INTRODUCTION

Proteoglycans are complex molecules formed by a core protein to which one or more glycosaminoglycan (GAG)¹ chains are linked. This basic definition, although true, hides the molecular complexity shown by these molecules. They encompass an exceptionally large range of structures involving different core proteins, different classes of GAGs, and different numbers and lengths of individual GAG chains. Other post-translation modifications such as N- and O-glycosylation increase the complexity of these molecules (for review see Refs. 1 and 2).

The biological functions of proteoglycans are numerous. They have been involved in several biological effects (1, 3-5), such as extracellular matrix (ECM) assembly (6) and cell surface-ECM receptors for growth factors and hormones (2, 5, 7) or have had a role in biological processes such as cell-cell recognition (8) and control of cell growth (9). The fact that several ECM proteins, such as fibronectin (10), laminin (11), thrombospondin (12), vitronectin (13), type IV collagen (14), and tenascin (15), have GAG binding sites adds credence to the postulated multiple roles of proteoglycans. Supporting the idea of proteoglycans as ECM receptors, syndecan type I binds fibronectin, thrombospondin, collagens (5), and tenascin (16); the heparan sulfate proteoglycan of Schwann cells binds laminin (17); a cell surface chondroitin sulfate proteoglycan is apparently involved in cell adhesion to laminin (18); and a cell surface phosphatidyl inositol-anchored heparan sulfate proteoglycan mediates melanoma cell adhesion to fibronectin (19). Strong corroboration for these proteoglycan-ECM interactions comes from the presence of a heparan sulfate proteoglycan that co-localizes with ₁ integrins as a widespread component of focal adhesion (20).

Among the several ECM molecules that bind proteoglycans, the role of fibronectin should be emphasized not only because of its GAG binding domains but also because of the adhesive properties conferred to this molecule by these domains together with the RGD cell-binding fragment (21-23). Cells devoid of proteoglycans or bearing proteoglycans with altered GAG chains have a reduced capacity of adhesion to fibronectin and have a defective focal adhesion plaque formation in response to this molecule (24, 25).

The best studied receptors for fibronectin that bear adhesiveness and focal adhesion plaque formation are integrins that are / heterodimers widely expressed by almost all animal cells (26, 27). Integrins represent good examples of how post-translational modifications can alter the structure of a molecule, thus modulating its biological activity. Integrin glycosylations represent a kind of regulation by which a wide variety of these receptors have their specificity and affinity modulated in several cell lines (28-30). However, the versatility of cells to modulate the binding properties of integrins is not restricted to glycosylation. Integrin functions can be modulated by acylation of membrane lipid (31), by divalent metal binding (32), and, for the cytoplasmic domain, by tyrosine phosphorylation, which is the best understood example of this type of biological modification, especially in leukocytes and platelets (27, 33).

In the present study, we characterize ₅₁ integrin as a part-time proteoglycan containing both heparan and chondroitin sulfate, which per se could affect cell adhesion to both fibronectin RGD and GAG binding domains.

EXPERIMENTAL PROCEDURES

Human fibronectin was purified from fresh plasma (obtained from Hospital A. C. Camargo, São Paulo, Brazil) by gelatin affinity chromatography as described (34). Monoclonal antibody A-1A5 that recognizes the ₁ integrin subunit (35) and B-5G10 that reacts with the ₄ integrin subunit (36) were provided by Dr. Martin E. Hemler (Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA). Monoclonal antibody II-F5, which specifically recognizes the ₃ integrin subunit (37), was a gift from Dr. Renata Pasqualini (The Burnham Institute, San Diego, CA). Monoclonal antibodies against ₂ integrin subunit CLB-thromb/4 and ₅ integrin chain SAM-1 were purchased from the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (Amsterdam, the Netherlands). Rabbit polyclonal antibody against the cytoplasmic domain of the ₇ integrin subunit (38) was a gift from Dr. Stephen J. Kaufman (Department of Cell and Structural Biology, University of Illinois, Urbana, IL), and rabbit polyclonal antiserum against the ₅₁ integrin molecule (RB3847) that in immunoblotting reacts only with the ₁ integrin subunit² was provided by Dr. Kenneth M. Yamada (National Institute for Dental Research, Bethesda, MD). Monoclonal antibody against chondroitin sulfate chains (CS-56) was purchased from Sigma.

A human melanoma cell line (Mel-85) was provided by Dr. Stephan Carrel (Ludwig Institute for Cancer Research, Lausanne, Switzerland). A human osteosarcoma cell line (MG-63) was given by Dr. Eva Engvall (Burnham Institute, San Diego, CA), and a human colon adenocarcinoma cell line (HCT-8) was given by Dr. M. M. Brentani (Department of Oncology, School of Medicine, São Paulo University, Brazil). All cells were grown in RPMI 1640 medium (Sigma) supplemented with 10% fetal calf serum (Cultilab, Campinas, Brazil) and gentamicin (50 µg/ml) at 37 °C, 5% CO₂ in humidified conditions. Cells were harvested using divalent cation-free phosphate-buffered saline containing 2 mM EDTA. For [³⁵S]sulfate incorporation, cells were labeled in the presence of sodium [³⁵S]sulfate (240 µCi/ml of medium) for 24 h.

Cell surface expression of ₁ integrin heterodimers in Mel-85 cells was probed through immunoprecipitation reactions of cells that were surface labeled (using [¹²⁵I]iodine) by the lactoperoxidase-H₂O₂ method as described previously (39). After washing, cells were solubilized by lysis buffer (50 mM Tris-HCl, pH 7.3, 1% Triton X-100, 50 mM NaCl, 5 mM CaCl₂, 5 mM MgCl₂, 1 mM phenylmethanesulfonyl fluoride, and 2 µg/ml of aprotinin) for 15 min at 4 °C. The extract was clarified by centrifugation for 10 min at 13,000 - g, and the supernatant was preincubated with either normal mouse or rabbit serum followed by precipitation with protein A-Sepharose (Sigma). Mel-85 extract (at the same mass of protein, 1 mg) was incubated respectively with antibodies against different integrin subunits (as shown above), and for B-5G10 (an IgG₁ molecule), rabbit IgG was preincubated against mouse IgG followed by protein A-Sepharose. Affinity beads were washed with lysis buffer, and the immunoprecipitates were eluted by boiling for 5 min with Laemmli buffer.

[³⁵S]Sulfate-labeled Mel-85 cell extracts were immunoprecipitated using the same mono- or polyclonal antibodies as above. Immunoprecipitates were analyzed by 7.5% SDS-PAGE (40) followed by electrotransference onto nitrocellulose membranes (41) and exposure to x-ray films (Kodak, Rochester, NY). The same procedure was used to study HCT-8 and MG-63 cell extracts with an anti-₅ integrin antibody.

The [³⁵S]sulfate-labeled immunoprecipitates obtained with anti-₅ integrin monoclonal antibody were incubated with 4 M NaCl, 4 M MgCl₂, 6 M guanidine HCl, and 8 M urea for 2 h at 37 °C. Mixtures were then boiled for 5 min, and ₅₁ integrin was separated from Sepharose beads by centrifugation for 1 min at 13,000 × g. Supernatants were dialyzed against water, concentrated in a speed vaccum concentrator, subjected to 7.5% SDS-PAGE under nonreducing conditions, and electrotransferred onto nitrocellulose membranes that were then exposed to x-ray films at room temperature for 10 days.

Fibronectin-affinity chromatography of [³⁵S]sulfate-labeled Mel-85 cell extract was performed using purified human plasma fibronectin coupled to CNBr-activated Sepharose (Pharmacia Biotech Inc.) as detailed elsewhere (30).

Gel Electrophoresis, Purification of ₅ and ₁ Integrin Subunits, and Immunoblotting

SDS gel electrophoresis was performed as described (40). Samples under reducing or nonreducing conditions were analyzed on 5 or 7.5% polyacrylamide gels, and proteins were transferred overnight to nitrocellulose filters as described (41). Molecular mass markers (myosin, 205 kDa; -galactosidase, 116 kDa; and phosphorylase b, 98 kDa; albumin, 67 kDa) were purchased from Sigma. For two-dimensional electrophoresis, samples were separated in the first dimension by isoelectric focusing (42) using an ampholyte gradient (pH 4.0-6.5, six parts and pH 3.0-10.0, four parts, Pharmacia) followed by 7.5% SDS-PAGE in nonreducing conditions.

Glycosaminoglycan analysis was performed using agarose gel electrophoresis in 0.05 M 1,3-diaminopropane acetate buffer pH 9.0 (Aldrich). After the electrophoretic run, compounds were precipitated in the gel using 0.1% Cetavlon for 2 h at room temperature (43). After drying, the gel was stained with toluidine blue and exposed to x-ray films (X-Omat, Kodak) for 10 days at room temperature. GAG standards used were heparan sulfate from bovine pancreas (44), dermatan sulfate from pig skin, and chondroitin sulfate from shark cartilage (Seikagaku, Kogyo Co., Tokyo, Japan).

To study the specific pattern of glycosylation of ₅ and ₁ integrin subunits, [³⁵S]sulfate-labeled Mel-85 cell lysate was immunoprecipitated using a monoclonal antibody against the ₅ integrin subunit as already described, and the precipitate was submitted to a preparative 7.5% SDS-PAGE under nonreducing conditions using prestained -galactosidase (116 kDa) that comigrates with the ₁ integrin subunit as a standard. Autoradiography of separated ₅ and ₁ subunits was done in identical conditions as above and used as a guide. Gel pieces were then cut off in the positions of separated ₅ and ₁ subunits, and proteins were excised and eluted from the gel by incubation in 50 mM Tris-HCl, pH 7.3, containing 0.1% Triton X-100 overnight at 4 °C. The mixtures were then filtered through 0.45-µm filters (Nalgene, Rochester, NY) to remove polyacrylamide, and the solutions containing extracted proteins were dialyzed against water and concentrated 20-fold. Purified ₅ and ₁ integrin subunits were then submitted to -elimination reaction to release free GAG chains, which were incubated with chondroitinase ABC, heparitinases I and II, or a mixture of these enzymes (see below), and the digests were analyzed by agarose gels.

Immunoblotting reactions using Rb3847 (a rabbit polyclonal antibody that only reacts with the ₁ integrin subunit but not with the denatured ₅ integrin subunit) and a monoclonal antibody against chondroitin sulfate chains (CS-56) were performed as described previously (30).

Chemical -Elimination and Enzyme Digestions

The GAG chains from [³⁵S]sulfate-labeled ₅₁ integrin, purified by immunoprecipitation using a monoclonal antibody against the ₅ integrin subunit, were liberated by digestion of the protein core using excess proteinase-K (50 µg; Sigma) at 58 °C overnight or by a -elimination reaction (treatment overnight at 37 °C with 0.1 M NaOH in the presence of 2 M NaBH₄; Sigma). The products obtained were analyzed by agarose gel electrophoresis. -Eliminated materials were submitted to digestion with chondroitinase ABC from Proteous vulgaris (Seikagaku, Kogyo Co, Tokyo, Japan), heparitinases from Flavobacterium heparinum (45), or all enzymes and analyzed by agarose gel electrophoresis.

RESULTS

Sodium [³⁵S] Sulfate Labeling of ₁ Dimer Integrin

Because integrins are substrates for several different post-translational modifications, we decided to determine whether they could function as substrates for sulfation. We decided to address this question using [³⁵S]sulfate labeling of the cells, immunoprecipitation, and blotting experiments. As shown in Fig. 1, Mel-85 cells in culture efficiently incorporate [³⁵S]sulfate. The cell lysate was submitted to immunoprecipitation with a monoclonal antibody against the ₁ integrin subunit, and a [³⁵S]sulfated ₁ integrin molecule dimer was detected. This suggests that ₁ integrin is a substrate for post-translational sulfation.

There is a sulfated ₁ integrin in Mel-85 cells.

₅₁ Is the Only Sulfated Integrin in Mel-85 Cells

After the demonstration that ₁ dimer integrin is a sulfated molecule, our next experimental procedure was to identify the  subunit complementing the ₁ subunit in this particular integrin heterodimer. To investigate this, Mel-85 cells were surface radiolabeled with [¹²⁵I]iodine by the lactoperoxidase method or metabolically labeled with [³⁵S]sulfate. Both cell lysates were immunoprecipitated with antibodies against different integrin subunits. We can see in the [¹²⁵I]iodine-labeled immunoprecipitates (Fig. 2A) the presence of ₁, ₂, ₃, ₄, ₅, ₇, and probably ₁ subunit, a 200-kDa signal (Fig. 2A, lane 1) that could be precipitated with the ₁ subunit. Neither cell flow cytometry nor immunoprecipitation showed detectable levels of the ₆ integrin subunit in Mel-85 cells (data not shown). Interestingly, Fig. 2B shows that only ₅ and the corresponding ₁ subunit are sulfated. These findings suggested that ₅₁ integrin is a facultative sulfated ₁ integrin molecule because none of the other ₁ integrin molecules incorporated [³⁵S]sulfate.

The ₅₁ integrin is a facultative sulfated molecule.

It is known that after reduction of the disulfide bonds by -mercaptoethanol (chemical reduction), the ₅ integrin subunit comigrates with the ₁ subunit (36). The immunoprecipitates obtained using monoclonal antibodies to ₁ and ₅ integrin subunits or a polyclonal antibody against the ₅₁ integrin dimer from a [³⁵S]sulfate-labeled Mel-85 cell lysate were then subject to chemical reduction. As shown in Fig. 2C, after chemical reduction the immunoprecipitates reveal just one band in the gel, confirming that ₅₁ integrin is a sulfated molecule. It is also known that the ₅₁ integrin has fibronectin as the only ECM ligand (27, 46). Fig. 2D shows that after elution from a fibronectin affinity chromatography ₅₁ integrin is detected as a sulfated molecule.

₅₁ Integrin Is the Only Sulfated Molecule; No Other Sulfated Molecule Co-precipitates with Integrin

Data in the literature describe proteoglycans as ₅₁ integrin-associated molecules that complement the requirements involved in cell adhesion to fibronectin. In addition, in melanoma cells a heparan sulfate proteoglycan of 150/175 kDa has been described to bind fibronectin (19, 22, 47, 48). We cannot therefore discard the possibility of a physical association between a third molecule that comigrates with integrin subunits, masking the sulfated signals in the autoradiograms. To rule out this possibility the same [³⁵S]sulfate-labeled Mel-85 cell extract was again immunoprecipitated by specific monoclonal antibodies to ₅ and ₁ integrin subunits and now submitted to a two-dimensional electrophoresis (Fig. 3, A and B). We can observe that the immunoprecipitation reactions using either anti-₁ antibody or anti-₅ antibody show only a sulfated signal of ₅₁ dimer.

No other sulfated molecule co-precipitates with ₅₁ integrin.

To corroborate the findings described above and demonstrate that the sulfate groups in ₅₁ integrin are covalently linked and not adsorbed to this molecule or to the beads during immunoprecipitation, this integrin was immunoprecipitated from a [³⁵S]sulfate-labeled Mel-85 cell. After washing with phosphate-buffered saline, the beads were submitted to different conditions of elution such as high ionic strength (4 M NaCl) and chaotrophic agents (6 M guanidine HCl, 4 M MgCl₂, 8 M urea). After boiling, the precipitates were dialyzed against water, submitted to 7.5% SDS-PAGE, and transferred onto nitrocellulose filters, which were then exposed to an x-ray film (Fig. 3C). These results show that the ₅₁ integrin bears covalently linked sulfate groups.

₅₁ Integrin Is a Hybrid Proteoglycan

Proteoglycans represent the best characterized sulfated molecules containing serine-linked sulfated GAG chains as a result of post-translational modifications of the protein core (2, 7, 10). To determine the site of sulfate substitution in the ₅₁ integrin dimer, [³⁵S]sulfate-labeled Mel-85 cell lysate was immunoprecipitated using antibody against the ₅ subunit and subjected to -elimination. This procedure cleaves GAG chains from the protein core. As shown in Fig. 4A (lane 2), ₅₁ integrin after -elimination showed two sulfated bands that comigrate electrophoretically with chondroitin and heparan sulfate standards. An identical result was obtained when [³⁵S]sulfated ₅₁ integrin was submitted to proteinase-K digestion (a serine protease of broad specificity) (Fig. 4A, lane 3). These experiments suggest that ₅₁ integrin is a hybrid chondroitin/heparan sulfate proteoglycan. This was further investigated by degradation of the -eliminated material from ₅₁ integrin with specific enzymes that degrade chondroitin sulfate (chondroitinase ABC) and heparan sulfate (heparitinases type I and II). We can see that under these conditions, both sulfated bands were completely degraded by the specific enzymes (Fig. 4B), demonstrating that ₅₁ integrin is in fact a hybrid chondroitin/heparan sulfate proteoglycan.

The ₅₁ integrin is a hybrid proteoglycan.

Both ₅ and ₁ Integrin Subunits Have Chondroitin and Heparan Sulfate Chains

Because in Mel-85 cells ₅ and ₁ subunits of integrin contain sulfate, our next goal was to determine the specific pattern of glycosylation, that is, to which subunit chondroitin and heparan sulfates are linked. Two complementary approaches based on immunologic specificities were used. Fist, ₅₁ integrin was immunoprecipitated from a ³⁵S-labeled Mel-85 cell extract as described above. After separation by polyacrylamide gel electrophoresis, the immunoprecipitate was exposed to an x-ray film, blotted, and reacted with a monoclonal antibody specific for chondroitin sulfate chains (Fig. 5A). We can see that both integrin subunits bear chondroitin sulfate chains. Also, Mel-85 cell lysate was immunoprecipitated with a anti-chondroitin sulfate monoclonal antibody and blotted with a polyclonal antiserum against the ₁ integrin subunit (Fig. 5B). Interestingly, we can see that only the 116-kDa ₁ integrin subunit, which corresponds to the completely glycosylated form, has chondroitin sulfate chains. In contrast, the pre-₁ integrin chain (100 kDa), which corresponds to a high mannose structure, has no chondroitin sulfate chains. Pre-₁ integrin shows the glycosylation profile of a protein that has not crossed the Golgi. Because synthesis of GAG occurs in the Golgi, the absence of chondroitin sulfate in the pre-₁ integrin should be expected (49). This finding was also substantiated by results shown in Fig. 1 in which the pre-₁ integrin subunit, although coprecipitated, is not sulfated. These results demonstrate that the integrin dimer ₅₁ is a proteoglycan and that in Mel-85 cells both integrin subunits have covalently linked chondrotin sulfate chains.

Both ₅ and ₁ integrin subunits have chondroitin and heparan sulfate.

As a second approach we have used successive immunoprecipitation reactions to isolate ₅₁ integrin from a ³⁵S-labeled Mel-85 cell lysate. The purified ₅₁ integrin was submitted to preparative polyacrylamide gel electrophoresis. Using an autoradiogram of the gel and prestained molecular mass standards as guides, separated ₅ and ₁ subunits were removed from the gel and subjected to -elimination to obtain GAG free chains. These chains were analyzed by agarose gel electrophoresis before and after treatment with chondroitinase ABC, heparitinases, and a mixture of these enzymes (Fig. 5C). The result obtained from this last set of experiments support the concept that ₅₁ integrin is a proteoglycan. It shows that both subunits contain chondroitin sulfate and heparan sulfate, thereby demonstrating that ₅₁ integrin is a hybrid chondroitin/heparan sulfate proteoglycan.

Sulfate Incorporation in ₅ Integrins Is a Conserved Phenomenon

Because all experiments described so far were performed using the human melanoma cell line Mel-85, which has a neuro-ectodermic origin, we decided to investigate whether this ₅₁ integrin post-translational modification was also present in cell lines of endodermic (HCT-8 cells) and mesodermic (MG-63 cells) origin. Cells were labeled with [³⁵S]sulfate, and lysates were immunoprecipitated with monoclonal antibodies against the ₅ integrin subunit and analyzed by SDS-PAGE followed by electroblotting onto nitrocellulose. A polyclonal antibody against the ₁ integrin subunit (Fig. 6A) was used to detect the integrin. We can see that both cells display the [³⁵S]sulfate incorporation into the ₅ integrin subunit. The results indicate that GAG substitution of ₅₁ integrin has been conserved, suggesting its biological significance. However, in the case of the MG-63 cells, only the ₅ subunit was labeled; the ₁ subunit found in MG-63 cells did not contain sulfate. To corroborate the results described in Fig. 6A and provide more evidence that the glycosylation of integrin ₅₁ integrin as a proteoglycan is maintained in different tissues, we submitted [³⁵S]sulfate-labeled ₅₁ integrin obtained from HCT-8 and MG-63 cell extracts to a -elimination reaction. Products were analyzed by agarose gel electrophoresis. As shown in Fig. 6B, we can see that heparan and chondroitin sulfates are present in ₅₁ integrins of endodermic and mesodermic origins, as observed for neuro-ectodermic cells. The results not only confirm the conservative proteoglycan nature of ₅₁ integrin from different origins but also indicate a similar glycosylation pattern.

Sulfate incorporation in ₅ integrins is a conservative phenomenon.

DISCUSSION

Working with the human melanoma cell line Mel-85, we have described ₅₁ integrin as a hybrid chondroitin/heparan sulfate proteoglycan. Based on immunoprecipitation reactions from cell lysates that were cell surface labeled with [¹²⁵I]iodine or metabolically labeled with [³⁵S]sulfate, we were able to detect ₅₁ integrin as the only sulfated integrin compared with other _(s)1 heterodimers present in Mel-85 cells. Sulfation of ₅₁ integrin was confirmed not only by immunological methods but also by fibronectin affinity chromatography, two-dimensional electrophoresis, and reduction of disulfide bonds of the ₅₁ heterodimer leading to comigration of both ₅ and ₁ integrin subunits, characteristic of this integrin as described (36). Based on different procedures such as chemical deglycosylation by -elimination, proteinase-K digestion, immunological methods, and susceptibility to chondroitinase ABC and heparitinases, we were able to confirm this integrin as a proteoglycan. These results raise the important question of which mechanisms determine ₅₁ as the only sulfated integrin. Why are ₁ subunits not sulfated in other ₁ heterodimers? The existence of alternative splicing for the ₁ integrin subunit as described (50) (reviewed in Ref. 27) could explain in part such differences. However, because glycosylation of cell surface proteoglycans is restricted to extracellular domains (2) and the ₁ integrin subunit has only alternatively spliced cytoplasmic domains (27) this mechanism does not explain our findings. Oligomerization of integrin heterodimers is an event that occurs during transit through the endoplasmic reticulum (28) and precedes glycosaminoglycan biosynthesis, which occurs during transit through the Golgi (2). Perhaps the best explanation for the part-time proteoglycan nature of ₅₁ integrin is that the conformation of the heterodimer exposes the serine residues that are acceptors for the GAG chains, which does not happen with other ₁ heterodimers. This conformational hypothesis is consistent with the lack of a consensus sequence for proteoglycan biosynthesis initiation (1, 2) and by the experiments performed with decorin, a proteoglycan in which the primary structure of the protein core surrounding the sugar acceptor serine residue can be changed without appreciable modification in the glycosaminoglycan (51). Considering the findings described above, it is possible to assume that the same conformational folding of ₅₁ that makes this integrin capable of recognizing the RGD peptide only in fibronectin among several other ECM molecules (52) is also responsible for a specific GAG synthesis that complements the molecular requirements involved in the interaction of this integrin with fibronectin.

The possibility that other integrins can also be sulfated is not ruled out by the present study because we could not detect ₆₁ integrin in Mel-85 cells. This is an integrin that binds laminin, a molecule with a GAG binding domain spatially close to the E8 domain corresponding to the ₆₁ integrin binding site (53). Furthermore, the structural relationship of the ₅ chain with _IIb and _v (reviewed in Ref. 27) could suggest other integrin heterodimers as putative acceptors for GAG addition. Interestingly, Hayashi, Madri, and Yurchenco (54) have shown that endothelial cell interaction with the basement membrane proteoglycan (perlecan) occurs between the core protein of perlecan and ₁ and ₃ integrins, an interaction partially RGD-independent and modulated by GAGs. The ₁ integrin heterodimer involved in this adhesion resembles the ₅₁ molecule and the ₃ integrin, the _v3 vitronectin receptor (54).

The present study suggests for the first time that integrins such as ₅₁ may have two extracellular binding sites that play a role in fibronectin binding. Previous studies have implicated a specific involvement of the heparin binding site of fibronectin with cell adhesion. These data were based on the fact that the purified fibronectin fragment containing only the heparin binding domain without the RGD peptide can promote adhesion in several different cell models (21, 23). Because our work describes ₅₁ integrin as a part-time proteoglycan compared with other ₁ dimers, we can postulate that the fibronectin-₅₁ integrin interaction, which occurs primarily through the RGD peptide in fibronectin, is complemented and stabilized by the secondary interactions of ₅₁ chondroitin or heparan sulfate chains with the fibronectin heparin binding domains. The possibility that ₅₁ integrin, an integrin that binds only fibronectin, has chondroitin and heparan sulfate chains interacting with the fibronectin heparin binding domains is suggested by the facts that during ECM assembly the fibronectin heparin binding domain can also bind chondroitin sulfate or dermatan sulfate proteoglycans (10) and that soluble proteoglycans can inhibit cell adhesion to fibronectin (reviewed in Ref. 22) and by the existence of nonintegrin fibronectin receptors like CD44 (a chondroitin sulfate proteoglycan) and a heparan sulfate proteoglycan (19, 48) as well as by the recent finding that monoclonal antibodies raised against the fibronectin heparin binding domain (Hep II/IIICS) inhibit cell adhesion and also partially inhibit integrin binding to fibronectin (55). A model is postulated in which the RGD and heparin binding sites in fibronectin, although linearly separated, are spatially close due to fibronectin folding. It is thus possible to assume that cell surface proteoglycans and integrin cooperativity during cell adhesion can really be achieved in the case of ₅₁ integrin by two binding sites in the integrin molecule that bind RGD peptide and GAG binding domains in fibronectin.