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Address correspondence to Jean E. Schwarzbauer, Princeton University, Department of Molecular Biology, Princeton, NJ 08544-1014. Tel.: (609) 258-2893. Fax: (609) 258-1035. E-mail: jschwarzbauer{at}molbio.princeton.edu
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Abstract |
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Key Words: fibronectin; matrix assembly; type III repeats; RGD sequence; self-association
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Introduction |
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FNFN interactions are also important for fibril formation. The major site of interaction is the NH2-terminal assembly domain which consists of repeats I15 and binds FN and many other molecules (Mosher, 1989; Hynes, 1990; Schwarzbauer, 1991). Other FN binding sites have been localized to the first one or two type III repeats (Morla and Ruoslahti, 1992; Aguirre et al., 1994; Hocking et al., 1994; Ingham et al., 1997), the cell binding repeat III10 (Hocking et al., 1996), and the COOH-terminal heparin binding domain (III1214) (Bultmann et al., 1998). Each of these sites interacts with the NH2-terminal assembly domain.
Results from binding, inhibition, and matrix assembly studies show that FN fibrils form via a multistep process (McKeown-Longo and Mosher, 1983; Schwarzbauer and Sechler, 1999). During the initiation stage of assembly, integrin binding immobilizes dimeric FN and promotes formation of deoxycholate (DOC)-soluble fibrils in a process that depends on the NH2-terminal assembly domain. Mutations that affect the RGD cell binding sequence or the NH2-terminal domain ablate fibril formation (Schwarzbauer, 1991; Sottile et al., 1991; Sechler et al., 1996; Sottile and Mosher, 1997). Assembly then progresses into a growth phase that involves incorporation of additional FN dimers into nascent fibrils, fibril elongation, and conversion of fibrils into a DOC-insoluble form. The matrix is further stabilized as DOC-insoluble FN is formed into high molecular mass multimers.
We have shown previously that a recombinant FN (recFN) lacking the first seven type III repeats (FNIII17) is able to form a fibrillar matrix, albeit at an altered rate (Sechler et al., 1996). It appears that this set of seven repeats, or a subset of them, has a regulatory role in FN assembly. In this study, recFNs containing overlapping deletions across this region were tested for the ability to form fibrils, DOC-insoluble matrices, and high molecular mass multimers. Surprisingly, deletion of repeat III1, a site proposed to be essential for assembly, had no detrimental effects on assembly. Similarly, relatively large deletions of up to four type III repeats, as well as displacement of the cell binding domain toward the NH2 terminus, caused no deficiencies in matrix formation. However, deletions that included repeat III2 reduced the assembly of a DOC-insoluble matrix and blocked fibril elongation. Binding studies using recombinant fragments showed that III2, but not III1, has FN binding activity. Our results indicate that repeat III2 is a key element in the regulation of FNFN interactions during matrix assembly.
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Results |
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Fibril growth depends on repeats III12
Because III12 has FN binding activity (Aguirre et al., 1994) and a proteolytic fragment containing III1 plus part of III2 inhibits incorporation of FN into matrix (Chernousov et al., 1991; Morla and Ruoslahti, 1992), we generated FNIII12 to test whether these two repeats together comprise a matrix regulatory region. Immunofluorescence analysis of FN assembled by CHO
5 cells showed that early during assembly, FN
III12 formed aggregates on the cell surface (Fig. 5 A) similar to, but much smaller than, those formed by FN
III17 (Sechler et al., 1996). As assembly progressed, FN
III12 formed mainly short fibrils between cells although occasional long thin fibrils were also visible. An extensive fibrillar network was never observed even after prolonged incubations. Levels of DOC-insoluble material were significantly reduced, especially at later times of assembly, and no high molecular mass multimers were observed (Fig. 5 B). Incubations with higher concentrations of FN
III12 did not increase the amount of DOC-insoluble matrix (unpublished data). Unlike FNs lacking the RGD sequence, FN
III12 was not deficient in binding to cells. In fact, substantially more FN
III12 than native FN was isolated as cell-associated DOC-soluble material. This indicates that FN
III12 can efficiently bind to the cell surface to initiate assembly, but is defective in the growth phase when conversion from DOC-soluble to -insoluble matrix occurs.
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Repositioning of III910 cell binding domain
The cell binding domain consisting of an RGD sequence and synergy site in repeats III910 is essential for initiation of FN matrix assembly by 5ß1 integrin (Sechler et al., 1996, 1997). Deletions within repeats III17 position the cell binding domain closer to the NH2-terminal assembly domain and could affect recFN fibril formation. To eliminate the possibility that changes in the domain organization can alter FN assembly, we created FNrIII45/910. Repeats III45 were replaced with III910 and the RGD sequence was deleted from its native position within the cell binding domain (Fig. 1). Therefore, the only functional III910 pair is in the position normally occupied by III45. CHO
5 cells efficiently assembled FNrIII45/910 into a DOC-insoluble fibrillar matrix identical to that of native FN at all time points (Fig. 8, AC). Cell cycle progression by CHO
5 cells assembling either FN or FNrIII45/910 was identical, as were the levels of focal adhesion kinase phosphorylation (unpublished data). These results demonstrate that the location of the cell binding domain is not restricted to the center of the molecule and that displacement toward the NH2 terminus does not reduce FN function in matrix assembly or its ability to influence cell cycle progression.
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Discussion |
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Our data with FNIII12 and FN
III25 show that repeat III2 participates in the growth phase of FN assembly. Assembly was initiated by both of these recFNs but DOC-insoluble matrix formation was blocked. Together with data showing a direct interaction between III2 and the NH2-terminal assembly domain of FN, our matrix assembly results indicate that this FN binding site plays a key role in FNFN interactions during fibril assembly. Repeats III23 have been shown previously to interact with repeats III1214, an interaction that appears to contribute to the formation of the compact conformation of soluble FN (Johnson et al., 1999). Thus, the III2 module may participate in matrix assembly through interactions with several sites on FN. These interactions may promote elongation by aligning fibrils into a stable, uniform structure that can then be converted into a DOC-insoluble form. In the absence of this repeat, fibrils begin to form but become stalled during elongation and are inefficiently converted into the DOC-insoluble matrix. DOC-insolubility of FN fibrils appears to occur through hydrophobic proteinprotein interactions that resist SDS denaturation (Chen and Mosher, 1996). Perhaps III2 participates in the formation of that hydrophobic interface.
Others have shown the presence of a cryptic FN binding site in III1 that is exposed by denaturation (Hocking et al., 1994; Ingham et al., 1997). The apparent affinity of 70 kD for III2 is higher than that reported for 70 kD binding to heat-denatured III1 (Hocking et al., 1994). The identification of two distinct sites indicates that the III12 segment contains more than one FN binding site, the site in III2 that is critical for fibril assembly and a cryptic site in III1 that is dispensable for this process. It is also possible that repeats III12 act as a functional unit to regulate fibril assembly and promote elongation. For example, interactions between III1 and III2 could regulate the accessibility of an FN binding site. Previous studies lend support to the idea of cooperation between these repeats. Both repeats were required for formation of an in vitro ternary complex with heat-denatured III10 and the NH2-terminal 70-kD fragment (Hocking et al., 1996). III2 has also been proposed to contribute to interactions between III1 and the COOH-terminal heparin binding domain (Bultmann et al., 1998). Furthermore, the reduced secretion of recFNs lacking III2 or carrying another type III repeat in place of III2 indicates that interactions between adjacent repeats contribute to domain structure and stability. III1-specific inhibitory peptides and antibodies have been described (Chernousov et al., 1987, 1991; Morla and Ruoslahti, 1992). If III1 and III2 do indeed function together, these inhibitory reagents may exert their effects indirectly through disruption of activities mediated by the adjacent III2 module.
Regulated assembly depends in part on conformational changes in the FN molecule. Accumulating evidence indicates that soluble FN dimers must be converted from a compact inactive form into an "unfolded" activated form in order for assembly to proceed (Alexander et al., 1979; Williams et al., 1982; Erickson and Carrell, 1983; Rocco et al., 1983; Ugarova et al., 1995; Schwarzbauer and Sechler, 1999). In vivo, integrin binding induces FN activation and this may expose the III2 binding site, allowing intermolecular interactions between cell surfacebound FNs. Whereas recFNs lacking III2 are able to initiate assembly and form short fibrils, the significantly reduced levels of DOC-insoluble FNIII12 suggest that in the absence of the III2 binding site, this recFN cannot effectively participate in the essential FNFN interactions needed for fibrillogenesis. FN molecules can also be induced to associate in solution by the addition of a peptide corresponding to part of the III1 module (Morla et al., 1994). This treatment may expose the III2 binding site by local perturbation of intramolecular interactions involving this region of the molecule.
Comparison of the progression of assembly by FNIII12 with FN
III17 shows that both initially form short stitches around cell peripheries and connect to adjacent cells. FN
III17 then forms aggregates that prematurely become insoluble in DOC (Sechler et al., 1996). These aggregates can apparently be remodeled by binding to adjacent cells and getting stretched into fibrils. In this way, FN
III17 forms a relatively normal-appearing fibrillar matrix. On the other hand, FN
III12 forms only a few small, DOC-soluble aggregates that can be converted into predominantly short fibrils. FN
III12 does not accumulate in DOC-insoluble material, nor does it form an extensive fibrillar matrix. The differences between assembly of FN
III12 and FN
III17 suggests that repeats III37 contribute to the progression of FN fibril formation. A few activities have been mapped to the III37 region. Repeats III46 can bind to heparin and DNA under low salt conditions (Hynes, 1990). Cryptic binding sites within repeat III5 have been reported for activated
4ß1 and
4ß7 integrins (Moyano et al., 1997) as well as for repeat III1 (Hocking et al., 1996). Repeat III1 can also bind to repeat III7 (Ingham et al., 1997). Thus, it is possible that during assembly this region of FN interacts with cell surface or matrix proteins or glycosaminoglycans, and that these interactions may help to control fibril formation.
FNIII12 and FN
III17 probably also differ in the alignment of FN dimers into fibrils. For example, binding of the NH2-terminal assembly domain of one FN
III17 dimer to the COOH-terminal heparin domain of another (Bultmann et al., 1998) would align their cell binding domains relatively close to each other. This juxtaposition could result in increased clustering of integrins and more stable contacts between matrix and cytoskeleton, giving the strong connections needed to remodel aggregates into fibrils and form DOC-insoluble material. Tension applied to FN fibrils has been predicted to cause slight unfolding of type III repeats (Erickson, 1994; Krammer et al., 1999), and this might allow the formation of SDS-resistant proteinprotein interactions (Chen and Mosher, 1996). On the other hand, the inclusion of III37 may yield a potentially different organization of both cell surface receptors and cytoskeletal elements, thus precluding the formation of a stable matrix.
Clearly, multiple options exist for establishing FNFN interactions during matrix assembly. For example, the NH2-terminal assembly domain is required throughout the assembly process, whereas the III2 module participates after initiation during a phase of fibril growth. This indicates that different FN binding sites have distinct temporal and spatial roles, and suggests that control of domain-specific FN interactions may play an important role in regulating the structural and functional organization of the FN matrix.
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Materials and methods |
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FN cDNA constructions and recombinant protein production
All recFNs were expressed with baculovirus vector pVL1392. Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs, Inc. Oligonucleotide primers were prepared by the Synthesis and Sequencing Facility (Princeton University, Princeton, NJ).
All deletions were created by PCR amplification of rat FN cDNA. A KpnI site was engineered into each oligonucleotide to join the regions spanning each deletion. PCR amplification for 25 cycles was performed for all constructions under the following conditions: 95°C, 30 s; 60°C, 60 s; and 72°C, 60 s. PCR products were digested with flanking enzymes and inserted into the FN cDNA using convenient restriction sites. The following 5' and 3' primers were used to generate the indicated deletions (base positions of the primers within the FN cDNA are in parentheses and base changes to introduce the Kpn I sites are underlined): III1: GGGGTACCTGTGCCTGGGTA (1831-1812), CTGGTACCAGCAACACAGTG (2116-2130);
III47: CGGGTACCTCATCGGATCGT (2721-2702), GCGGTACCTCCTCCCACGGA (4065-4084);
III25: TGTTGCTGGGTACCGGTGTG (2124-2105), CCTGGTACCTCTGCGCTCCA (3248-3267). pVL1392 FN
III12 was prepared by ligating a fragment from FN
III1 with a PCR-amplified fragment made using the primer CTGGTACCGCACCTGATGCGCCTCCAG (2424-2442). pVL1392 FN
III45 was created by ligating a 5' fragment from pVL1392 FN
III47 with a 3' fragment from pVL1392 FN
III25. To generate pVL1392 FNrIII45/910, a segment spanning repeats III910 was amplified using 5' primer GAGGTACCGGACTCCCCAACTGGTTTTG (4552-4570) and 3' primer GAGGTACCGCTGTTTGATAATTGATGGAAACTGGC (5098-5073) containing KpnI sites (underlined). The resulting KpnI fragment was then inserted at the engineered KpnI site in pVL1392 FN
III45 and a segment encoding an RGD deletion was inserted into the cell binding domain (Schwarzbauer, 1991). All regions obtained from PCR products were verified by DNA sequence analysis.
Recombinant baculoviruses were created and recombinant proteins and rat pFN were purified as described (Sechler et al., 1996, 1997). Yields of recFNs were at least 0.8 mg/100 ml of culture supernatant. The identity of FNIII1 and FN
III12 recombinant baculoviruses was further confirmed using DNA isolated from infected High Five insect cells as template for PCR amplification with flanking primers. Correctly sized PCR products were obtained as analyzed by PAGE before and after restriction with KpnI.
Immunofluorescence
As described in Sechler et al. (1996), CHO5 and AtT-20
5 cells were seeded in medium containing FN-depleted serum and plated onto glass coverslips in a 24-well dish or four-well chamber slides at a concentration of 1.5 x 105 and 4 x 105, respectively. After an overnight incubation, fresh medium was added along with either pFN or purified recFN and incubated for the times indicated. Cell layers were fixed with 3.7% formaldehyde and stained with a 1:1,000 dilution of IC3 ascites in PBS with 2% ovalbumin followed by a 1:400 dilution of fluorescein-conjugated goat antimouse secondary antibody. Stained cells were mounted with FluoroGuard (Bio-Rad Laboratories), and fibrils were visualized with a Nikon Optiphot-2 microscope. Images were collected with a DEI-750 cooled CCD camera (Optronics Engineering) and transferred to a Macintosh G3 computer with an LG3 frame grabber (Scion Corp.) and Adobe Photoshop v. 5.0.
Isolation and detection of DOC-soluble and -insoluble matrix
DOC-soluble and -insoluble material was isolated from CHO5 cells cultured in a 24-well dish with pFN or recFNs as described above. After the indicated time periods, cells were washed with serum-free DME and lysed with 200 µl of DOC lysis buffer (2% deoxycholate, 0.02 M Tris-HCl, pH 8.8, 2 mM phenylmethylsulfonyl fluoride, 2 mM EDTA, 2 mM iodoacetic acid, and 2 mM N-ethylmaleimide) per well. Lysates were separated into DOC-soluble and -insoluble fractions that were analyzed by SDS-PAGE. Immunodetection was performed as described (Sechler et al., 1996) using ascites fluid from rat FNspecific monoclonal antibody IC3 at a dilution of 1:1,000. Immunoblots were developed with Super Signal chemiluminescence reagents (Pierce Chemical Co.). Band intensities were quantified at two exposure times using IPLab software (Mac v. 3.5; Scanalytics, Inc.).
Expression of bacterial fusion proteins and FN binding assays
Rat FN cDNA fragments encoding repeat III1 or III2 were inserted into pMAL-cRI (New England Biolabs, Inc.) for expression as MBP fusion proteins. III1 spanned amino acid positions 604700 (TYP ... TTS) and III2 extended from residue 701 to 808 (AST ... QTT). BamHI sites and XbaI sites were engineered at the 5' and 3' ends, respectively. PCR amplification using primers homologous to sequences flanking the 17amino acid linker (701717) was used to replace these residues (AST ... APF) with a KpnI site to generate III12L. Escherichia coli TB1 cells expressing individual MBP fusion proteins were lysed with B-PER (Pierce Chemical Co.) and proteins were purified by amylose resin affinity chromatography following the manufacturer's recommendations. Rat pFN, recombinant 70-kD fragment, MBP, and MBP-III12 were purified as described previously (Aguirre et al., 1994).
Solid phase binding assays were performed essentially as described by Aguirre et al. (1994). Fusion proteins containing III1, III2, III12, and III12L were immobilized on Nunc Maxisorp microtiter plates by overnight incubation at the indicated concentrations. Relative amounts of immobilized proteins were determined by ELISA with an anti-MBP antiserum. After washing and blocking with 1% BSA in PBS, wells were incubated with rat pFN at 50 µg/ml or NH2-terminal 70-kD fragment of FN at 30 µg/ml for 2 h at room temperature. Bound FN and 70 kD were detected and quantified by ELISA using antirat FN monoclonal antibody 5G4 or anti70-kD polyclonal antiserum R457.
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Footnotes |
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T. Murata's present address is School of Dentistry, Niigata University, 2-5274 Gakkou-Machi Dori, Niigata-Si, 951-8514 Japan.
* Abbreviations used in this paper: DOC, deoxycholate; FN, fibronectin; pFN, plasma FN; recFN, recombinant FN; MBP, maltose-binding protein; RGD, arg-gly-asp.
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Acknowledgments |
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This work was supported by a grant from the National Institutes of Health (CA44627 to J.E. Schwarzbauer). J.L. Sechler was a recipient of a New Jersey Commission on Cancer Research postdoctoral fellowship. T. Murata was supported by the Japan Society for the Promotion of Science.
Submitted: 7 February 2001
Revised: 12 July 2001
Accepted: 13 July 2001
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