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

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.     

Platelet glycoprotein IIb-IIIa-like proteins mediate endothelial cell attachment to adhesive proteins and the extracellular matrix

The adherence of human umbilical vein endothelial (HUVE) cells to adhesive matrix proteins was examined to determine if cell attachment and spreading were mediated by the glycoprotein (GP) IIb-IIIa complex on endothelial cells. The HUVE cells adhered well to glass slides that had been coated with fibronectin, vitronectin, fibrinogen, or von Willebrand factor but failed to adhere to albumin-coated or to uncoated slides. The HUVE cell attachment and spreading on vitronectin, fibrinogen, and von Willebrand factor were greatly inhibited by a GP IIb-IIIa monoclonal antibody (7E3). In contrast, HUVE cell attachment to fibronectin was not inhibited by 7E3 but was inhibited by a fibronectin-receptor antibody (alpha GP140), which had no effect on cell attachment to the other adhesive proteins. The 7E3 antibody, but not alpha GP140, disrupted HUVE cell monolayers by detaching cells from their naturally occurring extracellular matrix. These data indicate that platelet GP IIb-IIIa-like proteins mediate the adherence of HUVE cells to specific adhesive proteins and to the extracellular matrix.     

The vitronectin receptor alpha v beta 3 binds fibronectin and acts in concert with alpha 5 beta 1 in promoting cellular attachment and spreading on fibronectin

The vitronectin receptor (alpha v beta 3) is a member of the integrin superfamily of adhesive protein receptors that mediate a wide spectrum of adhesive cellular interactions, including attachment to vitronectin, von Willebrand factor, fibrinogen, and thrombospondin. We have studied the binding of fibronectin to the purified vitronectin receptor, and the role of this receptor in the attachment of cells to fibronectin. A solid-phase microtiter assay was developed to investigate the binding properties of the vitronectin receptor. Purified alpha v beta 3 bound fibronectin with high affinity in a saturable, divalent cation- dependent manner. Binding was inhibited by soluble vitronectin, by RGD- containing peptides, and by LM609, a monoclonal antibody against the vitronectin receptor known to inhibit the binding of adhesive proteins to alpha v beta 3. Immunoinhibition experiments showed that M21 human melanoma cells, which express the fibronectin receptor, alpha 5 beta 1, as well as alpha v beta 3, used both of these integrins to attach and spread on fibronectin. In support of this finding, M21-L cells, a variant cell line that specifically lacks alpha v beta 3 but expresses alpha v beta 1, attached and spread poorly on fibronectin. In addition, alpha v beta 3 from surface-labeled M21 cells was retained, and selectively eluted by RGDS from a fibronectin affinity column. These results indicate that alpha v beta 3 acts in concert with alpha 5 beta 1 in promoting fibronectin recognition by these cells. We conclude that fibronectin binds to the alpha v beta 3 vitronectin receptor specifically and with high affinity, and that this interaction is biologically relevant in supporting cell adhesion to matrix proteins.     

Immobilized serotonin: a novel substrate for cell culture

Baby hamster kidney cells, bovine aortal endothelial cells, bovine smooth muscle cells, and chick embryo fibroblasts were all observed to attach and grow on serotonin which had been immobilized by covalent coupling to agarose beads. While growth and morphology of cells on immobilized serotonin appeared normal, a change in cell function may have occurred since the pattern of polypeptides expressed by these cells was different from that of cells grown on two other substrates: immobilized fibronectin and tissue culture plastic. By changing the composition of the fetal calf serum proteins in the growth medium it was shown that cells attach directly to immobilized fibronectin without mediation by medium components. In contrast, cells were found not to attach directly to immobilized serotonin but to attach indirectly via factors absorbed onto immobilized serotonin from fetal calf serum. The major component of this cell attachment activity was shown not to be fibronectin and was identified following separation by SDS-PAGE, electroblotting, and cell binding on nitrocellulose filters. The cell attachment activity compromises a major protein species of Mr 70,000 which is the molecular size of the recently identified serum spreading factor also called vitronectin.     

Adsorption from fetal calf serum of collagen-like proteins which bind fibronectin and promote cell attachment

Baby hamster kidney cells were seeded onto Western blots of fetal serum proteins which had been extracted from several foreign surfaces. This revealed that the major cell adhesive proteins adsorbed onto these surfaces from fetal serum were (1) fibronectin of Mr 220,000 Da and (2) vitronectin of Mr 65,000 and 78,000 Da. Two minor bands of cell attachment were observed at Mr 153,000 and Mr 134,000 Da in the fetal serum proteins extracted from heparin-agarose and serotonin-agarose. However, by exposing the Western blots of separated proteins to a second round of serum proteins, prior to cell blotting, very strong cell adhesive bands were revealed at Mr 153,000, 134,000, and 120,000 Da. By (i) modifying the composition of the serum proteins used to treat the Western blots, (ii) using specific antibodies to fibronectin, and (iii) using radiolabeled fibronectin, it was conclusively demonstrated that the new cell adhesive bands owed their increased cell attachment activity to secondary binding of fibronectin. The new bands were shown (i) to be trypsin sensitive and collagenase sensitive and therefore to be collagen-like proteins and (ii) to react negatively in immunoblots using anti-fibronectin, anti-vitronectin, anti-fibrinogen, anti-fetuin or anti-thrombospondin. In SDS-PAGE (i) the Mr 120,000-Da protein comigrated with the alpha 2-chain of Type I collagen, (ii) the Mr 134,000-Da protein comigrated with the alpha 1-chain of Type I collagen, and (iii) the Mr 153,000-Da protein comigrated with the pN-alpha 1-chain of Type III collagen. Since the novel collagen-like proteins acted as strong sites of cell attachment on nitrocellulose blots by binding fibronectin, they might well promote cell attachment on the foreign surfaces from which they were extracted.     

Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization

We have developed two rat mAbs that recognize different subunits of the human fibroblast fibronectin receptor complex and have used them to probe the function of this cell surface heterodimer. mAb 13 recognizes the integrin class 1 beta polypeptide and mAb 16 recognizes the fibronectin receptor alpha polypeptide. We tested these mAbs for their inhibitory activities in cell adhesion, spreading, migration, and matrix assembly assays using WI38 human lung fibroblasts. mAb 13 inhibited the initial attachment as well as the spreading of WI38 cells on fibronectin and laminin substrates but not on vitronectin. Laminin-mediated adhesion was particularly sensitive to mAb 13. In contrast, mAb 16 inhibited initial cell attachment to fibronectin substrates but had no effect on attachment to either laminin or vitronectin substrates. When coated on plastic, both mAbs promoted WI38 cell spreading. However, mAb 13 (but not mAb 16) inhibited the radial outgrowth of cells from an explant on fibronectin substrates. mAb 16 also did not inhibit the motility of individual fibroblasts on fibronectin in low density culture and, in fact, substantially accelerated migration rates. In assays of the assembly of an extracellular fibronectin matrix by WI38 fibroblasts, both mAbs produced substantial inhibition in a concentration-dependent manner. The inhibition of matrix assembly resulted from impaired retention of fibronectin on the cell surface. Treatment of cells with mAb 16 also resulted in a striking redistribution of cell surface fibronectin receptors from a streak-like pattern to a relatively diffuse distribution. Concomitant morphological changes included decreases in thick microfilament bundle formation and reduced adhesive contacts of the streak-like and focal contact type. Our results indicate that the fibroblast fibronectin receptor (a) functions in initial fibroblast attachment and in certain types of adhesive contact, but not in the later steps of cell spreading; (b) is not required for fibroblast motility but instead retards migration; and (c) is critically involved in fibronectin retention and matrix assembly. These findings suggest a central role for the fibronectin receptor in regulating cell adhesion and migration.     

Evidence that type 1 plasminogen activator inhibitor binds to the somatomedin B domain of vitronectin.

The interaction between type 1 plasminogen activator inhibitor (PAI-1) and fragments of vitronectin (Vn) was investigated. The PAI-1-binding domain was not destroyed when Vn was cleaved by treatment with either acid or CNBr. Acid-cleaved Vn was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by PAI-1 ligand binding. The smallest fragment (Mr 40,000) that retained PAI-1 binding function was sequenced and shown to contain the NH2 terminus of the molecule. Further cleavage of this fragment by treatment with CNBr generated a Mr 35,000 fragment (Pro52-Asp239) that did not interact with PAI-1, and a Mr 6,000 NH2-terminal fragment (Asp1-Met51) that spanned the somatomedin B domain and contained the RGD (cell binding) sequence. The purified Mr 6,000 fragment competed with immobilized Vn for PAI-1 binding, and formed complexes with activated PAI-1. These complexes could be immunoprecipitated by antibodies to PAI-1. Synthetic peptides containing the RGD sequence had no effect on the binding of this fragment to PAI-1. These results suggest that the cell-binding and PAI-1 binding sequences of Vn occupy distinct regions in the NH2-terminal somatomedin B domain of the molecule.     

Inhibition of in vitro tumor cell invasion by Arg-Gly-Asp-containing synthetic peptides [published erratum appears in J Cell Biol 1989 Jun;108(6):following 2546]

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.     

A rapid and convenient preparation of [4-3H]NADP and stereospecifically tritiated NADP3H

The glass-binding properties of a number of purified glycoproteins capable of promoting attachment and spreading of a variety of types of animal cells in culture have been examined. Two such factors in human serum, fibronectin and serum spreading factor, exhibited strong affinities for glass beads and could be eluted from glass-bead columns under similar conditions. A number of other glycoproteins of human serum that do not promote cell adhesion did not bind to glass beads under conditions that resulted in binding of serum spreading factor or fibronectin. At a sufficiently low ratio of serum volume to glass-bead volume, human serum could be simultaneously depleted of serum spreading factor, fibronectin, and cell spreading-promoting activity by glass-bead affinity chromatography. Laminin, another cell spreading-promoting glycoprotein, possessed glass-binding properties similar to those of serum spreading factor and fibronectin while chondronectin, a fourth cell spreading-promoting factor of more limited specificity of biological activity and distribution in vivo, did not exhibit a strong interaction with glass beads under the same conditions. These observations suggest that glass-bead column affinity chromatography may prove useful as a general method for isolation and study of glycoprotein factors promoting attachment and spreading of cells in culture.     

Adhesion of Human Glioma Cell Lines to Fibronectin, Laminin, Vitronectin and Collagen I Is Modulated by Gangliosides in vitro

Adhesion of eight cell lines, derived from human gliomas of different histological types, to fibronectin, collagen I, vitronectin, and laminin was investigated in vitro. The glioma cell lines were found to attach to these substrates to different extents. Interestingly, all cell lines strongly attached to laminin. In addition, glioma cell adhesion was found to be dose dependent. Moreover, adhesion of three cell lines to fibronectin and collagen I was partially inhibited and to vitronectin completely prevented by GRGDTP peptide, indicating the involvement of integrin receptors in glioma cell adhesion. We have demonstrated, recently, that gangliosides play an important role in promoting glioma cell invasion of the reconstituted basement membrane, Matrigel, in vitro. In order to study the mechanism of action of gangliosides in this process, the role of six gangliosides (GM1, GM3, GD3, GD1a, GD1b, and GT1b) in cell adhesion to the four proteins was investigated in three cell lines. Although all gangliosides, with the exception of GM3, were found to enhance cell adhesion to these proteins to different extents, GD3 proved to be the most effective adhesion-promoting ganglioside in all three cell lines. GM3 was found to inhibit cell adhesion to the four proteins in one cell line but enhanced cell adhesion in two other cell lines. The three cell lines were found to express both GD3 and gangliosides recognised by the A2B5 antibody. Furthermore, adhesion of the three cell lines to fibronectin, vitronectin, laminin, and collagen I was inhibited by incubation with A2B5, demonstrating the involvement of intrinsic cell membrane gangliosides in adhesion of glioma cells to these proteins. Taken together with the observation that gangliosides modulate integrin receptor function, these data suggest that gangliosides may play a central role in the control of the adhesive and invasive properties of human glioma cells.     

Constitutive αVβ3 integrin-mediated adhesion of human lymphoid B cells to vitronectin substrate

Adherence to cells and matrices participates in lymphocyte migration and tissue localization and contributes to the regulation of growth and differentiation of the lymphoid cells. The adherence is mainly mediated by three families of cell-surface proteins: integrins, immunoglobulin (Ig)-related molecules, and selectins. Integrins recognize Ig-related molecules such as ICAMs as well as fibronectin (FN), vitronectin (VN), and other matrix proteins. In this study, the in vitro adhesive properties of two Epstein—Barr virus-carrying B lymphoblastoid cell lines, IB-4 and NAD-20, were compared. IB-4 cells grow as a monolayer in contrast to NAD-20 cells, which grow as cell clusters. IB-4 cells were found to adhere to the tissue culture vessel through a component of the fetal bovine serum. By using blocking monoclonal antibodies to cell-surface molecules and serum proteins, IB-4 cells were found to use αVβ3 integrin (CD51/CD61) and serum VN as the adhesive molecules. αVβ3 integrin also mediated adhesion of IB-4 cells to human serum VN and to purified VN and FN. This constitutive adherence was not enhanced by phorbol ester treatment and was inhibited by RGD-containing peptides, in contrast to the homotypic adhesion of NAD-20 cells, which was mediated by β2 integrin CD11a/CD18 and its ligand ICAM-1 (CD54). Since VN is a component of both lymphoid tissue matrix and plasma, adhesion to this protein may affect functions and activities of B lymphocytes.     

Detachment of cells from culture substrate by soluble fibronectin peptides

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.     

Amino acid sequence of the alpha subunit of human leukocyte adhesion receptor Mo1 (complement receptor type 3)

Mo1 (complement receptor type 3, CR3; CD11b/CD18) is an adhesion-promoting human leukocyte surface membrane heterodimer (alpha subunit 155 kD [CD11b] noncovalently linked to a beta subunit of 95 kD [CD18]). The complete amino acid sequence deduced from cDNA of the human alpha subunit is reported. The protein consists of 1,136 amino acids with a long amino-terminal extracytoplasmic domain, a 26-amino acid hydrophobic transmembrane segment, and a 19-carboxyl-terminal cytoplasmic domain. The extracytoplasmic region has three putative Ca2+-binding domains with good homology and one with weak homology to the "lock washer" Ca2+-binding consensus sequence. These metal-binding domains explain the divalent cation-dependent functions mediated by Mo1. The alpha subunit is highly homologous to the alpha subunit of leukocyte p150,95 and to a lesser extent, to the alpha subunit of other "integrin" receptors such as fibronectin, vitronectin, and platelet IIb/IIIa receptors in humans and position-specific antigen-2 (PS2) in Drosophila. Mo1 alpha, like p150, contains a unique 187-amino acid stretch NH2-terminal to the metal-binding domains. This region could be involved in some of the specific functions mediated by these leukocyte glycoproteins.     

Routine heat inactivation of serum reduces its capacity to promote cell attachment

Summary Heat inactivation (56°C for 40 min) of bovine calf serum was shown to diminish its capacity to promote the attachment of cells to plastic or glass surfaces. This effect was not observed in stationary cultures (culture dishes) but became manifest under conditions in which the cells were subjected to a small amount of liquid shear force, i.e. by growing cells in roller bottles or culture tubes. Of four cell lines tested on bovine calf serum (SV-BHK, BALB-3T3, CV-1, and FS-4) SV-BHK and CV-1 cells showed the greatest sensitivity to loss of attachment-promoting activity. Fetal bovine serum also seemed to be affected by heat inactivation but to a lesser degree than bovine calf serum. Treatment of vessel surfaces with either unheated calf serum or specific attachment factors (gelatin, poly-d-lysine, and fibronectin) greatly increased cell attachment in the presence of heat inactivated serum. Heat inactivation did not seem to affect the ability of cells to grow after attachment. Of the four cell lines tested, the normal human fibroblast line (FS-4) was shown to be most effective at conditioning medium and restoring its capacity to promote the attachment of all four cell lines. This research was supported in part by VECOL, Inc., Bogata, Columbia and by grant SRC 5 U24 RR02557-02 from the National Institutes of Health, Bethesda, MD.     

Vitronectin production by human yolk sac carcinoma cells resembling parietal endoderm

Normal mesenchymal cells, normal epithelial cells and many transformed epithelial cells require serum attachment factors and extracellular matrix proteins for growth and differentiation in vitro, and recent evidence strongly supports a role for extracellular matrix molecules in the regulation of cell movement in vivo during early embryogenesis. We previously described the isolation and characterization of cell lines representative of three types of stem cells most commonly found in human adult testicular teratomas, namely embryonal carcinoma cells, yolk sac carcinoma cells resembling visceral endoderm and yolk sac carcinoma cells resembling parietal endoderm (endodermal sinus tumour cells). Of these three cell types, only endodermal sinus tumour cells, which show particularly malignant behaviour in vivo, have no serum requirement for attachment and growth in vitro. Supernatants from endodermal sinus tumour cells support the attachment of embryonal carcinoma cells in serum-free medium. We demonstrate here that endodermal sinus tumour cells, but not other cell types isolated from testicular teratomas, secrete the serum attachment protein, vitronectin (also known as serum-spreading factor, S-protein or epibolin), as well as fibronectin, laminin and type IV collagen, into serum-free medium. Purified vitronectin from medium conditioned by endodermal sinus tumour cells supported both attachment and spreading of embryonal carcinoma cells in vitro, whereas cells attached but did not spread properly on surfaces coated with fibronectin or laminin. Peptides containing the RGD cell recognition sequence common to many attachment proteins blocked attachment of endodermal sinus tumour cells to untreated tissue-culture plastic in serum-free medium. The results suggest a possible role for vitronectin in regulating cell motility and growth in early development, and in the invasion and spread of teratomas in vivo.     

Identification of the disulfide bonds in the recombinant somatomedin B domain of human vitronectin

The NH(2)-terminal somatomedin B (SMB) domain (residues 1-44) of human vitronectin contains eight Cys residues organized into four disulfide bonds and is required for the binding of type 1 plasminogen activator inhibitor (PAI-1). In the present study, we map the four disulfide bonds in recombinant SMB (rSMB) and evaluate their functional importance. Active rSMB was purified from transformed Escherichia coli by immunoaffinity chromatography using a monoclonal antibody that recognizes a conformational epitope in SMB (monoclonal antibody 153). Plasmon surface resonance (BIAcore) and competitive enzyme-linked immunosorbent assays demonstrate that the purified rSMB domain and intact urea-activated vitronectin have similar PAI-1 binding activities. The individual disulfide linkages present in active rSMB were investigated by CNBr cleavage, partial reduction and S-alkylation, mass spectrometry, and protein sequencing. Two pairs of disulfide bonds at the NH(2)-terminal portion of active rSMB were identified as Cys(5)-Cys(9) and Cys(19)-Cys(21). Selective reduction/S-alkylation of these two disulfide linkages caused the complete loss of PAI-1 binding activity. The other two pairs of disulfide bonds in the COOH-terminal portion of rSMB were identified as Cys(25)-Cys(31) and Cys(32)-Cys(39) by protease-generated peptide mapping of partially reduced and S-alkylated rSMB. These results suggest a linear uncrossed pattern for the disulfide bond topology of rSMB that is distinct from the crossed pattern present in most small disulfide bond-rich proteins.     

Structure of the platelet membrane glycoprotein IIb. Homology to the alpha subunits of the vitronectin and fibronectin membrane receptors

The platelet membrane glycoprotein IIb X IIIa heterodimer complex (GPIIb X IIIa) is the platelet receptor for adhesive proteins, containing binding sites for fibrinogen, von Willebrand factor, and fibronectin on activated platelets. GPIIb X IIIa also appears to be a member of a family of membrane adhesive protein receptors that plays a major role in cell-cell and cell-matrix interactions. GPIb is the larger component of this platelet receptor and is composed of two disulfide-linked subunits. In this report we describe the analysis of cDNA clones for human GPIIb that were isolated from a lambda gt11 expression library prepared using RNA from HEL cells. A total of 3.3 kilobases of cDNA was sequence, revealing a continuous open reading frame encoding both GPIIb subunits. The cDNA encodes 1039 amino acids: 137 constituting the smaller subunit, 871 constituting the larger subunit, and 30 constituting an NH2-terminal signal peptide. No homology was found between the larger and smaller subunits. The smaller subunit contains a 26-residue hydrophobic sequence near its COOH terminus that represents a potential transmembrane domain. Four stretches of 12 amino acids present in the larger subunit are homologous to the calcium binding sites of calmodulin and troponin C. Northern blot analysis using HEL cell RNA indicated that the mature mRNA coding for GPIIb is 4.1 kilobases in size. A comparison of the GPIIb coding region with available cDNA sequences of the alpha-chains of the vitronectin and fibronectin receptors revealed 41% DNA homology and 74% and 63% amino acid homology, respectively. Our data establish the amino acid sequence for the human platelet glycoprotein IIb and provide additional evidence for the existence of a family of cellular adhesion protein receptors.     

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.     

Alignment of biologically active domains in the fibronectin molecule

Gelatin-binding material was isolated from a human plasma cryoprecipitate by affinity chromatography on gelatin-Sepharose. Individual fragments of fibronectin with Mr = 170,000, 100,000, and 80,000 and a mixture of fragments with Mr = 205,000 and 190,000 (200K fraction) were isolated from this material. These fragments reacted with antifibronectin and with antibodies to a gelatin-binding Mr = 70,000 tryptic fragment of fibronectin. They all shared the same NH2-terminal amino acid sequence. The 205K and 190K fragments bound also to heparin-Sepharose, whereas the smaller fragments did not. The 200K fraction and the 170K fragment mediated cell attachment when used to coat plastic, whereas the 100K and 80K fragments were inactive in this assay. Further digestion of the 205K and 190K fragments with chymotrypsin yielded separate sets of smaller fragments that bound to either gelatin-Sepharose or heparin-Sepharose, as well as fragments that did not show either of these binding activities but mediated cell attachment. Since the NH2-terminal ends of the 205K, 190K, 100K, and 80K fragments are the same, the results define the order of the active sites in the fibronectin molecule as gelatin-binding site, cell attachment site, and heparin-binding site.     

Differential properties of attachment of human fibroblasts to various extracellular matrix proteins

Human diploid fibroblasts (TIG-3) were shown to attach and spread onto substrata coated with collagen, fibronectin, laminin and vitronectin. The cell attachment to these proteins required divalent cations. Mg2+ stimulated the cell attachment to all the proteins, while Ca2+ alone was not effective for the attachment to collagen and laminin. A mild trypsin treatment had prevented cells from attaching to the laminin, while it had no effect on the attachment to the other proteins. The fibronectin fragment, which retained cell binding activity, inhibited the cells from attaching and spreading onto fibronectin, but it did not cause any inhibition on the other proteins. The synthetic peptide GRGDSP inhibited the cells from attaching and spreading onto fibronectin and vitronectin, while it did not cause any inhibition on collagen and laminin. In attempts to isolate distinct receptors for these proteins, we were able to purify proteins very similar to the fibronectin and vitronectin receptors of human placenta. Based on the differential properties of the attachment of TIG-3 cells to these proteins and biochemical data, we indicate that human diploid fibroblasts have distinctive binding sites (receptors) for collagen, fibronectin, laminin and vitronectin.