The cytoplasmic domain of C-CAM1 tumor suppressor is necessary and sufficient for suppressing the tumorigenicity of prostate cancer cells.

We have previously shown that C-CAM1 cell adhesion molecule can suppress the growth of prostate cancer cells in vivo. In this study, we determined the minimal domain of C-CAM1 that is required for its tumor-suppressive activity. DU145 prostate cancer cells were infected with recombinant adenoviruses containing various C-CAM1 mutant genes, and the effects of the mutant C-CAM1 proteins on the growth of DU145 cells were assessed in a nude-mice xenograft model. Deletion of C-CAM1's cytoplasmic domain, which is not required for its adhesion activity, abolished the growth-suppressive activity, whereas deletion of the adhesion domain did not. This observation suggests that C-CAM1's extracellular domain may be not essential for its tumor suppressive activity. Indeed, we found that expression of the C-CAM1 cytoplasmic domain alone led to growth suppression of DU145 cells. These results suggest that the cytoplasmic domain of C-CAM1 is necessary and sufficient for its growth-suppressive function.     

Identification of regions and residues in feline junctional adhesion molecule required for feline calicivirus binding and infection

The feline junctional adhesion molecule A (fJAM-A) is a functional receptor for feline calicivirus (FCV). fJAM-A is a member of the immunoglobulin superfamily (IgSF) and consists of two Ig-like extracellular domains (D1 and D2), a membrane-spanning domain, and a short cytoplasmic tail. To identify regions of fJAM-A that interact with FCV, we purified recombinant fJAM-A ectodomain and D1 and D2 domains. We found that preincubation of FCV with the ectodomain or D1 was sufficient to inhibit FCV infection in plaque reduction assays. In enzyme-linked immunosorbent assays, FCV binding to fJAM-A ectodomain was concentration dependent and saturable; however, FCV bound D1 alone weakly and was unable to bind D2. To characterize FCV binding to surface-expressed fJAM-A, we transfected truncated and chimeric forms of fJAM-A into a nonpermissive cell line and assayed binding by flow cytometry. Only D1 was necessary for FCV binding to cells; all other domains could be replaced. Using a structure-guided mutational approach, we identified three mutants of fJAM-A within D1 (D42N, K43N, and S97A) that exhibited significantly decreased capacities to bind FCV. In contrast to our finding that D1 mediated FCV binding, we found that all domains of fJAM-A were necessary to confer susceptibility to FCV infection. Furthermore, surface expression of fJAM-A was not sufficient to permit FCV infection by all of the isolates we investigated. This indicates that (i) other cellular factors are required to permit productive FCV infection and (ii) individual FCV isolates differ in the factors they require.     

Molecular basis for the homophilic activated leukocyte cell adhesion molecule (ALCAM)-ALCAM interaction

Activated leukocyte cell adhesion molecule (ALCAM/CD166), a member of the immunoglobulin superfamily with five extracellular immunoglobulin-like domains, facilitates heterophilic (ALCAM-CD6) and homophilic (ALCAM-ALCAM) cell-cell interactions. While expressed in a wide variety of tissues and cells, ALCAM is restricted to subsets of cells usually involved in dynamic growth and/or migration processes. A structure-function analysis, using two monoclonal anti-ALCAM antibodies and a series of amino-terminally deleted ALCAM constructs, revealed that homophilic cell adhesion depended on ligand binding mediated by the membrane-distal amino-terminal immunoglobulin domain and on avidity controlled by ALCAM clustering at the cell surface involving membrane-proximal immunoglobulin domains. Co-expression of a transmembrane ALCAM deletion mutant, which lacks the ligand binding domain, and endogenous wild-type ALCAM inhibited homophilic cell-cell interactions by interference with ALCAM avidity, while homophilic, soluble ligand binding remained unaltered. The extracellular structures of ALCAM thus provide two structurally and functionally distinguishable modules, one involved in ligand binding and the other in avidity. Functionality of both modules is required for stable homophilic ALCAM-ALCAM cell-cell adhesion.     

An octapeptide in the juxtamembrane domain of VE-cadherin is important for p120ctn binding and cell proliferation

The cadherins are a family of adhesive proteins involved in cell-cell homophilic interactions. VE-cadherin, expressed in endothelial cells, is involved in morphogenesis, regulation of permeability, and cellular proliferation. The cytoplasmic tails of cadherins contain two major domains, the juxtamembrane domain that plays a role in the intercellular localization of the protein and also serves for binding of p120ctn, and a C-terminal domain that associates with beta- or gamma-catenin. A highly conserved region present in the juxtamembrane domain of the cadherins has been shown to be necessary for p120ctn binding in E-cadherin. Using a mutant VE-cadherin lacking a highly conserved octapeptide, we demonstrated that it is required for p120ctn binding to VE-cadherin as determined by immunoprecipitation and colocalization studies. By immunofluorescence, this mutant protein has a topographical distribution similar to that of the wild-type VE-cadherin and, therefore, we conclude that the topographical distribution of VE-cadherin is independent of this motif. In addition, although cell-cell association is present in cells expressing this mutant form of VE-cadherin, we found that the strength of adhesion is decreased. Finally, our results for the first time demonstrate that the interaction of VE-cadherin with p120 catenin plays an important role in cellular growth, suggesting that the binding of p120 catenin to cadherins may regulate cell proliferation.     

Structural Requirements for Outside-In and Inside-Out Signaling by Drosophila Neuroglian,a Member of the L1 Family of Cell Adhesion Molecules

Expression of the Drosophila cell adhesion molecule neuroglian in S2 cells leads to cell aggregation and the intracellular recruitment of ankyrin to cell contact sites. We localized the region of neuroglian that interacts with ankyrin and investigated the mechanism that limits this interaction to cell contact sites. Yeast two-hybrid analysis and expression of neuroglian deletion constructs in S2 cells identified a conserved 36-amino acid sequence that is required for ankyrin binding. Mutation of a conserved tyrosine residue within this region reduced ankyrin binding and extracellular adhesion. However, residual recruitment of ankyrin by this mutant neuroglian molecule was still limited to cell contacts, indicating that the lack of ankyrin binding at noncontact sites is not caused by tyrosine phosphorylation. A chimeric molecule, in which the extracellular domain of neuroglian was replaced with the corresponding domain from the adhesion molecule fasciclin II, also selectively recruited ankyrin to cell contacts. Thus, outside-in signaling by neuroglian in S2 cells depends on extracellular adhesion, but does not depend on any unique property of its extracellular domain. We propose that the recruitment of ankyrin to cell contact sites depends on a physical rearrangement of neuroglian in response to cell adhesion, and that ankyrin binding plays a reciprocal role in stabilizing the adhesive interaction.     

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.     

N-glycosylation is required for human CD2 immunoadhesion functions.

The T-lymphocyte glycoprotein receptor, CD2, mediates cell-cell adhesion by binding to the surface molecule CD58 (LFA-3) on many cell types including antigen presenting cells. Two domains comprise the CD2 extracellular segment, with all adhesion functions localized to the amino-terminal domain that contains a single N-glycosylation site at Asn65. We have defined an important role for the N-linked glycans attached to Asn65 of this domain in mediating CD2-CD58 interactions and also characterize its N-glycotype structure. Analysis of deglycosylated soluble recombinant CD2 as well as a mutant transmembrane CD2 molecule containing a single Asn65-Gln65 substitution demonstrates that neither deglycosylated CD2 nor the mutant CD2 transmembrane receptor binds CD58 or monoclonal antibodies directed at native CD2 adhesion domain epitopes. Electrospray ionization-mass spectrometry demonstrates that high mannose oligosaccharides ((Man)nGlcNAc2, n = 5-9) are the only N-glycotypes occupying Asn65 when soluble CD2 is expressed in Chinese hamster ovary cells. Based on a model of human CD2 secondary structure, we propose that N-glycosylation is required for stabilizing domain 1 in the human receptor. Thus, N-glycosylation is essential for human CD2 adhesion functions.     

The transmembrane domain of podoplanin is required for its association with lipid rafts and the induction of epithelial-mesenchymal transition

Podoplanin is a transmembrane glycoprotein that is upregulated in cancer and was reported to induce an epithelial-mesenchymal transition (EMT) in MDCK cells. The promotion of EMT was dependent on podoplanin binding to ERM (ezrin, radixin, moesin) proteins through its cytoplasmic (CT) domain, which led to RhoA-associated kinase (ROCK)-dependent ERM phosphorylation. Using detergent-resistant membrane (DRM) assays, as well as transmembrane (TM) interactions and ganglioside GM1 binding, we present evidence supporting the localization of podoplanin in raft platforms important for cell signalling. Podoplanin mutant constructs harbouring a heterologous TM region or lacking the CT tail were unable to associate with DRMs, stimulate ERM phosphorylation and promote EMT or cell migration. Similar effects were observed upon disruption of a GXXXG motif within the TM domain, which is involved in podoplanin self-assembly. In contrast, deletion of the extracellular (EC) domain did not affect podoplanin DRM association. Together, these data suggest that both the CT and TM domains are required for podoplanin localization in raft platforms, and that this association appears to be necessary for podoplanin-mediated EMT and cell migration.     

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.     

Attachment, spreading and locomotion of avian neural crest cells are mediated by multiple adhesion sites on fibronectin molecules.

Cellular adhesion to fibronectin (FN) can be mediated by several sequences located in different portions of the molecule. In human FN, these are: (i) the bipartite RGDS domain containing the RGDS cell-binding sequence functioning in synergy for full cellular adhesion with a second site (termed here the synergistic adhesion site) and (ii) the recently characterized CS1 and REDV adhesion sites within the alternatively-spliced type III homology-connecting segment. Using specific adhesive ligands and inhibitory probes, we have examined the role of each of these domains in the adhesion, spreading, and motility of avian neural crest cells in vitro. Both the RGDS domain and the CS1 adhesion site were found to promote attachment of neural crest cells, but only the RGDS domain supported their spreading. However, the RGDS sequence could mediate both attachment and spreading efficiently only when it was associated with the synergistic adhesion site. In migratory assays, it was found that both the RGDS domain and the CS1 site are required in association, each with functional specificity, to permit effective locomotion of neural crest cells. The REDV adhesion site was apparently not recognized by avian neural crest cells, presumably because this sequence is absent from chicken FN. Finally, it was found that recognition of both the RGDS domain and CS1 binding site by neural crest cells involved receptors belonging to the integrin family. From these results, we conclude that neural crest cells can interact with several binding sites of FN molecules, and use them for distinct functions. Our results also suggest the possibility of an instructive role for FN in the control of adhesive and migratory events during embryonic development.     

Structural requirements for CD43 function

The regulation of T cell activation and adhesion by CD43 (leukosialin, sialophorin) has been thought to be mainly a function of the large size and negative charge of the extracellular domain of the protein. In this work, we demonstrate that the cytoplasmic tail is both necessary and sufficient for the negative regulatory effect of CD43 on cell-cell adhesion. Expression of mutant CD43 proteins in primary T cells from CD43-deficient mice demonstrated that the antiproliferative effect of CD43 is also dependent upon the cytoplasmic tail. In contrast, Ab-mediated costimulation through CD43 does not require the intracellular domain of CD43. These data demonstrate that CD43 primarily serves as a negative regulator of T cell activation and adhesion, and that this is mediated not exclusively by passive effects of the extracellular domain, but requires participation of the cytoplasmic tail, perhaps through interactions with the cytoskeleton, or alternatively, active regulation of intracellular signaling pathways.     

Ankyrin-binding domain of CD44(GP85) is required for the expression of hyaluronic acid-mediated adhesion function

GP85 is one of the most common hemopoietic isoforms of the cell adhesion molecule, CD44. CD44(GP85) is known to contain at least one ankyrin-binding site within its 70 aa cytoplasmic domain and to bind hyaluronic acid (HA) with its extracellular domain. In this study we have mapped the ankyrin-binding domain of CD44(GP85) by deleting various portions of the cytoplasmic region followed by expression of these truncated cDNAs in COS cells. The results of these experiments indicate that the ankyrin-binding domain resides between amino acids 305 and 355. Biochemical analyses, using competition binding assays and a synthetic peptide (NGGNGT-VEDRKPSEL) containing 15 aa between aa 305 and aa 320, support the conclusion that this region is required for ankryin binding. Furthermore, we have constructed a fusion protein in which this 15 aa sequence of CD44(GP85) is transplanted onto another transmembrane protein which does not bind ankyrin. Our results show that this fusion protein acquires the ability to bind ankyrin confirming that the sequence (306NGGNGTVEDRKPSE320L) is a critical part of the ankryin-binding domain of CD44(GP85). In addition, we have demonstrated that deletion of this 15 aa ankyrin-binding sequence from CD44(GP85) results in a drastic reduction (> or = 90%) of HA-binding and HA-mediated cell adhesion. These findings strongly suggest that ankyrin binding to the cytoplasmic domain of CD44(GP85) plays a pivotal role in regulating hyaluronic acid-mediated cell-cell and cell- extracellular matrix interactions.     

Interaction between CD44 and the repeat domain of ankyrin promotes hyaluronic acid-mediated ovarian tumor cell migration

The adhesion molecule, CD44, interacts with ankyrin within its cytoplasmic domain and binds to hyaluronic acid (HA) at its extracellular domain. In this study, we focused on the functional domain in ankyrin (in particular, the ankyrin repeat domain [ARD]) responsible for CD44 binding and its role in regulating HA-mediated ovarian tumor cell function. Using recombinant fragments of ankyrin (e.g., ARD and subdomain 1 [S1, aa1-aa217], subdomain 2 [S2, aa218-aa381], subdomain 3 [S3, aa382-aa612], and subdomain 4 [S4, aa613-aa834]) and in vitro binding assays, we determined that the S2 but not S1, S3, or S4 of ARD is the primary ankyrin binding region for CD44. Microinjection of antiglutathione S-transferase (GST)-tagged S2 or GST-tagged ARD fusion protein into CD44-positive ovarian tumor cells (e.g., SKOV3 cell line) promotes ankyrin association with CD44 in plaque-like structures and membrane projections. Additionally, we demonstrated that transfection of SKOV3 cells with S2cDNA or ARD cDNA results in an upregulation of HA-mediated tumor cell migration. Taken together, we believe that the S2 of the ARD plays a pivotal role in the direct binding to CD44 and promotes the cytoskeleton activation required for HA-mediated function such as ovarian tumor cell migration.     

Separating the adhesive and signaling functions of the Fat and Dachsous protocadherins

The protocadherins Fat (Ft) and Dachsous (Ds) are required for several processes in the development of Drosophila, including controlling growth of imaginal discs, planar cell polarity (PCP) and the proximodistal patterning of appendages. Ft and Ds bind in a preferentially heterophilic fashion, and Ds is expressed in distinct patterns along the axes of polarity. It has thus been suggested that Ft and Ds serve not as adhesion molecules, but as receptor and ligand in a poorly understood signaling pathway. To test this hypothesis, we performed a structure-function analysis of Ft and Ds, separating their adhesive and signaling functions. We found that the extracellular domain of Ft is not required for its activity in growth, PCP and proximodistal patterning. Thus, ligand binding is not necessary for Ft activity. By contrast, the extracellular domain of Ds is necessary and sufficient to mediate its effects on PCP, consistent with the model that Ds acts as a ligand during PCP. However, we also provide evidence that Ds can regulate growth independently of Ft, and that the intracellular domain of Ds can affect proximodistal patterning, both suggestive of functions independent of binding Ft. Finally, we show that ft mutants or a dominant-negative Ft construct can affect disc growth without changes in the expression of wingless and Wingless target genes.     

Neuregulin-heparan-sulfate proteoglycan interactions produce sustained erbB receptor activation required for the induction of acetylcholine receptors in muscle

Neuregulins bind to and activate members of the EGF receptor family of tyrosine kinases that initiate a signaling cascade that induces acetylcholine receptor synthesis in the postsynaptic membrane of neuromuscular synapses. In addition to an EGF-like domain, sufficient for receptor binding and tyrosine auto-phosphorylation, many spliced forms also have an IG-like domain that binds HSPGs and maintains a high concentration of neuregulin at synapses. Here, we show that the IG-like domain functions to keep the EGF-like domain at sufficiently high concentrations for a sufficiently long period of time necessary to induce acetylcholine receptor gene expression in primary chick myotubes. Using recombinant neuregulins with and without the IG-like domain, we found that IG-like domain binding to endogenous HSPGs produces a 4-fold increase in receptor phosphorylation. This enhancement of activity was blocked by soluble heparin or by pretreatment of muscle cells with heparitinase. We show that at least 12-24 h of neuregulin exposure was required to turn on substantial acetylcholine receptor gene expression and that the erbB receptors need to be kept phosphorylated during this time. The need for sustained erbB receptor activation may be the reason why neuregulins are so highly concentrated in the extracellular matrix of synapses.     

Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells

Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomitantly inducing the loss of Nanog and Oct4 self-renewal markers. Conversely, reducing fibronectin production by mES cells growing on a feeder-free gelatin substrate caused loss of cell adhesion, decreased integrin signaling, and decreased expression of self-renewal markers. These effects were reversed by providing the cells with exogenous fibronectin, thereby restoring adhesion to the gelatin substrate. Interestingly, mES cells do not adhere directly to the gelatin substrate, but rather adhere indirectly through gelatin-bound fibronectin, which facilitates self-renewal via its effects on cell adhesion. These results provide new insights into the mechanism of regulation of self-renewal by growth on a gelatin-coated surface. The effects of increasing or decreasing fibronectin levels show that self-renewal depends on an intermediate level of cell-fibronectin interactions. By providing cell adhesive signals that can act with other self-renewal factors to maintain mES cell pluripotency, fibronectin is therefore a necessary component of the self-renewal signaling pathway in culture.     

Effect of FGF-binding protein 3 on vascular permeability

Fibroblast growth factor-binding protein 1 (FGF-BP1 is BP1) is involved in the regulation of embryonic development, tumor growth, and angiogenesis by mobilizing endogenous FGFs from their extracellular matrix storage. Here we describe a new member of the FGF-BP family, human BP3. We show that the hBP3 protein is secreted from cells, binds to FGF2 in vitro and in intact cells, and inhibits FGF2 binding to heparin. To determine the function of hBP3 in vivo, hBP3 was transiently expressed in chicken embryos and resulted in > 50% lethality within 24 h because of vascular leakage. The onset of vascular permeability was monitored by recording the extravasation kinetics of FITC-labeled 40-kDa dextran microperfused into the vitelline vein of 3-day-old embryos. Vascular permeability increased as early as 8 h after expression of hBP3. The increased vascular permeability caused by hBP3 was prevented by treatment of embryos with PD173074, a selective FGFR kinase inhibitor. Interestingly, a C-terminal 66-amino acid fragment (C66) of hBP3, which contains the predicted FGF binding domain, still inhibited binding of FGF2 to heparin similar to full-length hBP3. However, expression of the C66 fragment did not increase vascular permeability on its own, but required the administration of exogenous FGF2 protein. We conclude that the FGF binding domain and the heparin binding domain are necessary for the hBP3 interaction with endogenous FGF and the activation of FGFR signaling in vivo.     

Phosphoinositide binding by the disabled-1 PTB domain is necessary for membrane localization and Reelin signal transduction

Disabled-1 (Dab1) is an essential adaptor protein that functions in the Reelin signaling pathway and is required for the regulation of neuronal migration during embryonic development. Dab1 interacts with NPXY motifs in the cytoplasmic tails of the lipoprotein receptors ApoER2 and very low density lipoprotein receptor through an amino-terminal phosphotyrosine binding (PTB) domain. Binding of Reelin to these receptors leads to tyrosine phosphorylation of Dab1 and the initiation of a signaling cascade that results in remodeling of the cytoskeleton. Structural and biochemical studies of the Dab1 PTB domain have demonstrated that this domain binds to both the NPXY peptide motif in the lipoprotein receptor tails as well as to the head group of phosphoinositide 4,5-P2 through energetically independent mechanisms. Here we have investigated how phosphoinositide binding by the Dab1 PTB domain influences Reelin signal transduction. Our findings in cultured primary neurons that have been transduced with lentiviral constructs expressing mutant Dab1 forms reveal that phosphoinositide binding by the Dab1 PTB domain is necessary for proper membrane localization of Dab1 and for effective transduction of a Reelin signal.