Syndecan-4 deficiency impairs focal adhesion formation only under restricted conditions

Two domains of fibronectin deliver two different but cooperative signals required for focal adhesion formation. The signal from the cell-binding domain is mediated by integrins, whereas the signal from the heparin-binding domain is recognized by heparan sulfate proteoglycans, of which syndecan-4 has been hypothesized to be involved in focal adhesion formation. We generated mice deficient in syndecan-4 to study its role directly. Even in fibroblasts from syndecan-4-deficient mice, focal adhesions were formed, and actin fibers terminated normally at focal adhesions when they were cultured on coverslips coated with fibronectin or with a mixture of its cell-binding and heparin-binding fragments. However, when the cells were cultured on the cell-binding fragment and the heparin-binding fragment was added to the medium, focal adhesion formation was impaired in the syndecan-4 null fibroblasts as compared with that in wild-type cells. Therefore, syndecan-4 is essential for promoting focal adhesion formation only when the signal of the heparin-binding domain of fibronectin is delivered as a soluble form, most probably from the apical surface. When the signal is delivered as a substratum-bound form, other molecule(s) also participate(s) in the signal reception.     

Modulation of haptotactic migration of metastatic melanoma cells by the interaction between heparin and heparin-binding domain of fibronectin

We utilized recombinant fibronectin polypeptides with cell-binding domain and heparin-binding domains (referred to as C-274 and H-271, respectively) and their fusion polypeptide (CH-271) to examine the role of sulfated polysaccharide heparin and/or the functional domains of fibronectin in modulating tumor cell behavior. Both C-274 and CH-271 polypeptides with cell-binding domains promoted the adhesion and migration of B16-BL6 melanoma cells, whereas H-271 did not. Heparin bound to the immobilized polypeptides with heparin-binding domain (H-271, CH-271, and a mixture of C-274 and H-271 or fibronectin) but did not affect the tumor cell adhesion to the substrates. At the same time, heparin or two monoclonal antibodies against the heparin-binding domain were able to inhibit the haptotactic migration to CH-271 or fibronectin, though not to C-274 or a mixture of C-274 and H-271. This suggests that although heparin did not affect tumor cell adhesion to the cell-binding domain near the heparin-binding domain in CH-271 or fibronectin, it did lead to a modulation of cell motility. It seems likely that the regulatory mechanism may depend on interaction between heparin-like molecules on the cell surface and the heparin-binding domain in fibronectin, rather than on simple steric hindrance or on the masking of the cell-binding domain caused by the binding of heparin to heparin-binding domain.     

Molecular interactions between fibronectin and integrins during mouse blastocyst outgrowth

To investigate the mechanism of trophoblast adhesion to fibronectin, we cultured blastocysts in serum-free medium on proteolytic fibronectin fragments containing its major functional domains, and localized fibronectin-binding integrins in outgrowing trophoblast cells by immunofluorescent staining. Outgrowth comparable to that obtained with intact fibronectin was observed using a 120 kD chymotryptic fragment containing the central cell-binding domain (FN-120) and the Arg-Gly-Asp (RGD) recognition sequence. A 40 kD COOH-terminal chymotryptic fragment of fibronectin containing both a heparin-binding region and an alternate (non-RGD) cell-binding site was inactive in supporting trophoblast adhesion. Three synthetic peptides derived from the heparin-binding domain, including the CS1 alternate cell-binding site, were also unable to promote trophoblast cell adhesion. A 75 kD recombinant protein, ProNectin F, containing 13 copies of the cell recognition epitope of fibronectin, Val-Thr-Gly-Arg-Gly-Asp-Ser-Pro-Ala-Ser, vigorously supported blastocyst outgrowth. Blastocyst outgrowth was not significantly different when surfaces were precoated with cellular fibronectin, which contains an alternatively spliced type III repeat and is the form actually encountered in vivo. Several putative fibronectin receptors were localized in trophoblast outgrowths by immunofluorescent labeling. Antibodies reactive with integrin subunits α3, α5, αllb, αv, β1 and β3, but not α4, all bound to trophoblast cells. Antibodies raised against either the β1 or β3 integrin subunits significantly inhibited fibronectin-mediated outgrowth. These findings demonstrate the key role of the central cell-binding domain of fibronectin in trophoblast adhesion, and suggest four RGD-binding integrins, α3β1, α5β1, αllbβ3, and αvβ3, that could mediate trophoblast adhesion in vitro and may play an important role during implantation. © 1995 Wiley-Liss, Inc.     

Fibronectin fibril formation involves cell interactions with two fibronectin domains

Fibronectin fragments and domain-specific antibodies have been used to study the mechanism by which cells reorganize exogenous fibronectin substrata into fibrils. Fibroblasts prevented from protein synthesis, and hence not secreting endogenous fibronectin or other matrix components, reorganized exogenous fibronectin substrata into arrays resembling the matrix of normally cultured cells. Cells also formed fibrils from substrata containing mixtures of cell- and either of two different heparin-binding fibronectin fragments but not from either fragment alone. The gelatin-binding fragment alone or in conjunction with the cell-binding fragment did not promote fibril formation. Antibodies recognizing cell- and either heparin- or the gelatin-binding domains labeled fibrils formed by cells under normal culture conditions or when a substratum of intact fibronectin was used as the sole exogenous source. However, only antibodies recognizing the cell- or either heparin-binding fragment reduced fibrillogenesis from intact fibronectin substrates when added during cell spreading. These data suggest that formation of fibronectin fibrils can occur at the cell surface and that membrane components recognizing the cell- and the heparin-binding domains in fibronectin may cooperate in the assembly process     

Adhesion of glycosaminoglycan-deficient chinese hamster ovary cell mutants to fibronectin substrata

We have examined the role of cell surface glycosaminoglycans in fibronectin-mediated cell adhesion by analyzing the adhesive properties of Chinese hamster ovary cell mutants deficient in glycosaminoglycans. The results of our study suggest that the absence of glycosaminoglycans does not affect the initial attachment and subsequent spreading of these cells on substrata composed of intact fibronectin or a fibronectin fragment containing the primary cell-binding domain. However, in contrast to wild-type cells, the glycosaminoglycan- deficient cells did not attach to substrate composed of a heparin- binding fibronectin fragment. Furthermore, the wild-type but not the glycosaminoglycan-deficient cells formed F-actin-containing stress fibers and focal adhesions on substrata composed of intact fibronectin. We propose, therefore, that cell surface proteoglycan(s) participate in the transmembrane linking of intracellular cytoskeletal components to extracellular matrix components which occurs in focal adhesions.     

Molecular mechanisms of avian neural crest cell migration on fibronectin and laminin

We have examined the molecular interactions of avian neural crest cells with fibronectin and laminin in vitro during their initial migration from the neural tube. A 105-kDa proteolytic fragment of fibronectin encompassing the defined cell-binding domain (65 kDa) promoted migration of neural crest cells to the same extent as the intact molecule. Neural crest cell migration on both intact fibronectin and the 105-kDa fragment was reversibly inhibited by RGD-containing peptides. The 11.5-kDa fragment containing the RGDS cell attachment site was also able to support migration, whereas a 50-kDa fragment corresponding to the adjacent N-terminal portion of the defined cell-binding domain was unfavorable for neural crest cell movement. In addition to the putative "cell-binding domain," neural crest cells were able to migrate on a 31-kDa fragment corresponding to the C-terminal heparin-binding (II) region of fibronectin, and were inhibited in their migration by exogenous heparin, but not by RGDS peptides. Heparin potentiated the inhibitory effect of RGDS peptides on intact fibronectin, but not on the 105-kDa fragment. On substrates of purified laminin, the extent of avian neural crest cell migration was maximal at relatively low substrate concentrations and was reduced at higher concentrations. The efficiency of laminin as a migratory substrate was enhanced when the glycoprotein occurred complexed with nidogen. Moreover, coupling of the laminin-nidogen complex to collagen type IV or the low density heparan sulfate proteoglycan further increased cell dispersion, whereas isolated nidogen or the proteoglycan alone were unable to stimulate migration and collagen type IV was a significantly less efficient migratory substrate than laminin-nidogen. Neural crest cell migration on laminin-nidogen was not affected by RGDS nor by YIGSR-containing peptides, but was reduced by 35% after addition of heparin. The predominant motility-promoting activity of laminin was localized to the E8 domain, possessing heparin-binding activity distinct from that of the N-terminal E3 domain. Migration on the E8 fragment was reduced by greater than 70% after addition of heparin. The E1' fragment supported a minimal degree of migration that was RGD-sensitive and heparin-insensitive, whereas the primary heparin-binding E3 fragment and the cell-adhesive P1 fragment were entirely nonpermissive for cell movement.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of cell substrate adhesion: effects of small galactosaminoglycan-containing proteoglycans.

Cell adhesion is a process which is initiated by the attachment of cells to specific sites in adhesive matrix proteins via cell surface receptors of the integrin family. This is followed by a reorganization of cytoskeletal elements which results in cell spreading and the formation of focal adhesion plaques. We have examined the effects of a class of small galactosaminoglycan-containing proteoglycans on the various stages of cell adhesion to fibronectin-coated substrates. Our results indicate that dermatan sulfate proteoglycans (DSPGs) derived from cartilage, as well as other related small proteoglycans, inhibit the initial attachment of CHO cells and rat embryo fibroblasts to substrates composed of the 105-kD cell-binding fibronectin fragment, but do not affect cell attachment to intact fibronectin. Although this effect involves binding of DSPGs to the substrate via the protein core, the intact proteoglycan is necessary for the observed activity. Isolated core proteins are inactive. The structural composition of the galactosaminoglycan chain does not appear to be functionally significant since both chondroitin sulfate and various dermatan sulfate proteoglycans of this family inhibit cell attachment to the fibronectin fragment. Neither the percentage of cells spread nor the mean area of spread cells adhering to substrates of intact fibronectin was significantly affected by the DSPGs. However, significantly fewer cells formed focal adhesions in the presence of DSPGs as compared with untreated control cells. These results suggest that the binding of small galactosaminoglycan-containing proteoglycans to a fibronectin substrate may affect several stages in the cell adhesion process.     

Effects of different fragments of the fibronectin molecule on latex bead translocation along neural crest migratory pathways

Previous studies from this laboratory have utilized latex beads as probes of embryonic migratory pathways. After microinjection into embryos at the time of neural crest migration, uncoated latex polystyrene beads were found to translocate to ventral sites and to settle in the vicinity of endogenous neural crest derivatives. However, latex beads coated with fibronectin did not translocate ventrally, but remained associated with cells surrounding the implantation site. Fibronectin is a large glycoprotein with a variety of biological activities and multiple binding domains. Here, the binding activities which might be responsible for immobilization of the fibronectin-coated beads are examined. Latex beads were coated with three types of fragments of the fibronectin molecule representing different functional domains: (i) a 66-kDa fragment containing collagen-binding activity; (ii) a mixture of 45- and 32-kDa fragments containing heparin-binding activity; and (iii) a 120-kDa fragment containing cell-binding activity. The beads coated with fibronectin fragments were injected into the newly formed trunk somites of avian embryos. After injection, beads coated with either the heparin- or the collagen-binding domain translocated ventrally and distributed analogously to uncoated latex beads. In contrast, the majority of beads coated with the fibronectin cell-binding domain did not translocate but remained associated with dermamyotomal cells surrounding the injection site. The cell-binding fragment, however, was not as effective as the intact fibronectin molecule in preventing translocation of the beads. The results suggest that the cell-binding domain is primarily responsible for restriction of fibronectin beads from the ventral neural crest pathway. Because intact fibronectin is more effective at immobilizing beads than is the cell-binding fragment, other binding domains of fibronectin, more efficient coating with intact fibronectin, or crosslinking of intact fibronectin molecules may also play some role in immobilization of the beads at the implantation site.     

Human fibronectin contains distinct adhesion- and motility-promoting domains for metastatic melanoma cells

The active migration of tumor cells through extracellular matrices has been proposed to play a role in certain aspects of metastasis. Metastatic tumor cells migrate in vitro in response to substratum-bound adhesive glycoproteins such as fibronectin. The present studies use affinity-purified proteolytic fragments of fibronectin to determine the nature of adhesion- and/or motility-promoting domains within the protein. Two distinct fragments were identified with cell adhesion-promoting activities. By a number of criteria, the adhesive activity promoted by these two fragments was distinct. One fragment, a 75-kD tryptic fragment purified by monoclonal antibody chromatography, promoted the adhesion, spreading, and haptotactic motility of melanoma cells. Experiments using a synthetic cell attachment peptide in solution indicated that at least part of the attachment activity exhibited by the 75-kD fragment is mediated by the sequence arg-gly-asp-ser. It was not possible to demonstrate migration-stimulating activity using a small (11.5 kD) peptic fragment containing this sequence (Pierschbacher, M.D., E. G. Hayman, and E. Ruoslahti, 1981, Cell, 26:259-267) suggesting that another cell-binding activity within the 75 kD fragment distinct from arg-gly-asp-ser might be required for motility. The second fragment that stimulated melanoma adhesion was a 33-kD tryptic/catheptic carboxyl-terminal heparin-binding fragment, which is localized to the A chain of fibronectin. This fragment promotes adhesion and spreading but not the motility of these cells. Melanoma adhesion to this heparin-binding fragment was sensitive to the effects of cycloheximide, which contrasted adhesion to the haptotaxis-promoting fragment. Importantly, these studies illustrate that haptotaxis in response to fibronectin is not due to simple adhesion gradients of this protein. The results are discussed in light of a model for multiple distinct cell surface constituents mediating cell adhesion and motility on fibronectin.     

Selective interaction of peripheral and central nervous system cells with two distinct cell-binding domains of fibronectin

Mechanisms of cell interaction with fibronectin have been studied with proteolytic fibronectin fragments that have well-defined ligand binding properties. Results of a previous study (Rogers, S. L., J. B. McCarthy, S. L. Palm, L. T. Furcht, and P. C. Letourneau, 1985, J. Neurosci., 5:369-378) demonstrated that (a) central (CNS) and peripheral (PNS) nervous system neurons adhere to, and extend neurites on a 33-kD carboxyl terminal fibronectin fragment that also binds heparin, and (b) neurons from the PNS, but not the CNS, have stable interactions with a 75-kD cell-binding fragment and with intact fibronectin. In the present study domain-specific reagents were used in inhibition assays to further differentiate cell surface interactions with the two fibronectin domains, and to define the significance of these domains to cell interactions with the intact fibronectin molecule. These reagents are (a) a soluble synthetic tetrapeptide Arg-Gly-Asp-Ser (RGDS; Pierschbacher, M. D., and E. Ruoslahti, 1984, Nature (Lond.), 309:30-33) representing a cell-binding determinant in the 75-kD fragment, and (b) an antibody raised against the 33-kD fragment that binds specifically to that fragment. Initial cell attachment to, and neurite extension upon, fibronectin and the two different fragments was evaluated in the presence and absence of the two reagents. Attachment of both PNS and CNS cells to intact fibronectin was reduced in the presence of RGDS, the former more so than the latter. In contrast, the antibody to the 33-kD fragment did not affect attachment of PNS cells to fibronectin, but significantly decreased attachment of CNS cells to the molecule. RGDS inhibited attachment of CNS cells to the molecule. RGDS inhibited attachment of both cell types to the 75-kD fragment to a greater degree than it did attachment to the intact molecule. Cell interaction with the 33-kD fragment was not affected by RGDS. Reduction of neurite lengths (determined after 24 h of culture) by the domain-specific reagents paralleled the reduction in initial adhesion to each substratum. Therefore, it appears that (a) both PNS and CNS cells have receptors for each cell-binding domain of fibronectin, (b) the receptor(s) for the two domains are distinct, with attachment to the 33-kD fragment being independent of RGDS, and (c) the relative importance of each domain to cell interaction with intact fibronectin is different for CNS and PNS cells.     

Influence of decorin on fibroblast adhesion to fibronectin.

Decorin is a ubiquitous small dermatan sulfate proteoglycan carrying a single glycosaminoglycan chain. It is known for its ability to bind, via its core protein, to interstitial collagens. Decorin was purified from the secretions of cultured human skin fibroblasts under non-denaturing conditions. The intact proteoglycan and its glycosaminoglycan-free core protein were tested for their interference with fibroblast adhesion to a fibronectin substrate. Concentrations of 40 nmoles or more of hexuronic acid/ml of decorin or equivalent amounts of core protein inhibited cell adhesion. Inhibition was caused by an interaction of core protein with fibronectin and not by masking of the fibronectin receptor. When cell-binding fragments of fibronectin were used as substrates, a similar inhibition of cell adhesion by decorin core protein was found, and in vitro assays demonstrated an interaction of core protein with the cell-binding domain of fibronectin. Decorin core protein also inhibited the low degree of cell adhesion to heparin-binding fragments on the N-terminus and near the C-terminus of the fibronectin molecules.     

A synthetic peptide from the COOH-terminal heparin-binding domain of fibronectin promotes focal adhesion formation.

Cell adhesion to extracellular matrix molecules such as fibronectin involves complex transmembrane signaling processes. Attachment and spreading of primary fibroblasts can be promoted by interactions of cell surface integrins with RGD-containing fragments of fibronectin, but the further process of focal adhesion and stress fiber formation requires additional interactions. Heparin-binding fragments of fibronectin can provide this signal. The COOH-terminal heparin-binding domain of fibronectin contains five separate heparin-binding amino acid sequences. We show here that all five sequences, as synthetic peptides coupled to ovalbumin, can support cell attachment. Only three of these sequences can promote focal adhesion formation when presented as multicopy complexes, and only one of these (WQPPRARI) retains this activity as free peptide. The major activity of this peptide resides in the sequence PRARI. The biological response to this peptide and to the COOH-terminal fragment may be mediated through cell surface heparan sulfate proteoglycans because treatment of cells with heparinase II and III, or competition with heparin, reduces the response. Treatment with chondroitinase ABC or competition with chondroitin sulfate does not.     

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.     

Rat plasma fibronectin contains two distinct chemotactic domains for fibroblastic cells

Mechanism of fibronectin (FN)-induced chemotaxis of fibroblastic cells has not been fully understood. The present study was performed to establish a molecular nature of the chemotactic region of rat plasma FN. The chemotactic dose-response pattern of intact FN for mouse embryo fibroblastic cells, NIH-L13 cells, which was represented as a "bell-shape" curve with a maximum activity at around 50 nM, changed to a "biphasic" mode through a proteolysis with thermolysin. Two distinct chemotactic components were isolated from the thermolytic fragments. One component, a fragment with a molecular mass of 110-150 kDa, was estimated to contain the central cell-binding domain and the carboxyl-terminal heparin-binding domain of the intact FN molecule. Cell migration stimulated by the 110-150-kDa fragment increased successively in a dose-dependent manner, and the capability to promote the migration was much higher than that of the intact FN (over 2-fold). The second chemotactic component, a fragment with a molecular mass of 21 kDa, was shown to reside in the carboxyl-terminal fibrin-binding domain. The 21-kDa fragment produced a bell-shape dose-response pattern, being consistent with the intact FN, whereas a maximum response occurred at a 100-fold lower concentration (0.5 nM) than that of the intact FN molecule. At higher concentrations, this fragment revealed an inhibitory activity for the cell migration in response to the 110-150-kDa fragment. No significant molecular interaction between these two active components was observed by polyacrylamide gel electrophoresis under nondenaturing conditions, suggesting that the 21-kDa fragment may act directly on the cell to inhibit the cell migration. These results suggest that rat plasma FN contains at least two chemotactically active components that regulate cooperatively chemotactic migration of fibroblastic cells.     

Demonstration of high affinity fibronectin receptors on rat hepatocytes in suspension

A cell-binding peptide (Mr = 85,000) which lacks the gelatin- and heparin-binding domains, was purified from trypsin-digested fibronectin. Preincubation of rat hepatocytes in suspension with the peptide, inhibited initial attachment of the cells to immobilized fibronectin while attachment to immobilized laminin and collagen was unaffected. 125I-labeled 85-kDa peptide bound to the cells in suspension at 4 degrees C in a time-dependent, saturable, and partially reversible reaction. Scatchard analysis of the binding data indicated a single class of receptors with a Kd of 1.7 X 10(-8) M. The number of binding-sites was approximately 2.8 X 10(5)/cell. Unlabeled 85-kDa peptide inhibited the binding of 125I-labeled 85-kDa peptide 30-fold more effectively than intact fibronectin. These results provide direct evidence for the presence of a domain in the fibronectin molecule which has or may acquire a high affinity for receptors involved in adhesion of hepatocytes to immobilized fibronectin.     

Adhesion of lymphoid cells to the carboxyl-terminal heparin-binding domains of fibronectin

Previously, we have shown that some lymphoid cell lines adhere to fibronectin (FN)-coated substratum, whereas others do not. In this study, the adhesion of five adherent lymphoid cell lines to different FN domains was examined. These cell lines ranged in their adherence to substratum coated with FN, the cell-binding domain (CBD) fragment, or the heparin-binding domain (HBD) fragments. None of the cell lines adhered to substratum coated with the gelatin-binding domain fragment. Three of the lymphoid cell lines adhered preferentially to HBD over CBD, whereas two other lymphoid cell lines and BHK fibroblasts adhered preferentially to CBD. These results suggest that two distinct adhesive interactions occur between cells and FN and that the pattern of interaction varies among cell types. Using MOPC 315 (which adheres preferentially to HBD) as a cell model to study the cell-HBD interaction, the HBD-promoted adhesion was found to be independent of the RGD sequence and could be inhibited by anti-FN antibodies. Moreover, the MOPC 315-HBD interaction had the following characteristics: (1) adhesion was temperature dependent, (2) presence of divalent cations was necessary, (3) integrity of cellular microfilaments but not microtubules was required, (4) inhibition of protein synthesis abolished adhesion, (5) pretreatment of cells with trypsin inhibited adhesion, and (6) the adhesion was mediated by the carboxyl-terminal HBD.     

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.     

Immunological cross-reactivity between human tripeptidyl peptidase II and fibronectin.

Tripeptidyl peptidase II (TPP II) is a large intracellular exopeptidase with an active site of the subtilisin type. Affinity-purified hen antibodies against human erythrocyte TPP II cross-reacted with fibronectin in an immunoblot analysis. Furthermore, antibodies against human fibronectin cross-reacted with TPP II. Antibodies against a 65 kDa cell-binding fragment of fibronectin specifically reacted with TPP II, whereas antibodies against the collagen-binding domain, the main heparin-binding domain or the N-terminal fibrin-binding domain did not react. Moreover, the affinity-purified antibodies against TPP II reacted with a 105 kDa cell-binding fragment of fibronectin but not with the fibrin-binding domain or the collagen-binding domain. When native TPP II was dissociated into smaller units through dialysis against a dilute Tris buffer, it could be digested by chymotrypsin into three stable fragments of 70 kDa, 42 kDa and 20 kDa. It could be demonstrated that the 42 kDa fragment was specifically recognized by antibodies against the 65 kDa cell-binding fragment of fibronectin. Furthermore, labelling with di-[3H]isopropyl phosphorofluoridate and N-terminal sequence determination showed that the 70 kDa fragment contained the active-site serine residue. In conclusion, our findings suggest that one domain of the TPP II molecule bears structural resemblance to a cell-binding fragment of fibronectin.     

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.