Characterization of a 140-kD avian cell surface antigen as a fibronectin-binding molecule

A 140,000-D protein cell surface antigen (140k) complex has been implicated in fibronectin-mediated cell-substratum attachment. We have used three different experimental systems to evaluate the hypothesis that this 140k complex can function as a fibronectin receptor. A monoclonal antibody that binds to the 140k complex specifically inhibits the direct binding of 3H-labeled 75,000-D fibronectin cell-binding fragment (f75k) to chicken embryo fibroblasts in suspension. The 140k complex is retarded in its passage through an affinity column consisting of immobilized f75k, and this interaction is specifically inhibited by a synthetic peptide that contains the fibronectin cell-recognition signal sequence. Finally, exogenous purified 140k complex inhibits the attachment and spreading of chicken embryo fibroblasts on fibronectin-coated substrates. Thus, our results indicate that the 140k complex can bind directly to fibronectin and is likely to be a fibronectin receptor for chicken cells.     

The interaction of fibronectin fragments with fibroblastic cells

We have examined the interaction of the purified cell-binding domain of fibronectin with fibroblastic baby hamster kidney cells. When the cell-binding region of fibronectin is part of a large 75,000-dalton fragment, the direct binding of the tritium-labeled fragment to cells in suspension can be observed. There is a single class of 10(5) sites/cell with an apparent dissociation constant of 4 X 10(-7) M. When the cell-binding region is part of a smaller 11,500-dalton fragment, an interaction with cells can only be observed indirectly via inhibition assays. The apparent affinity of this fragment for the cell surface fibronectin receptor is low. This 11,500-dalton fragment competitively inhibits both the direct binding of soluble [3H]fibronectin to cells in suspension and the spreading of cells on fibronectin-coated substrates, suggesting that the fragment binds to the same receptor site as intact fibronectin. Possible models describing the mechanism of the interaction of fibronectin with its receptor are proposed.     

The interaction of plasma fibronectin with fibroblastic cells in suspension

We have examined the interaction of soluble plasma [3H]fibronectin with fibroblastic cells in suspension. Fibronectin labeled by reductive methylation binds to baby hamster kidney cells in serum-free medium in a time-dependent manner at 4, 22, and 37 degrees C, with half-maximal binding occurring in 12-15 min at 22 degrees C. The binding is saturable and reversible. At least 90% of the cell-associated fibronectin is external to the plasma membrane, as judged by trypsin susceptibility of the bound radioactivity. Scatchard analysis of the concentration dependence of binding indicates the presence of a single class of binding sites, even at low input concentrations of fibronectin. There are approximately 5 +/- 1 X 10(5) sites/cell with an apparent dissociation constant of 8.0 +/- 0.5 X 10(-7) M; thus, the binding of soluble fibronectin to these cells is of moderate affinity. This putative fibroblast fibronectin receptor is resistant to trypsin in the presence of physiological concentrations of divalent cations but is susceptible to trypsin in the presence of 5 mM EDTA. Binding of 0.1 mg/ml [3H]fibronectin is 60-80% inhibited by 8 mg/ml unlabeled fibronectin and 95% inhibited by 1 mg/ml purified 75-kDa fibronectin cell-binding domain, but is unaffected by 1 mg/ml 44-kDa collagen-binding domain or 5 mg/ml ovalbumin. The binding parameters determined in this study further define the fibroblast cell-surface fibronectin receptor.     

Peptide inhibitors of fibronectin, laminin, and other adhesion molecules: unique and shared features

Synthetic peptides can specifically inhibit the function of certain adhesive glycoproteins in vitro and in vivo. We have compared the relative activities of a set of six variant synthetic peptides based on the sequence of fibronectin in terms of their ability to inhibit the interactions of fibroblasts with fibronectin, spreading factor/vitronectin, laminin, and native collagen gels. BHK (baby hamster kidney) and chick embryo fibroblasts spreading on these adhesive molecules displayed distinctive patterns of sensitivity to inhibition by this panel of peptides, which depended on the adhesive molecule rather than the cell type. For fibronectin, Gly-Arg-Gly-Asp-Ser was considerably more active than Arg-Gly-Asp-Ser, whereas these two peptides displayed little difference in activity in inhibiting cell adhesion to spreading factor. For both proteins, the inverted peptide sequence Ser-Asp-Gly-Arg was also moderately active, whereas closely related peptides containing a transposition, a deletion, or a single, conserved amino acid substitution were much less active. For inhibiting interactions with laminin or native type I collagen gels, Gly-Arg-Gly-Asp-Ser was only weakly active, but the inverted peptide Ser-Asp-Gly-Arg unexpectedly continued to display inhibitory activity for both attachment proteins in both cell types. Our results indicate that different adhesive processes depend on distinct peptide recognition events by a cell. However, there may be a possible common denominator among attachment proteins in a moderate sensitivity to Ser-Asp-Gly-Arg. Our study also underscores the importance of examining a full set of peptide analogs when these novel inhibitors are used to characterize biological processes.     

Identification of two distinct regions of the type III connecting segment of human plasma fibronectin that promote cell type-specific adhesion

The principal region of the human plasma fibronectin molecule mediating the adhesion of melanoma cells appears to be the alternatively spliced type III connecting segment (IIICS (Humphries, M. J., Akiyama, S. K., Komoriya, A., Olden, K., and Yamada, K. M. (1986a) J. Cell Biol., in press]. A series of overlapping synthetic peptides spanning the entire IIICS (CS peptides) were examined for their effects on B16-F10 melanoma cell adhesion to the parent fibronectin molecule. Two nonadjacent CS peptides, designated CS1 and CS5, were inhibitory. In contrast, neither inhibited fibronectin-mediated spreading of fibroblastic baby hamster kidney cells. When N-terminal cysteine derivatives of the CS peptides were conjugated to IgG by covalent cross-linking with N-succinimidyl-3(2-pyridyldithio)propionate, both the CS1 and CS5 conjugates promoted B16-F10 melanoma cell spreading. All conjugates were inactive for spreading of baby hamster kidney cells, confirming the cell type specificity of the IIICS adhesion site. Determination of the amounts of CS peptide required to support melanoma cell adhesion revealed that the activity of CS1 was only 2.4-fold lower than that of the intact fibronectin molecule. CS5 was approximately 320-fold less active than fibronectin, suggesting that the CS1 region may be the major site of interaction with the melanoma cell surface. The adhesion-promoting activities of CS1-IgG and CS5-IgG were additive as were the inhibitory activities of the free peptides for B16-F10 cell spreading on fibronectin. These findings suggest that both regions of the IIICS can function separately or together in mediating the interaction of melanoma cells with fibronectin. Since CS1 and CS5 are each found in separate alternatively spliced regions of the IIICS, it is conceivable that the adhesion-promoting activity of fibronectin for different cell types may be under complex regulation.     

Synergism between membrane gangliosides and Arg-Gly-Asp-directed glycoprotein receptors in attachment to matrix proteins by melanoma cells

The identification of specific cell surface glycoprotein receptors for Arg-Gly-Asp-containing extracellular matrix proteins such as fibronectin has focused attention on the role of gangliosides in this process. Is their involvement dependent or independent of the protein receptors? In attachment assays with cells from a human melanoma cell line, titration experiments with an antibody (Mel 3) with specificity for the disialogangliosides GD2 and GD3, used together with a synthetic peptide containing the cell binding sequence Arg-Gly-Asp, show that their joint effect is synergistic. Both the Mel 3 antibody and the synthetic peptide individually cause rapid detachment of melanoma cells from fibronectin substrate but, when used together, much smaller concentrations of both are required to achieve the same effect. The Mel 3 antibody was not nonspecifically reducing receptor binding to the Arg- Gly-Asp sequence since, in binding assays with radiolabeled peptide performed with cells in suspension, very little peptide is bound by the melanoma cells under these conditions but addition of Mel 3, an antibody of IgM isotype, causes a two- to threefold increase in specific binding. The simplest interpretation of these data is that the Mel 3 antibody is causing sufficient clustering of membrane gangliosides in local areas and producing a favorably charged environment to facilitate peptide binding by specific glycoprotein receptors.     

Evidence that arginyl-glycyl-aspartate peptides and fibrinogen gamma chain peptides share a common binding site on platelets

Synthetic peptides corresponding to the carboxyl terminus of the fibrinogen gamma chain inhibit the binding of fibrinogen, fibronectin, and von Willebrand factor to platelets, yet the active decapeptide sequence has only been found in fibrinogen to date. In contrast, all three proteins contain Arg-Gly-Asp sequences, and peptides containing Arg-Gly-Asp are potent inhibitors of their binding to activated platelets. We have analyzed the relationship between these peptide sets by direct binding assays. H12 (gamma 400-411) inhibited the binding of an Arg-Gly-Asp-containing peptide to platelets with similar dose response to inhibition of fibronectin binding. We have previously reported that GPIIb-IIIa binds to immobilized Arg-Gly-Asp peptides and can be eluted by Arg-Gly-Asp-containing peptides in solution. Both H12 and L10 (gamma 402-411) completely eluted GPIIb-IIIa bound to immobilized Arg-Gly-Asp peptides. Conversely, when GPIIb-IIIa was bound to immobilized L10, either L10 or an Arg-Gly-Asp peptide could elute it. Peptide specificity was established by the failure of Gly-Arg-Gly-Glu-Ser-Pro or acetylated L10 to elute GPIIb-IIIa from the immobilized peptides. These results indicate that the two peptide sets interact with the same receptor which contains GPIIb-IIIa.     

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.     

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.     

Effects of modifications of the RGD sequence and its context on recognition by the fibronectin receptor

The receptor for fibronectin is a member of the integrin superfamily of cell surface adhesion receptors, many of which recognize the sequence RGD in their ligands. We have developed sensitive enzyme-linked and radioreceptor assays to examine the ligand specificity of the fibronectin receptor. The fibronectin receptor bound only to fibronectin of the various Arg-Gly-Asp (RGD)-containing proteins tested. The smallest amount of receptor detectable in the assay was about 10 ng. Mn2+ enhanced the binding of the receptor to fibronectin 3-10-fold as compared to Ca2+ and Mg2+. Scatchard analysis of the saturation plot from the radioreceptor assay gave a dissociation constant (Kd) of 3 x 10(-8) M for the binding of fibronectin receptor to fibronectin in the presence of Mn2+. Inhibition experiments showed that the affinities of the ligands for the receptor decreased in the order of fibronectin approximately 110-kDa fibronectin fragment greater than GRGDSP peptide greater than 11.5-kDa fragment. Peptides not containing an RGD were several hundred to several thousand-fold less inhibitory than GRGDSP. These included the closely related peptides GRADSP and GRGESP, as well as three peptides containing the reverse sequence DGR. A peptide from the fibrinogen gamma-chain, KQAGDV, which had about 0.5% of the inhibitory activity of the standard GRGDSP peptide, was the most active peptide not containing an RGD. These results document the exquisite specificity of the fibronectin receptor for the RGD sequence.     

Glycosylation of the basic fibroblast growth factor receptor. The contribution of carbohydrate to receptor function

We have examined the glycosylation of the basic fibroblast growth factor (bFGF) receptor to determine whether carbohydrates contribute to receptor structure and function. Using a combination of cross-linking and radioreceptor assays, we demonstrated that the two bFGF receptors in baby hamster kidney cells have protein cores of 100 and 125 kDa. They are glycosylated to high mannose forms of 115 and 140 kDa and further processed to their mature forms of 130 and 150 kDa. Because peptide:N-glycosidase F, but not endo-alpha-N-acetylgalactosamidase can reduce the size of the bFGF receptors, the carbohydrate residues of the receptor appear all N-linked. The inability of deglycosylated receptors to bind 125I-bFGF supports the notion that the carbohydrate residues are required for receptor function. Furthermore, the capacity of the wheat germ agglutinin lectin to inhibit 125I-bFGF binding and the biological activity of bFGF suggests that N-acetylglucosamine residues are functionally significant components of the receptor.     

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.     

Analysis of Hormone–Receptor Interaction Sites Using Synthetic Peptides: Receptor Binding Regions of the α-Subunit of Human Choriogonadotropin

Synthetic peptides are valuable tools for determining sites of interaction between hormones and their receptors. We have learned much about linear receptor binding regions of the glycoprotein hormone human choriogonadotropin (hCG) using synthetic peptides corresponding to its primary sequence. Of paramount importance in any study using synthetic peptides as a tool to investigate protein structure are an efficient means of peptide purification and a definitive measure of peptide purity and composition. Purification is easily achieved for all but the most hydrophobic peptides using preparative reverse-phase HPLC. Of the methods available for analysis of peptide purity, mass spectrometry is perhaps the most useful and often most rapid approach. Other essential components of studies involving synthetic peptides and hormone binding are reproducible hormone labeling, receptor preparations, and bioassays. The ability of peptides to compete with hCG for binding to specific receptors is tested in radioreceptor binding assays and bioassays with transformed Leydig cell lines. These cells express the lutropin receptor that is coupled to a measurable endpoint such as cAMP or steroid production. The conditions for these assays are optimized for rapid and accurate measurement of peptide activity. Since the three-dimensional structure of hCG is not known, a systematic approach to the identification of potential receptor binding sites is advocated. First, a comprehensive analysis using synthetic overlapping peptides of uniform length that span the entire sequence of the α-subunit is employed. This approach is an effective means for surveying the entire subunit for receptor binding sites. Next, the boundaries of the active regions are delimited by a series of nested peptides sequentially shortened in length. The importance of each residue within the delimited binding regions is then studied using a series of peptides containing single alanine substitutions. Finally, modifications to enhance activity of the synthetic peptides are further made on the basis of data from alanine substitution studies, circular dichroic analysis, and molecular modeling. These studies provide valuable information to aid in the design of synthetic hormone analogs and for further analysis of the structure–function of hCG via site-directed mutagenesis.     

Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion

We have compared the molecular specificities of the adhesive interactions of melanoma and fibroblastic cells with fibronectin. Several striking differences were found in the sensitivity of the two cell types to inhibition by a series of synthetic peptides modeled on the Arg-Gly-Asp-Ser (RGDS) tetrapeptide adhesion signal. Further evidence for differences between the melanoma and fibroblastic cell adhesion systems was obtained by examining adhesion to proteolytic fragments of fibronectin. Fibroblastic BHK cells spread readily on fl3, a 75-kD fragment representing the RGDS-containing, "cell-binding" domain of fibronectin, but B16-F10 melanoma cells could not. The melanoma cells were able to spread instead on f9, a 113-kD fragment derived from the large subunit of fibronectin that contains at least part of the type III connecting segment difference region (or "V" region); f7, a fragment from the small fibronectin subunit that lacks this alternatively spliced polypeptide was inactive. Monoclonal antibody and fl3 inhibition experiments confirmed the inability of the melanoma cells to use the RGDS sequence; neither molecule affected melanoma cell spreading, but both completely abrogated fibroblast adhesion. By systematic analysis of a series of six overlapping synthetic peptides spanning the entire type III connecting segment, a novel attachment site was identified in a peptide near the COOH- terminus of this region. The tetrapeptide sequence Arg-Glu-Asp-Val (REDV), which is somewhat related to RGDS, was present in this peptide in a highly hydrophilic region of the type III connecting segment. REDV appeared to be functionally important, since this synthetic tetrapeptide was inhibitory for melanoma cell adhesion to fibronectin but was inactive for fibroblastic cell adhesion. REDV therefore represents a novel adhesive recognition signal in fibronectin that possesses cell type specificity. These results suggest that, for some cell types, regulation of the adhesion-promoting activity of fibronectin may occur by alternative mRNA splicing.     

Monoclonal antibody characterization of two distant sites required for function of the central cell-binding domain of fibronectin in cell adhesion, cell migration, and matrix assembly

Site-directed mutagenesis studies have suggested that additional peptide information in the central cell-binding domain of fibronectin besides the minimal Arg-Gly-Asp (RGD) sequence is required for its full adhesive activity. The nature of this second, synergistic site was analyzed further by protein chemical and immunological approaches using biological assays for adhesion, migration, and matrix assembly. Fragments derived from the cell-binding domain were coupled covalently to plates, and their specific molar activities in mediating BHK cell spreading were compared with that of intact fibronectin. A 37-kD fragment purified from chymotryptic digests of human plasma fibronectin had essentially the same specific molar activity as intact fibronectin. In contrast, other fragments such as an 11.5-kD fragment lacking NH2-terminal sequences of the 37-kD fragment had only poor spreading activity on a molar basis. Furthermore, in competitive inhibition assays of fibronectin-mediated cell spreading, the 37-kD fragment was approximately 325-fold more active than the GRGDS synthetic peptide on a molar basis. mAbs were produced using the 37-kD protein as an immunogen and their epitopes were characterized. Two separate mAbs, one binding close to the RGD site and the other to a site approximately 15 kD distant from the RGD site, individually inhibited BHK cell spreading on fibronectin by greater than 90%. In contrast, an antibody that bound between these two sites had minimal inhibitory activity. The antibodies found to be inhibitory in cell spreading assays for BHK cells also inhibited both fibronectin-mediated cell spreading and migration of human HT-1080 cells, functions which were also dependent on function of the alpha 5 beta 1 integrin (fibronectin receptor). Assembly of endogenously synthesized fibronectin into an extracellular matrix was not significantly inhibited by most of the anti-37-kD mAbs, but was strongly inhibited only by the antibodies binding close to the RGD site or the putative synergy site. These results indicate that a second site distant from the RGD site on fibronectin is crucial for its full biological activity in diverse functions dependent on the alpha 5 beta 1 fibronectin receptor. This site is mapped by mAbs closer to the RGD site than previously expected.     

Inhibition of Cell Surface Binding of Fibronectin and Fibronectin-Promoted Cell Migration by Synthetic Peptides in Sea Urchin Primary Mesenchyme Cells In Vitro

The role of a site of fibronectin molecule in the cell binding and cell migration was examined in vitro using sea urchin primary mesenchyme cells and synthetic peptides that contain a particular amino acid sequence, Arg-Gly-Asp-Ser.
The binding of fibronectin to the cell surface was inhibited by addition of larger synthetic peptides, Gly-Arg-Gly-Asp-Ser-Pro-Cys (HP) or Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro (DP), but not by a smaller synthetic peptide Gly-Arg-Gly-Asp-Ser (PP). The inhibition was recovered by addition of excess amount of fibronectin to the medium.
The fibronectin-promoted cell migration, by contraries, was conspicuously inhibited by addition of the PP to the cell culture medium which has alrady contained sufficient amount of fibronectin for the migration, but not so obviously by addition of the larger peptides, the HP or the DP. The inhibition was also recovered by addition of excess amount of fibronectin.
These results indicated that fibronectin utilizes Arg-Gly-Asp-Ser in the molecule as an active cell binding and a cell migration promotion site. Slightly different effects seen between the smaller peptide and the larger ones were discussed.     

The fibronectin receptor on mammalian erythroid precursor cells: characterization and developmental regulation

The plasma membrane of murine erythro-leukemia (MEL) cells contains a 140-kD protein that binds specifically to fibronectin. A 125I-labeled 140-kD protein from surface-labeled uninduced MEL cells was specifically bound by an affinity matrix that contained the 115-kD cell binding fragment of fibronectin, and specifically eluted by a synthetic peptide that has cell attachment-promoting activity. The loss of this protein during erythroid differentiation was correlated with loss of cellular adhesion to fibronectin. Both MEL cells and reticulocytes attached to the same site on fibronectin as do fibroblasts since adhesion of erythroid cells to fibronectin was specifically blocked by a monoclonal antibody directed against the cell-binding fragment of fibronectin and by a synthetic peptide containing the Arg-Gly-Asp-Ser sequence found in the cell-binding fragment of fibronectin. Erythroid cells attached specifically to surfaces coated either with the 115-kD cell-binding fragment of fibronectin or with the synthetic peptide-albumin complex. Thus, the erythroid 140-kD protein exhibits several properties in common with those described for the fibronectin receptor of fibroblasts. We propose that loss or modification of this protein at the cell surface is responsible for the loss of cellular adhesion to fibronectin during erythroid differentiation.     

A unique sequence of the laminin alpha 3 G domain binds to heparin and promotes cell adhesion through syndecan-2 and -4

Laminin-5, consisting of the alpha 3, beta 3, and gamma 2 chains, is localized in the skin basement membrane and supports the structural stability of the epidermo-dermal linkage and regulates various cellular functions. The alpha chains of laminins have been shown to have various biological activities. In this study, we identified a sequence of the alpha 3 chain C-terminal globular domain (LG1-LG5 modules) required for both heparin binding and cell adhesion using recombinant proteins and synthetic peptides. We found that the LG3 and LG4 modules have activity for heparin binding and that LG4 has activity for cell adhesion. Studies with synthetic peptides delineated the A3G75aR sequence (NSFMALYLSKGR, residues 1412--1423) within LG4 as a major site for both heparin and cell binding. Substitution mutations in LG4 and A3G75aR identified the Lys and Arg of the A3G75aR sequence as critical for these activities. Cell adhesion to LG4 and A3G75aR was inhibited by heparitinase I treatment of cells, suggesting that cell binding to the A3G75aR site was mediated by cell surface heparan sulfate proteoglycans. We showed by affinity chromatography that syndecan-2 from fibroblasts bound to LG4. Solid-phase assays confirmed that syndecan-2 interacted with the A3G75aR peptide sequence. Stably transfected 293T cells with expression vectors for syndecan-2 and -4, but not glypican-1, specifically adhered to LG4 and A3G75aR. These results indicate that the A3G75aR sequence within the laminin alpha 3 LG4 module is responsible for cell adhesion and suggest that syndecan-2 and -4 mediate this activity.     

Identification of a novel recognition sequence for the integrin alpha 4 beta 1 in the COOH-terminal heparin-binding domain of fibronectin.

The type III connecting segment of fibronectin contains two cell binding sites, represented by the peptides CS1 and CS5, that are recognized by the integrin receptor alpha 4 beta 1. Using assays measuring the spreading of A375-SM human melanoma cells, we now report that the adhesion promoting activity of a 29 kDa protease fragment of fibronectin containing the COOH-terminal heparin-binding domain (HepII), but lacking CS1 and CS5, is completely sensitive to anti-alpha 4 and anti-beta 1 antibodies, suggesting that HepII contains a third alpha 4 beta 1-binding sequence. Examination of the primary structure of HepII revealed a sequence with homology to CS1. A 19mer peptide spanning this region (designated H1) was found to support cell spreading to the same level as the 29 kDa fragment. H1-dependent adhesion was completely sensitive to anti-alpha 4 and anti-beta 1 antibodies. When soluble peptides were tested for their ability to block cell spreading on the 29 kDa fragment, a 13mer peptide comprising the central core of H1 was found to be completely inhibitory. The active region of H1 was localized to the pentapeptide IDAPS, which is homologous to LDVPS from the active site of CS1. Taken together, these results identify a novel peptide sequence in the HepII region of fibronectin that supports alpha 4 beta 1-dependent cell adhesion.     

Control of AKR fibroblast phenotype by fibronectin: Regulation of cell-surface fibronectin binding receptor by fibronectin

Results of previous studies show that the expression of fibronectin and its cell-surface fibronectin binding receptor is coregulated in 3-methylchloranthrene transformation of normal AKR-2B cells to form AKR-MCA cells and in N, N,-dimethylformamide (DMF) induction of differentiation of transformed AKR-MCA cells (1990, J. Cell. Physiol., 143:445). In this study, we tested the corgulation hypothesis by transfection experiments using an antisense fibronectin expression vector. We determined the effect of antisense fibronectin RNA expression on untransformed AKR-2B cells, and on the responses of transformed AKR-MCA cells to DMF treatment. Expression of antisense fibronectin RNA in AKR-2B cells down-modulated fibronectin production, reduced adhesion to extracellular fibronectin, and altered cellular morphology Saturation binding and Scatchard analyses using radiolabelled fibronectin revealed a concurrent down-modulation of cell-surface fibronectin binding sites, but the binding affinity of the receptor for the ligand was not affected. Immunoblotting and immunostaining revealed down-modulation of the expression of α5β1 integrins. Expression of antisense fibronectin RNA in AKR-MCA cells down-modulated the ability of DMF to restore normal fibronectin production, cell-surface fibronectin binding receptor, adhesion to extracellular fibronectin, and cellular morphology. These studies show that both fibronectin and its cell-surface fibronectin binding receptor were tightly regulated during transformation and induction of differentiation in these cells, that the ligand and its cell-surface fibronectin binding receptor worked together to bring about phenotypic changes, and that fibronectin production regulated the expression of its cell-surface fibronectin binding receptor. © 1994 Wiley-Liss, Inc.