fibronectin self-association is mediated by complementary sites

THE JOURNAL OF BIO~GICAL CHEMISTRY Vol. 269, No. 45, Issue of November 11, pp. 27863-27868, 1994 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Fibronectin Self-association Is Mediated by Complementary Sites within the Amino-terminal One-third of the Molecule*

(Received for publication, July 21, 1994, and in revised form, August 30, 1994)

Karen M. Aguirre$, Richard J. McCormick, and Jean E. Schwarzbauers From the Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014

The formation of a fibrillar fibronectin (FN) extracellular matrix requires self-association of FN dimers. In this report, we show that the major sites for self-association are the amino-terminal repeats I,, and the first type I11 repeats. Recombinant FNs and fragments were generated by baculovirus expression of cysteine-rich domains and by bacterial expression of type I11 repeats as fusion proteins with maltose binding protein. When recombinant polypeptides were immobilized on microtiter wells, FN bound to 70-kDa amino-terminal fragment and to fusion proteins containing repeats III,, and 111,, but not to other type I11 repeats. Similar results were obtained with a gel overlay assay. Binding was concentration-dependent and saturable. The amino-terminal binding site for III,, was further localized to repeats I14. Therefore, at least two different sites for FN-FN interaction reside near the amino terminus of the molecule. A model for the regulation of FN matrix assembly is proposed based on intramolecular interactions between these amino-terminal sites.

Fibronectin (FN)’ is a large secreted glycoprotein found as a major soluble component of blood and as an insoluble protein in the tissue extracellular matrix (1, 2). F N is secreted as a disulfide-bonded dimer and is subsequently assembled into a fibrillar matrix via interactions with cell surface integrin receptors, with other matrix components, and with itself (3, 4). Matrix assembly requires FN self-association involving localized regions highly specialized for FN-FN interactions. How- ever, the mechanism of dimer self-association to form fibrils has not been well defined.

Two regions of FN have been implicated in both FN binding and matrix assembly. The 70-kDa amino-terminal region is essential for matrix assembly (5-71, and within this region, type I repeats I,, confer F N binding activity (7-9). McKeown- Longo and Mosher ( 5 ) initially showed saturable binding of the 70-kDa amino-terminal fragment to fibroblast monolayers, and a fragment containing repeats 11-5 can be covalently cross- linked to FN at the cell surface (10, 11). Deletion of any of the

Grant CA44627 and a grant from the March of Dimes Birth Defects * This work was supported in part by National Institutes of Health

Foundation (to J. E. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

lowship and a fellowship from the New Jersey Commission on Cancer $ Recipient of a Minority Access to Research Careers predoctoral fel-

Research. Present address: Trudeau Institute, Saranac Lake, NY. 8 Established Investigator of the American Heart Association. To

whom correspondence should be addressed. The abbreviations used are: FN, fibronectin; pFN, plasma F N cFN,

cellular FN; mAb, monoclonal antibody; MBP, maltose binding protein; PAGE, polyacrylamide gel electrophoresis; CAPS, 3-(cyclohexylami- no)propanesulfonic acid; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin.

five repeats drastically reduced the ability of this domain to bind to fibroblasts (12). The amino-terminal domain was also required for de novo assembly of newly synthesized recombinant FNs into the extracellular matrix (7, 13). Excess 70-kDa fragment as well as antibodies against repeats I,, also inhibit matrix assembly (5 , 6). A second site of FN binding has been identified in a fragment spanning the first two type I11 repeats (III1-.J adjacent to the gelatin binding domain (14, 15). Simi- larly, a 56-kDa fragment containing the gelatin binding site plus repeats 1111-2 and antibodies that recognize repeat 111, or the adjacent repeat I, can inhibit FN binding and matrix formation (16, 17). Other regions that are involved in matrix assembly include the cell binding domain (18-20) and the carboxyl-terminal cysteines that form the interchain disulfide bonds in the FN dimer (7).

Although at least two regions have been shown to have FN binding activity, their complementary sites of interaction within the molecule have not been identified. Given that each of the FN binding domains is also involved in matrix assembly, it seems likely that FN self-association is an integral part of this assembly process. Here, we extend our previous studies of FN matrix assembly to include the identification of several sites involved in FN self-association. Recombinant FNs and fragments generated using bacterial and baculovirus expression systems have been used in several different binding assays to show that the amino-terminal matrix assembly domain (Il5) can bind to the first two type I11 repeats. A model is presented to explain the role of these sites in the regulation of matrix assembly at the cell surface.

EXPERIMENTAL PROCEDURES FN cDNA Constructions-Fusions of maltose binding protein (MBP)

and FN were prepared by insertion of FN cDNAs into the BamHI and XbaI sites of p W - c R I (New England Biolabs). In all cases, the XbaI site introduced a termination codon (TCXGA) at the end of the insert. The cDNAinserts have termini as follows where the base positions refer to sites within the rat FN cDNA sequence (GenBank accession number X15906): III,, 1808-3498; 1111-2, 1808-2407; IIIGI1, 3254-5154; 1118-15, 4239-6860; 1111~16, 5154-6860.

Properly folded disulfide-bonded fragments of FN were prepared using the baculovirus insect cell expression system (21). 70-kDa and FNAIIIl_, cDNA constructs in retroviral vectors have been previously described (7). The cDNAs were transferred intact into baculovirus vectors. BV-7OM was generated by insertion of a 1.8-kilobase cDNA into BamHI to XbaI-digested pVL1393 vector. BV-FNAIII,_, was expressed using pVL1392 and encodes the amino-terminal 70-kDa and the carboxyl-terminal half but lacks repeats III,_,. This recombinant FN was previously called Il&l10 (7).

Cells and Viruses-SF9 fall army worm ovary cells were grown in Grace’s insect medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Hyclone, Logan, UT), TC yeastolate, TC lactalbumin hydrolysate (Difco Laboratories, Detroit, MI), and penicillin/ streptomycin. Two different protocols were used to generate recombinant baculoviruses. For BV-70kD, SF9 cells were infected with wild type virus followed by transfection with pVL1393-7OkD (22). BV- FNAIII,_, recombinant baculovirus was generated by cotransfection of SF9 cells with wild type baculovirus DNA (Invitrogen) plus pVL1392-

27863

27864 Fibronectin Self-association FNAIIIl-,. Recombinant viruses were isolated from transfection supernatants by limiting dilution cloning, and high titer stocks were prepared by amplification as described (21).

Mouse AtT20 pituitary cells expressing FNAIII,_, were grown in a 50:50 mixture of Ham's F12 and Dulbecco's modified Eagle's medium supplemented with 20 mM HEPES, pH 7.4, 4 m L-glutamine, 10% Nu-serum (Collaborative Research), 5% heat-inactivated horse serum (Hyclone Labs), and 0.25 mg/ml Geneticin (Life Technologies, Inc.). Mouse SVT2 cells expressing recombinant FNA16-1117 (previously called II4/CI1O) were grown as previously described (7). The Rat 1 fibroblast line was cultured in 5% bovine serum in Dulbecco's modified Eagle's medium.

Protein Purification and SDS-PAGE-SF9 cells were infected with BV-70kD or BV-FNAIII,_, recombinant virus stock at a multiplicity of infection between 2 and 10. Culture supernatants were collected 5 days postinfection, and recombinant polypeptides were purified by gelatin- agarose affinity chromatography. To obtain pure FNAIIIl-, from AtT20 cell medium, AtT2O cells expressing FNAIII,-, were grown in serum depleted of pFN. Cellular FN (cFN) was purified from Rat 1 fibroblast- conditioned medium, and pFN was purified from fresh rat plasma, outdated human plasma, or bovine serum, again by gelatin-agarose affinity chromatography (23).

SVT2 cells expressing FNA1,-111, were grown in spinner flasks on microcarrier beads (ICN, Irvine, CA) as recommended by the manufacturer using Dulbecco's modified Eagle's medium plus 10% bovine serum depleted of pFN. FNAIe-II17 was concentrated and partially purified from cell-conditioned medium by 40% ammonium sulfate precipitation. Solubilized precipitated proteins were dialyzed into 20 lll~ Tris-HCI, pH 8.0,lOO mM NaCl, 2 mM EDTAand subjected to heparin-agarose affinity chromatography. Specifically, bound proteins were eluted with 800 mM NaCl in Tris-EDTA, and peak fractions were dialyzed into 20 mM Tris- HCI, pH 8.0, 100 nm NaCI, 2 mM EDTA. Contaminating SVT2 FN was removed by gelatin-agarose affinity chromatography.

Maltose binding protein (MBP)-FN fusion proteins were purified from Escherichia coli HBlOl bacterial extracts using amylose resin affinity chromatography as described by the manufacturer (New Eng- land Biolabs). MBP-IIIl~15 fusion protein did not bind efficiently to the amylose resin and was purified instead by heparin-agarose affinity chromatography and elution with 1 M NaCl(24). Column fractions containing fusion proteins were identified by SDS-PAGE and Coomassie Blue staining. In some cases, protein identification was confrmed by immunoblotting with an anti-FN polyclonal antiserum (25). All fusion proteins were dialyzed into 0.02 M Tris-HCI, pH 8, 0.05 M NaCl and stored at -80 "C. Several analyses were used to show that type I11 repeats expressed in bacteria behave in a manner similar to intact FN: MBP-III,,, and MBP-IIIl%15 bind heparin and are eluted from a heparin-agarose column with the same concentration of NaCl as native FN? and the timing and extent of 3T3 fibroblast adhesion and spreading on MBP-III,,, is similar to intact FN.

Proteins were analyzed by SDS-PAGE and immunoblotting as previously described (25). To monitor purity, proteins were visualized by silver staining. In immunoblots, proteins were detected using either an anti-FN antiserum (25) or, for MBP-fusion proteins, with an anti"BP antiserum (see below).

Gel Overlay Binding Assay-0.346 pg of FN or recombinant FNs were electrophoresed through SDS-polyacrylamide gels and transferred to nitrocellulose. Immobilized proteins were denatured in 2.5 M guanidine HCl in bufferA (0.02 M Tris-HC1, pH 7.5,0.15 M NaCl, 0.1% Tween 20) at room temperature for 1 h and then renatured by multiple 10-min incubations in buffer A plus 0.25 mg of BSA/ml. After incubation with gentle shaking in buffer A containing 50 pgml human pFN for 2 h at room temperature, the filter was washed, and bound FN was detected by immunoblotting with an anti-human FN monoclonal antibody (Clone 111, Telios Pharmaceuticals, San Diego, CA) used at a dilution of 1:10,000. Biotinylated goat anti-mouse second antibody and streptavidin-horseradish peroxidase (Life Technologies, Inc.) were followed by chemiluminescence reagents (DuPont NEN). Signals were detected by exposure to XAR film and quantitated by scanning densitometry using a Bio-Rad densitometer. No binding of mAb Clone I11 to filter-bound proteins was detected in the absence of added FN. The relative amounts of protein in each lane were determined by comparison with silver- stained gels of the same samples.

Antibodies and EL1SA.s-Relative amounts of immobilized proteins and levels of specific protein bound to immobilized protein were determined by ELISA using one of these antibodies. Anti-70kD is a rabbit

F. Barkalow, unpublished observations.

polyclonal antiserum raised against BV-70kD and was used at 1:lOOO dilution. mAb 5G4 is a rat-specific anti-FN antibody that recognizes an epitope in repeats III,,-ll. mAb 5G4 culture supernatant was used at 1:50. Anti-MBP polyclonal antiserum is a rabbit serum prepared against an MBP-SPARC domain IV fusion protein (26) using the same protocol as described for FN (25). This antiserum was used at a dilution of 1:lOOO and has no reactivity against FN. Anti-FN antiserum was previously described (25). An anti-human FN mAb, Clone 111, was pur- chased from Telios Pharmaceuticals and was used at 1:lOOO. ELISAs were carried out using biotinylated second antibodies, streptavidin P-galactosidase, and p-nitrophenyl-p-D-galactopyranoside substrate at the recommended dilutions (Life Technologies, Inc.), and color was measured at 405 nm using a Bio-Rad ELISA plate reader.

plates (ICN) or Maxisorp plates (Nunc Inter Med, Denmark) were Microtiter Binding Assays-96-well non-tissue culture microtiter

coated at 4 "C overnight using solutions of FN, recombinant FNs, fusion proteins, or BSA diluted in 0.05 M Tris-HC1, pH 8.8, 0.05 M NaCl or in 0.01 M CAPS, pH 11,0.15 M NaCl at final coating concentrations ranging from 0.2 to 30 pg/ml as indicated. Coating concentrations were adjusted to yield approximately equimolar amounts of immobilized proteins. Wells were blocked with 2 mg of heat-denatured BSA/ml in phosphate- buffered saline (1 h, 37 "C) and then washed three times with phos-

pFN, cFN, BV-70kD, FNAIII,-,, or FNA16-II17 at the indicated concen- phate-buffered saline. Immobilized proteins were then incubated with

trations between 0.2 and 100 pg/ml in 0.25 mg of BSA/ml of phosphate- buffered saline. Bound proteins were detected using segment-specific or monoclonal antibodies followed by biotinylated goat anti-rabbit or anti- mouse IgG and streptavidin p-galactosidase. For each microtiter assay, a parallel set of wells was coated, and the amount of immobilized protein was quantitated by ELISA with one or more of the antibodies described above to ensure approximately equal amounts were added to each well. As negative controls, wells were coated with BSA and MBP, and values are expressed as the absorbance at 405 nm minus the BSA or MBP background as indicated. BSA background values were between 0.11 and 0.14 A405 units.

RESULTS Expression of Recombinant FN Fragments-As a first step

toward identifying sites of FN self-association, bacterial MBP fusion proteins were constructed containing overlapping segments of type I11 repeats. The regions expressed as MBP-FN fusion proteins are illustrated in Fig. 1 and include repeats I1Il+ III,, IIIGl1 (cell binding domain), IIIG15 (cell + heparin domains), and III,,,, (heparin domain). The alternatively spliced segments, EIIIB, EIIIA, and the V region fall after repeats 111,, 11111, and 11114, respectively, and were not tested in these experiments. MBP-111,,, and MBP-IIIl~l, represent the VO variant of FN. MBP fusion proteins were purified from bacterial lysates via amylose resin affinity chromatography or by heparin-agarose affinity chromatography in the case of

To examine the interactions of segments containing intra- chain disulfide bonds, the amino-terminal 70-kDa fibrinl collagen binding fragment (BV-70kD) and a larger recombinant FN lacking repeats III,, (FNAIII,.J (Fig. 1) were expressed using the baculovirus insect cell system. FNAIIIl-7 was also expressed using mouse AtT2O pituitary cells. These recombinant polypeptides were purified from cell culture supernatants by gelatin-agarose affinity chromatography. The recombinant polypeptide FNA16-111, was expressed in mouse SVT2 cells.

Protein purities were assessed by SDS-PAGE. Several contaminating bands were present in the MBP fusion proteins and appeared to be MBP degradation products as they were recog- nized by an anti-MBP antiserum in immunoblots (data not shown). Intact fusion proteins represented at least 80% of the total protein as determined by scanning densitometry of chemiluminescence signals from immunoblots.

Identification of FN Binding Sites-FN-FN interactions were first examined using a gel overlay assay. Plasma and cellular FNs were electrophoresed non-reduced alongside BV- FNAIIIl-7, AtT20-FNAIII1_,, BV-70kD, and BSA and transferred to nitrocellulose. After denaturation with guanidine HC1

MBP-IIIl%lw

Fibronectin Self-association Matrix Assembly Flbrin Collagen W1-7) Cell HeDarin Rbrln

FIG. 1. Structure of FN and recombinant fragments. FN is composed of three types of repeating units that com- prise domains for matrix assembly and binding to a variety of molecules such as collagen/gelatin, cell surface receptors, and heparin. The repeats that make up the different MBP fusion proteins, the baculovirus generated BV-70kD and the larger recombinant FNs, FNAIII1-, and FNA16-II17, are illustrated.

ElIIB EIIIA v120 ss

ss

mllmkcm

and renaturation in Tris-buffered saline plus Tween-20 and BSA, immobilized polypeptides were incubated with a solution containing 50 1.18 of human FN/ml. FN that specifically bound to the immobilized proteins was detected with an anti-human FN mAb and chemiluminescence reagents. Fig. 2 shows that the human FN bound to pFN, cFN, FNAIII,-,, and 70kD. No binding to the BSA control was detected. Similar gel overlay experiments were performed with two MBP fusion proteins, and FN binding to MBP-111,, but not MBP-IIIl~15 was observed. That both MBP-111,, and 70kD support binding while spanning different portions of the molecule shows that there is more than one FN binding site.

Although binding can be measured and quantitated using the gel overlay assay, we wanted to use an assay that did not require prior denaturation of the proteins. Therefore, for the next series of experiments, a microtiter plate binding assay was used. Microtiter wells were coated using 0.2-1 pg/ml solutions of rat pFN, cFN, BV-70kD, and BSA. Coating concentrations were adjusted to yield equimolar amounts of immobilized protein as measured by ELISA with anti-70kD antiserum. Fol- lowing incubation with human pFN, a species-specific mAb was used to detect bound human pFN. Nonspecific binding to BSA was subtracted from the other AdO5 values. Fig. 3 shows that intact FN bound specifically to the FN and 70kD sub- strates. Levels of binding to 70kD were routinely higher than for intact FN.

Binding to MBP fusion proteins was performed in essentially the same manner. Fusion proteins were immobilized and incubated with rat pFN; bound FN was detected with an anti-rat FN mAb (5G4) (Fig. 4). FN bound to MBP-111,, and MBP-III,,, although binding to the larger fusion protein was somewhat better. No significant binding to the other type I11 repeats nor to MBP alone was observed. In another type of assay, 111,, expressed by in vitro translation bound specifically to FN immobilized on gelatin-agarose beads (data not shown). Taken together, the results of several different assays localize two FN binding sites, one in the amino-terminal 70-kDa fragment and the other within the first two type I11 repeats. Both of these regions have been previously implicated in FN self-association

A direct comparison of FN binding to 70kD and MBP-111,, was carried out using wells coated with equal masses of these two proteins alongside BSA and MBP. As expected, FN bound to both 70kD and MBP-111,, but did not bind to MBP or BSA (Fig. 5). The amount of FN bound to 70kD was about twice that bound to MBP-III,,. Although this difference could be due to differ- ences in coating efficiencies between these two proteins, we routinely observed higher binding between 70kD and FN than be-

(5, 7-9, 14, 15).

7 l-- PFN cFN FNAIIII-7 70kD BSA

FIG. 2. FN binding using a gel overlay assay. 0.3-0.6 pg of rat pFN, cFN, FNAII1,-, from AtT2O cells ( lef t ) and from baculovirus- infected insect cells (right) were electrophoresed through a 5% polyacrylamide-SDS gel under nonreducing conditions. A 7% polyacrylamide-SDS gel was used for 0.5 pg of nonreduced BV-70kD and BSA. After transfer to nitrocellulose, blots were incubated with a 50 pg/ml solution of human pFN in buffer A plus BSA. Specifically bound protein was detected with a human-specific anti-FN mAb and chemiluminescence reagents.

1

" 1 ? 0.6

T

PFN cFN 70kD

Immobilized protein FIG. 3. Binding of FN to FN and 70kD. Microtiter plate wells were

coated with 0.2-1 pg/ml solutions of rat pFN, cFN, BV-70kD fragment, or BSAto give equimolar amounts of protein coated. After blocking with BSA, wells were incubated with a 30 pg/ml solution of human pFN, and bound protein was measured by ELISA using an anti-human-specific mAb. Background binding to BSA-coated wells was subtracted. Values are the average of three experiments.

tween MBP-111,, and FN. This finding could suggest that the 70kD-FN interaction has a higher affinity or that the site in 111,, is not totally accessible as previously proposed (15, 17). 70kD and III,, Are Complementary Binding Sites-The ini-

tial results demonstrated that FN binds to amino-terminal 70kD and repeats 1111-2 and III,+. To localize complementary binding sites within FN, similar experiments were performed using recombinant fragments. BV-70kD binding to varying amounts of MBP fusion proteins was measured by ELISA with an anti-70kD antiserum. BV-70kD bound to MBP-111,, and MBP-III,-, in a concentration-dependent manner (Fig. 6). No

27866 Fibronectin Self-association

1 T

MBP 1111.6 1111.2 1116.11 II112.15

Immobilized protein

FIG. 4. Binding of FN to type I11 repeats. MBP fusion proteins containing FN type 111 repeats were coated onto microtiter plate wells using 2 pg/ml solutions of protein. After incubation with a 30 pg/ml solution of rat pFN, specific binding was detected with a rat-specific mAb, 5G4. Results are the average of two experiments. Significant binding of FN to 1111-2 and III,, was observed. Similar results were obtained with higher amounts of immobilized fusion proteins.

I 0.84 7-

7 o k ~ I I I ~ . ~ MBP

Immobilized protein

FIG. 5. Comparison of FN binding to 70kD and 111, Wells were coated with solutions containing 2 pglml of BV-70kD, MBP-III,,, MBP, and BSAfollowed by incubation with a 30 pglml solution ofhuman pFN. Specifically bound FN was detected with the anti-human mAb. Non- specific binding to BSA was subtracted.

MBP II11.6 1111.2 1118.15 11&.11 lII12.15

Immobilized protein FIG. 6. 70kD binding to fusion proteins. Fusion proteins were

coated onto wells using two different protein concentrations, 2 (dark bars) and 0.4 (light bars) pg/ml, and incubated with a 30 pg/ml solution of BV-70kD. Specific binding was detected with the anti-70kD antiserum. Clearly, BV-70kD binds to III,, and III,, but not to the other fusion proteins.

specific interactions were detected between 70kD and MBP or the segments spanning the carboxyl-terminal heparin and cell binding domains. Therefore, among the type I11 repeats, the primary 70-kDa binding site resides within the first two. Sat-

0 20 40 60 80 100

pgYml 70kD FIG. 7. 70kD-III,, binding curve. Increasing concentrations of BV-

70kD (from 1 to 100 pg/ml) were incubated with wells coated with 2 pg/ml MBP-III,, (0) or MBP (0). Bound 70kD was measured using an anti-70kD antiserum. Binding reached saturation in the presence of about 25 pg of 70kD/ml.

urable binding was observed for the interaction between BV- 70kD and MBP-111,. When increasing concentrations of 70kD were incubated with constant amounts of MBP-111,,, a plateau in the level of 70kD binding was reached at about 25 pg/ml (Fig. 7). Background binding to MBP was significantly lower. These experiments identify complementary specific binding sites within the amino-terminal 70kD and the first two type I11 repeats.

Previously reported affinity chromatography results demonstrated that the first five type I repeats I,, within the 70-kDa region have FN binding activity (7). I t seemed likely, therefore, that repeats I,, form the III,, binding site within 70kD. The following experiment confirms that hypothesis. MBP, MBP- 11114, and MBP-III,, were immobilized and incubated with pFN, cFN, FNAIII,-7, and FNA16-II17, a recombinant FN containing repeats I,, but lacking the gelatin binding domain and repeats 1111-7 (see Fig. 1). All of these proteins bound to III,, and IIIl-z but not to MBP or BSA (Fig. 8). We have thus localized two major complementary sites of interaction between FN molecules to repeats I,, and III,-,.

An amino-terminal domain containing all five repeats I,, is essential for FN matrix assembly (7, 12). To show that the intact domain is also required for binding, repeat I, was deleted from FNA16-II17, yielding a recombinant protein with only four type I repeats in this domain. Binding of this protein to 111,, and III,, was significantly reduced (data not shown). There- fore, similar molecular structures are required for binding the first type I11 repeats and for assembly of a fibrillar matrix.

DISCUSSION In this report, we have demonstrated that the amino-temi-

nal matrix assembly domain encompassing the first five type I repeats binds to another amino-terminal region, the first several type I11 repeats. A variety of binding assays and different combinations of recombinant FN fragments were used to show that the interaction is specific. Type I11 repeats outside of the first six did not support FN binding or binding to the amino- terminal 70-kDa fragment. This does not rule out a role for these repeats in FN-FN interactions but simply indicates that any interactions involving these repeats are not detected with the assays used here.

Fibronectin Se

pFN cF'N FNAIII1., FNAk-111,

Protein in solution FIG. 8. Localization of III,, binding site in 70kD. MBP-111,,

(dark bars), MBP-III,, (lzght bars), MBP, and BSA were immobilized using 2 pg/ml solutions. Wells were then incubated with solutions containing approximately 30 pg/ml rat pFN, cFN, FNAIIIl-7, and FNAI, 111,. Specific binding was detected by ELISA using a rat-specific mAb, 5G4, and MBP background was subtracted. Equivalent binding to both MBP-111,, and MBP-III,, was observed. III,, values are the average of two experiments.

Both 70kD and III,-, have been implicated in FN matrix assembly. There is a very well documented role for the amino- terminal I,, domain (reviewed in Ref. 3). FN fragments lacking these repeats (5) or recombinant proteins with deletions in the amino-terminal segment (7, 12, 13) are not assembled into a fibrillar matrix. Antibody and fragment blocking experiments (5,6,27) and cross-linking studies (10,111 also demonstrate an essential role for this region in FN matrix assembly. Similar approaches have been used to show that repeats 1111-2 are also involved in formation of an FN matrix. Chernousov et al. have shown that a 56-kDa gelatin-binding fragment containing the first two type I11 repeats can inhibit FN incorporation into the matrix (17), and monoclonal antibodies against this region block the assembly process (16). Various-sized fragments containing repeats III,, also bind directly to intact FN (14, 15). However, this site appears to be cryptic and, within certain contexts, does not interact with FN (17, 28).

While repeats I,, play a central role in FN matrix assembly, repeats 111, and 111, are clearly not essential for this process. Recombinant FNs lacking repeats 1111-7 but containing the rest of FN including the amino-terminal 11-5 region are very effb ciently assembled into a disulfide-bonded fibrillar matrix (7). This observation rules out a crucial role for these type I11 repeats in formation of a disulfide-bonded matrix. Rather, it appears that repeats 1111-2 play an accessory role in this process. We did not look for interactions with the carboxyl-terminal type I repeats. However, Sottile and Mosher (29) have recently demonstrated that recombinant FNs lacking repeats I,,,, are efficiently incorporated into the extracellular matrix.

What might be the purpose of having two FN binding sites, one essential and one not, at the same end of the molecule? We propose that these sites have a regulatory role as well as a role in assembly. The amino-terminal domains could regulate FN self-association by forming intramolecular interactions, which would sequester these sites. Release of the I,, domain from binding to III,, would then be induced by an appropriate signal making these sites available for forming intermolecular FN-FN interactions. In our experiments, we routinely observed higher levels of binding of fragments or recombinant FNs lacking the 111,-2 site than we did for intact FN. In fitting with our model, this observation suggests that the sites in the intact molecule were not always available for binding. Also in support of this model are results from numerous biophysical and biochemical studies showing a folded, compact structure for FN in solution

If-association 27867

(30-37). In particular, many of the models for the structure of an FN dimer include folding over of the amino-terminal region to form interdomain interactions with other parts of the molecule (8, 31, 34, 36, 38, 39). This compact structure can be un- folded to an extended form by changes in pH or ionic strength (31-34, 36). The physiological requirement for such a regulatory mechanism is illustrated by circulating pFN, which does not form insoluble fibrils in blood even though it is present at a relatively high concentration.

What factors could encourage a conformational change as a prerequisite for matrix assembly in vivo? FN matrix assembly requires participation by live cells and is initiated by binding of FN at the cell surface (see Refs. 3 and 40). This immobilization involves a5pl integrin recognition of the central cell binding domain and the RGD sequence (20). Once tethered at the cell surface, interactions with other FN dimers promote fibril formation. However, if intramolecular interactions are blocking the amino-terminal binding sites, then a5pl recognition would have to initiate a process to shift the equilibrium toward the dissociated state and make the sites available for intermolecular interactions. Such a process might involve binding by ad- ditional cell surface molecules (5) or long range conformational changes. A highly cooperative melting transition in intact FN has been attributed to the 110-kDa central cell binding domain (391, a domain that extends to within the first several type I11 repeats. Furthermore, regions as far amino-terminal as repeat 111, have been shown to synergize with the RGD sequence in repeat 111,, for optimal interactions with cell surface receptors (41,42). Clearly, long range effects are involved in FN function and may be a central part of matrix assembly as well. Compe- tition by binding domains on adjacent molecules could provide an alternative mechanism for disrupting intramolecular interactions. Morla et aZ. (43) have recently shown that FN becomes more adhesive and apparently more closely resembles matrix FN when incubated with a fragment from the first type I11 repeat. Perhaps this fragment is acting by increasing the avail- ability of these amino-terminal binding sites.

Upon opening up of the FN binding sites by integrin binding, I,, and III,, can then participate in FN-FN interactions. Ac- cumulation of FN at the cell surface by integrin clustering would juxtapose these and other weaker binding sites and al- low multiple FN-FN interactions to form and stabilize the fibril. One interaction outside of the amino-terminal region was detected in our studies, i.e. binding of FN to the alternatively spliced V r e g i ~ n . ~ "his interaction was significantly weaker than interactions involving the amino-terminal sites, making it unlikely that the V region is a major site for nucleating FN-FN interactions. However, it could play an important role in align- ment of FN subunits during fibrillogenesis when local concentrations of FN are increased. We would also predict that there are many other sites of FN-FN interaction that are too weak to be detected biochemically but are able to play a role in fibril assembly when the local FN concentration is high and when FN is in the appropriate conformation.

Other extracellular matrix molecules are almost certainly involved in FN matrix assembly. For example, the addition of collagen or a collagen fragment (CB7) results in a reorganiza- tion and increased accumulation of FN fibrils at the surface of collagen-deficient MOV-13 cells (44). The collagedgelatin binding domain lies right between the two complementary binding sites described here, providing an optimal location for collagen binding to disengage any intramolecular interactions and thus increase matrix assembly. Williams et al. (31) have shown par- tial unfolding of FN upon binding of the CB7 collagen fragment. In addition to collagen, heparin and anti-111, Fab fragments,

K. M. Aguirre and J. E. Schwarzbauer, unpublished observations.

27868 Fibronectin Se two types of molecules that directly interact with the III,, region, have been shown to increase FN and/or 70kD binding to fibroblasts (5, 17).

In summary, FN self-association and matrix assembly involve interactions between two amino-terminal domains. Other interdomain interactions probably enhance the assembly process, and binding to other matrix components, e.g. collagen or proteoglycans, could also serve to control or modify assembly. Further experiments are needed to sort out other types of interactions that are at play during formation of this important structure.

Acknowledgments-We thank Carol Ryan for excellent technical assistance and David Castle for generously providing the AtT20 cell line. We are also grateful to Marty Marlow and the Dept. of Molecular Biology monoclonal antibody facility for preparation of the 5G4 anti-FN monoclonal used in these studies.

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