Search PubMed⌕ Search

Biomedical subjects

E Ruoslahti

Publications and source records attributed to E Ruoslahti.

At least 163 records · Page 9Linked to original sources
PubMed1988

Regulation of the fibronectin receptor affinity by divalent cations.

The cell surface receptor for fibronectin is a heterodimeric membrane protein that recognizes an Arg-Gly-Asp sequence in fibronectin and that requires cations such as Mg2+ or Ca2+ for binding to fibronectin. The divalent cation requirements of this receptor were analyzed by measuring attachment of receptor liposomes to ligand-coated surfaces in the presence of different cations. The most striking effect observed was produced by Mn2+, which increased the binding of the receptor liposomes to fibronectin 2-3-fold over their binding in buffers containing Ca2+ and Mg2+. The binding activities of two related adhesion receptors, the vitronectin receptor and platelet GP IIb-IIIa, were supported but not enhanced by Mn2+. Two observations suggest that Mn2+ can compete with Ca2+ for the same cation-binding sites of the receptor. First, Mn2+ could still enhance fibronectin receptor binding activity even in the presence of 10-fold higher concentrations of Ca2+ or Mg2+. Second, Mn2+ inhibited the binding of radioactive Ca2+ to the alpha subunit of the receptor. The increased fibronectin receptor activity in the presence of Mn2+ appeared to be due to an increase in the affinity of the receptor for the Arg-Gly-Asp sequence because a 110-kDa cell attachment fragment and a synthetic hexapeptide containing the Arg-Gly-Asp sequence inhibited liposome binding more effectively in the presence of Mn2+ than in the presence of Ca2+/Mg2+. The affinity for the peptide was affected more than the affinity for the fragment, indicating that Mn2+ also induces a change in receptor specificity. Increased receptor binding in the presence of Mn2+ was also apparent in affinity chromatography of the fibronectin receptor on the 110-kDa fibronectin fragment; Mn2+ improved the yield of the receptor 4-fold. Mn2+ similarly increased the number of receptor-fibronectin complexes in preparations analyzed by electron microscopy. These results show that exogenous influences can modulate the affinity and specificity with which the fibronectin receptor binds to its ligands.

Calcium↗
PubMed1988

The human laminin receptor is a member of the integrin family of cell adhesion receptors.

A receptor for the adhesive basement membrane protein, laminin, was isolated from human glioblastoma cells by affinity chromatography on laminin. This receptor has a heterodimeric structure similar to that of receptors for other extracellular matrix proteins such as fibronectin and vitronectin. Incorporation of the laminin receptor into liposomal membranes makes it possible for liposomes to attach to surfaces coated with laminin. The receptor liposomes also attached to some extent to surfaces coated with fibronectin, but not with other matrix proteins. These properties identify the laminin receptor as a member of the integrin family of cell adhesion receptors.

Antigens, Surface↗
PubMed1988

An adhesion variant of the MG-63 osteosarcoma cell line displays an osteoblast-like phenotype.

MG-63 human osteosarcoma cells were selected for attachment and growth in increasing concentrations of a synthetic peptide containing the cell attachment-promoting Arg-Gly-Asp (RGD) sequence derived from the cell-binding region of fibronectin. Cells capable of attachment and growth in 5 mM concentrations of a peptide having the sequence Gly-Arg-Gly-Asp-Ser-Pro overproduce the cell surface receptor for fibronectin. No increase in fibronectin receptor gene copy number was detected by Southern blot analysis. The peptide-resistant MG-63.3A cells look very different from the MG-63 cells and resemble osteocytes. The resistant cells also grow more slowly than MG-63 cells. The enhanced expression of the fibronectin receptor on the resistant cells indicates that cells can regulate the amount of this receptor on their surface in response to environmental factors and that this may affect the phenotypic properties of the cell. MG-63.3A cells differ from MG-63 cells in their ability to form a calcified matrix in vitro and in their increased synthesis of type I collagen. The MG-63.3A cells synthesize 50-100-fold less prostaglandin E2, a mediator of bone resorption, than MG-63 cells. There is an overall down-regulation of chondroitin sulphate proteoglycans in MG-63.3A cells. These results are consistent with the hypothesis that such proteoglycans interfere with calcium phosphate deposition and with the observation that chondroitin sulphate is increased in a wide variety of neoplasms but is absent or in small amounts in normal tissue. We conclude that MG-63.3A cells represent a more differentiated cell type with osteoblast-like properties.

Cell Adhesion↗
PubMed1988

The neurite-promoting domain of human laminin promotes attachment and induces characteristic morphology in non-neuronal cells.

The interaction of cells with laminin and laminin fragments was studied in short-term cell attachment assays. Neurite-promoting chymotrypsin fragments of laminin were isolated using a monoclonal antibody which blocks neurite outgrowth on laminin. The fragments were shown, by electron microscopy after rotary shadowing and by immunological reactivity with different monoclonal antibodies, to contain only the distal end of the long arm. These fragments promoted the attachment and spreading of glioma, sarcoma, carcinoma, muscle, and endodermal cells to the same extent as intact laminin. The attachment was unaffected by peptides containing the RGD sequence. The morphology of the cells on the chymotrypsin fragments was indistinguishable from that on intact laminin but different from the morphology of the same cells on fibronectin. Light microscopy and scanning electron microscopy showed extensive process formation on laminin but not on fibronectin suggestive of increased cell motility in response to laminin. We conclude that the neurite-promoting domain of laminin contains a major site of interaction for non-neuronal cells and that this site induces a cellular response in certain non-neuronal cells that is unique to laminin.

Animals↗
PubMed1988

Inhibition of in vitro tumor cell invasion by Arg-Gly-Asp-containing synthetic peptides.

The interaction of cells with extracellular matrix components such as fibronectin, vitronectin, and type I collagen has been shown to be mediated through a family of cell-surface receptors that specifically recognize an arginine-glycine-aspartic acid (RGD) amino acid sequence within each protein. Synthetic peptides containing the RGD sequence can inhibit these receptor-ligand interactions. Here, we use novel RGD-containing synthetic peptides with different inhibition properties to investigate the role of the various RGD receptors in tumor cell invasion. The RGD-containing peptides used include peptides that inhibit the attachment of cells to fibronectin and vitronectin, a peptide that inhibits attachment to fibronectin but not to vitronectin, a cyclic peptide with the opposite specificity, and a peptide, GRGDTP, that inhibits attachment to type I collagen in addition to inhibiting attachment to fibronectin and vitronectin. The penetration of two human melanoma cell lines and a glioblastoma cell line through the human amniotic basement membrane and its underlying stroma was inhibited by all of the RGD-containing peptides except for the one that inhibits only the vitronectin attachment. Various control peptides lacking RGD showed essentially no inhibition. This inhibitory effect on cell invasion was dose-dependent and nontoxic. A hexapeptide, GRGDTP, that inhibits the attachment of cells to type I collagen in addition to inhibiting fibronectin- and vitronectin-mediated attachment was more inhibitory than those RGD peptides that inhibit only fibronectin and vitronectin attachment. Analysis of the location of these cells that were prevented from invading indicated that they attached to the amniotic basement membrane but did not proceed further into the tissue. These results suggest that interactions between RGD-containing extracellular matrix adhesion proteins and cells are necessary for cell invasion through tissues and that fibronectin and type I collagen are important for this process.

Amino Acid Sequence↗
PubMed1988

Cell surface distribution of fibronectin and vitronectin receptors depends on substrate composition and extracellular matrix accumulation.

We used antibodies against the alpha subunits of the human fibronectin receptor (FNR) and vitronectin receptor (VNR) to localize simultaneously FNR and VNR at major substrate adhesion sites of fibroblasts and melanoma cells with double-label immunofluorescence microscopy. In early (2-6-h) serum-containing cultures, both FNR and VNR coaccumulated in focal contacts detected by interference reflection microscopy. Under higher resolution immunoscanning electron microscopy, FNR and VNR were also observed to be distributed randomly on the dorsal cell surface. As fibronectin-containing extracellular matrix fibers accumulated beneath the cells at 24 h, FNR became concentrated at contacts with these fibers and was no longer detected at focal contacts. VNR was not observed at matrix contacts but remained strikingly localized in focal contacts of the 24-h cells. Since focal contacts represent the sites of strongest cell-to-substrate adhesion, these results suggest that FNR and VNR together play critical roles in the maintenance of stable contacts between the cell and its substrate. In addition, the accumulation of FNR at extracellular matrix contacts implies that this receptor might also function in the process of cellular migration along fibronectin-containing matrix cables. To define the factors governing accumulation of FNR and VNR at focal contacts, fibroblasts in serum-free media were plated on substrates coated with purified ligands. Fibronectin-coated surfaces fostered accumulation of FNR but not VNR at focal contacts. On vitronectin-coated surfaces, or substrata derivatized with a tridecapeptide containing the cell attachment sequence Arg-Gly-Asp, both FNR and VNR became concentrated at focal contacts. These observations suggest that the availability of ligand is critical to the accumulation of FNR and VNR at focal contacts, and that FNR might also recognize substrate-bound vitronectin.

Cell Adhesion↗
PubMed1987

Influence of stereochemistry of the sequence Arg-Gly-Asp-Xaa on binding specificity in cell adhesion.

Peptides containing the tripeptide sequence Arg-Gly-Asp can duplicate or inhibit the cell attachment-promoting effects of fibronectin and vitronectin. Peptides analogous to a prototype peptide, Gly-Arg-Gly-Asp-Ser-Pro-Cys, the sequence of which was taken from the cell attachment site of fibronectin, were assayed for their relative abilities to inhibit the attachment of cells to a fibronectin or vitronectin substrate. A peptide having the L-Arg residue replaced with D-Arg showed no difference in this capacity, whereas substituting Gly with D-Ala or L-Asp with D-Asp resulted in completely inactive peptides. Replacement of L-Ser with D-Ser drastically reduced the influence that the resulting peptide had on the vitronectin interaction, but this peptide showed little difference in its effect on the binding of cells to fibronectin when compared with the prototype peptide. Furthermore, substitution of the Ser with L-Asn resulted in a peptide that had an apparent increased preference for the fibronectin receptor and decreased preference for the vitronectin receptor. Conversely, threonine in this position gave a peptide with increased preference for the vitronectin receptor, whereas L-Pro in this position gave a completely inactive peptide. Finally, by cyclicizing the prototype peptide to restrict its conformational flexibility, a peptide was obtained that was a much improved inhibitor of attachment of cells to vitronectin and yet nearly inactive with respect to the interactions of cells with fibronectin substrates. These studies lend support to the hypothesis that different Arg-Gly-Asp-directed adhesion receptors can recognize differences in the conformation and environment of the Arg-Gly-Asp tripeptide, and they establish the feasibility of obtaining synthetic probes that are more selective for individual receptors than are the peptides modeled after the natural sequences of adhesive extracellular matrix molecules.

Amino Acid Sequence↗
PubMed1987

New perspectives in cell adhesion: RGD and integrins.

Rapid progress has been made in the understanding of the molecular interactions that result in cell adhesion. Many adhesive proteins present in extracellular matrices and in the blood contain the tripeptide arginine-glycine-aspartic acid (RGD) as their cell recognition site. These proteins include fibronectin, vitronectin, osteopontin, collagens, thrombospondin, fibrinogen, and von Willebrand factor. The RGD sequences of each of the adhesive proteins are recognized by at least one member of a family of structurally related receptors, integrins, which are heterodimeric proteins with two membrane-spanning subunits. Some of these receptors bind to the RGD sequence of a single adhesion protein only, whereas others recognize groups of them. The conformation of the RGD sequence in the individual proteins may be critical to this recognition specificity. On the cytoplasmic side of the plasma membrane, the receptors connect the extracellular matrix to the cytoskeleton. More than ten proved or suspected RGD-containing adhesion-promoting proteins have already been identified, and the integrin family includes at least as many receptors recognizing these proteins. Together, the adhesion proteins and their receptors constitute a versatile recognition system providing cells with anchorage, traction for migration, and signals for polarity, position, differentiation, and possibly growth.

Amino Acid Sequence↗
PubMed1987

Amino acid sequence of the vitronectin receptor alpha subunit and comparative expression of adhesion receptor mRNAs.

The amino acid sequence of the vitronectin receptor alpha subunit deduced from cDNA is presented. The sequence defines a 1047-amino-acid polypeptide precursor with a putative signal sequence, a large extracellular domain with several sites homologous to calcium binding sites in other proteins, a transmembrane domain, and a 32-amino-acid cytoplasmic domain. The 7-kilobase vitronectin receptor alpha subunit mRNA was found to be expressed in all cell lines examined, including endothelial cells, K562 and HEL leukemia cells, and osteosarcoma cells. In the two leukemia cell lines, the expression of the vitronectin receptor mRNA, as well as that of the fibronectin receptor, was enhanced in the presence of phorbol ester, a treatment known to increase the adhesiveness of these cells. The HEL cells were the only ones among the cell lines tested that also contained the mRNA of the platelet adhesion receptor alpha subunit, glycoprotein IIb. The expression of glycoprotein IIb was slightly enhanced by treatment of the cells with phorbol ester. These results complete the partial cDNA sequence of the vitronectin receptor alpha subunit published previously (Suzuki, S., Argraves, W. S., Pytela, R., Arai, H., Krusius, T., Pierschbacher, M. D., and Ruoslahti, E. (1986) Proc. Natl. Acad. Sci. U.S.A., 83, 8614-8618), confirm that the vitronectin receptor, and not IIb, is expressed in endothelial cells, and show that changes in the level of its expression correlate with changes in cell adhesiveness.

Amino Acid Sequence↗
PubMed1987

A fibroblast chondroitin sulfate proteoglycan core protein contains lectin-like and growth factor-like sequences.

We have isolated cDNA clones that code for a proteoglycan-related polypeptide with unique properties. A lambda gt11 expression library made from human fibroblast mRNA was screened with an antiserum made against a proteoglycan fraction from human fetal membranes. One group of positive clones revealed an open reading frame coding for 685 amino acids from the COOH terminus of a polypeptide. This amino acid sequence contains a domain that is strongly homologous with the COOH-terminal core protein domain of the large aggregating cartilage proteoglycan. This domain also contains sequences that are homologous with vertebrate lectins that bind terminal galactosyl, N-acetyl-glucosaminyl or mannosyl residues. On the NH2-terminal side of the lectin-like domain the cDNA-derived amino acid sequence contains two epidermal growth factor-related segments. The cDNA clones were shown to belong to a chondroitin sulfate proteoglycan by using antisera made against two peptides predicted from the cDNA sequence. These antisera were reactive with a proteoglycan fraction from fibroblasts after chondroitinase treatment of the fraction but not after treatment with heparinase or no treatment. Among the several polypeptides reactive with the anti-peptide antibodies the largest one, corresponding to a molecular weight of about 400,000, is likely to be the intact core protein, whereas the smaller polypeptides may be processing products or products of artifactual proteolysis. These results show that the amino acid sequence belongs to a proteoglycan core protein, and the sequence, therefore, provides a molecular definition to this proteoglycan. The lectin-related and growth factor-like sequences in the core protein of this proteoglycan suggest that it may play a role in intercellular signaling.

Aggrecans↗
PubMed1987

Immunochemical and amino-terminal sequence comparison of two cytoadhesins indicates they contain similar or identical beta subunits and distinct alpha subunits.

Platelet membrane glycoprotein (GP) IIb-IIIa is functionally and antigenically related to proteins present on many cell types, suggesting that it is a member of the proposed cytoadhesin family of membrane proteins. We have compared the purified tissue vitronectin receptor (VnR) with GP IIb-IIIa. Anti-VnR immunoprecipitated GP IIb-IIIa and a related endothelial cell protein. In immunoblots, GP IIIa reacted with anti-VnR and the beta subunit of the VnR reacted with poly and monoclonal anti-GP IIIa. In contrast, the alpha subunit of the VnR failed to react either with a polyclonal anti-GP IIb or with monoclonal anti-GP IIb. Furthermore, the amino-terminal sequence of GP IIIa and the beta subunit of VnR were identical at determined residues while the alpha subunit and the GP IIb were different, but showed 33% identity. These data indicate the identity or close homology of GP IIIa and the beta subunit of the VnR. In contrast, the alpha subunit and GP IIb are distinct polypeptides which may be homologous. Since GP IIb-IIIa and the VnR differ in ligand recognition specificity, the data also suggest that this specificity may be governed by the alpha subunit of cytoadhesins.

Amino Acid Sequence↗
PubMed1987

Identification and synthesis of a recognition signal for the attachment of glycosaminoglycans to proteins.

Comparison of the amino acid sequences of three different proteoglycan core proteins reveals a 12-amino acid sequence that is about 50% homologous among these proteoglycans. In each of the proteoglycans, this sequence surrounds the serine-glycine dipeptide in which the serine is known or presumed to be substituted with a chondroitin/dermatan sulfate glycosaminoglycan chain. Peptides containing this sequence from two proteoglycans were examined for their ability to serve as acceptors for xylosyltransferase, the enzyme that begins the assembly of glycosaminoglycan chains. Those peptides corresponding to amino acid sequences known to contain glycosaminoglycan-substituted serine residues in the protein were efficient xylosyltransferase acceptors, whereas peptides from sequences with no glycosaminoglycan-substituted serine residues were not. Amino acid substitutions at four critical sites in the acceptor peptides showed that single substitutions could completely abolish acceptor activity or greatly reduce it. The results suggest that the proteoglycan recognition consensus sequence for the attachment of glycosaminoglycans to core proteins consists of acidic amino acids closely followed by the tetrapeptide Ser-Gly-Xaa-Gly, where Xaa is any amino acid. The signal appears to be contained in the primary sequence information. In this regard it resembles a number of other signals for protein processing and intracellular routing.

Amino Acid Sequence↗
PubMed1987

A cell surface receptor complex for collagen type I recognizes the Arg-Gly-Asp sequence.

To isolate collagen-binding cell surface proteins, detergent extracts of surface-iodinated MG-63 human osteosarcoma cells were chromatographed on affinity matrices of either type I collagen-Sepharose or Sepharose carrying a collagen-like triple-helical peptide. The peptide was designed to be triple helical and to contain the sequence Arg-Gly-Asp, which has been implicated as the cell attachment site of fibronectin, vitronectin, fibrinogen, and von Willebrand factor, and is also present in type I collagen. Three radioactive polypeptides having apparent molecular masses of 250 kD, 70 kD, and 30 kD were distinguishable in that they showed affinity toward the collagen and collagen-like peptide affinity columns, and could be specifically eluted from these columns with a solution of an Arg-Gly-Asp-containing peptide, Gly-Arg-Gly-Asp-Thr-Pro. These collagen-binding polypeptides associated with phosphatidylcholine liposomes, and the resulting liposomes bound specifically to type I collagen or the collagen-like peptide but not to fibronectin or vitronectin or heat-denatured collagen. The binding of these liposomes to type I collagen could be inhibited with the peptide Gly-Arg-Gly-Asp-Thr-Pro and with EDTA, but not with a variant peptide Gly-Arg-Gly-Glu-Ser-Pro. We conclude from these data that these three polypeptides are membrane molecules that behave as a cell surface receptor (or receptor complex) for type I collagen by interacting with it through the Arg-Gly-Asp tripeptide adhesion signal. The lack of binding to denatured collagen suggests that the conformation of the Arg-Gly-Asp sequence is important in the recognition of collagen by the receptor complex.

Amino Acid Sequence↗
PubMed1987

An Arg-Gly-Asp-directed receptor on the surface of human melanoma cells exists in an divalent cation-dependent functional complex with the disialoganglioside GD2.

The disialogangliosides GD2 and GD3 play a major role in the ability of human melanoma cells to attach to Arg-Gly-Asp-containing substrates such as fibronectin and vitronectin, since pretreatment of these cells with monoclonal antibodies to the oligosaccharide of GD2 and GD3 can inhibit their attachment and spreading on such adhesive proteins. This report demonstrates that human melanoma cells (M21) synthesize and express a glycoprotein receptor that shares antigenic epitopes with the vitronectin receptor on human fibroblasts and is capable of specifically recognizing the Gly-Arg-Gly-Asp-Ser-Pro sequence. In the presence of calcium, GD2, the major ganglioside of M21 cells, colocalized with this receptor on the surface of human melanoma cells and their focal adhesion plaques as demonstrated by double-label transmission immunoelectron microscopy and indirect immunofluorescence. Biochemical evidence is presented indicating that the vitronectin receptor on M21 human melanoma cells contains associated calcium and GD2. This ganglioside copurified with the glycoprotein receptor for vitronectin on affinity columns containing either an Arg-Gly-Asp-containing peptide, concanavalin A, or lentil lectin. This major Arg-Gly-Asp-directed receptor on M21 cells could be metabolically labeled with 45Ca2+. Chelation of this ion with EDTA caused the dissociation of GD2 from the receptor and rendered the remaining glycoprotein incapable of binding to an Arg-Gly-Asp-containing peptide. Reconstitution experiments demonstrated a requirement for calcium, and not magnesium, for receptor binding to Arg-Gly-Asp and indicated that addition of ganglioside can enhance this interaction.

Amino Acid Sequence↗