Heparin-binding Domain Of Fibronectin Research Articles

Functional differences between integrin α4 and integrins α5/αv in modulating the motility of human oral squamous carcinoma cells in response to the V region and heparin-binding domain of fibronectin

The high-affinity heparin-binding domain and the V region of fibronectin (FN) mediate invasion and migration of human oral squamous cell carcinoma (SCC) cells. We investigated the role of the α4, α5, and αv integrin receptors—which are central to mediating interactions with these domains of FN—in regulating SCC cell migration. SCC cells expressed the α4, α5, and αv integrin subunits on their surface, although α4 expression was low. Treatment with recombinant FN proteins containing an alternatively spliced V region (V+) and either an unmutated (H+) or a mutated, nonfunctional high-affinity heparin-binding domain (H−) increased expression of α5 and αv and cell motility. Antisense α5 or αv oligonucleotides inhibited cell motility stimulated by FN proteins, as did blocking antibodies to α5 or αv. Blocking antibodies to α5 increased αv and α4 levels, and blocking antibodies to αv increased the levels of α5 and α4, without increasing cell motility. In contrast, an antisense α4 oligonucleotide and α4-blocking antibodies increased cell motility, especially migration stimulated by V+H+ and V+H− FN proteins. α4-Blocking antibodies alone increased motility, probably by inducing α5 and αv expression. Transfection with α4 cDNA decreased cell motility and α5 and αv expression. Thus, the increased motility induced by the FN protein is probably mediated by αv and α5, whereas α4 downregulates this process in a transdominant fashion.

Integrin α5β1 supports the migration of Xenopus cranial neural crest on fibronectin

During early embryonic development, cranial neural crest cells emerge from the developing mid- and hindbrain. While numerous studies have focused on integrin involvement in trunk neural crest cell migration, comparatively little is known about mechanisms of cranial neural crest cell migration. We show that fibronectin, but not laminin, vitronectin, or type I collagen can support cranial neural crest cell migration and segmentation in vitro. These behaviors require both the RGD and “synergy” sites located within the central cell-binding domain of fibronectin. While these two sites are sufficient for cranial neural crest cell migration, we find that the second Heparin-binding domain of fibronectin can provide additional support for cranial neural crest cell migration in vitro. Finally, using a function blocking monoclonal antibody, we show that cranial neural crest cell migration on fibronectin requires the integrin α5β1.

ADAM13 Disintegrin and Cysteine-rich Domains Bind to the Second Heparin-binding Domain of Fibronectin

ADAM13 is a member of the disintegrin and metalloprotease protein family that is expressed on cranial neural crest cells surface and is essential for their migration. ADAM13 is an active protease that can cleave fibronectin in vitro and remodel a fibronectin substrate in vivo. Using a recombinant secreted protein containing both disintegrin and cysteine-rich domains of ADAM13, we show that this "adhesive" region of the protein binds directly to fibronectin. Fibronectin fusion proteins corresponding to the various functional domains were used to define the second heparin-binding domain as the ADAM13 binding site. Mutation of the syndecan-binding site (PPRR --> PPTM) within this domain abolishes binding of the recombinant disintegrin and cysteine-rich domains of ADAM13. We further show that the adhesive disintegrin and cysteine-rich domain of ADAM13 can promote cell adhesion via beta(1) integrins. This adhesion requires integrin activation and can be prevented by antibodies to the cysteine-rich domain of ADAM13 and beta(1) integrin. Finally, wild type, but not the E/A mutant of ADAM13 metalloprotease domain, can be shed from the cell surface, releasing the metalloprotease domain associated with the disintegrin and cysteine-rich domains. This suggests that ADAM13 shedding may involve its own metalloprotease activity and that the released protease may interact with both integrins and extracellular matrix proteins.

Gene transfer of a fibronectin peptide inhibits leukocyte recruitment and suppresses inflammation in mouse collagen-induced arthritis.

Cell adhesion plays an essential role in arthritis by recruiting and retaining leukocytes in the joint. Fibronectin, a major extracellular matrix component in synovium, plays a central role in cell-cell and cell-matrix interactions through ligation of cell surface integrins. The present study was designed to determine the effects of gene transfer of a 15-amino acid peptide derived from the 33-kd carboxy-terminal cell and heparin-binding domain of fibronectin (FN-C/H-II) on established arthritis in mice. Plasmid DNA encoding a FN-C/H-II minigene under control of the cytomegalovirus promoter was injected intravenously into mice with established collagen-induced arthritis, and the effects on leukocyte adhesion and recruitment to the joints was determined. Following injection, circulating FN-C/H-II could be detected for at least 5 days. Treated mice demonstrated a marked reduction in progression of arthritis. Not only was disease progression halted, but a significant improvement in joint swelling was observed within 2 days of treatment. Leukocyte adhesion and recruitment were inhibited by FN-C/H-II, both in vitro and in vivo. Histologic evaluation revealed a marked reduction in infiltration of both neutrophils and lymphocytes into synovium, persisting for at least 10 days. These results suggest that antagonism of cell adhesion by soluble fibronectin peptides may provide an approach to attenuating chronic arthritis.

Regulation of heparan sulfate proteoglycan nuclear localization by fibronectin.

Heparan sulfate proteoglycans (HSPG) regulate multiple cellular processes and mediate the cellular uptake of numerous molecules. While heparan sulphate glycosaminoglycan chains are known to modulate receptor binding of several heparin-binding proteins, here we show that distinct extracellular matrices direct HSPG to the nucleus. We analyzed HSPG localization in primary corneal fibroblasts, cultured on fibronectin or collagen type I matrices, using confocal laser scanning microscopy and cell fractionation. Image analysis revealed that the nuclear localization of HSPG core proteins was greater when cells were cultured on fibronectin versus collagen. Matrices containing the heparin-binding domain of fibronectin, but not the integrin-activating domain, demonstrated increased nuclear staining of core proteins. Furthermore, activation of protein kinase C with phorbol 12-myristate 13-acetate inhibited nuclear targeting of HSPG in cells on fibronectin, whereas inhibition of protein kinase C with Ro-31-8220 greatly enhanced nuclear localization of HSPG in cells on both collagen and fibronectin. We propose a matrix-dependent mechanism for nuclear localization of cell surface HSPG involving protein kinase C-mediated signaling. Nuclear localization of HSPG might play important roles in regulating nuclear function.

Mutations in the Heparin Binding Domain of Fibronectin in Cooperation with the V Region Induce Decreases in pp125FAKLevels Plus Proteoglycan-mediated Apoptosis via Caspases

Intact fibronectin (FN) protects cells from apoptosis. When FN is fragmented, specific domains induce proteinase expression in fibroblasts. However, it is not known whether specific domains of FN can also regulate apoptosis. We exposed fibroblasts to four recombinant FN fragments and then assayed for apoptosis using criteria of cellular shape change, condensed nuclear morphology, and DNA fragmentation. The fragments extended from the RGD-containing repeat III10 to III15; they included (V(+)) or excluded (V(-)) the alternatively spliced V region and contained either a mutated (H(-)) or an unmutated (H(+)) heparin binding domain. Only the V(+)H(-) fragment triggered decreases in pp125(FAK) levels and apoptosis, which was rescued by intact FN and inhibitors of caspase-1 and caspase-3. This apoptotic mechanism was mediated by a chondroitin sulfate proteoglycan, since treating cells with chondroitin sulfate or chondroitinase reversed the apoptotic cell shape changes. The alpha4 integrin receptor may also be involved, since using a blocking antibody to alpha4 alone induced apoptotic cell shape changes, whereas co-treatment with this antibody plus V(+)H(+) reversed these effects. These results demonstrate that the V and heparin binding domains of FN modulate pp125(FAK) levels and regulate apoptosis through a chondroitin sulfate proteoglycan- and possibly alpha4 integrin-mediated pathway, which triggers a caspase cascade.

Fibronectin Type III5 Repeat Contains a Novel Cell Adhesion Sequence, KLDAPT, Which Binds Activated α4β1 and α4β7 Integrins

The region of fibronectin encompassing type III repeats 4-6 contains a low affinity heparin binding domain, but its physiological significance is not clear. We have studied whether this domain is able to interact with cells as already shown for other heparin binding domains of fibronectin. A computer search based on homologies with known active sites in fibronectin revealed the sequence KLDAPT located in FN-III5. A synthetic peptide containing this sequence induced lymphoid cell adhesion upon treatment with the activating anti-beta1 monoclonal antibody (mAb) TS2/16 or with Mn2+, indicating that KLDAPT was binding to an integrin. A recombinant fragment containing repeat III5 (FN-III5) also mediated adhesion of TS2/16/Mn2+-treated cells while the FN-III6 fragment did not. Soluble KLDAPT peptide inhibited cell adhesion to FN-III5 as well as to a 38-kDa fibronectin fragment and VCAM-1, two previously known ligands for alpha4beta1 integrin. KLDAPT also competed with the binding of soluble alkaline phosphatase-coupled VCAM-Ig to Mn2+-treated alpha4beta1. Furthermore, mAbs anti-alpha4 and anti-alpha4beta7, but not mAbs to other integrins, inhibited cell adhesion to FN-III5 and KLDAPT. These results therefore establish a cell adhesive function for the FN-III5 repeat and show that KLDAPT is a novel fibronectin ligand for activated alpha4 integrins.

Novel Synthetic Peptides From the C-terminal Heparin Binding Domain of Fibronectin With Heparin Binding Activity

To identify a minimal peptide ligand that participates in recognition of heparin, we synthesized peptides extending a 29-kDa fragment in the C -terminal heparin binding domain of fibronectin. We obtained evidence that two peptides (designated D5 and D1) exhibit inhibitory activity on binding of the 29-kDa fragment to heparin-Sepharose CL-6B. In dose inhibitory studies, peptide D5 showed the potent inhibitory activity with IC 50 of 210 ± 37 μ M . A new heparin binding site in the C -terminal heparin binding domain of fibronectin is demonstrated and provides a rationale for understanding the mechanism of cell adhesion and spreading.

A 31-kDa recombinant fibronectin cell-binding domain fragment: its binding to receptor, cell adhesive activity, and fusion proteins.

The binding of fibronectin to fibronectin receptor was studied using a recombinant 31-kDa cell-binding domain fragment of fibronectin (C279), which consisted of three type III repeats (III8-III9-III10). Fibronectin receptor in several cell lysates was bound to a column of C279-immobilized Sepharose HP and obtained in a highly purified form by elution with a synthetic peptide, GRGDSP. alpha 5 beta 1-Integrin was detected in the GRGDSP-eluted fraction by immunoblotting. The cell-adhesive activity of C279 was inhibited by GRGDSP peptide, an anti-integrin a5 subunit antibody, and an anti-integrin beta 1 subunit antibody. The cell adhesion of fusion proteins of the 31-kDa fragment with biologically interesting polypeptides (heparin-binding domain of fibronectin, and basic fibroblast growth factor) was also studied. In the presence of an anti-integrin a5 subunit antibody, human fibrosarcoma HT-1080 cells attached to the fusion protein containing fibroblast growth factor, giving rise to changes the morphology of the attached cells. The cell adhesion of C279 was inhibited by GRGDSP peptide but that of the fusion protein with the heparin-binding domain of fibronectin was not completely inhibited by the peptide. These results suggest that these biologically interesting polypeptides contribute to the cell adhesion of the fusion proteins.

Role of the heparin binding domain of fibronectin in attachment and spreading of human bone-derived cells

Human bone-derived cells are known to attach and spread on surfaces which have been precoated with fibronectin, but the contributions made by specific domains of the molecule have not yet been defined. Here we refer to the osteoblast-like cells as human bone cells. We have determined the relevance of separate regions of fibronectin, particularly the heparin-binding region, for the initial attachment and spreading of these cells. Human bone cells attached to fragments from each of the cell- and heparin-binding regions of fibronectin, but failed to attach to a fragment from the gelatin-binding region. Bovine corneal epithelial cells, which were included as an example of an alternative primary cell strain, attached to the cell-binding fragment but showed no specific short-term attachment to the heparin or gelatin-binding fragments. Monoclonal antibody MAb17, which binds to the cell binding region of fibronectin, partially inhibited the attachment of both human bone cells and corneal epithelial cells to intact fibronectin when present at 50 micrograms/ml and reduced human bone cell attachment to the cell-binding region fragment of fibronectin. Monoclonal antibody, MAb 32, which binds to the heparin-binding region of fibronectin, failed to inhibit attachment of the human bone cells to fibronectin but reduced the attachment of these cells to the heparin-binding region fragment. Heparin and chondroitin sulphate were able to inhibit human bone cell attachment to the heparin-binding fragment of fibronectin but had no effect on their attachment to intact fibronectin or the cell-binding region of fibronectin. Immunofluorescent staining and confocal microscopy showed extensive spreading and actin filament formation when human bone cells were cultured on intact fibronectin. Cells cultured on the heparin-binding fragment showed only minimal spreading coinciding with less extensive actin filament organisation. On the cell-binding fragment of fibronectin more spreading was seen than on the heparin-binding fragment but it was not as extensive as on intact fibronectin. Taken together, these data suggest that human bone cells, unlike bovine corneal epithelial cells, have an attachment mechanism for the heparin-binding region of fibronectin. Attachment to this region is probably mediated by cell surface proteoglycans. However, interaction with the cell-binding domain is required for effective cell spreading of human bone cells on fibronectin during the first 90 minutes after seeding into culture.

IL-3 increases surface proteoglycan synthesis in haemopoietic progenitors and their adhesiveness to the heparin-binding domain of fibronectin.

Haemopoietic progenitor cells (HPC) synthesize and accumulate a single type of membrane-associated chondroitin sulphate proteoglycan (MA-PG), which participates in HPC adhesiveness to fibronectin by interacting with its heparin-binding domain. Shortly after incubating cells with IL-3, we observed an increase in MA-PG synthesis in the multipotent (FDCP-mix) but not in the bipotent (FDCP-1) progenitor cell line. The charge density, hydrodynamic size, nature of the glycosaminoglycan (GAG) chains and stability of MA-PG from IL-3-treated and non-treated FDCP-mix cells were the same, suggesting that IL-3 affects the amount of MA-PG. The latter was evaluated by flow cytometry using monoclonal antibodies to the core protein and GAG residues. In all cases the mean fluorescence intensities were higher for IL-3-treated than for untreated cells. Cell adhesion studies to dishes coated with the fibronectin 40 kD fragment, containing the heparin-binding domain, demonstrated that adhesiveness of IL-3-treated cells was higher than that of untreated cells. These results suggest that in multipotent haemopoietic cells IL-3 regulates the amount of membrane-associated proteoglycans, which in turn modify the adhesive interactions of cells with the heparin-binding domain of fibronectin.

Fibronectin attachment activates the NF-kappa B p50/p65 heterodimer in fibroblasts and smooth muscle cells.

The nuclear factor kappa B, NF-kappa B, is activated by numerous inflammatory mediators, regulating biological responses that in part are dependent on the extracellular matrix. Analyses of the influence of cell-matrix interaction on NF-kappa B activation revealed that attachment to fibronectin caused an increase in NF-kappa B activity corresponding to 3-fold the level in cells plated on bare plastic. This effect was dependent on the degree of attachment and appeared to involve both the RGD motif of the cell-binding domain, and the heparin-binding domain of fibronectin. Fibronectin attachment specifically caused activation of the p50/p65 heterodimeric form of NF-kappa B, resulting in an increase of this complex to a level similar to that induced by cytokine stimulation. These data show that cell attachment has a pronounced influence on the level of NF-kappa B activity, affecting specific dimeric complexes, and suggest this to be a pathway through which effects depending on cell-matrix interaction can regulate cell behavior and cytokine responses.

Isolation and partial characterization of a cell-surface heparan sulfate proteoglycan from embryonic rat spinal cord.

Cell-surface heparan sulfate proteoglycans (HSPGs) are potential mediators of neuronal cell adhesion, spreading, and neurite outgrowth on various extra-cellular matrix molecules. One possible site of HSPG attachment is a heparin binding domain of fibronectin, which is present in the synthetic peptide FN-C/H II. In this study, HSPGs extracted from embryonic rat spinal cord by detergent were purified by ion-exchange chromatography, gel filtration, and affinity chromatography on an agarose column coupled with FN-C/H II conjugated to ovalbumin (OA). Heparitinase treatment of the iodinated HSPG fraction led to the appearance of a major protein core with a molecular size of 72 kDa, as determined by reducing SDS-PAGE. The intact proteoglycan has a molecular size of approximately 150-165 kDa, containing heparan sulfate glycosaminoglycan chains of about 10-15 kDa. Anti-HSPG antibodies recognized the 72 kDa core protein by immunoblotting, and stained the surface of spinal cord neurons, oligodendrocytes, and a subset of astrocytes. These results identify a cell-surface HSPG that may mediate neuron-substratum or neuron-glia interactions in embryonic central nervous system.

Modulation of tissue plasminogen activator-catalyzed plasminogen activation by synthetic peptides derived from the amino-terminal heparin binding domain of fibronectin.

Fibronectin is a multidomain adhesive glycoprotein found in plasma, interstitial connective tissue, and basement membrane. Diverse biological activities have been associated with the fibronectin molecule including cell adhesion, cell migration, wound healing, hemostasis, and oncogenic transformation. Binding sites for heparin, fibrin, gelatin/collagen, and cells have been localized to various structural domains of the molecule. In addition, fibronectin also binds both plasminogen and tissue plasminogen activator (t-PA) via a 55-kDa amino-terminal fragment (Moser, T.L., Enghild, J.J., Pizzo, S.V., and Stack, M.S. (1993) J. Biol. Chem. 268, 18917-18923). Although intact fibronectin does not enhance the rate of t-PA-catalyzed plasminogen activation, a mixture of proteolytically degraded fibronectin fragments stimulates the activation reaction, resulting in an 11-fold increase in the kcat/Km. Based on these observations, we have synthesized a variety of peptides derived from the plasminogen/t-PA binding region of fibronectin and determined the effect of these peptides on the initial rate kinetics of plasminogen activation by t-PA as well as on plasmin and t-PA amidolytic activity. Here we report that a specific octapeptide, SRNRCNDQ-NH2, consisting of residues 196-203 of the fibronectin molecule is a potent stimulator of t-PA-catalyzed plasminogen activation, resulting in a 15-fold increase in the kcat/Km of the activation reaction.

Platelet interactions with fibronectin: divalent cation-independent platelet adhesion to the gelatin-binding domain of fibronectin

Divalent cation-dependent platelet adhesion to fibronectin (FN) is mediated by the integrin receptors alpha 5 beta 1 (GP Ic-IIa) and alpha IIb beta 3 (GP IIb-IIIa), which recognize the RGD (Arg-Gly-Asp) sequence in the cell-binding domain. However, FN can also support divalent cation-independent platelet adhesion. To determine which domain of FN mediates divalent cation-independent adhesion, proteolysis with thermolysin and affinity chromatography were used to isolate the cell-binding, gelatin-binding, and heparin-binding domains of FN. Unactivated and thrombin-activated platelets adhered to intact FN and the 45-Kd gelatin-binding domain in the presence of either Ca2+ or EDTA. Platelet spreading was mediated only by the 105-Kd cell-binding domain and required divalent cations. The heparin-binding domains did not support platelet adhesion. Reduction of intrachain disulfide bonds or removal of carbohydrate side chains on the gelatin-binding domain did not alter the ability to support platelet adhesion. Divalent cation-independent adhesion to the 45-Kd gelatin-binding domain was not inhibited by RGDS (Arg-Gly-Asp-Ser) synthetic peptides or monoclonal antibodies (MoAbs) directed against known platelet receptors. We conclude that platelets can adhere but not spread on the gelatin-binding domain of FN by a novel divalent cation-independent mechanism.

Platelet Interactions With Fibronectin: Divalent Cation-Independent Platelet Adhesion to the Gelatin-Binding Domain of Fibronectin

Abstract Divalent cation-dependent platelet adhesion to fibronectin (FN) is mediated by the integrin receptors alpha 5 beta 1 (GP Ic-IIa) and alpha IIb beta 3 (GP IIb-IIIa), which recognize the RGD (Arg-Gly-Asp) sequence in the cell-binding domain. However, FN can also support divalent cation-independent platelet adhesion. To determine which domain of FN mediates divalent cation-independent adhesion, proteolysis with thermolysin and affinity chromatography were used to isolate the cell-binding, gelatin-binding, and heparin-binding domains of FN. Unactivated and thrombin-activated platelets adhered to intact FN and the 45-Kd gelatin-binding domain in the presence of either Ca2+ or EDTA. Platelet spreading was mediated only by the 105-Kd cell-binding domain and required divalent cations. The heparin-binding domains did not support platelet adhesion. Reduction of intrachain disulfide bonds or removal of carbohydrate side chains on the gelatin-binding domain did not alter the ability to support platelet adhesion. Divalent cation- independent adhesion to the 45-Kd gelatin-binding domain was not inhibited by RGDS (Arg-Gly-Asp-Ser) synthetic peptides or monoclonal antibodies (MoAbs) directed against known platelet receptors. We conclude that platelets can adhere but not spread on the gelatin- binding domain of FN by a novel divalent cation-independent mechanism.

33-kDa C-terminal heparin binding fragment of fibronectin promotes attachment and spreading of hepatocytes

The possibility of interaction of hepatocytes with the heparin binding domain of Fibronectin was examined. Rat hepatocytes adhered to coverslips coated with the 33-kDa heparin binding fragment of the C-terminal region of plasma fibronectin. When different concentrations of the heparin binding fragment were used to coat coverslips and used as substratum, cell attachment showed saturation kinetics. Half the maximum attachment was observed at 30–40 min after seeding of cells. The cells became flat after 2–3 h indicating that they spread on the heparin binding domain as they do on intact fibronectin. Among the different glycosaminoglycans tested, maximum inhibition of attachment was observed for heparin. However it was not possible to completely inhibit attachment even at high concentrations. These results indicate that hepatocytes interact with fibronectin not only through the Arg-Gly-Asp-containing cell binding fragment, but also through the heparin binding domain of fibronectin and, further, that there exist heparin-dependent and heparin-independent mechanisms of interaction of cells with the 33-kDa heparin binding fragment of fibronectin

Keratinocyte Migration Is Partially Supported by the Cell-Binding Domain of Fibronectin and Is RGDS-Dependent

Fibronectin (FN) plays a key role in cell attachment, embryonic development, and wound healing. In this respect, it is known that FN promotes keratinocyte migration. The aim of this study was to examine specific FN domains (120-kD cell-binding fragment, 45-kD collagen fragment, and 40-kD heparin fragment) and a biologically active peptide within the molecule (RGDS) for their ability to influence human keratinocyte (HK) locomotion. HKs were plated on gold salts coated with different substrates (type IV collagen, FN with or without the RGDS peptide, and the three FN fragments). After 20 h, locomotion tracks were quantified by computer-assisted image analysis that determines the area of each microscopic field occupied by migration tracks, a so-called migration index (MI). MIs on type IV collagen and FN were 39.14 +/- 2.8% and 30 +/- 0.4%, respectively. The maximal MIs on the collagen-binding domain and heparin-binding domain of FN were similar to our negative controls (plastic and albumin): 3 +/- 1%. In contrast, the maximal MI on the cell-binding fragment of FN was 18.45 +/- 2.1%. The effect of the cell-binding domain on keratinocyte motility was found to be dose dependent. Moreover, we could specifically inhibit the FN-driven locomotion using the RGDS sequence contained in the cell-binding fragment. We did not observe a synergistic effect (i.e., a higher MI) when we added the three fragments in a same dish. These results suggest i) that the cell-binding fragment of FN partially supports HK locomotion, ii) that other untested FN domain(s) should act in synergy with the cell-binding fragment to promote keratinocyte locomotion, or alternatively iii) that the FN function of promoting cell migration resides within the FN cell-binding domain, but the proper presentation of this domain to the cell requires an intact, native FN molecule, and iv) that the RGDS sequence is essential for HK movement.

Localization of two binding domains for thrombospondin within fibronectin

Thrombospondin is a major glycoprotein of the platelet α-granule and is secreted during platelet activation. Several protease-resistant domains of thrombospondin mediate its interactions with components of the extracellular matrix including fibronectin, collagen, heparin, laminin, and fibrinogen. Thrombospondin, as well as fibronectin, is composed of several discretely located biologically active domains. We have characterized the thrombospondin binding domains of plasma fibronectin and determined the binding affinities of the purified domains; fibronectin has at least two binding sites for thrombospondin. Thrombospondin bound specifically to the 29-kDa amino-terminal heparin binding domain of fibronectin as well as to the 31-kDa non-heparin binding domain located within the larger 40-kDa carboxy-terminal fibronectin domain generated by chymotrypsin proteolysis. Platelet thrombospondin interacted with plasma fibronectin in a specific and saturable manner in blot binding as well as solid-phase binding assays. These interactions were independent of divalent cations. Thrombospondin bound to the 29-kDa fibronectin heparin binding domain with a K d of 1.35 × 10 −9 m. The K d for the 31-kDa domain of fibronectin was 2.28 × 10 −8 m. The 40-kDa carboxy-terminal fragment bound with a K d of 1.65 × 10 −8 m. Heparin, which binds to both proteins, inhibited thrombospondin binding to the amino-terminal domain of fibronectin by more than 70%. The heparin effect was less pronounced with the non-heparin binding carboxy-terminal domain of fibronectin. By contrast, the binding affinity of the thrombospondin 150-kDa domain, which itself lacked heparin binding, was not affected by the presence of heparin. Based on these data, we conclude that thrombospondin binds with different affinities to two distinct domains in the fibronectin molecule.

A cell-surface heparan sulfate proteoglycan mediates neural cell adhesion and spreading on a defined sequence from the C-terminal cell and heparin binding domain of fibronectin, FN-C/H II

FN-C/H II is a heparin binding synthetic peptide from the C-terminal cell and heparin binding domain of fibronectin (FN) that mediates neuronal cell adhesion, spreading, and neurite outgrowth. Cellular interactions with FN-C/H II are inhibited by soluble heparin, suggesting that a cell-surface proteoglycan may mediate interactions with FN-C/H II (Haugen et al., 1990). To test this hypothesis further, heparan sulfate (HS) or chondroitin sulfate (CS) was removed from the cell surface by enzyme treatment. Heparitinase but not chondroitinase treatment of cells inhibited rat B104 neuroblastoma cell adhesion and spreading on FN-C/H II. Additionally, heparitinase treatment decreased the spreading of cells on the 33/66 kDa fragments containing the C-terminal heparin binding domain of FN. Furthermore, antibodies generated against a mouse melanoma HS proteoglycan (HSPG) inhibited B104 cell adhesion to FN-C/H II and the 33/66 kDa FN fragments. 35S-HSPG isolated from B104 cells directly bound to FN-C/H II both in solid phase assays and by affinity chromatography, but failed to bind to a control peptide from this region, CS1. The binding of 35S-HSPG was predominantly mediated by the HS and not the core protein of the HSPG. SDS-PAGE of iodinated HSPG demonstrated a single 78 kDa core protein following heparitinase digestion, which migrated at 51 kDa under nonreducing conditions. Anti-HSPG antibodies recognized the 78 kDa core protein by immunoblotting, and stained the surface of rat B104 neuroblastoma cells and cells of the primary neonatal rat nervous system. These results identify a cell-surface HSPG that likely mediates neuronal cell binding interactions with FN-C/H II.