fibronectin's amino-terminal matrix assembly site is located within

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 263, No. 36, Issue of December 26, pp. 19602-19609,1988 Printed in U.S.A.

Fibronectin’s Amino-terminal Matrix Assembly Site Is Located within the 29-kDa Amino-terminal Domain Containing Five Type I Repeats*

(Received for publication, June 29, 1988)

Bradley J. QuadeS and John A. McDonald8 From the Respiratory and Critical Care Division, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 631 10

Fibronectin is organized into disulfide cross-linked, insoluble pericellular matrix fibrils by fibroblasts in vitro. Two sites, the Arg-Gly-Asp-Ser-containing cell attachment domain and a site located in the first 70 kDa of fibronectin, are required for matrix assembly. The first 70 kDa of fibronectin contain two structural motifs termed type I and type I1 homologies, which are repeated nine and two times, respectively. Previous work has implicated the amino-terminal region and the carboxyl terminus containing three type I repeats in matrix assembly, suggesting that type I repeats pos- sess binding activity essential for fibronectin matrix assembly. To test this hypothesis, we developed a sensitive capture immunoassay to quantify insoluble matrix fibronectin and tested a panel of fibronectin fragments, containing all of the type I repeats found in the intact protein, for their ability to inhibit matrix assembly. Only fragments containing the first five type I repeats inhibited fibronectin matrix assembly, although sequences carboxyl-terminal to this domain en- hanced this activity. Additional evidence for the specific recognition of the amino-terminal type I repeats by matrix assembling cells was found when the reversible, detergent-sensitive binding of a 12’I-labeled fragment containing the first five type I repeats (29 kDa) to cell monolayers was studied. Only monolayers of cell lines that incorporate fibronectin into a fibrillar matrix specifically bound 12’I-labeled 29 kDa. Binding of the radiolabeled amino-terminal fragment to matrix- forming cells was inhibited by unlabeled fragments containing the first five type I repeats but not by unlabeled fragments containing the remaining seven type I repeats. Matrix assembly is therefore not a generalized property of type I repeats. Rather, a critical site is located within the first 29 kDa of fibronectin.

Fibronectins, glycoproteins created by alternative splicing of a single gene product (1-8), are present in blood (9,101 and in some extracellular matrices (11, 12). Insoluble fibronectin appears to provide a substrate for cell migrations in embryo- genesis (13-20) and wound repair (21, 22) and may provide a scaffold for the organization of other matrix components (23-

* This work was supported by National Institutes of Health Grant 9 RO1 GM 38276-02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported in part by NIH Research Service Award GM 07200 from the National Institute of General Medical Science.

3 To whom reprint requests should be addressed Box 8052, Wash- ington University School of Medicine, 660 S. Euclid, St. Louis, MO 63110.

26). In vitro, fibroblasts organize fibronectins into disulfide cross-linked matrix fibrils similar to the matrices found in vivo (23-26). The mechanism by which a fibronectin matrix is created by cultured fibroblasts remains poorly delineated (reviewed in Ref. 27). To better understand this mechanism, we have attempted to define the site(s) involved.

Fibronectin is composed of three different types of homologous amino acid sequences known as type I, 11, and I11 repeats (4, 28). These homologies occur in other proteins; e.g. a single type I repeat is present in tissue-type plasminogen activator (31), and type I1 repeats are found in bovine seminal plasma proteins PDC-109 and BSP-A3 (32-34) and in the mannose 6-phosphate/insulin-like growth factor I1 receptor (35,36). In fibronectin, groups of these repeats separated by nonhomologous stretches fold into separate domains defined by their insensitivity to proteolysis and their affinities for different ligands (37). For example, recombinant type I1 repeats appear to provide the collagen binding activity (38) found within proteolytic fragments containing those repeats (29). A model, modified from Gutman and Kornblihtt et al. (7), relating biologically active domains to the homologous repeats is illus- trated in Fig. 5.

At least two sites in fibronectin appear important in matrix assembly (39-43). The first is the Arg-Gly-Asp-Ser-containing cell attachment site of fibronectin that binds to a specific fibronectin receptor complex on the cell surface (42, 44-47). A second site is located somewhere within the amino-terminal 70-kDa portion of fibronectin (39-43). Unlike the cell adhesive sequence, the specific structural features contained within the amino terminus responsible for matrix assembly are not known.

The amino-terminal 70 kDa of fibronectin contains two domains of 29 and 40 kDa. The amino-terminal 29-kDa fibrin/ heparin binding domain contains five consecutive type I repeats (4, 30). The carboxyl-terminal collagen binding domain contains one type I, two type 11, and three type I repeats in that order. Because fragments including the carboxyl-terminal type I repeats also bind to cell monolayers, it has been suggested that matrix assembly may be a general property of the type I motif (40). This is an attractive concept, as weak binding activity of each type I repeat would be increased by the multiple copies of this motif present in the amino- and carboxyl-terminal domains (8). If matrix assembly is a general property of type I repeats, the carboxyl-terminal type I repeats should inhibit matrix assembly. Furthermore, participation of the carboxyl-terminal type I repeats in matrix assembly would suggest that amino-terminal sites interact with carboxyl-terminal sites. To test this, we developed a sensitive immunoassay for endogenous fibronectin incorporation into the pericellular matrix of cultured human fibroblasts and examined a panel of fibronectin fragments for their ability to inhibit matrix assembly.

19602

Fibronectin Matrix Assembly 19603

Using this assay, we establish that the carboxyl-terminal type I repeats are not active, and that a principal matrix assembly site is located within the first five type I repeats of the 70-kDa domain. The remaining four type I repeats (or possibly type I1 repeats) in the amino terminus contribute to binding affinity but do not function alone. Moreover, the 29- kDa fragment containing the first five type I repeats bound only to monolayers of cell lines that incorporated fibronectin into a fibrillar matrix. This binding was competed by unlabeled fragments containing the first five type I repeats, but not by fragments containing other type I repeats.

We conclude that matrix assembly is not a general property of type I repeats, but rather is a specific function of a site or sites contained within the first five type I repeats. This suggests two classes of cellular interactions with fibronectin. Cells possessing amino-terminal and Arg-Gly-Asp-Ser-binding sites, e.g. fibroblasts, can both deposit and adhere to fibronectin matrices, whereas cells lacking the amino-terminal binding site but possessing the Arg-Gly-Asp-Ser-dependent receptor only adhere to fibronectin. This scheme allows considerable flexibility in cellular interactions with fibronectin and may have evolved to prevent inadvertent deposition of fibronectin matrices within the vasculature.

EXPERIMENTAL PROCEDURES

Materials-Fibronectin-rich by-product of factor VI11 preparation ("SLIME A"), was a gift of Armour Pharmaceutical. Nitrocellulose transfer membranes and goat anti-rabbit IgG horseradish peroxidase conjugate were obtained from Bio-Rad; Centrex" (0.2-pm cellulose acetate) microfilters from Schleicher & Schuell; ABTS' peroxidase substrate and stop solution from Kirkegaard & Perry Laboratories; Linbro tissue culture and nonsterile polystyrene 96-well plates from Flow Laboratories; BCA protein assay reagent and IODO-BEADSe from Pierce Chemical Co.; porcine pancreatic elastase from Elastin Products; and carrier-free Na'*'I from Du Pont-New England Nu- clear. All other organic and biochemicals were purchased from Sigma unless otherwise stated.

Purification of Fibronectin and Fragments-Plasma fibronectin was prepared from outdated plasma and SLIME A by a modification of the method of Engvall et al. (48,49). Frozen SLIME A was dissolved by gently stirring at 37 'C for 16 h in 0.3 M glycine, 0.15 M NaC1, 10 mM KB citrate, 20 mM EDTA, 20 mM e-aminocaproic acid, 2 mM PMSF, 0.02% NaN3 at 37 "C after adjusting pH to 8.0. After centrif- ugation (5000 X g, 15 min, 20 "C), fibronectin was isolated from the supernatant by gelatin-Sepharose chromatography.

A panel of fragments containing all of the type I repeats present in fibronectin were prepared for study; the nomenclature and origin of each fragment is shown in comparison to a model of fibronectin's structure (Fig. 5). To verify the purity of isolated fragments, 1-pl aliquots were analyzed by SDS-PAGE (50) in the presence of DTT on 8 to 25% gradient gels using the Pharmacia LKB Biotechnology Inc. PhastSysteme and Tris/tricine as running buffer. Gels were stained with Coomassie Blue R 350 (Fig. 1). Protein concentrations were determined by Am and BCA protein assay (51).

The amino-terminal 70-kDa fragment (Fig. 1, lane d ) was prepared by digestion with PMSF-treated cathepsin D (37 "C, 16 h, 1:9000) followed by gelatin-Sepharose and Sephacryl S-200 HR (Pharmacia) chromatography in 50 mM Tris, 500 mM NaCI, 5 mM EDTA, 1 mM PMSF at pH 7.4 (40). The 70-kDa fragment was subfragmented into an amino-terminal 29-kDa fragment and a collagen binding 40-kDa fragment by incubation with pancreatic elastase (37 "C, 1 h, 1:5000) (42).

The amino-terminal 29-kDa and the collagen binding 40-kDa fragments (Fig. 1, lanes b and c) were prepared as previously described

The abbreviations used are: ABTS, 2,2'-azino-di-[3-ethylbenz- thiazoline-6-sulfonate]; BCA, bicinchoninic acid; PMSF, phenyl- methanesulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate-poly- acrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; PBS, Dulbecco's phosphate-buffered saline; DTT, dithio- threitol; BSA, bovine serum albumin; Adlo, the absorbance measured at 410 nm; Ki, concentration of fragment causing half-maximal inhibition of matrix assembly.

110

94 - 68 - 43 - Ir

31 --

a b c d e f g

L "V 4"

- I

14.4 - - c -

FIG. 1. Fragments of fibronectin containing type I repeats. Fibronectin fragments of fibronectin were prepared as described under "Experimental Procedures." Lane a, standards (0.1 pg each): lysozyme, carbonic anhydrase, ovalbumin, bovine serum albumin, phosphorylase a, and the 110-kDa thermolytic cell binding fragment of fibronectin. Lane b, amino-terminal 29-kDa fragment produced by pancreatic elastase digestion. When compared to a full size SDS- PAGE gel (not shown) using Tris/glycine as electrophoresis buffer (50), the 29-kDa fragment migrated anomalously fast with an apparent mass of 24 kDa when electrophoresed on the PhastSysteme. Lane c, collagen binding 40-kDa elastase fragment. Lane d, amino-terminal, collagen binding 70-kDa fragment produced by cathepsin D digestion. Lane e, heparin binding, carboxyl-terminal catheptic fragment denoted 65/75 kDa. In the absence of DTT, this fragment migrated as a broad band at 140 kDa (not shown). Lane f, a mixture of fragments produced by extensive chymotryptic digestion of the 65/75-kDa fragment. This mixture, denoted CHT 65/75 in Fig. 5, contains three heparin binding fragments of approximately 31,34, and 40 kDa and one non-heparin binding fragment of 15 kDa. Lane g, a 15-kDa non- heparin binding fragment was purified from CHT 65/75.

(48). When only the 29-kDa fragment was required, the gelatin- Sepharose step was omitted. Material not retained by DEAE-Sepha- cel was chromatographed on CM-Sepharose with a linear gradient of 0 to 500 mM NaCl in 50 mM Tris, pH 7.4. A 16-kDa contaminant contained 4% of the total Coomassie Blue staining material in the 29-kDa fragment prepared from fibronectin when analyzed by SDS- PAGE/laser densitometry (LKB Ultroscan XL). Preparations of the 29-kDa fragment lack this contaminant when purified from elastase digests of the 70-kDa fragment and were equally inhibitory; the 16- kDa contaminant did not affect the activity of 29-kDa fragment preparations in matrix assembly assays (data not shown). The 29- kDa fragment was stored at 4 "C below its PI, pH 8.2 (52), in either 100 mM sodium acetate, pH 4.5, or PBS.

The carboxyl-terminal disulfide cross-linked dimer of 65 and 75 kDa (Fig. 1, lane e) was purified from the gelatin-Sepharose nonbound fraction of the catheptic digest by heparin-agarose (Sigma type 11) chromatography (53). The bound fragment was eluted with a gradient of 0 to 500 mM NaCl in 50 mM Tris, pH 7.4. The 65/75-kDa dimer was subjected to chymotryptic cleavage (37 "C, 1 h, 1:50,000) into 65- and 75-kDa monomeric fragments, or using more extensive chymotryptic cleavage (37 "C, 1 h, 1:500) into a variety of smaller fragments (Fig. 1, lanef). The 65/75-kDa dimeric fragment migrated as a single band at 140 kDa when analyzed by SDS-PAGE in the absence of DTT, the 65- and 75-kDa monomeric fragments migrated separately at 65 and 75 kDa, respectively in the absence of D'IT (not shown). Thus, although each chain of this fragment has a cysteine residue, there was no detectable intermolecular or intramolecular disulfide cross-linking involving the free sulfhydryl group.

A 15-kDa fragment (Fig. 1, lane g) beginning in the first of three carboxyl-terminal type I repeats was purified from a chymotryptic digest (37 "C, 8 h, 1:1000) of the 65/75-kDa fragment. The non- heparin binding 15-kDa fragment was separated from three larger heparin binding fragments by affinity chromatography on heparin- agarose. After dialysis (Spectra Por 3 membrane) against 20 mM Tris, pH 7.4, the nonbound protein was further purified by DEAE-Sephacel chromatography. A gradient of 0 to 500 mM NaCl resolved two minor peaks and a major peak containing approximately 75% of the re- covered material. The major peak contained the 15-kDa fragment. Vapor-phase sequencing of this fragment was performed by the Pro- tein Chemistry Laboratory in the Department of Biochemistry, Washington University School of Medicine, using an Applied Biosys- tems 470A Sequencer. The detected sequence, Ala-Val-Gly-Asp- Glu(Trp/Tyr)Glu-Arg-Met, accounted for roughly 80% of the material loaded. This sequence matches the published sequence (4) starting

19604 Fibronectin Matrix Assembly

at residue number 2155 and follows a tyrosine at residue 2154. Antibody Purification-Polyclonal rabbit anti-human plasma fi-

bronectin IgG was affinity purified as described (54). N294, an anti- human fibronectin mouse monoclonal IgG inhibiting cell adhesion promoted by the Arg-Gly-Asp-Ser site (42), was purified from ascites by octanoic acid precipitation (55).

Cell Culture-Diploid human fetal lung fibroblasts, IMR-90 (CCL 186), were obtained from the American Type Culture Collection (Rockville, MD) and cultured as described (39). For matrix assembly experiments, trypsin-treated cells (approximately 2 X lO'/well) were plated in 96-well microtiter plates, allowed to adhere for 2 h, and medium changed to control or test media (100 pl/well). Test media were prepared by dialysis of fibronectin fragments against DMEM, addition of 10% fetal bovine serum, and filter sterilization using Centrex" units. Fibronectin content of triplicate wells was analyzed after 48 to 72 h as below.

Other cells tested for 29-kDa binding included Swiss albino 3T6 fibroblasts (CCL 96), HT-1080 fibrosarcoma cells (CCL 121), NRK- 49F fibroblasts (CRL 1570), and CHO-K1 Chinese hamster ovary cells (CCL 61, American Type Culture Collection). L cells (aprt- tk-) were a gift of Dr. John Lowe, Howard Hughes Medical Institute, University of Michigan. CEF-14 chick embryo fibroblasts were prepared from 14-day chick embryos (Ken-Roy Hatchery, Berger, MO) as described (56). All cell lines were grown as described (39) except for the CHO-K1 cells which were supplemented with 1 mM L-proline.

Quantification of Endogenous Fibronectin Matrix Assembly-Fibro- nectin-rich isolated matrices were prepared by sequential detergent extractions (24, 57). IMR-90 cell layers were aspirated and washed with 1) PBS; 2) 3% Triton X-100 in PBS; 3) 100 pg/ml DNase I in 50 mM Tris, pH 7.4,lO mM MnClZ, 1 M NaCI; and 4) 2% deoxycholate in 50 mM Tris, pH 8.8,lO mM EDTA. Washes were done at 20 "C for 1 to 3 min in the presence of 1 mM PMSF. Isolated matrices were solubilized by trituration with 1% SDS and 5 mM DTT in 50 mM Tris, pH 8.2, 5 mM EDTA, 150 mM NaCl and heated to 37 'C for 30 min. After cooling to 20 "C, iodoacetamide was added to 13 mM. Samples were diluted 1:4 with 1.25% Triton X-100, 50 mM Tris, pH 7.4, 190 mM NaCl, 5 mM EDTA to form mixed micelles (58). To quantify total fibronectin present in cell layers, parallel wells were rinsed with PBS three times before extraction with SDS/DTT as above.

The capture immunoassay was modified from Refs. 59 and 60. Polystyrene assay plates were coated with N294 anti-human fibronectin mouse monoclonal IgG, remaining protein binding sites were blocked with heat-denatured bovine serum albumin (BSA) in 100 mM NaHC03, pH 9.0, and plates were rinsed twice with 0.2% SDS, 1.0% Triton X-100 in 50 mM Tris, pH 7.4, 5 mM EDTA, 180 mM NaCl (buffer A). Samples were then diluted in buffer A to fibronectin concentrations approximately in the midrange of the standard curve. Standard curves were based on plasma fibronectin treated identically to unknowns.

Solubilized fibronectin was captured by immunoadsorption to the N294-coated plates for 20 h at 4 "C. Between steps, assay plates were washed with buffer B (1 mg/ml BSA, 20 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween-20). Bound fibronectin was detected by incubation with 0.5 pg/ml rabbit anti-fibronectin IgG followed by incubation with a 1:3000 dilution of rabbit specific, goat anti-rabbit IgG peroxidase conjugate in the immunoassay wash buffer. After rinsing five times with buffer B, bound peroxidase-conjugate was detected color- imetrically with ABTS substrate at 410 nm on a Dynatek Minireader I1 after addition of a SDS stop solution.

As a control for cell number/well, total protein present in aliquots of the whole cell layer extracts was measured using the BCA protein assay reagent (51) and BSA (fraction V) standards.

Binding of Radiolabeled Amino-terminal 29 kDa to Cell Layers- The binding of amino-terminal 29-kDa fragment to cells was measured as described (40). The 29-kDa fragment (0.5 mg in 0.5 ml of PBS) was iodinated by adding to 1 mCi of NalZ5I and IODO-BEADS" in a siliconized glass tube for 6 min at 20 "C, followed by centrifuga- tion (5 min, 1,200 X g) through a 3-ml G-10 Sephadex column equilibrated with PBS to remove free iodine. The product had a specific activity of 200,000 to 300,000 cpm/pg and was a single band when autoradiographed following SDS-PAGE. Greater than 95% of the collected counts were precipitable with 10% cold trichloroacetic acid. Binding experiments were carried out on cells plated in triplicate, confluent wells of 96- or 24-well tissue culture plates. After washing with 2 mg/ml BSA/DMEM three times, cell layers were incubated with 5 pg/ml 1Z61-labeled 29-kDa fragment in 2 mg/ml BSA/DMEM. After incubation (37 "C, 1 h, 5% COZ), the cell layers

were washed five times. Bound counts were solubilized with either 1 N NaOH or 1% solutions of SDS or Triton X-100.

RESULTS

Capture Immunoassay for Insoluble Fibronectin Matrix- We combined several techniques (58, 59) to quantify matrix fibronectin. Fibronectin was solubilized with SDS in the presence of DTT and carboxymethylated with iodoacetamide. The soluble fibronectin was quantified using a capture immunoassay. The capture antibody, N294, was selected from six monoclonal antibodies (42) based on: 1) high affinity for detergent-solubilized, reduced, and carboxymethylated fibronectin; 2) lack of recognition of fibronectin fragments used in these experiments (42); and 3) no binding to bovine plasma fibronectin present in the media (data not shown). These characteristics are demonstrated in a sample standard curve shown in Fig. 2 4 . Because the sandwich assay is limited by the affinity of the capture antibody, standard curves were linearized as a double reciprocal plot of l/ng fibronectin versus 1/A410 (Fig. 2B).

Structure-Function Relationships-The amino-terminal 70-kDa fragment inhibits fibronectin incorporation into the cell layer and into the detergent-resistant, "isolated" pericellular matrix (40,42) (Fig. 3A). In control cultures, 60% of the cell-associated fibronectin is detergent-insoluble, similar to values obtained with metabolic labeling (42). At the highest concentration of 70-kDa fragment tested (900 pg/ml), no fibronectin was incorporated into the deoxycholate-insoluble pool, and fibronectin accumulation in the cell layer was 90% inhibited. The fibronectin missing from the matrix is dis- placed into the medium (42). Total cell layer protein did not vary with the addition of any of the tested fragments (data not shown).

To test the role of the type I repeats in matrix assembly, we prepared fibronectin fragments containing all of the type I repeats (Fig. 1). First, we tested the inhibitory activity of type I repeats from both domains of the 70-kDa fragment by comparing intact 70-kDa fragment with 70-kDa fragment cleaved into the 29-kDa fibrin binding and 49-kDa gelatin binding domains by elastase. The dose-response curves for

A B I

0 0 100 200 300 0 1 2 3 4 5

ng added per well 1 / Absorbance 4 10 nm

FIG. 2. Standard curve for the capture immunoassay for fibronectin. A, plasma fibronectin was diluted into the extraction buffer containing 1% SDS with 5 mM DTT (closed circles) or without DTT (open circles) and heated at 37 "C for 30 min. The amino- terminal 70-kDa fragment of fibronectin was diluted into extraction buffer without DTT (closed squares). After addition of iodoacetamide, samples diluted to a final concentration of 0.2% SDS and 1.0% Triton X-100 were assayed as described under "Experimental Procedures." The fibronectin, but not the 70-kDa fragment, was captured by the mouse monoclonal antibody N294; the captured fibronectin was detected by incubation with an affinity purified rabbit anti-fibronectin IgG followed by a goat anti-rabbit IgG/horseradish peroxidase conjugate. Allo of the peroxidase reaction product was measured. B, to facilitate analysis, data were replotted as a double reciprocal plot (r > 0.98).


both preparations were similar (Fig. 3). Thus, if type I repeats present in the 29-kDa and the 40-kDa fragments are both active, they need not be on the same polypeptide chain. We next compared inhibition by the 29-kDa amino-terminal, the 40-kDa collagen binding domain, and the intact 70-kDa fragment (Fig. 4). By a double-reciprocal plot (not shown), K, values for inhibition of fibronectin incorporation into deoxycholate-insoluble matrix were estimated as 7 f 3 X M for 70 kDa, 3 f 2 X M for 29 kDa, and >2.5 X M for 40 kDa (Fig. 5). There was a consistent 3- to &fold difference between the Ki for the 29-kDa fragment and the 70-kDa fragment. These data are consistent with a critical matrix

A B - ~~~~~~ ~ ~~~

t P oo

300 600 900 ‘ 0 300 600 900 ugh1 Intact 70KD ugh1 Fragmented 70KD

FIG. 3. Inhibition of fibronectin (FN) matrix assembly by amino-terminal fibronectin fragments. IMR-90 fetal lung fibroblasts were plated into 96-well tissue culture plates and allowed to adhere. After 2 h, fresh medium containing either intact amino- terminal 70-kDa fragment ( A ) or 70-kDa fragment which had been completely digested into an amino-terminal 29-kDa and a 40-kDa collagen fragment with pancreatic elastase ( B ) was added. After 48 h, the confluent cells were washed with PBS. The total cell layers (squares) and deoxycholate-insoluble isolated matrices (circles) were extracted with 1% SDS and 5 mM DTT and assayed as described under “Experimental Procedures” and in Fig. 2. Under control con- ditions, cell layer fibronectin was 1.1% of the total cell layer protein.

2o 0 L 0 -C+J 100 200 300

Fragment (MI x lo7 FIG. 4. Inhibition of fibronectin matrix assembly by frag-

ments of fibronectin containing type I homologies. IMR-90 fetal lung fibroblasts were plated in microtiter wells and grown in the presence of fragments of fibronectin containing type I repeats. The amino-terminal fragments studied were the 70-kDa fragment (open circles), the 29-kDa fragment (open squares), and the 40-kDa fragment (closed squares). The carboxyl-terminal fragments studied included the disulfide cross-linked 65/75-kDa fragment (open triangles), the monomeric mixture of 65-kDa and 75-kDa fragments (closed triangles), chymotryptic subfragments of the 65/75 kDa (CHT 65/75, closed circles), and a 15-kDa fragment beginning in the carboxyl- terminal fibrin-binding domain (inverted, closed triangle). After 48 h, deoxycholate-insoluble matrices prepared from confluent cell layers were extracted with 1% SDS and 5 mM DTT and assayed by the capture immunoassay for fibronectin. The results of several experiments were compared by calculating the percent inhibition, defined as 100 x (1 - (ng of fibronectin/experimental well/ng of fibronectin/ control well)).

~i (MI X 107

70 kDa 7

PPE 70 30

29 kDa 30

40 kDa ,250

65/75kDa ,200 TT

65 & 75 kDa k150

CHT 65/75 ,150 = = 15 kDa ,300 -

EO I, W l YlCS I1

5-w 1 m h 0 - 0 d f i h o G & c o o n LI PII u sn-

MATRIX ASSEMBLY COLLAQCN anam HEPARIN F ~ B R * I FIBRrd

*PARIN

FIG. 5. The structure-function relationship for type I repeats and fibronectin matrix assembly. The results of matrix assembly experiments described in Figs. 3 and 4 are summarized in the central table. The primary structure of fibronectin fragments and mixtures of fragments containing type I repeats (horizontal lines) are compared to a model of fibronectin (bottom) described in Ref. 7. PPE 70 is the mixture of 29- and 40-kDa fragments generated by the digestion of the 70-kDa fragment with pancreatic elastase. 65/75-kDa is a disulfide cross-linked (vertical lines) dimeric fragment. The monomeric mixture of 65 and 75-kDa fragments was prepared by limited chymotryptic digestion; further digestion with chymotrypsin produced a mixture of subfragments designated CHT 65/75. A 15- kDa fragment beginning at residue 2155 in the first of three carboxyl- terminal type I repeats was also tested. The three homologous sequences repeated within fibronectin are denoted as type I (solid boxes), type I1 (stippled boxes), and type 111 (open boxes).

assembly site contained within the first five type I repeats, with additional affinity provided by carboxyl-terminal sequences in the 40-kDa fragment. Moreover, type I repeats in the 40-kDa domain are inactive alone, and the same Ki is obtained for 29-kDa fragment alone (Fig. 4) and for the elastase-generated mixture (Fig. 3). Although a tryptic 40- kDa collagen binding fragment has been suggested to have cell layer binding activity (40), the amino-terminal 31-kDa and the collagen binding 40-kDa fragments prepared by tryptic digestion of the 70-kDa fragment did not differ from their respective elastase-generated fragments in their capacity to inhibit matrix assembly. The 31-kDa fragment inhibited matrix assembly with a K; similar to that of the 29-kDa fragment preparation; the tryptic 40-kDa fragment did not inhibit (data not shown). The basis of the 3- to &fold increase in Ki upon losing the four type I repeats in the 40 kDa is unclear. The acidic PI (52) and/or the glycosylation (61) of the 40-kDa domain may stabilize the matrix assembly site within the basic 29-kDa domain. Alternatively, sequence present near the cleavage site between the two domain may be important.

These results argue against a general matrix assembly property of amino-terminal type I homologues. However, three additional type I repeats are found in fibronectin’s carboxyl terminus. To determine if this region inhibited matrix assembly, we purified the dimeric carboxyl-terminal, heparin binding fragment (denoted 65/75 kDa). 65/75 kDa had no effect in the matrix assembly assay (Figs. 4 and 5). Because the dimeric 65/75-kDa fragment could contain cryptic inhibitory activity, it was tested as an equimolar mixture of 65- and 75- kDa monomers produced by limited chymotrypsin digestion. Neither this mixture of monomers nor a mixture of subfragments produced by more extensive chymotrypsin digestion inhibited fibronectin matrix assembly. Finally, we purified a 15-kDa subfragment beginning in the first of the three carboxyl-terminal type I repeats at residue 2155 from a digest of


the 65/75 heparin binding fragment. Because each type I repeat is predicted to be about 5 kDa, the 15-kDa fragment contains most of the last three type I repeats. The 15-kDa fragment lacked inhibitory activity at concentrations as high as 6.7 X M. Additionally, we tested the 65/75-kDa dimer in a qualitative immunofluorescence assay (42) and found no morphological effect (not shown). We therefore conclude that the carboxyl-terminal type I repeats do not contain an important matrix assembly site.

Binding of 1251-labeled 29-kDa Fragment to Cell Mono- layers-Based on the experiments described above, we rea- soned that the 29-kDa fragment containing the first five type I repeats would bind to cell lines assembling a fibronectin- based matrix. If binding was mediated by a specific feature of this fragment rather than a property common to all type I repeats, only the unlabeled fragment would compete for binding to the cell layer. Fragments containing the remaining type I repeats would not compete with the labeled amino-terminal 29-kDa fragment for cell layer binding. Furthermore, cell lines that do not assemble a fibronectin matrix might not bind the 29-kDa fragment.

Fibronectin matrix-forming cells including IMR-90 fetal lung fibroblasts, Swiss albino 3T6 fibroblasts, CEF-14 chick embryo fibroblasts, and NRK-49F fibroblasts were tested. Three cell lines that do not assemble a fibronectin matrix were also studied. In the absence of dexamethasone, HT-1080 fibrosarcoma cells do not bind radiolabeled 70-kDa fragment (62). CHO-K1 cells do not synthesize nor assemble fibronectin, but are able to adhere to fibronectin-coated substrates using Arg-Gly-Asp-X-dependent receptors (63). L cells, a spingle-shaped permanent mouse cell line, synthesize and secrete nearly as much fibronectin as the 3T6 cell line by metabolic labeling and SDS-PAGE analysis, but fibronectin is found diffusely over the cell surface when stained with anti- fibronectin IgG-Texas Red conjugates (not shown).

Radiolabeled 29-kDa fragment bound specifically to cell layers of matrix-forming cell lines, and the binding was inhibited only by fragments containing the first five type I repeats (Fig. 6). Neither unlabeled 40-kDa collagen binding fragment nor carboxyl-terminal 65/75-kDa dimeric fragment competed with the labeled amino-terminal 29-kDa fragment. Previous work demonstrated that high concentrations of 40-kDa tryptic

: looK 75

I

I 0 ""-L , -J".. - A 0 100 200 300

Unlabeled fragment (MI x lo7 FIG. 6. The binding of the '261-labeled 29-kDa fragment is

not competed by unlabeled fragments containing the other type I repeats. IMR-90 were grown to confluence in 96-well plates and incubated 1 h at 37 "C in 5% COa in the presence of 5 pg/ml lZ5I- labeled 29 kDa (2 X M) and increasing amounts of unlabeled fragments containing type I repeats: amino-terminal 29-kDa fragment (solid squares), amino-terminal 70-kDa fragment (open circles), collagen binding 40-kDa fragment (open squares), and the carboxyl- terminal heparin binding 65/75-kDa dimeric fragment (open triangle). Similar results were obtained for Swiss 3T6 cells (not shown).

1 ooT---

- 75 e c

E 25

Unlabeled 29 kDa fragment (MI x lo7

FIG. 7. The amino-terminal 29-kDa fragment of fibronectin binds to monolayers of cell lines, which organize a fibronectin matrix. Confluent monolayers were incubated with 5 pg/ml '251-labeled 29 kDa (2 X lo" M) and increasing amounts of unlabeled 29-kDa fragment for 1 h at 37 "C. When plated in a 96-well plate, IMR-90 (solid circles) specifically bound 53 fmol of radiolabeled fragment, Swiss albino 3T6 (solid triangle) bound 48 fmol, and CEF- 14 (solid squares) bound 102 fmol of labeled fragment. Binding of radiolabeled fragment by NRK-49F (open circles) was comparable to that of 3T6 cells. To compare results from the different cell lines, the results are expressed as the percentage of counts bound in the absence of unlabeled fragment. L, CHO, and HT-1080 cells (all represented by the open b o x ) are lines which do not assemble a fibronectin matrix. When L, CHO, and HT-1080 cells were grown to confluence, the amount of labeled fragment bound by these monolayers in the absence of unlabeled 29-kDa fragment was similar to the nonspecific binding observed with 3T6 cells.

collagen binding fragment inhibited binding of labeled 70- kDa fragment to cell monolayers (40). A difference in activity between fragments generated with elastase and trypsin would suggest that the missing sequence forms an important part of the matrix assembly site; however, such a difference was not found. Unlabeled tryptic amino-terminal 31-kDa fragment inhibited binding of labeled 29-kDa fragment and tryptic 40- kDa fragment did not (data not shown). The 40-kDa and 65/ 75-kDa fragments increased binding of the labeled 29-kDa fragment. This increase is possibly a nonspecific effect of increasing protein concentration. Alternatively, interactions between the active amino-terminal domain and carboxyl- terminal regions of fibronectin, lacking activity of their own, might enhance the binding of labeled 29-kDa fragment. How- ever, similar concentrations of 40-kDa and 65/75-kDa fragment preparations did not change the amount of endogenous fibronectin accumulated in the matrix (Fig. 4). Thus, similar to matrix assembly, binding of the amino-terminal domain to matrix-organizing cell monolayers is not a general property of the type I motif.

In addition, only cell lines that assemble fibronectin matrices bound radiolabeled 29-kDa fragment (Fig. 7). Typically a few tenths of a percent of the total counts added to each well were bound. Several factors could account for this. 1) Affinity: at 5 gg/ml, the molar concentration (2 X M) of the radiolabeled fragment is about 1 order of magnitude lower than the Ki (3 X M) determined in the assembly assay. 2) Iodination damage: the type I repeat has a conserved tyrosine in its consensus sequence (29,30). Labeling at lysines by the Bolton-Hunter method, however, did not significantly alter the outcome (not shown). 3) Occupied binding sites: additional binding sites might be occupied by the fibronectin present in the endogenous matrix. Binding was specific, be-


cause binding of the labeled fragment was competed by 100- fold excess of unlabeled 29-kDa fragment. Furthermore, the binding of labeled 29-kDa fragment to the cell layer was qualitatively similar to that reported for the larger 70-kDa fragment (40): 1) binding of the labeled 29-kDa fragment to cell layers was reversible (e.g. tlhaff = 0.5 h for 3T6 cells); 2) bound counts could be completely released by a 1% solution of SDS or Triton X-100 without DTT; and 3) binding of the radiolabeled 29-kDa fragment to cells was abolished when cells were resuspended by mild trypsin treatment (not shown).

DISCUSSION

Localization of Matrix Assembly Site in Fibronectin-A site critical for matrix assembly is contained within the first 29 kDa of fibronectin, a proteolytic domain composed of five type I repeats. The 29-kDa fragment and the overlapping 70- kDa fragment inhibited cellular fibronectin incorporation into the pericellular matrices of human fetal lung fibroblasts with a Ki of 3 and 0.7 p ~ , respectively. This difference in K , may be explained by the presence of an additional “affinity” site in the 40-kDa domain or by stabilization of the site in the 29- kDa domain by the 40-kDa domain. In contrast, fragments containing the remaining seven type I repeats, 40 kDa, 65/75 kDa, or subfragments of 65/75 kDa, were not inhibitory at the highest concentrations tested (3 mg/ml). Their Ki, while not directly determined, were larger by more than 1 order of magnitude.

McKeown-Longo and Mosher (40) found that all three regions containing type I repeats contributed to the binding of labeled fibronectin to fibroblast monolayers. The 70-kDa fragment was the most active fragment, but fragments containing the last three carboxyl-terminal type I repeats also bound to fibroblast monolayers (40). Furthermore, the 40- kDa collagen binding domain and a carboxyl-terminal tryptic 31-kDa fibrin binding domain competed with radiolabeled 70- kDa fragment or fibronectin for binding (40). The discrepancy between these and the present results may be a consequence of the assay chosen, namely binding versus inhibition of assembly. Although binding is necessary for matrix assembly, not all binding interactions may be relevant. McKeown-Longo and Mosher also examined the ability of 70-kDa amino- terminal fragment and a large “fingerless” fibronectin fragment lacking type I repeats to inhibit accumulation of lZ5I- fibronectin in the matrix. Only the 70-kDa fragment pre- vented the transfer of exogenous radiolabeled fibronectin from a detergent-soluble to a detergent-insoluble form in the presence of a preexisting matrix.

The binding of the 29-kDa fragment to fibroblast cell layers is consistent with inhibition of matrix assembly by a compet- itive mechanism. Moreover, unlabeled fragments containing type I repeats other than the first five found in the 29-kDa fragment, i.e. the collagen binding fragment and the carboxyl- terminal heparin binding fragment, did not compete with the labeled 29-kDa fragment for binding to cell monolayers. Fur- ther support for the conclusion that carboxyl-terminal type I repeats are not sufficient for matrix assembly comes from work with recombinant forms of fibronectin (64). Truncated fibronectin lacking the amino terminus, termed deminectins, appropriately form dimers via carboxyl-terminal interchain disulfides. Deminectin homodimers are not incorporated into the pericellular matrix. In contrast, deminectin-fibronectin heterodimers are assembled into a detergent-resistant form.

The Structure-Function Relationships of Matrix Assembly and Type I Repeats-The 29-kDa fragment contains five type I repeats and nonhomologous sequences at both termini. Reduction and carboxymethylation abolishes the inhibitory

activity of the 70-kDa catheptic fragment (Ref. 40 and data not shown). The 4 cysteines in each type I repeat form two disulfide bonds which configure the repeat into a large loop/ small loop motif (28, 30). Thus, sensitivity to reduction/ carboxymethylation suggests that the matrix assembly site arises from a structure more complex than that of the sequence. The double loop motif is likely integral to the structure of the matrix assembly site. If this generic disulfide- bonded structure is found in several domains, why do only the most amino-terminal five type I repeats block function in matrix assembly? We offer two possible explanations.

First, generation of a matrix assembly site could result from consecutive type I repeats, each binding weakly. Receptor- mediated erythrocyte invasion by malarial merozoites is an example of such a model. The Plasmodium falciparum protein GBP-130, the receptor for erythrocyte glycophorin, contains 11 highly conserved 50-residue repeats, and the binding of recombinant GBP-130 to glycophorin correlates with the number of repeats present in the recombinant protein (65). However, our results do not suggest a simple correlation between number of repeats and activity in the inhibitory assay. The 70-kDa fragment has nearly twice as many type I repeats as the 29 kDa, but the difference in Ki is only a factor of 4. Furthermore, the lack of effect with the purified 40-kDa collagen binding fragment and the 65/75-kDa fragment (containing two sets of three consecutive repeats) stands against a straightforward numerical relationship. Obviously, we can- not exclude a model where a minimum number (five) of contiguous type I repeats are required for activity.

A more likely explanation is that specific information con- stituting the matrix assembly site exists in one or more of the first five type I repeats, analogous to the cell adhesive site of fibronectin. There are many type I11 repeats but only one contains the Arg-Gly-Asp-Ser sequence that is essential, though not sufficient, for high affinity recognized by the fibronectin receptor complex (66, 67). Another pertinent example of the single-site model comes from the superfamily of complement binding proteins (68). These proteins contain 3 to 36 copies of a 60-residue motif with 4 highly conserved cysteines forming two disulfide bonds in each unit. But the presence of the motif is not sufficient for C3b/C4b binding as three proteins with multiple copies of this motif (interleukin- 2 receptor, 2; p subunit of factor XIII, 5; p2 glycoprotein I, 10) do not interact with complement proteins. Moreover, not all copies of the 60-residue repeat present in a given complement binding protein, factor H, are functional (69-73). Like factor H, the type I repeats present in active fragments of fibronectin have no apparent distinguishing differences within them- selves or with repeats of inactive fragments (see Fig. 2 of Petersen and Skorstengaard (30)).

Mechanistic Implications-Understanding the structural aspects of fibronectin’s matrix assembly site is critical to understanding the mechanism by which fibroblasts organize soluble fibronectin into an insoluble pericellular matrix. A possible function for the matrix assembly site is fibronectin- fibronectin association. Lack of participation of carboxyl- terminal repeats in matrix assembly rules out mechanisms which include amino-to-carboxyl-terminal or carboxyl-to-carboxyl-terminal interactions. Although direct determination of the structure of fibronectin fibers has not been possible, fibronectin polymerization appears to be mediated by amino- to-amino-terminal associations.

The cell-dependent assembly mechanism (74) can be viewed as a multistep process involving fibronectin’s interaction with at least two cell surface components. A critical interaction takes place between fibronectin and the Arg-Gly-Asp-Ser-


1.

2.

3.

4.

5.

6.

7.

8. 9.

10. 11. 12.

13. 14.

15.

16.

17. 18. 19.

20.

21.

22. 23.

24.

25.

26. -

REFERENCES

Schwarzbauer, J . E., Tamkun, J. W . , Lemischka, I. R., and Hynes,

Tamkun, J. W . , Schwarzbauer, J . E., and Hynes, R. 0. (1984)

Kornblihtt, A. R., Vibe-Pedersen, K., and Baralle, F. E. (1984)

Kornblihtt, A. R., Umezawa, K., Vibe-Pedersen, K., and Baralle,

Paul, J. I., Schwarzbauer, J. E., Tamkun, J. W . , and Hynes, R. 0. (1986) J. Biol. Chem. 2 6 1 , 12258-12265

Zardi, L., Carnemolla, B., Siri, A., Petersen, T. E., Paolella, G., Sebastio, G., and Baralle, F. E. (1987) EMBO J. 6, 2337-2342

Gutman, A., and Kornblihtt, A. R. (1987) Proc. Natl. Acad. Sci.

Hynes, R. (1985) Annu. Reu. Cell Biol. 1,67-90 Mosesson, M. W . , and Umfleet, R. A. (1970) J. Biol. Chem. 245 ,

Mosher, D. F. Annu. Reu. Med. 3 5 , 561-575 Stenman, S., and Vaheri, A. (1978) J. Exp. Med. 147,1054-1064 Vartio, T., Laitinen, L., Narvanen, O., Cutolo, M., Thornell, L.-

E., Zardi, L., and Virtanen, I. (1987) J. Cell Sci. 8 8 , 419-430 Duband, J. L., and Thiery, J. P. (1982) Deu. Biol. 9 4 , 337-350 Rovasio, R. A., Delouvee, A., Yamada, K. M., Timpl, R., and

Boucaut, J. C., and Darribere, T. (1983) Cell Tissue Res. 2 3 4 ,

Boucaut, J. C., Darribere, T., Boulekbache, H., and Thiery, J. P.

Bronner-Fraser, M. (1986) Deu. Biol. 117 , 528-536 Grabel, L. B., and Watts, T. D. (1987) J. Cell Biol. 105,441-448 Thiery, J. P., Duband, J . L., and Tucker, G. C. (1985) Annu. Reu.

McClay, D. R., and Ettensohn, C. A. (1987) Annu. Reu. Cell Biol.

Grinnell, F., Billingham, R. E., and Burgess, L. (1981) J. Znuest.

Grinnell, F. (1984) J. Cell Biochem. 2 6 , 107-116 Vaheri, A,, Kurkinen, M., Lebto, V. P., Linder, E., and Timpl, R.

Hedman, K., Kurkinen, M., Alitalo, K., Vaheri, A., Jobansson,

Hedman, K., Johansson, S., Vartio, T., Kjellen, L., Vaheri, A.,

Hedman, K., Vartio, T., Johansson, S., Kjellen, L., Hook, M.,

R. 0. (1983) Cell 35,421-431

Proc. Natl. Acad. Sci. U. S. A . 8 1 , 5140-5144

Nucleic Acids Res. 12 , 5853-5868

F. E. (1985) EMBO J. 4 , 1755-1759

U. S. A. 84,7179-7182

5728-5736

Thiery, J. P. (1983) J. Cell Biol. 9 6 , 462-473

135-145

(1984) Nature 307 , 364-367

Cell Biol. 1 , 91-113

3 , 319-345

Dermatol. 7 6 , 181-189

(1978) Proc. Natl. Acad. Sci. U. S. A. 75,4944-4948

S., and Hook, M. (1979) J. Cell Biol. 81,83-91

and Hook, M. (1982) Cell 2 8 , 663-671

dependent fibronectin receptor (42). The other interaction is mediated by the amino-terminal type I repeats. Detergent- extractable binding of lZ5I-labeled 70-kDa fragment to fibroblast cell layers suggests that the interaction with the amino terminus also occurs at or near the cell surface (40, 62, 75). The magnitude of the binding constant for radiolabeled 70- kDa fragment binding to cell layers (4 X lo-’ M (62)) and the inhibition constant reported here (7 x lov7 M ) imply an interaction with an affinity as high as the fibronectin-fibronectin cell adhesive receptor association (4 x M (76)). The amino-terminal “receptor” is unidentified at present, but McKeown-Longo and Etzler have proposed a 48- and an 18- kDa protein as candidates (62, 77). Gangliosides also specifically bind the amino terminus and may also be a candidate for the fibronectin matrix assembly receptor (78). Cell surface proteoglycans are unlikely participants because cell lines bear- ing proteoglycans on their cell surfaces (e.g. HT-1080) did not bind labeled 29-kDa fragment.* The specific function of the matrix assembly receptor as well as the relationship between the matrix assembly receptor and the adhesive receptor complex remain issues for further study.

Acknowledgments-We thank Drs. Samuel Santoro, Thomas Bir- kenmeier, and Charlotte Roberts and Thomas Broekelmann for their helpful discussions during the preparation of this manuscript.

~

M. Hook, personal communication.

Linker, A., Salonen, E. M., and Vaheri, A. (1984) EMEO J. 3 , 581-584

27. McDonald, J . A. (1988) Annu. Reu. Cell Biol. 4 , 183-207 28. Petersen, T. E., Thgersen, H. C., Skorstengaard, K., Vibe-Ped-

ersen, K., Sahl, P., Sottrup-Jensen, L., and Magnusson, S. (1983) Proc. Natl. Acad. Sci. U. S. A. 8 0 , 137-141

29. Skorstengaard, K., Thgersen, H. C., and Petersen, T. E. (1984) Eur. J. Eiochem. 140, 235-243

30. Petersen, T. E., and Skorstengaard, K. (1985) in Plasma Fibro- rzectin (McDonagb, J., ed.) pp. 7-29, Marcel Dekker, New York

31. Banyai, L., Varadi, A., and Patthy, L. (1983) FEBS Lett. 163 ,

32. Baker, M. E. (1985) Biochem. Biophys. Res. Commun. 130,1010- 1014

33. Esch, F. S., Ling, N. C., Bohlen, P., Ying, S. Y., and Guillemin, R. (1983) Biochem. Biophys. Res. Commun. 113,861-867

34. Seidah, N. G., Manjunath, P., Rochemont, J., Sairan, M. R., and Chretien, M. (1987) Biochem. J. 243 , 195-203

35. Lobel, P., Dahms, N. M., and Kornfeld, S. (1988) J. Biol. Chem.

36. Tong, P. Y., Tollefsen, S. E., and Kornfeld, S. (1988) J. Biol. Chem. 263,2585-2588

37. Ruoslahti, E., Hayman, E. G., Engvall, E., Cothran, W . C., and Butler, W . T. (1981) J. Biol. Chem. 2 5 6 , 7277-7281

38. Owen, R. J., and Baralle, F. E. (1986) EMEO J. 6, 2825-2830 39. McDonald, J. A., Kelley, D. G., and Broekelmann, T. J. (1982) J.

40. McKeown-Longo, P. J., and Mosher, D. F. (1985) J. Cell Biol.

41. Spiegel, S., Yamada, K. M., Hom, B. E., Moss, J., and Fishman, P. H. (1986) J. Cell Biol. 102,1898-1906

42. McDonald, J . A., Quade, B. J., Broekelmann, T. J., Lachance, R., Forsman, K., Hasegawa, E., and Akiyama, S. (1987) J. Biol. Chem. 262,2957-2967

43. Chernousov, M. A., Faerman, A. I., Frid, M. G., Printseva, 0. Y., and Koteliansky, V. E. (1987) FEBS Lett. 2 1 7 , 124-128

44. Ruoslahti, E., and Pierschbacher, M. D. (1986) Cell 44 , 517-518 45. Pytela, R., Pierschbacher, M. D., and Ruoslahti, E. (1985) Cell

46. Ruoslahti, E., and Pierschbacher, M. D. (1987) Science 238,491-

47. Hynes, R. 0. (1987) Cell 48,549-554 48. McDonald, J. A,, and Kelley, D. G. (1980) J. Biol. Chem. 2 5 5 ,

49. Engvall, E., Bell, M. L., and Ruoslahti, E. (1981) Coll. Reht. Res.

50. Laemmli, U. K. (1970) Nature 227,680-685 51. Smith, P. K., Drohn, R. I., Hermanson, G . T., Mallia, A. K.,

Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Anal. Biochem. 150, 76-85

52. Seidl, M., and Hormann, H. (1983) Hoppe-Seyler’s 2. Physiol. Chem. 364,83-92

53. Richter, H., and Hormann, H. (1982) Hoppe Seyler’s 2. Physiol. Chem. 363,351-364

54. Villiger, B., Kelley, D. G., Engleman, W . , Kuhn, C., 111, and McDonald, J. A. (1981) J. Cell Biol. 9 0 , 711-720

55. McKinney, M. M., and Parkinson, A. (1987) J. Zmmunol. Methods

56. Freshney, R. I. (1987) in Culture of Animal Cells. A Manual of

57. Carey, D. J., Eldridge, C. F., Cornbrooks, C. J., Timpl, R., and

58. Anderson, D. J., and Blobel, G. (1983) Methods Enzymol. 9 6 ,

59. Butler, J . E., Peterman, J. H., Suter, M., and Dierks, S. E. (1987)

60. Brown, E. J., Bohnsack, J. F., O’Shea, J. J., and McGarr, J.

61. Bernard, B. A,, Yamada, K. M., and Olden, K. (1982) J. Biol.

62. McKeown-Longo, P. J., and Etzler, C. A. (1987) J. Cell Biol. 104 ,

63. Brown, P. J., and Juliano, R. L. (1985) Science 228,1448-1451 64. Schwarzbauer, J. E., Mulligan, R. C., and Hynes, R. 0. (1987)

65. Kochan, J., Perkins, M., and Ravetch, J. V. (1986) Cell 44,689-

37-41

263,2563-2570

Cell Biol. 9 2 , 485-492

100,364-374

40,191-198

497

8848-8858

1,505-516

96,271-278

Basic Technique. Alan R. Liss, Inc., New York

Bunge, R. P. (1983) J. Cell Biol. 97,473-479

111-120

Fed. Proc. 46,2548-2556

(1987) Mol. Zmmunol. 2 4 , 221-230

Chem. 257,8549-8554

601-610

Proc. Natl. Acad. Sci. U. S. A. 8 4 , 754-758

696


66. Pierschbacher, M., and Ruoslahti, E. (1984) Nature 309, 30-33 72. Kristensen, T., and Tack, B. F. (1986) Proc. Natl. Acad. Sci. 67. Obara, M., Kang, M. S., Rocherdufour, S., Kornblihtt, A., Thiery, U. S. A . 83,3963-3967

J. p., and Yamada, K. M. (1987) FEBS Lett. 213, 261-264 73. Schulz, T. F., Schwable,W., Stanley, K. K., Weib, E., andDierich, 68. Reid, K. B. M., Bentley, D. R., Campbell, R. D., Chung, L. P., M. P. (1986) Eur. J. Zmmunol. 16, 1351-1355

Sim, R. B., Kristensen, T., and Tack, B. F. (1986) Zmmunol. 74. Carter* w. G. (lgU) J. 999 lo5-'14 Today 7,230-234 75. Pesciotta Peters, D. M., and Mosher, D. F. (1987) J. Cell Biol.

69. Hang, K., Kinoshita, T., y., and Inoue, K. (1982) J. 76. Akiyama, S. K,, Hasegawa, E., Hasegawa, T., and Yamada, K. 104, 121-130

Immunol. 129,647-652 70. Alsenz, J., Lambris, J. D., Schulz, T. F., and Dierich, M. P. (1984) 77. M ~ K ~ ~ ~ ~ - L ~ ~ ~ ~ , p. J. (1987) J , cell B ~ L , 105, 44a

Biochem. J . 224, 389-398 78. Thompson, L. K., Horowitz, P. M., Bentley, K. L., Thomas, D. 71. Alsenz, J., Schulz, T. F., Lambris, J. D., Sim, R. B., and Dierich, D., Alderete, J. F., and Klebe, R. J. (1986) J. Bwl. Chem. 261,

M. P. (1985) Biochem. J. 232, 841-850 5209-5214

M. (1985) J. Biol. Chem. 260,13256-13260

fibronectin's amino-terminal matrix assembly site is located within

Documents