heparitin-sulfate and arginyl-glycyl-aspartyl-serine

Previous studies have demonstrated that the alpha v beta 5 integrin receptor functions in the endocytosis and degradation of matrix-bound vitronectin by human skin fibroblasts (Panetti, T. S., and McKeown-Longo, P. J. (1993) J. Biol. Chem. 268, 11988-11993; Panetti, T. S., and McKeown-Longo, P. J. (1993) J. Biol. Chem. 268, 11492-11495). These earlier studies demonstrated that vitronectin degradation was inhibited by either antibodies to the beta 5 integrin or exogenous heparin, suggesting that both integrin receptors and cell surface heparan sulfate proteoglycans are involved in the endocytosis and degradation of vitronectin. The present study was done to define intracellular signaling pathways involved in endocytosis of vitronectin and to evaluate the relative contribution of cell surface heparan sulfate proteoglycans and the alpha v beta 5 integrin in the activation of these signaling pathways. The addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, to monolayers of human skin fibroblasts, increased vitronectin degradation. Staurosporine and calphostin C, inhibitors of protein kinase C, blocked internalization and subsequent degradation of vitronectin, while KT5720, an inhibitor of protein kinase A, had no effect on the degradation of vitronectin. PMA was also able to reverse the inhibition of vitronectin degradation seen when cells were pretreated with heparinase or incubated with exogenous heparin. In contrast, the inhibitory effect of either RGD peptides or anti-alpha v beta 5 antibodies on vitronectin degradation were not overcome by the addition of PMA. These data suggest that the internalization of vitronectin from the matrix is mediated by the alpha v beta 5 integrin following activation of protein kinase C.

Topics: Biological Transport; Cells, Cultured; Endocytosis; Fibroblasts; Glycoproteins; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Integrins; Naphthalenes; Oligopeptides; Protein Kinase C; Proteoglycans; Receptors, Vitronectin; Signal Transduction; Skin Physiological Phenomena; Tetradecanoylphorbol Acetate; Vitronectin

Perlecan has been previously been shown to support attachment of a wide variety of cells through interactions of its core protein with the cell surface. The core protein domains involved in cell adhesion are, however, unknown. The laminin-like domain III of murine perlecan contains an RGDS sequence and is a likely candidate for supporting integrin-mediated cell attachment. We made a cDNA construct corresponding to domain III and containing an in frame signal peptide at the 5' end as well as in frame a stop codon at the 3' end by using cDNA clones to perlecan. The construct was inserted into the pRC/CMV vector and transfected into HT1080 cells, and the secreted recombinant domain III, a 130-kDa protein, was purified from the medium. The size of proteolytic fragments produced by digestion with V8 protease as well as analysis of the rotary shadowed image of the recombinant protein indicated it was produced in a native conformation. Recombinant domain III coated on tissue culture dishes, supports adhesion of an epithelial-like mouse mammary tumor cell line MMT 060562 in a dose-dependent manner. This interaction was inhibited specifically by the RGDS synthetic peptide and intact perlecan, but not laminin. This domain III RGD-dependent cell attachment activity indicates a role for perlecan in integrin-mediated signaling.

Topics: Amino Acid Sequence; Cell Adhesion; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Molecular Sequence Data; Oligopeptides; Protein Structure, Secondary; Proteoglycans; Recombinant Proteins; Transfection; Tumor Cells, Cultured

Subclones of F11 neuronal hybrid cells (neuroblastoma x dorsal root ganglion neurons) have segregated differing and/or overlapping neuritogenic mechanisms on three substrata--plasma fibronectin (pFN) with its multiple receptor activities, cholera toxin B subunit (CTB) for binding to ganglioside GM1, and platelet factor-4 (PF4) for binding to heparan sulfate proteoglycans. In this study, specific cell surface receptor activities for the three substrata were tested for their modulation during neuritogenesis by several experimental paradigms, using F11 subclones representative of three differentiation classes (neuritogenic on pFN only, on CTB only, or on all three substrata). When cycloheximide was included in the medium to inhibit protein synthesis during the active period, neurite formation increased significantly for all subclones on all three substrata, virtually eliminating substratum selectivity for differentiation mediated by cell surface integrin, ganglioside GM1, or heparan sulfate proteoglycans. Therefore, one or more labile proteins (referred to as disintegrins) must modulate functions of matrix receptors (e.g., integrins) mediating neurite formation. To verify whether cycloheximide-induced neuritogenesis was also regulated by integrin interaction with cell surface GM1, two approaches were used. When (Arg-Gly-Asp-Ser)-containing peptide A was added to the medium, it completely inhibited cycloheximide-induced neuritogenesis on all three substrata of all subclones, indicating stringent requirement for cell surface integrin function in these mechanisms. In contrast, when CTB or a monoclonal anti-GM1 antibody was also added to the medium, cycloheximide-induced neuritogenesis was amplified further on pFN and sensitivity to peptide A inhibition was abolished. Therefore, in some contexts ganglioside GM1 must complex with integrin receptors at the cell surface to modulate their function. These results also indicate that (a) cycloheximide treatment leads to loss of substratum selectivity in neuritogenesis, (b) this negative regulation of neurite outgrowth is affected by integrin receptor association with labile regulatory proteins (disintegrins) as well as with GM1, and (c) complexing of GM1 by multivalent GM1-binding proteins shifts neuritogenesis from an RGDS-dependent integrin mechanism to an RGDS-independent receptor mechanism.

Topics: Amino Acid Sequence; Animals; Axons; Cell Differentiation; Cell Line; Cholera Toxin; Chondroitin Sulfate Proteoglycans; Cycloheximide; Fibronectins; G(M1) Ganglioside; Ganglia, Spinal; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Hybrid Cells; Integrins; Mice; Molecular Sequence Data; Neuroblastoma; Neurons; Oligopeptides; Platelet Factor 4; Rats; Receptors, Fibronectin; Receptors, Immunologic

We have examined the molecular interactions of avian neural crest cells with fibronectin and laminin in vitro during their initial migration from the neural tube. A 105-kDa proteolytic fragment of fibronectin encompassing the defined cell-binding domain (65 kDa) promoted migration of neural crest cells to the same extent as the intact molecule. Neural crest cell migration on both intact fibronectin and the 105-kDa fragment was reversibly inhibited by RGD-containing peptides. The 11.5-kDa fragment containing the RGDS cell attachment site was also able to support migration, whereas a 50-kDa fragment corresponding to the adjacent N-terminal portion of the defined cell-binding domain was unfavorable for neural crest cell movement. In addition to the putative "cell-binding domain," neural crest cells were able to migrate on a 31-kDa fragment corresponding to the C-terminal heparin-binding (II) region of fibronectin, and were inhibited in their migration by exogenous heparin, but not by RGDS peptides. Heparin potentiated the inhibitory effect of RGDS peptides on intact fibronectin, but not on the 105-kDa fragment. On substrates of purified laminin, the extent of avian neural crest cell migration was maximal at relatively low substrate concentrations and was reduced at higher concentrations. The efficiency of laminin as a migratory substrate was enhanced when the glycoprotein occurred complexed with nidogen. Moreover, coupling of the laminin-nidogen complex to collagen type IV or the low density heparan sulfate proteoglycan further increased cell dispersion, whereas isolated nidogen or the proteoglycan alone were unable to stimulate migration and collagen type IV was a significantly less efficient migratory substrate than laminin-nidogen. Neural crest cell migration on laminin-nidogen was not affected by RGDS nor by YIGSR-containing peptides, but was reduced by 35% after addition of heparin. The predominant motility-promoting activity of laminin was localized to the E8 domain, possessing heparin-binding activity distinct from that of the N-terminal E3 domain. Migration on the E8 fragment was reduced by greater than 70% after addition of heparin. The E1' fragment supported a minimal degree of migration that was RGD-sensitive and heparin-insensitive, whereas the primary heparin-binding E3 fragment and the cell-adhesive P1 fragment were entirely nonpermissive for cell movement.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Binding Sites; Cell Movement; Cells, Cultured; Chondroitin Sulfate Proteoglycans; Collagen; Fibronectins; Heparan Sulfate Proteoglycans; Heparin; Heparitin Sulfate; Laminin; Membrane Glycoproteins; Membrane Proteins; Neural Crest; Oligopeptides; Peptide Fragments; Platelet Factor 4; Quail

Human neuroblastoma cells (Platt and La-N1) adhere and extend neurites on a ganglioside GM1-binding substratum provided by cholera toxin B (CTB). These adhesive responses, similar to those on plasma fibronectin (pFN), require the mediation of one or more cell-surface proteins [G. Mugnai and L. A. Culp (1987) Exp. Cell Res. 169, 328]. The involvement of two pFN receptor molecules in ganglioside GM1-mediated responses on CTB have now been tested. In order to test the role of cellular FN binding to its glycoprotein receptor integrin, a soluble peptide containing the Arg-Gly-Asp-Ser (RGDS) sequence was added to the medium. It did not inhibit attachment on CTB but completely inhibited formation of neurites; in contrast, the RGDS peptide minimally inhibited attachment or neurite formation on pFN. Once formed, neurites on CTB became resistant to the peptide. In order to test the role of cell-surface heparan sulfate proteoglycan (HS-PG), two approaches were used. First, the HS-binding protein platelet factor-4 (PF4) was used to dilute CTB or pFN on the substratum or, alternatively, added to the medium. Diluting the substratum ligand with PF4 had no effects on attachment on either CTB or pFN. However, neurite formation on CTB was readily inhibited and on pFN partially inhibited; the effects of PF4 were far greater than a similar dilution with nonbinding albumin. When PF4 was added to the medium of cells, attachment on either substratum was unaffected as was neurite outgrowth on pFN, revealing differences in PF4's inhibition as the substratum-bound or medium-borne component. In contrast, PF4 in the medium at low concentrations (1 microgram/ml) was highly inhibitory for neurite formation on CTB. The second approach utilized the addition of bovine cartilage dermatan sulfate proteoglycan (DS-PG), shown to bind to pFN as well as to substratum-bound CTB by ELISA, or cartilage chondroitin sulfate/keratan sulfate proteoglycan (CS/KS-PG) to the substratum or to the medium. At low concentrations, DS-PG but not CS/KS-PG actually stimulated neurite formation on CTB while at higher concentrations DS-PG completely inhibited attachment and neurite formation. While DS-PG partially inhibited attachment on pFN, it had no effect on neurite formation of the attached cells. Neuroblastoma cells adhered to some extent to substrata coated only with DS-PG, indicating "receptors" for PGs that permit stable interaction.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Axons; Cell Adhesion; Cholera Toxin; Chondroitin Sulfate Proteoglycans; Dermatan Sulfate; Enzyme-Linked Immunosorbent Assay; Fibronectins; G(M1) Ganglioside; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Neuroblastoma; Neurons; Oligopeptides; Platelet Aggregation Inhibitors; Proteoglycans; Receptors, Fibronectin; Receptors, Immunologic; Tumor Cells, Cultured