistant memory formation, and Klg expression increases upon LTM induction. LTM formation also requires activity of the Notch cell-surface receptor. Although defects in Notch have been implicated in memory loss because of Alzheimer's disease, downstream signaling linking Notch to memory have not been determined. Strikingly, we found that Notch activity increases upon LTM induction and regulates Klg expression. Furthermore, Notch-induced enhancement of LTM is disrupted by a klg mutation. We propose that Klg is a downstream effector of Notch signaling that links Notch activity to memory.

The Plane Facts of PCP in the CNS

Planar cell polarity (PCP) pathways have been defined by their ability to direct the development of obviously polarized cellular architectures. Recent studies indicate that PCP pathways also regulate aspects of cell morphology that are not restricted to the plane of the epithelium. In the developing nervous system, PCP-mediated changes in the cytoskeleton are fundamental to neuronal migration, neuronal polarity, axon guidance, and dendritic arborization, highlighting the importance of "planar polarity" genes for defining the shape of a neuron in all dimensions.

Signaling Pathways Involved in NCAM-Induced Neurite Outgrowth

The neural cell adhesion molecule, NCAM, plays important roles in both the developing and adult brains. NCAM, originally, was described as a homophilic cell adhesion molecule abundantly expressed in the central nervous system. This view has been challenged, however, during the last two decades, and NCAM now is considered to be an inducer of complex intracellular signaling cascades in response to extracellular cues. Homo and heterophilic NCAM interactions result in the induction of neurite outgrowth, neuronal survival, and astrocyte proliferation. Here, we review the available data regarding NCAM-induced intracellular signaling and focus on the most intensively studied cellular response, neurite outgrowth.