Showing papers by "Michael D. Pierschbacher published in 1985"

The effect of Arg-Gly-Asp-containing peptides on fibrinogen and von Willebrand factor binding to platelets.

Abstract: The Arg-Gly-Asp sequence resides in the cell attachment region of fibronectin. Arg-Gly-Asp-containing peptides support fibroblast attachment, inhibit fibroblast adhesion to fibronectin, and inhibit fibronectin binding to thrombin-stimulated platelets. In view of the similarities between the binding of fibronectin, fibrinogen, and von Willebrand factor to stimulated platelets, we have examined the effects of Arg-Gly-Asp-containing peptides on the interaction of these latter two adhesive proteins with platelets. Gly-Arg-Gly-Asp-Ser-Pro was used as a prototype peptide, and this hexapeptide inhibited fibrinogen binding to ADP and thrombin-stimulated platelets in the 10-200 microM range. The inhibition exceeded 90% at high concentrations of peptide and was observed in the presence of either calcium or magnesium. Platelet aggregation was also inhibited by the peptide in this dose range. The hexapeptide inhibited fibrinogen binding to platelets with receptors fixed in an exposed state, indicating direct interference with the ligand-platelet interaction. The peptide was 1/2 to 1/3rd as potent in inhibiting fibrinogen as fibronectin binding to platelets, but fibrinogen and von Willebrand factor binding were inhibited to an identical extent. Conservative amino acid substitutions for the arginine, glycine, or aspartic acid markedly reduced inhibitory activity and the Asp-Gly-Arg sequence was inactive. These results indicate that Arg-Gly-Asp-containing peptides can inhibit the binding of the three adhesive proteins to stimulated platelets, establishing a basic common feature between the interaction of these molecules with platelets.

A 125/115-kDa cell surface receptor specific for vitronectin interacts with the arginine-glycine-aspartic acid adhesion sequence derived from fibronectin

Abstract: Affinity chromatography was used to identify a cell surface receptor for the adhesive protein vitronectin Detergent extracts of human osteosarcoma (MG-63) cells were chromatographed on either vitronectin-Sepharose or Sepharose linked to the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro, which includes the fibronectin cell attachment sequence Arg-Gly-Asp Two cell surface proteins with apparent molecular mass of 125 and 115 kDa bound to both columns and were specifically eluted with a solution containing the Gly-Arg-Gly-Asp-Ser-Pro peptide These proteins could be incorporated into phosphatidylcholine liposomes and mediated the specific binding of these liposomes to vitronectin but not to fibronectin In contrast, liposomes containing a previously identified 140-kDa fibronectin receptor, which interacts with the Arg-Gly-Asp sequence in fibronectin, did not bind to vitronectin Thus, the fibronectin and vitronectin receptors each recognize the Gly-Arg-Gly-Asp-Ser-Pro peptide but exhibit mutually exclusive reactivities toward fibronectin and vitronectin These receptors appear to belong to a family of proteins that mediate cell substratum adhesion via related but subtly different specificities

Complete amino acid sequence of human vitronectin deduced from cDNA. Similarity of cell attachment sites in vitronectin and fibronectin.

Abstract: cDNA clones for vitronectin, a cell adhesion-promoting plasma and tissue protein, were isolated from a lambda gt11 library containing cDNA inserts made from human liver mRNA. The library was screened with anti-vitronectin antibodies and the positive clones were further identified with synthetic oligonucleotide probes deduced from the partial amino acid sequence of vitronectin. Nucleotide sequence analysis showed that the largest insert was 1545 bp long and contained the whole sequence corresponding to plasma vitronectin. It showed that vitronectin contains the entire 44-amino acid somatomedin B peptide at its NH2 terminus and, near its COOH terminus, a 34-amino acid glycosaminoglycan binding site in which half of the amino acids are basic residues. Three potential carbohydrate attachment sites are present in the sequence. An Arg-Gly-Asp sequence, which has previously been shown to be the cell attachment site in fibronectin, was found in vitronectin immediately after the NH2-terminal somatomedin B sequence. No other homologies with fibronectin were found. The Arg-Gly-Asp sequence appears to constitute the cell attachment site of vitronectin, since it is in the region where we have previously localized the cell attachment site, its presence correlate with cell attachment activity among the insert-coded polypeptides, and because previous results have shown that synthetic peptides containing the Arg-Gly-Asp sequence inhibit the cell attachment function of vitronectin. The discovery of an Arg-Gly-Asp cell attachment site in a protein with a known cell attachment function emphasizes the general importance of this sequence in cell recognition.

Detachment of cells from culture substrate by soluble fibronectin peptides.

Abstract: The synthetic cell attachment-promoting peptides from fibronectin (Pierschbacher, M. D., and E. Ruoslahti, 1984, Nature (Lond.)., 309:30-33) were found to detach cultured cells from the substratum when added to the culture in a soluble form. Peptides ranging in length from tetrapeptide to heptapeptide and containing the active L-arginyl-glycyl-L-aspartic acid (Arg-Gly-Asp) sequence had the detaching activity, whereas a series of different peptides with chemically similar structures had no detectable effect on any of the test cells. The Arg-Gly-Asp-containing peptides caused detachment of various cell lines of different species and histogenetic origin. Studies with defined substrates showed that the active peptides could inhibit the attachment of cells to vitronectin in addition to fibronectin, indicating that vitronectin is recognized by cells through a similar mechanism as fibronectin. The peptides did not inhibit the attachment of cells to collagen. However, cells cultured on collagen-coated plastic for 24-36 h, as well as cells with demonstrable type I or type VI collagen in their matrix, were susceptible to the detaching effect of the peptides. These results indicate that the recognition mechanism(s) by which cells bind to fibronectinand vitronectin plays a major role in the substratum attachment of cells and that collagens may not be directly involved in cell-substratum adhesion. Since vitronectin is abundant in serum, it is probably an important component in mediating the attachment of cultured cells. The independence of the effects of the peptide on the presence of serum and the susceptibility of many different cell types to detachment by the peptide show that the peptides perturb an attachment mechanism that is intrinsic to the cells and fundamentally significant to their adhesion.

Molecular cloning and sequence analysis of a chondroitin sulfate proteoglycan cDNA

Abstract: We report the identification and DNA sequence of a chondroitin sulfate proteoglycan core protein cDNA. A cDNA clone, pPG1, was selected from a rat yolk sac tumor poly(A)+RNA-derived cDNA library by using synthetic oligonucleotides predicted from the NH2-terminal peptide sequence of the mature chondroitin sulfate proteoglycan. The resulting sequence analysis demonstrated that the 874-base-pair pPG1 clone contained the complete coding region of the mature proteoglycan core protein as well as 5' and 3' flanking sequences. The 104 amino acid proteoglycan core protein sequence reveals that the core protein is composed of three regions, the most striking of which is the central 49 amino acid region composed of alternating serine and glycine residues. This region clearly functions as the acceptor site for the attachment of chondroitin sulfate side chains. The serine-glycine repeat region is flanked by a 14 amino acid NH2-terminal region identical to the NH2-terminal sequence of the proteoglycan obtained by amino acid sequencing and a 41 amino acid COOH-terminal region. RNA transfer blot hybridizations of poly(A)+ mRNA from rat yolk sac tumor cells with nick-translated pPG1 reveal a single mRNA of approximately equal to 1300 nucleotides. The possibility of detecting mRNAs and genomic sequences for other proteoglycans with a serine-glycine repeat by using this cDNA clone is discussed.

Vitronectin specific cell receptor derived from mammalian mesenchymal tissue

Abstract: A method of isolating cell surface receptors utilizing a short peptide sequence bound to an affinity column. Cell surface receptors which bind selectively to the short peptide and which are specific to various adhesion proteins may be isolated therewith from various cell preparations. These receptors, whose functional integrity has been maintained by the presence of the peptide ligand, are incorporated into liposomes and used to deliver specific compounds inside the liposomes to select tissues containing the specific adhesion proteins.

The cell attachment determinant in fibronectin

Abstract: Fibronectin possesses a domain that interacts with cell surfaces. The ability of fibronectin to promote cell attachment can be duplicated with a short amino acid sequence, glycyl-L-arginyl-glycyl-L-aspartyl-L-serine, taken from that domain. The tripeptide Arg-Gly-Asp appears to be irreplaceable for maintenance of the activity of this peptide, whereas the serine residue can be replaced with some, but apparently not all, possible residues. This recognition sequence, or a closely related sequence, is present in a number of proteins other than fibronectin that interact with cells. These proteins include collagens, fibrinogen, thrombin, a bacterial surface protein, and two viral proteins, as well as discoidin-I, a protein implicated in the aggregation of Dictyostelium discoideum. A similar sequence is also repeated in some, but not all, fibronectin molecules, making it possible that some fibronectin molecules have more than a single cell attachment site. Synthetic peptides constructed from sequences taken from several of these other proteins have also been shown to promote cell attachment. The tripeptide sequence may, therefore, constitute an ancient cellular recognition mechanism common to many proteins.

Use of peptides in control of cell attachment and detachment

Abstract: A method of using synthetic cell attachment-promoting peptides from fibronectin to detach cultured cells from the substratum is described.