Michael D. Pierschbacher

Arg-Gly-Asp: A versatile cell recognition signal

Fibronectin is a large extracellular glycoprotein that can mediate adhesion of cells to the extracellular matrix. Study of the cellular recognition of fibronectin has recently led to the unexpected observation that, of the approximately 2,500 amino acids in the fibronectin polypeptide, three-an Arg-Gly-Asp (RGD) tripeptide-are crucial for its interaction with its cell surface receptor. Moreover, analysis of the cell attachment sites of several other proteins that interact with cell surfaces has implicated the same amino acid triplet. These data, summarized below, establish the RGD sequence as a basic unit of a widespread cellular recognition system. The Structure of the Cell Attachment Site in Fibronectin The cell attachment site within the fibronectin molecule has been identified, sequenced, and reproduced by peptide synthesis (Pierschbacher et al., PNAS 80, 1224-1227, 1983). Analysis of progressively smaller synthetic peptides has narrowed down the active site to a tetrapeptide, Arg-Gly-Asp-Ser (Pierschbacher and Ruoslahti, Nature 309,30-33,1984; Yamada and Kennedy, JCB 99,29-38, 1984). The arginine, glycine, and aspartic acid residues are essential for activity, while the serine residue can be replaced by other amino acids (Pierschbacher and Ruoslahti, PNAS 87, 5985-5988, 1984). In rat fibronectin, the expression of a second RGD sequence is controlled by alternative mRNA splicing (Schwarzbauer et al., Cell 35, 421-431, 1983) raising the possibility that some fibronectin molecules have two distinct cell attachment sites. The synthetic peptides containing the RGD sequence mediate cell attachment directly when presented to cells as an insoluble substrate, whereas in solution they can inhibit cell attachment to fibronectin. Surprisingly, the same peptides also inhibit the attachment of fibroblasts to a number of other proteins, including vitronectin. Vitronectin is the active component of serum spreading factor and is the S-protein of the complement membrane attack complex (Jenne and Stanley, EMBO J. 4,3153-3157, 1985). Its cell attachment-promoting activity has also been localized to an RGD sequence (Suzuki et al., EMBO J. 4, 25192524, 1985). Fibrinogen contains two RGD sequences and von Willebrand factor has at least one. The binding of each of these proteins to platelets is inhibited by RGDcontaining peptides (Gartner and Bennett, JBC 260, 11891-11894, 1985; Plow et al., PNAS 82, 8057-8061, 1985) indicating that this tripeptide is important for the function of each of these proteins in platelet adhesion. The RGD sequences in the various adhesive proteins are recognized by cell Minireview

Identification and isolation of a 140 kd cell surface glycoprotein with properties expected of a fibronectin receptor

Affinity chromatography was used to identify a putative cell surface receptor for fibronectin. A large cell-attachment-promoting fibronectin fragment was used as the affinity matrix, and specific elution was effected by using synthetic peptides containing the sequence Arg-Gly-Asp, which is derived from the cell recognition sequence in the fibronectin cell attachment site. A 140 kd protein was bound by the affinity matrix from octylglucoside extracts of MG-63 human osteosarcoma cells and specifically eluted with the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro. The 140 kd protein was labeled by cell surface specific radioiodination and became incorporated into liposomes at a high efficiency. Liposomes containing this protein showed specific affinity toward fibronectin-coated surfaces, and this binding could be selectively inhibited by the synthetic cell-attachment peptide but not by inactive peptides. Affinity chromatography on wheat germ agglutinin-Sepharose showed that the 140 kd protein is a glycoprotein and, in combination with the fibronectin fragment chromatography, gave highly enriched preparations of the 140 kd protein. These properties suggest that the 140 kd glycoprotein is a membrane-embedded cell surface protein directly involved in the initial step of cell adhesion to fibronectin substrates.

[46] Fibronectin: Purification, immunochemical properties, and biological activities

Publisher Summary Fibronectin is a cell-surface and blood glycoprotein that apparently mediates adhesion of cells to the extracellular matrix. Malignant cells tend to lack cell surface fibronectin, and this may contribute to their capacity for invasive and metastatic growth. Early isolation methods for plasma fibronectin made use of its propensity to precipitate with fibrinogen and some other proteins when plasma is allowed to stand in the cold. A large fraction of the plasma cryoprecipitate is fibronectin, and it can be purified from such precipitate by using a combination of differential precipitation steps and ion exchange chromatography. Fibronectin from the surface of cultured fibroblasts can be isolated by extraction with low concentrations of urea. Antifibronectin columns can also be used to isolate fibronectin from both plasma and cell cultures. These methods have now largely been replaced by affinity chromatography on gelatin-Sepharose. The methods used in immunochemical quantitation of fibronectin include radioimmunoassay, enzyme immunoassay, and rocket immunoelectrophoresis. Of these methods, enzyme linked immunosorbent assay is probably the most useful one.

Location of the cell-attachment site in fibronectin with monoclonal antibodies and proteolytic fragments of the molecule

Proteolytic fragments of human plasma fibronectin were used to identify monoclonal antibodies reacting with the various domains of fibronectin. One of these antibodies, which reacts with cell-attachment-promoting fragments of fibronectin, inhibits attachment of cells to fibronectin-coated surfaces. A cell-attachment-promoting, chymotryptic, 120 kilodalton fragment was cleaved further with pepsin into three main fragments. The smallest, 15 kilodalton fragment was purified by affinity chromatography on the cell-attachment-inhibiting antibody insolubilized on Sepharose. This fragment is active in promoting cell attachment but lacks the other known binding activities of fibronectin. It can be localized between the collagen-binding and heparin-binding domains, about 127 to 197 kilodaltons from the NH2 terminus of the polypeptide. These results show that the interaction of fibronectin with cells is restricted to a defined portion of the molecule and is independent of the direct involvement of the known affinities toward other macromolecules.

Vitronectin—A major cell attachment-promoting protein in fetal bovine serum☆

Bovine serum is a constituent of most media used for the culture of animal cells. The adhesion-promoting properties of serum are generally attributed to fibronectin, yet there have been frequent reports of other adhesion-promoting molecules in bovine serum. Using a technique in which adhesive proteins are visualized after separation by SDS-PAGE, we graphically confirm the presence of a second cell attachment protein in bovine serum and present the evidence that this molecule is the bovine equivalent of vitronectin. The molecular size of this protein is in the same range as the size of the adhesive human plasma protein, vitronectin. The bovine protein also shared with human vitronectin an affinity for glass, and it could be purified by a combination of glass bead and ion exchange chromatography. The isolated bovine protein had varying proportions of an 80 and a 65 kD polypeptide. It showed immunological cross-reactivity with anti-human vitronectin and with anti-human somatomedin B. Somatomedin B is a serum peptide which has a NH2-terminal sequence identical to that of human vitronectin. The identity of the bovine protein as vitronectin was established by showing that its NH2-terminal amino acid sequence is strongly homologous with those of human vitronectin and somatomedin B. Quantitation of the adhesive activities of fibronectin and vitronectin in bovine plasma and fresh serum showed that more activity is associated with vitronectin than with fibronectin. The preponderance of vitronectin was particularly clear in fetal bovine serum intended for cell culture. In various batches, cell attachment activity attributable to vitronectin was 8-16-fold greater than that of fibronectin, making vitronectin the main adhesive protein in routine cell culture media.

[27] Arginine-glycine-aspartic acid adhesion receptors

Publisher Summary This chapter discusses the isolation and properties of cell surface receptors for fibronectin and vitronectin. These receptors recognize the same arginine-glycine-aspartic acid (Arg-Gly-Asp) cell attachment determinant in their ligands. The cell attachment site of fibronectin can be isolated as an 11.5kDa peptic fragment of fibronectin which retains this cell attachment-promoting function. It is possible synthetically to reconstruct the cell attachment determinant that is contained within this natural fragment. By testing smaller and smaller synthetic fragments of fibronectin for their ability to support cell attachment, it can be shown that the minimum structural component of this cell attachment activity is composed of the tripeptide Arg-Gly-Asp. It appears then that a number of extracellular proteins are recognized by the cell surface via an Arg-Gly-Asp-dependent mechanism. Such recognition is mediated by receptors specific for the Arg-Gly-Asp sequence in fibronectin, vitronectin, and some other proteins. The discovery of the Arg-Gly-Asp cell adhesion system has considerably enhanced the opportunities for increasing our understanding of cell surface recognition phenomena. The availability of methods for the purification of the Arg-Gly-Asp receptors will now allow studies of their structure, distribution, and binding specificities.

Antifibrotic effect of decorin in a bleomycin hamster model of lung fibrosis

We reported previously that treatment with antibody to transforming growth factor-beta (TGF-beta) caused a marked attenuation of bleomycin (BL)-induced lung fibrosis (LF) in mice. Decorin (DC), a proteoglycan, binds TGF-beta and thereby down-regulates all of its biological activities. In the present study, we evaluated the antifibrotic potential of DC in a three-dose BL-hamster model of lung fibrosis. Hamsters were placed in the following groups: (1) saline (SA) + phosphate-buffered saline (PBS) (SA + PBS); (2) SA + DC; (3) BL + PBS; and (4) BL + DC. Under pentobarbital anesthesia, SA (4 mL/kg) or BL was instilled intratracheally in three consecutive doses (2.5, 2.0, 1.5 units/kg/4 mL) at weekly intervals. DC (1 mg/mL) or PBS was instilled intratracheally in 0.4 mL/hamster on days 3 and 5 following instillation of each dose of SA or BL. In week 4, hamsters received three doses of either DC or PBS every other day. The hamsters were killed at 30 days following the first instillation, and their lungs were appropriately processed. Lung hydroxyproline levels in SA + PBS, SA + DC, BL + PBS, and BL + DC groups were 965, 829, 1854, and 1387 microg/lung, respectively. Prolyl hydroxylase activities were 103, 289, and 193% of SA + PBS control in SA + DC, BL + PBS, and BL + DC groups, respectively. The myeloperoxidase activities in the corresponding groups were 222, 890, and 274% of control (0.525 units/lung). Intratracheal instillation of BL caused significant increases in these biochemical markers, and instillation of DC diminished these increases in the BL + DC group. DC treatment also caused a significant reduction in the infiltration of neutrophils in the bronchoalveolar lavage fluid (BALF) of hamsters in the BL + DC group. However, DC treatment had little effect on BL-induced increases in lung superoxide dismutase activity and lipid peroxidation and leakage of plasma proteins in the BALF of the BL + DC group. Hamsters in the BL + PBS group showed severe multifocal fibrosis and accumulation of mononuclear inflammatory cells and granulocytes. In contrast, hamsters in the BL + DC group showed mild multifocal septal thickening with aggregations of mononuclear inflammatory cells. Hamsters in both control groups (SA + PBS and SA + DC) showed normal lung structure. Frozen lung sections following immunohistochemical staining revealed an intense staining for EDA-fibronectin and collagen type I in the BL + PBS group as compared with all other groups. It was concluded that DC potentially offers a novel pharmacological intervention that may be useful in treating pulmonary fibrosis.

Integrin αIIbβ3 Inhibitor Preserves Microvascular Patency in Experimental Acute Focal Cerebral Ischemia

Background and Purpose—Platelets become activated and accumulate in brain microvessels of the ischemic microvascular bed after experimental focal cerebral ischemia. The binding of glycoprotein IIb/IIIa (integrin αIIbβ3) on platelets to fibrinogen is the terminal step in platelet adhesion and aggregation. This study tests the hypothesis that inhibition of platelet-fibrin(ogen) interactions may prevent microvascular occlusion after experimental middle cerebral artery occlusion (MCA:O). Methods—TP9201 is a novel Arg-Gly-Asp (RGD)-containing integrin αIIbβ3 inhibitor. Microvascular patency after 3-hour MCA:O and 1-hour reperfusion within the ischemic and nonischemic basal ganglia was compared in adolescent male baboons who received high-dose TP9201 (group A: IC80 in heparin, n=4), low-dose TP9201 (group B: IC30 in heparin, n=4), or no treatment (group C: n=4) before MCA:O. Results—After MCA:O, microvascular patency decreased significantly in group C. However, in the ischemic zones of groups A and B compared w...

Fibronectin: a molecule with remarkable structural and functional diversity

Fibronectin is a large, extracellular glycoprotein that binds to a variety of macromolecules including fibrin, collagen, cell surfaces, and glycosaminoglycans. Proteases cleave fibronectin into fragments which have one or more of these binding activities contained in molecular domains. The proteolytic fragments and monoclonal antibodies reacting with different domains have revealed the sequential ordering of the active sites in the molecule.

Comparison of two phosphoproteins in chicken bone and their similarities to the mammalian bone proteins, osteopontin and bone sialoprotein II

Two phosphorylated proteins of approximately 66 kDa and approximately 60 kDa mass with different DEAE-Sephacel elution patterns were isolated from chicken bone and were shown to be genetically distinct by both biochemical and immunological analysis. A tryptic peptide from the 60 kDa protein was identified that was similar to a sequence of the rat bone sialoprotein II. Both proteins showed RGD inhibited cell-attachment with the MG-63 osteosarcoma cell, and the approximately 66 kDa phosphoprotein appeared to promote cell adhesion better than human vitronectin. The two phosphoproteins appear to share functional and biochemical characteristics and to be homologous to the mammalian bone phosphoproteins, osteopontin and bone sialoprotein II.