Forced unfolding modulated by disulfide bonds in the Ig domains of a cell adhesion molecule.
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Cell adhesion molecules (CAMs) mediate cell attachment and stress transfer through extracellular domains. Here we forcibly unfold the Ig domains of a prototypical Ig superfamily CAM that contains intradomain disulfide bonds. The Ig domains of all such CAMs have conformations homologous to cadherin extracellular domains, titin Ig-type domains, and fibronectin type-III (FNIII) domains. Atomic force microscopy has been used to extend the five Ig domains of Mel-CAM (melanoma CAM)--a protein that is overexpressed in metastatic melanomas--under conditions where the disulfide bonds were either left intact or disrupted through reduction. Under physiological conditions where intradomain disulfide bonds are intact, partial unfolding was observed at forces far smaller than those reported previously for either titin's Ig-type domains or tenascin's FNIII domains. This partial unfolding under low force may be an important mechanism for imparting elasticity to cell-cell contacts, as well as a regulatory mechanism for adhesive interactions. Under reducing conditions, Mel-CAM's Ig domains were found to fully unfold through a partially folded state and at slightly higher forces. The results suggest that, in divergent evolution of all such domains, stabilization imparted by disulfide bonds relaxes requirements for strong, noncovalent, folded-state interactions. (+info)
HEMCAM/CD146 downregulates cell surface expression of beta1 integrins.
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HEMCAM/gicerin, an immunoglobulin superfamily protein, is involved in homophilic and heterophilic adhesion. It interacts with NOF (neurite outgrowth factor), a molecule of the laminin family. Alternative splicing leads to mRNAs coding for HEMCAM with a short (HEMCAM-s) or a long cytoplasmic tail (HEMCAM-l). To investigate the cellular function of these two variants, we stably transfected murine fibroblasts with either form of HEMCAM. Expression of each isoform of this protein in L cells delayed proliferation and modified their adhesion properties to purified extracellular matrix proteins. Expression of either HEMCAM-s or HEMCAM-l inhibited integrin-dependent adhesion and spreading of fibroblasts to laminin 1, showing that this phenomenon did not depend on the cytoplasmic region. By contrast, L-cell adhesion and spreading to fibronectin depended on the HEMCAM isoform expressed. Flow cytometry and immunoprecipitation studies revealed that the expression of HEMCAM downregulated expression of the laminin-binding integrins alpha3beta1, alpha6beta1 and alpha7beta1, and fibronectin receptor alpha5beta1 from the cell surface. Semi-quantitative PCR and northern blot experiments showed that the expression of alpha6beta1 integrin modified by HEMCAM occurred at a translation or maturation level. Thus, our data demonstrate that HEMCAM regulates fibroblast adhesion by controlling beta1 integrin expression. (+info)
Mel-CAM-specific genetic suppressor elements inhibit melanoma growth and invasion through loss of gap junctional communication.
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Normal human melanocytes are interspersed singly among keratinocytes along the basement membrane of the epidermis, whereas melanoma cells readily adhere to each other during invasion of the dermis or distant organs. The tumorigenic and metastatic phenotype of melanoma cells often correlates with increased expression of cell-cell and cell-matrix adhesion receptors. Mel-CAM (MCAM, MUC 18, CD146) is a cell-cell adhesion receptor highly expressed by melanoma cells but not normal melanocytes. We show here that inhibition of Mel-CAM expression in metastatic melanoma cells using genetic suppressor elements of Mel-CAM cDNA leads to inhibition of adhesion between melanoma cells and to downregulation of the tumorigenic phenotype. Growth was not inhibited in genetic suppressor elements-transduced melanoma cells cultured in monolayers but was inhibited when cells were maintained anchorage-independently in soft agar and greatly reduced in immunodeficient mice. A three-dimensional epidermal skin equivalent model demonstrated that Mel-CAM allows melanoma cells to separate from the epidermis and invade the basement membrane zone and dermis. However, melanoma cells with little or no Mel-CAM were poorly invasive, possibly due to their loss of gap junctional communication. These results suggest the multifunctional role of a melanoma-associated cell-cell adhesion receptor in tumor progression. (+info)
Identification of a role for the sialomucin CD164 in myogenic differentiation by signal sequence trapping in yeast.
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Determination and differentiation of skeletal muscle precursors requires cell-cell contact, but the full range of cell surface proteins that mediate this requirement and the mechanisms by which they work are not known. To identify participants in cell contact-mediated regulation of myogenesis, genes that encode secreted proteins specifically upregulated during differentiation of C2C12 myoblasts were identified by the yeast signal sequence trap method (K. A. Jacobs, L. A. Collins-Racie, M. Colbert, M. Duckett, M. Golden-Fleet, K. Kelleher, R. Kriz, E. R. La Vallie, D. Merberg, V. Spaulding, J. Stover, M. J. Williamson, and J. M. McCoy, Gene 198:289-296, 1997), followed by RNA expression analysis. We report here the identification of CD164 as a gene expressed in proliferating C2C12 cells that is upregulated during differentiation. CD164 encodes a widely expressed cell surface sialomucin that has been implicated in regulation of cell proliferation and adhesion during hematopoiesis. Stable overexpression of CD164 in C2C12 and F3 myoblasts enhanced their differentiation, as assessed by both morphological and biochemical criteria. Furthermore, expression of antisense CD164 or soluble extracellular regions of CD164 inhibited myogenic differentiation. Treatment of C2C12 cells with sialidase or O-sialoglycoprotease, two enzymes previously reported to destroy functional epitopes on CD164, also inhibited differentiation. These data indicate that (i) CD164 may play a rate-limiting role in differentiation of cultured myoblasts, (ii) sialomucins represent a class of potential effectors of cell contact-mediated regulation of myogenesis, and (iii) carbohydrate-based cell recognition may play a role in mediating this phenomenon. (+info)
Competing sorting signals guide endolyn along a novel route to lysosomes in MDCK cells.
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We have examined the trafficking of the mucin-like protein endolyn in transfected, polarized MDCK cells using biochemical approaches and immunofluorescence microscopy. Although endolyn contains a lysosomal targeting motif of the type YXXPhi and was localized primarily to lysosomes at steady state, significant amounts of newly synthesized endolyn were delivered to the apical cell surface. Antibodies to endolyn, but not lamp-2, were preferentially internalized from the apical plasma membrane and efficiently transported to lysosomes. Analysis of endolyn-CD8 chimeras showed that the lumenal domain of endolyn contains apical targeting information that predominates over basolateral information in its cytoplasmic tail. Interestingly, surface polarity of endolyn was independent of O-glycosylation processing, but was reversed by disruption of N-glycosylation using tunicamycin. At all times, endolyn was soluble in cold Triton X-100, suggesting that apical sorting was independent of sphingolipid rafts. Our data indicate that a strong, N-glycan-dependent apical targeting signal in the lumenal domain directs endolyn into a novel biosynthetic pathway to lysosomes, which occurs via the apical surface of polarized epithelial cells. (+info)
Identification of CD146 as a component of the endothelial junction involved in the control of cell-cell cohesion.
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CD146 is a cell-surface molecule belonging to the immunoglobulin superfamily and expressed in all types of human endothelial cells. Confocal and electron microscopic analysis of confluent human umbilical vein endothelial cells (HUVECs) were used to demonstrate that CD146 is a component of the endothelial junction. Double immunolabeling with vascular endothelial cadherin showed that CD146 is localized outside the adherens junction. Moreover, CD146 expression is not restricted to the junction, since part of the labeling was detectable at the apical side of the HUVECs. Interestingly, cell-surface expression of CD146 increased when HUVECs reached confluence. In addition, the paracellular permeability of CD146-transfected fibroblast cells was decreased compared with that of control cells. Finally, CD146 colocalized with actin, was partly resistant to Triton X-100 extraction, and had its expression altered by actin-disrupting agents, indicating that CD146 is associated with the actin cytoskeleton. These results show the regulated expression of CD146 at areas of cell-cell junction and strongly suggest involvement of CD146 as a mediator of cell-cell interaction. (+info)
Expression and adhesive ability of gicerin, a cell adhesion molecule, in the pock lesions of chorioallantoic membranes infected with an avian poxvirus.
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The expression and adhesive activities of gicerin, a cell adhesion protein, in the pock lesions on chicken chorioallantoic membranes (CAM) infected with an avian poxvirus were studied. In normal CAMs, gicerin was found on the flattened epithelial cells, and neurite outgrowth factor (NOF) was in the basement membrane. However, in the pock lesions on infected CAMs, gicerin was overexpressed on the cell membranes of hyperplastic epithelial cells forming thick epithelial layers. Neurite outgrowth factor was also found mainly in the basement membrane, but occasionally showed aberrant expression among hyperplastic cells. In vitro analyses, using the dissociated cells from pock lesions, demonstrated that an anti-gicerin polyclonal antibody inhibit cell aggregation activity and cell adhesion to NOF. These results suggest that gicerin might promote the cell-cell and cell-extracellular matrix protein bindings of the hyperplastic epithelial cells by its homophilic and heterophilic adhesive activities, and contribute to pock formation on the infected CAMs. (+info)
Melanoma Cell Adhesion Molecule (MCAM) expression in the myogenic lineage during early chick embryonic development.
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We describe the expression pattern of cMCAM, a cell adhesion molecule of the immunoglobulin superfamily, in early chick embryonic development by in situ hybridisation. An initial ectodermal domain of expression is subsequently expanded, and cMCAM is expressed in the neural crest cells, otic vesicle, heart primordium, notochord and endoderm. In addition, cMCAM expression localises in the myotome once the somite cells have been specified. An in vitro murine cellular system allowed us to confirm that MCAM expression coincides with the onset of myogenic cell determination. (+info)