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• W1986956955 abstract "The cartilage-specific (V + C)− fibronectin isoform does not efficiently heterodimerize with other V-region splice variants of fibronectin. To understand better the structural elements that determine this restricted dimerization profile, a series of truncated fibronectin expression constructs with various internal deletions in the V, III-15, or I-10 segments were constructed and co-transfected into COS-7 cells with either the V+C+ or the (V + C)− isoform. SDS-PAGE and immunoblot analyses of the resulting conditioned media suggest that the I-10 segment must either be present in both monomeric subunits of fibronectin or absent from both subunits for efficient dimerization to occur. Further studies suggest that the I-10 segment specifically, not simply a balanced number of type I repeats at the carboxyl terminus of each monomeric subunit, plays an important role in determining different fibronectin dimerization patterns. Neither I-11 nor I-12 could be substituted for segment I-10 without significantly reducing the formation of heterodimers. Therefore, absence of segment I-10 explains why (V + C)− fibronectin is not found in heterodimeric configurations with other native V-region splice variants in cartilage. The unique dimerization pattern of (V + C)− fibronectin does not prevent matrix formation yet is consistent with this isoform having specialized properties in situ that are important for either the structural organization and biomechanical properties of cartilage or the regulation of a chondrocytic phenotype. The cartilage-specific (V + C)− fibronectin isoform does not efficiently heterodimerize with other V-region splice variants of fibronectin. To understand better the structural elements that determine this restricted dimerization profile, a series of truncated fibronectin expression constructs with various internal deletions in the V, III-15, or I-10 segments were constructed and co-transfected into COS-7 cells with either the V+C+ or the (V + C)− isoform. SDS-PAGE and immunoblot analyses of the resulting conditioned media suggest that the I-10 segment must either be present in both monomeric subunits of fibronectin or absent from both subunits for efficient dimerization to occur. Further studies suggest that the I-10 segment specifically, not simply a balanced number of type I repeats at the carboxyl terminus of each monomeric subunit, plays an important role in determining different fibronectin dimerization patterns. Neither I-11 nor I-12 could be substituted for segment I-10 without significantly reducing the formation of heterodimers. Therefore, absence of segment I-10 explains why (V + C)− fibronectin is not found in heterodimeric configurations with other native V-region splice variants in cartilage. The unique dimerization pattern of (V + C)− fibronectin does not prevent matrix formation yet is consistent with this isoform having specialized properties in situ that are important for either the structural organization and biomechanical properties of cartilage or the regulation of a chondrocytic phenotype. Fibronectin (FN) 1The abbreviations used are: FNfibronectinDNsdeminectinsPCR-SOEingpolymerase chain reaction-sequence overlap extensionpFNrat plasma FNV-regionvariable region of fibronectin alternative splicing1The abbreviations used are: FNfibronectinDNsdeminectinsPCR-SOEingpolymerase chain reaction-sequence overlap extensionpFNrat plasma FNV-regionvariable region of fibronectin alternative splicing is an extracellular glycoprotein that has important roles in cell adhesion and migration, cell differentiation and proliferation, cell morphology and cytoskeletal organization, tissue remodeling and wound repair, and cancer progression (1Hynes R.O. Fibronectins. Springer-Verlag Inc., New York1990: 200-364Crossref Google Scholar). It is expressed by cells primarily in an anti-parallel dimeric configuration, composed of two 200–250-kDa monomeric subunits that are linked together by a pair of disulfide bonds near their carboxyl termini (2Skorstengaard K. Jensen M.S. Sahl P. Petersen T.E. Magnusson S. Eur. J. Biochem. 1986; 161: 441-453Crossref PubMed Scopus (151) Google Scholar, 3An S.S. Jimenez-Barbero J. Petersen T.E. Llinas M. Biochemistry. 1992; 31: 9927-9933Crossref PubMed Scopus (16) Google Scholar, 4Kar L. Lai C.S. Wolff C.E. Nettesheim D. Sherman S. Johnson M.E. J. Biol. Chem. 1993; 268: 8580-8589Abstract Full Text PDF PubMed Google Scholar). Dimers are formed in the endoplasmic reticulum and are subsequently transported through the Golgi and secreted (5Choi M.G. Hynes R.O. J. Biol. Chem. 1979; 254: 12050-12055Abstract Full Text PDF PubMed Google Scholar). A portion of secreted, soluble FN is captured by the cell surface in a reversible and saturable manner (6McKeown-Longo P.J. Mosher D.F. J. Cell Biol. 1985; 100: 364-374Crossref PubMed Scopus (247) Google Scholar, 7Quade B.J. McDonald J.A. J. Biol. Chem. 1988; 263: 19602-19609Abstract Full Text PDF PubMed Google Scholar, 8McKeown-Longo P.J. Mosher D.F. Mosher D.F. Fibronectin. Academic Press Inc., San Diego1989: 163-179Google Scholar), which is then irreversibly assembled into a fibrillar extracellular matrix that is insoluble in the detergent deoxycholate and consists of disulfide-stabilized multimers (8McKeown-Longo P.J. Mosher D.F. Mosher D.F. Fibronectin. Academic Press Inc., San Diego1989: 163-179Google Scholar, 9Mosher D.F. Sottile J., Wu, C. McDonald J.A. Curr. Opin. Cell Biol. 1992; 4: 810-818Crossref PubMed Scopus (136) Google Scholar, 10Sechler J.L. Corbett S.A. Wenk M.B. Schwarzbauer J.E. Ann. N. Y. Acad. Sci. 1998; 857: 143-154Crossref PubMed Scopus (61) Google Scholar, 11Schwarzbauer J.E. Sechler J.L. Curr. Opin. Cell Biol. 1999; 11: 622-627Crossref PubMed Scopus (247) Google Scholar). fibronectin deminectins polymerase chain reaction-sequence overlap extension rat plasma FN variable region of fibronectin alternative splicing fibronectin deminectins polymerase chain reaction-sequence overlap extension rat plasma FN variable region of fibronectin alternative splicing Alternative splicing of FN gene transcripts results in different protein isoforms. Four sites of alternative splicing have been reported, extra domain A, extra domain B, the variable (V or IIICS) region, and a cartilage-specific (C) region composed of nucleotides that encode protein segments III-15 and I-10. In adult canine and equine articular cartilage, 50–80% of the FN transcripts have an unique splicing pattern, designated (V + C)−, that deletes both the V and C regions (12MacLeod J.N. Burton-Wurster N. Gu D.N. Lust G. J. Biol. Chem. 1996; 271: 18954-18960Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Dimerization patterns of the (V + C)− isoform were studied under native conditions within canine articular cartilage and experimentally in COS-7, NIH-3T3, and CHO-K1 cell cultures (13Burton-Wurster N. Gendelman R. Chen H., Gu, D.-N. Tetreault J.W. Lust G. Schwarzbauer J.E. MacLeod J.N. Biochem. J. 1999; 341: 555-561Crossref PubMed Scopus (11) Google Scholar). In all systems, the (V + C)−isoform exists predominantly in a homodimeric configuration. Heterodimers composed of (V + C)− and the other V-region splice variants (V+C+ or V−C+) are either not observed or detected at only low levels. The homodimeric configuration of (V + C)−FN does not reflect the laws of random assortment (14Schwarzbauer J.E. Mulligan R.C. Hynes R.O. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 754-758Crossref PubMed Scopus (28) Google Scholar, 15Schwarzbauer J.E. Bioessays. 1991; 13: 527-533Crossref PubMed Scopus (119) Google Scholar). By using isoform-specific constructs, it was shown that this pattern results from a problem with heterodimer formation involving the (V + C)− isoform, rather than secretion (13Burton-Wurster N. Gendelman R. Chen H., Gu, D.-N. Tetreault J.W. Lust G. Schwarzbauer J.E. MacLeod J.N. Biochem. J. 1999; 341: 555-561Crossref PubMed Scopus (11) Google Scholar). Different patterns of alternative splicing and dimerization have been shown to influence FN solubility and matrix assembly (1Hynes R.O. Fibronectins. Springer-Verlag Inc., New York1990: 200-364Crossref Google Scholar, 16Yamada K.M. Schlesinger D.H. Kennedy D.W. Pastan I. Biochemistry. 1977; 16: 5552-5559Crossref PubMed Scopus (115) Google Scholar, 17Alexander S.S.J. Colonna G. Edelhoch H. J. Biol. Chem. 1979; 254: 1501-1505Abstract Full Text PDF PubMed Google Scholar, 18Guan J.-L. Trevithick J.E. Hynes R.O. J. Cell Biol. 1990; 110: 833-847Crossref PubMed Scopus (69) Google Scholar). FN dimers containing the extra domain A and extra domain B domains are incorporated more efficiently into pre-existing matrices (18Guan J.-L. Trevithick J.E. Hynes R.O. J. Cell Biol. 1990; 110: 833-847Crossref PubMed Scopus (69) Google Scholar). In addition, Ichihara-Tanaka et al. (19Ichihara-Tanaka K. Titani K. Sekiguchi K. J. Cell Sci. 1995; 108: 907-915Crossref PubMed Google Scholar) found that the segments III-15 and I-10 through I-12 are actively involved in FN matrix assembly, and deletion of even one of the type I modules reduces the matrix assembly activities. In the current study, we test the hypothesis that the restricted dimerization pattern of (V + C)− FN is related to the absence of III-15 and/or I-10 protein segments, and we study the matrix structure of this naturally occurring isoform. Deminectins (DNs) are amino-terminal truncations of rat FN extending from within segment III-8 to the carboxyl terminus (Fig. 1). Construction of DN1, DN2, and DN3 have been described previously, and they have been shown to model accurately the dimerization profiles of native full-length FN isoforms (13Burton-Wurster N. Gendelman R. Chen H., Gu, D.-N. Tetreault J.W. Lust G. Schwarzbauer J.E. MacLeod J.N. Biochem. J. 1999; 341: 555-561Crossref PubMed Scopus (11) Google Scholar, 14Schwarzbauer J.E. Mulligan R.C. Hynes R.O. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 754-758Crossref PubMed Scopus (28) Google Scholar, 20Schwarzbauer J.E. Spencer C.S. Wilson C.L. J. Cell Biol. 1989; 109: 3445-3453Crossref PubMed Scopus (70) Google Scholar). A series of new DN constructs, containing various V, III-15, and I-10 segment deletions, were made by PCR-sequence overlap extension (PCR-SOEing) (21Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6769) Google Scholar, 22Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 61-68Crossref PubMed Scopus (2614) Google Scholar). The procedure involved the generation of two PCR fragments. The first fragment extended from a 5′ PstI site in the region encoding III-12 to a targeted junction site defined by the desired construct. The 5′ end of the second fragment started from the same targeted junction site and extended to a 3′ SalI site downstream of nucleotides encoding the carboxyl terminus. Oligonucleotide primers used to generate the two PCR fragments were designed with overlaps in the targeted junction region between 18 and 24 nucleotides in length (Table I). This overlap allowed fusion of the two fragments in a third PCR that used only the 5′ (TGG TTC AGA CTG CAG TGA)- and 3′ (TCT AGA GTC GAC CCG G)-flanking primers. The resulting fusion product was digested with PstI and SalI and substituted for the corresponding PstI/SalI fragment of DN1. The only exception was construction of DN14, which required two independent PCR-SOEing steps (DN14a and DN14b in Table I). Oligonucleotide primers were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA), and PCR amplifications were carried out with Pfu DNA Polymerase (Stratagene, La Jolla, CA). Nucleotide identities of all new DNs were confirmed by bi-directional sequencing (Biotechnology Service, Cornell University, Ithaca, NY).Table IOverlapping primers used for the construction of individual DN cDNAs by PCR-SOEingDNsAntisense primer of upstream ampliconSense primer of downstream ampliconDN4GCA CCA TTT AGT GTT GCC TAC AGT AACGGC AAC ACT AAA TGG TGC CAT GAC AACDN5TTC GTT GAC TGT AGA GGC ATT TGG ATT GAGGCC TCT ACA GTC AAC GAA GGC CTG AADN6GCA CCA TTT AGT GTT GCC TAC AGT AAC AACGGC AAC ACT AAA TGG TGC CAT GAC AACDN7TTC GTT GAC TGT CTT TTT CCT CCC AATAAA AAG ACA GTC AAC GAA GGC CTG AACDN8GCA CCA TTT TGT AGA GGC ATT TGG ATT GAGGCC TCT ACA AAA TGG TGC CAT GAC AACDN9TGC TTC ATG AGA TGA ATC GCA TCT GAAGAT TCA TCT CAT GAA GCA ACG TGT TATDN10GCG CCA GCC GGG ATC GCA TTT GAA TTCTGC GAT CCC GGC TGG CGC TGT GAC AACDN13ATG GCA CCA TTC GGG ATC GCA TTT GAAAAA TGC GAT CCC GAA TGG TGC CAT GAC AACDN14aCGT TGC TTC ATG TGT CTT TTT CCT CCC AAT CAGAGG AAA AAG ACA CAT GAA GCA ACG TGT TAT GACDN14bATG GCA CCA TTC CCG CTG GCC CCC GAA ACAGGC CAG CGG GAA TGG TGC CAT GAC AACA series of DN constructs, containing various V, III-15, and I-10 segment deletions, were made by PCR-SOEing. The procedure involved the generation of two PCR fragments. The first amplicon extended from a 5′PstI site in the region encoding III-12 to a targeted junction site defined by the desired construct. The 5′ end of the second amplicon started from the same targeted junction site and extended to a 3′ SalI site downstream of nucleotides encoding the carboxyl terminus. Oligonucleotide primers used to generate the two PCR amplicons were designed with overlaps in the targeted junction region (in bold). This overlap allowed fusion of the two cDNA fragments in a third PCR that used only the 5′ (TGG TTC AGA CTG CAG TGA) and 3′ (TCT AGA GTC GAC CCG G)-flanking primers containing the PstI and SalI restriction sites, respectively. The resulting fusion product was digested with PstI and SalI and substituted for the corresponding PstI/SalI fragment of DN1. The only exception was construction of DN14, which required two independent PCR-SOEing steps (DN14a and DN14b). Open table in a new tab A series of DN constructs, containing various V, III-15, and I-10 segment deletions, were made by PCR-SOEing. The procedure involved the generation of two PCR fragments. The first amplicon extended from a 5′PstI site in the region encoding III-12 to a targeted junction site defined by the desired construct. The 5′ end of the second amplicon started from the same targeted junction site and extended to a 3′ SalI site downstream of nucleotides encoding the carboxyl terminus. Oligonucleotide primers used to generate the two PCR amplicons were designed with overlaps in the targeted junction region (in bold). This overlap allowed fusion of the two cDNA fragments in a third PCR that used only the 5′ (TGG TTC AGA CTG CAG TGA) and 3′ (TCT AGA GTC GAC CCG G)-flanking primers containing the PstI and SalI restriction sites, respectively. The resulting fusion product was digested with PstI and SalI and substituted for the corresponding PstI/SalI fragment of DN1. The only exception was construction of DN14, which required two independent PCR-SOEing steps (DN14a and DN14b). Each DN construct, both singly and in combination with the other DNs, was transiently transfected into COS-7 cells (American Type Culture Collection number 1651-CRL, Manassas, VA) using Superfect transfection reagents following the protocol recommended by the manufacturer (Qiagen, Valencia, CA). The amount of DNA used for each co-transfection was adjusted empirically to achieve approximately equal expression levels. Twenty four hours after transfection, the cells were re-fed with Dulbecco's modified Eagle's medium, 10% fetal bovine serum. Conditioned media were collected another 24 h later, clarified by centrifugation (175 × g), stabilized with protease inhibitors (0.3 mm benzamidine, 20 mm EDTA, 10 mm N- ethylmaleimide, 0.4 mm phenylmethylsulfonyl fluoride, final concentrations), and frozen at −20 °C pending analysis of dimerization patterns. Samples were subjected to electrophoresis and immunoblot analysis by heating at 95 °C for 5 min in the presence of 0.2% (w/v) SDS without β-mercaptoethanol. Heterodimers, homodimers, and monomers of recombinant DNs were separated according to size on 6% SDS-polyacrylamide gels in Tris glycine buffer at pH 8.6 (23Laemmli U.K. Nature. 1970; 277: 680-685Crossref Scopus (205492) Google Scholar), transferred onto nitrocellulose membranes (24Towbin H. Staehlin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44642) Google Scholar), and probed with a rat FN-specific monoclonal antibody IC-3 (25Castle A.M. Schwarzbauer J.E. Wright R.L. Castle J.D. J. Cell Sci. 1995; 108: 3827-3837Crossref PubMed Google Scholar) (a gift from Dr. Jean Schwarzbauer at Princeton University), followed by either a peroxidase-linked (Sigma) or a 35S-labeled anti-mouse IgG antibody (Amersham Biosciences). The binding epitope of IC-3 has been localized to the cell binding region within III-8 to III-11, 2J. Schwarzbauer, personal communication. which is common to all FN isoforms. Relative binding affinities of IC-3 to the DN constructs are equivalent to anti-human FN polyclonal IgG (ICN Pharmaceuticals Inc., Aurora, OH; data not shown). Peroxidase activity was detected by chemiluminescence (ECL Western blotting detection system, Amersham Biosciences), and 35S decay events were measured by a PhosphorImager (Fuji Photo Film Co., Ltd., Japan). Band intensities on radiograph films or PhosphorImager plates were quantified using MacBas software (Fuji Photo Film Co., Ltd.). Quantitative data obtained with the two protein detection strategies (ECL versus 35S-labeled secondary antibody) yielded comparable results (data not shown). Therefore, for DNs with higher level of expression, band intensities were quantified with the35S-labeled secondary antibody. The increased sensitivity of the ECL system was useful in samples where DN expression was reduced. Relative homodimer and heterodimer percentages were calculated with total band intensity of DN set at 100%. All quantitative data are presented as the mean ± S.D. of triplicate co-transfection experiments. Construction of recombinant baculovirus containing the rat V+C+ construct has been described previously (26Sechler J.L. Takada Y. Schwarzbauer J.E. J. Cell Biol. 1996; 134: 573-583Crossref PubMed Scopus (128) Google Scholar). Full-length (V + C)− FN cDNA was subcloned into the baculovirus vector PVL 1392 (BD PharMingen). Recombinant viruses were generated according to established procedures (27O'Reilly D.R. Miller L.K. Luckow V.A. Baculovirus Expression Vectors: A Laboratory Manual. Oxford University Press, New York1994: 124-138Google Scholar). To produce recombinant protein, 50% confluent T175 flasks of Sf21 cells that had been adapted for growth under serum-free conditions (gift from Dr. Ping Wang at Cornell University) were incubated with recombinant viruses for 2 h in Excell-400 medium (JRH Biosciences, Lenexa, KS). After re-feeding, conditioned media from infected cells were collected 48 h later and clarified by centrifugation (175 × g). The mixture of protease inhibitors (above) was added to inhibit proteolysis. Recombinant FN proteins were purified using affinity chromatography columns of gelatin-agarose (Amersham Biosciences), followed by heparin-agarose (Pierce) as described by Poulouin et al. (28Poulouin L. Gallet O. Rouahi M. Imhoff J.M. Protein Expression Purif. 1999; 17: 146-152Crossref PubMed Scopus (78) Google Scholar). The concentration of purified FN was determined by enzyme-linked immunosorbent assay using a polyclonal (goat) antiserum to human FN, followed by peroxidase-conjugated rabbit anti-goat IgG (ICN Pharmaceuticals Inc.) as described previously (29Wurster N.B. Lust G. Biochem. Biophys. Res. Commun. 1982; 109: 1094-1101Crossref PubMed Scopus (55) Google Scholar). FN null mouse embryonic fibroblasts (30Saoncella S. Echtermeyer F. Denhez F. Nowlen J.K. Mosher D.F. Robinson S.D. Hynes R.O. Goetinck P.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2805-2810Crossref PubMed Scopus (328) Google Scholar) were a gift of Dr. Deane Mosher (University of Wisconsin, Madison). The fibroblasts were plated into the wells (3 × 104cells/well) of immunofluorescence slides (Polysciences, Warrington, PA) in Dulbecco's modified Eagle's medium supplemented with 10% FN-depleted fetal bovine serum (31Engavall E. Ruoslathi E. Int. J. Cancer. 1977; 20: 1-5Crossref PubMed Scopus (1420) Google Scholar), glutamine (0.29 mg/ml), and penicillin (100 units/ml)/streptomycin (100 μg/ml). After a 3-h period for cell attachment and spreading, media were removed, and the cells were re-fed with Dulbecco's modified Eagle's medium containing 30 μg/ml of either rat plasma FN (pFN, Sigma), recombinant V+C+ FN, or recombinant (V + C)−FN. The cellular assembly of a FN matrix was allowed to proceed for 24 h. For immunofluorescence microscopy, cells were fixed with 3.5% paraformaldehyde for 15 min at 4 °C and incubated sequentially with a monoclonal antibody against rat FN (IC-3) in Tris-buffered saline containing 0.1% bovine serum albumin overnight at 4 °C and goat anti-mouse IgG conjugated to fluorescein isothiocyanate (Molecular Probe, Eugene, OR) overnight at 4 °C. After rinses, the slides were covered with prolong anti-fade mounting media (Molecular Probe). The pattern of fluorescence was assessed with an Olympus IX70 confocal microscope using an argon laser with 488 nm excitation and bandpass filters for collecting green fluorescence. Unlike the V+C+ and V−C+ FN isoforms that can heterodimerize efficiently with each other, the cartilage-specific (V + C)− FN isoform exists predominantly as homodimers within canine articular cartilage (13Burton-Wurster N. Gendelman R. Chen H., Gu, D.-N. Tetreault J.W. Lust G. Schwarzbauer J.E. MacLeod J.N. Biochem. J. 1999; 341: 555-561Crossref PubMed Scopus (11) Google Scholar). This dimerization pattern of (V + C)− FN is retained in experimental cell culture models (13Burton-Wurster N. Gendelman R. Chen H., Gu, D.-N. Tetreault J.W. Lust G. Schwarzbauer J.E. MacLeod J.N. Biochem. J. 1999; 341: 555-561Crossref PubMed Scopus (11) Google Scholar) and led to the hypothesis that absence of the III-15 and/or I-10 protein segments restricts heterodimerization with the other two V-region splice variants (V+C+ or V−C+). To test the hypothesis, an experimental system using truncated versions of FN, termed DNs, was utilized. In addition to DN1 representing the V+C+ isoform, DN2 representing the V−C+ isoform, and DN3 representing the (V + C)− isoform, five new DN constructs, DN4 to DN8, were made with various combinations of the V, III-15, and I-10 segments deleted (Fig. 1). Either DN1 or DN3 was transiently co-transfected into COS-7 cells with one of the other seven DNs. Homodimers, heterodimers, and monomers of expressed DNs in conditioned medium were separated by SDS-PAGE under non-reducing conditions and detected using immunoblots. Culture media from cells transfected with only single DNs were run in parallel to confirm the identity of homodimers. As illustrated in Fig. 2, there are two dimerization patterns observed following co-transfection of DN1 with other DNs. They either have the potential to form a heterodimer or remain only as their corresponding homodimers. For example, DN1 does not form heterodimers with DN6 (Fig. 2, panel a). When they are co-transfected into COS-7 cells, only bands corresponding to DN1 and DN6 homodimers are formed (Fig. 2, lane 2). In contrast, DN1 heterodimerizes efficiently with DN7 (Fig. 2, panel b). Co-transfection of these two DNs results in a large amount of heterodimers (Fig. 2, lane 5) that have an intermediate size between DN1 (Fig. 2, lane 4) and DN7 homodimers (Fig. 2,lane 6). Since DN1, DN4, and DN5 are very similar in size, DN1/DN4 and DN1/DN5 heterodimers could not be resolved by 6% SDS-PAGE. Therefore, full-length rat V+C+ FN (FN1) was co-transfected with DN4 or DN5. FN1 heterodimerizes with DN5 (Fig. 2,panel c) but not DN4 (not shown). Fig. 2 B summarizes the quantitative ratios of homodimer/heterodimer formation in each co-transfection combination with DN1 (or FN1). DN1 (or FN1) does not heterodimerize well with DN3, DN4, DN6, and DN8, whereas it heterodimerizes efficiently with DN2, DN5, and DN7. A similar experimental strategy was used to analyze the heterodimerization potential of DN3. Again, two general dimerization patterns are observed (Fig. 3). For example, DN3 heterodimerizes with DN4 (Fig. 3, panel a) and with DN6 (Fig. 3, panel c). Co-transfection of DN3 with either DN4 or DN6 results in a high level of intermediate-sized heterodimers. In contrast, DN3 does not heterodimerize with DN5 (Fig. 3, panel b). Co-transfected cells express only DN3 and DN5 homodimers (Fig. 3, lane 5). Fig. 3 B lists the relative percentages of DN3 homodimers, DN3/DN X heterodimers, and DN X homodimers. DN3 does not heterodimerize with DN1 and DN5 but heterodimerizes with the other DNs to varying degrees. Comparative heterodimerization patterns of DN1 and DN3 are summarized in Table II. The percentages of DN1/DN X and DN3/DN X heterodimers are scored in deciles by asterisks (≤10%, *; 11–20%, **; 21–30%, ***; 31–40%, ****; ≥41%, *****). The data indicate that isoforms that include segment I-10 heterodimerize preferentially with DN1, which also includes the I-10 segment. Similarly, isoforms that lack protein segment I-10 heterodimerize preferentially with DN3, which also lacks the I-10 segment. For each DN X, the level of DN1/DN X heterodimerization is significantly different (p < 0.05) than the level of DN3/DN X heterodimerization. The data also indicate that the presence or absence of protein segments V and III-15 does not influence substantially whether heterodimerization is favored with DN3 or DN1.Table IISummary of the dimerization patterns of DN1 and DN3 with co-transfected DNsDN XProtein segmentsDimerizationVIII-15I-10DN1/DN XDN3/DN XDN1+++******DN3−−−******DN2−++*******DN4++−* 2-aFull-length FN-1 was substituted for DN-1 to enable band resolution by SDS-PAGE.****DN5+−+***** 2-aFull-length FN-1 was substituted for DN-1 to enable band resolution by SDS-PAGE.*DN6−+−*****DN7−−+*********DN8+−−****The presence or absence of V, III-15, and I-10 protein segments in the various DN constructs is indicated by the + or − sign. Relative dimerization efficiencies (from Figs. 2 B and3 B) were scored in deciles by asterisks as indicated. Percent heterodimerization of DNs: *, ≤10%; **, 11–20%; ***, 21–30%; ****, 31–40%; *****, ≥41%. The data demonstrate the following: 1) a DN that lacks protein segment I-10 heterodimerizes preferentially with DN3, which also lacks the I-10 segment; 2) a DN that includes the I-10 segment heterodimerizes preferentially with DN1 (or FN1), which also includes the I-10 segment; and 3) the presence or absence of protein segment V and III-15 does not influence substantially whether heterodimerization is favored with DN3 or DN1.2-a Full-length FN-1 was substituted for DN-1 to enable band resolution by SDS-PAGE. Open table in a new tab The presence or absence of V, III-15, and I-10 protein segments in the various DN constructs is indicated by the + or − sign. Relative dimerization efficiencies (from Figs. 2 B and3 B) were scored in deciles by asterisks as indicated. Percent heterodimerization of DNs: *, ≤10%; **, 11–20%; ***, 21–30%; ****, 31–40%; *****, ≥41%. The data demonstrate the following: 1) a DN that lacks protein segment I-10 heterodimerizes preferentially with DN3, which also lacks the I-10 segment; 2) a DN that includes the I-10 segment heterodimerizes preferentially with DN1 (or FN1), which also includes the I-10 segment; and 3) the presence or absence of protein segment V and III-15 does not influence substantially whether heterodimerization is favored with DN3 or DN1. Is there something specific about protein segment I-10 that regulates the heterodimerization patterns of DN1 and DN3, or is it simply necessary to have a balanced number of type I repeats at the carboxyl terminus? For example, it could be necessary to have the same number of carboxyl-terminal type I repeats on each monomeric FN subunit for efficient interchain association or as structural elements for proper alignment. To answer this question, DN9 and DN10, which delete I-11 and I-12 protein segments, respectively, were prepared (Figs. 1and 4 B). DN4, DN9, and DN10 all contain V and III-15, but each has a different combination of two type I repeats at the carboxyl terminus. These constructs were co-transfected individually with DN3 into COS-7 cells. The results are shown in Fig. 4. If a balanced number of type I repeats at the carboxyl terminus determines heterodimerization efficiency, then DN3 would be expected to form similar amounts of heterodimers with DN4, DN9, and DN10. Although DN3 does heterodimerize with all three (Fig. 4 A), the levels of heterodimer formation are significantly different (Fig. 4 B). DN3 heterodimerizes most efficiently with DN4, both of which lack protein segment I-10. DN13 and DN14 were constructed to enable a parallel analysis with DN1. They are structurally similar to DN7, all three lacking V and III-15, but segment I-10 is removed and substituted with duplications of either I-11 (DN13) or I-12 (DN14) (Figs. 1 and5 B). As reported in Fig. 5, these changes greatly reduce the formation of DN1 heterodimers. Once again, efficient heterodimerization of different DN isoforms requires that both subunits have the same I-10 status. In this case, both DN1 and DN7 contain protein segment I-10 and heterodimerize most efficiently. The unique primary structure and restricted heterodimerization of (V + C)− FN raise the potential that this isoform may have different matrix assembly characteristics that influence the matrix organization and biomechanical properties of cartilage. To explore this possibility and compare FN matrix structure, pFN, recombinant V+C+ FN, and recombinant (V + C)−FN (30 μg/ml) were added to mouse embryo" @default.
• W1986956955 created "2016-06-24" @default.
• W1986956955 creator A5002677360 @default.
• W1986956955 creator A5022499603 @default.
• W1986956955 creator A5047457069 @default.
• W1986956955 creator A5072876010 @default.
• W1986956955 date "2002-05-01" @default.
• W1986956955 modified "2023-10-18" @default.
• W1986956955 title "Absence of the I-10 Protein Segment Mediates Restricted Dimerization of the Cartilage-specific Fibronectin Isoform" @default.
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