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• W2000201832 abstract "Cell surface molecules on adherent cells that bind 125I-labeled fibronectin or its 70-kDa N-terminal fragment were identified by cross-linking with factor XIIIa and by photoaffinity labeling. Such cross-linking caused the 70-kDa fragment to become associated irreversibly to cell layers and was greater in cells treated with lysophosphatidic acid, an enhancer of fibronectin assembly and strong modulator of cell shape. Cross-linking of the 70-kDa fragment with factor XIIIa was to molecules that migrated in discontinuous sodium dodecyl sulfate-polyacrylamide gels at the top of the 3.3% stacking gel and near the top of the separating gel. Estimated sizes of these large apparent molecular mass molecules (LAMMs) were ≫3 MDa and ~3 MDa. The label in 70-kDa fragment conjugated with 125I-sulfosuccinimidyl 2-(p-azidosalicylamido)-1,3′-dithiopropionate was associated with ≫3-MDa LAMMs without reduction and with ~3-MDa LAMMs after reduction and transfer of the cleavable label. The LAMMs were expressed on monolayer cells shortly after adherence, required both 1% Triton X-100 and 2 M urea for efficient extraction, and were susceptible to digestion with trypsin but not to cathepsin D digestion. Complexes of 125I-70-kDa fragment and LAMMs were also susceptible to limited acid digestion and Glu-C protease digestion but were not cleaved by chondroitin lyase or heparitinase. Neither the uncleaved complexes nor the cleavage products were immunoprecipitated with anti-fibronectin antibodies directed toward epitopes outside the 70-kDa region. Thus, cell surface molecules that are either very large or not dissociated in sodium dodecyl sulfate comprise the labile matrix assembly sites for fibronectin. Cell surface molecules on adherent cells that bind 125I-labeled fibronectin or its 70-kDa N-terminal fragment were identified by cross-linking with factor XIIIa and by photoaffinity labeling. Such cross-linking caused the 70-kDa fragment to become associated irreversibly to cell layers and was greater in cells treated with lysophosphatidic acid, an enhancer of fibronectin assembly and strong modulator of cell shape. Cross-linking of the 70-kDa fragment with factor XIIIa was to molecules that migrated in discontinuous sodium dodecyl sulfate-polyacrylamide gels at the top of the 3.3% stacking gel and near the top of the separating gel. Estimated sizes of these large apparent molecular mass molecules (LAMMs) were ≫3 MDa and ~3 MDa. The label in 70-kDa fragment conjugated with 125I-sulfosuccinimidyl 2-(p-azidosalicylamido)-1,3′-dithiopropionate was associated with ≫3-MDa LAMMs without reduction and with ~3-MDa LAMMs after reduction and transfer of the cleavable label. The LAMMs were expressed on monolayer cells shortly after adherence, required both 1% Triton X-100 and 2 M urea for efficient extraction, and were susceptible to digestion with trypsin but not to cathepsin D digestion. Complexes of 125I-70-kDa fragment and LAMMs were also susceptible to limited acid digestion and Glu-C protease digestion but were not cleaved by chondroitin lyase or heparitinase. Neither the uncleaved complexes nor the cleavage products were immunoprecipitated with anti-fibronectin antibodies directed toward epitopes outside the 70-kDa region. Thus, cell surface molecules that are either very large or not dissociated in sodium dodecyl sulfate comprise the labile matrix assembly sites for fibronectin. INTRODUCTIONFibronectin is a major extracellular protein that is necessary for normal embryogenesis (1George E.L. Georges-Labouesse E.N. Patel-King R.S. Rayburn H. Hynes R.O. Development. 1993; 119: 1079-1091Crossref PubMed Google Scholar). Fibronectin exists as a soluble protomer at near micromolar concentrations in blood plasma and other body fluids and in an insoluble multimeric form in extracellular matrix (2Hynes R.O. Fibronectins. Springer-Verlag Inc., New York1990Crossref Google Scholar, 3Mosher, D. F., (ed) (1989) Fibronectin, Academic Press, San Diego.Google Scholar). Insoluble tissue fibronectin has several likely biological functions, including promotion of cellular migration during embryogenesis and wound healing (4Colvin R.B. Mosher D.F. Fibronectin. Academic Press, New York1989: 213-254Google Scholar, 5Gailit J. Clark R.A.F. Curr. Opin. Cell Biol. 1994; 6: 717-725Crossref PubMed Scopus (375) Google Scholar, 6Thiery J.-P. Duband J.-L. Dufour S. Savagner P. Imhof B.A. Mosher D.F. Fibronectin. Academic Press, New York1989: 181-212Google Scholar). During gastrulation of Xenopus laevis, polarized fibronectin fibrils are thought to guide migrating mesoderm to its target region (7Winklbauer R. Nagel M. Dev. Biol. 1991; 148: 573-589Crossref PubMed Scopus (112) Google Scholar). Local perturbation of the fibronectin-rich extracellular matrix of Xenopus gastrulae correlates with localized randomization of left-right asymmetries later in development (8Yost H.J. Nature. 1992; 357: 158-161Crossref PubMed Scopus (114) Google Scholar).Since fibronectin is primarily functional in its insolubilized state, the polymerization of fibronectin has importance similar to that of the fibrinogen-to-fibrin conversion. There is no evidence that a modifying proteolytic event, as with the cleavage of fibrinogen to fibrin monomer, triggers polymerization of fibronectin. Unlike collagens and laminins (9Yurchenco P.D. O'Rear J. Curr. Opin. Cell Biol. 1994; 6: 674-681Crossref PubMed Scopus (259) Google Scholar), plasma fibronectin does not self-polymerize in physiologically relevant solutions. Strategies that have worked to induce self-polymerization of plasma fibronectin reproducibly include incubation with denaturants (10Mosher D.F. Johnson R.B. J. Biol. Chem. 1983; 258: 6595-6601Abstract Full Text PDF PubMed Google Scholar), reduction of disulfides (11Williams E.C. Janmey P.A. Johnson R.B. Mosher D.F. J. Biol. Chem. 1983; 258: 5911-5914Abstract Full Text PDF PubMed Google Scholar, 12Sakai K. Fujii T. Hayashi T. J. Biochem. (Tokyo). 1994; 115: 415-421Crossref PubMed Scopus (24) Google Scholar), incubation with peptides based on a specific sequence of fibronectin (13Morla A. Zhang Z. Ruoslahti E. Nature. 1994; 367: 193-196Crossref PubMed Scopus (265) Google Scholar), and exposure to shear forces at air-liquid or liquid-solid interfaces (14Ejim O.S. Blunn G.W. Brown R.A. Biomaterials. 1993; 14: 743-748Crossref PubMed Scopus (92) Google Scholar, 15Brown R.A. Blunn G.W. Ejim O.S. Biomaterials. 1994; 15: 457-464Crossref PubMed Scopus (60) Google Scholar). There is little passive accumulation of fibronectin in pre-existing extracellular matrix (16Barry E.L. Mosher D.F. J. Biol. Chem. 1988; 263: 10464-10469Abstract Full Text PDF PubMed Google Scholar, 17Carter W.G. J. Cell Biol. 1984; 99: 105-114Crossref PubMed Scopus (26) Google Scholar). Rather, assembly of fibronectin requires cells and takes place at specialized sites on cell surfaces (18Peters D.M. Mosher D.F. J. Cell Biol. 1987; 104: 121-130Crossref PubMed Scopus (62) Google Scholar). Binding to these sites is mediated by the N-terminal modules of fibronectin, especially modules I-1 through I-5 (19Sottile J. Schwarzbauer J. Selegue J. Mosher D.F. J. Biol. Chem. 1991; 266: 12840-12843Abstract Full Text PDF PubMed Google Scholar, 20McKeown-Longo P.J. Mosher D.F. J. Cell Biol. 1985; 100: 364-374Crossref PubMed Scopus (247) Google Scholar, 21Quade B.J. McDonald J.A. J. Biol. Chem. 1988; 263: 19602-19609Abstract Full Text PDF PubMed Google Scholar). A number of cells synthesize and secrete forms of fibronectin that are spliced differently from the plasma form, but all have the N-terminal modules. These cellular isoforms have a greater tendency for self-polymerization than plasma fibronectin (2Hynes R.O. Fibronectins. Springer-Verlag Inc., New York1990Crossref Google Scholar). Cultured fibroblasts, however, direct assembly of both plasma fibronectin (present in serum-containing culture medium) and fibroblast-derived, alternatively spliced fibronectin (17Carter W.G. J. Cell Biol. 1984; 99: 105-114Crossref PubMed Scopus (26) Google Scholar, 22Allio A.E. McKeown-Longo P.J. J. Cell. Physiol. 1988; 135: 459-466Crossref PubMed Scopus (24) Google Scholar, 23Peters D.M. Portz L.M. Fullenwider J. Mosher D.F. J. Cell Biol. 1990; 111: 249-256Crossref PubMed Scopus (71) Google Scholar). Thus, the rules for assembly seem the same for both plasma and cellular fibronectin. Reagents that block assembly of fibronectin in cell culture also block assembly of fibronectin in the blastula (24Darribère T. Guida K. Larjava H. Johnson K.E. Yamada K.M. Thiery J.-P. Boucaut J.-C. J. Cell Biol. 1990; 110: 1813-1823Crossref PubMed Scopus (103) Google Scholar, 25Darribère T. Koteliansky V.E. Chernousov M.A. Akiyama S.K. Yamada K.M. Thiery J.-P. Boucaut J.-C. Dev. Dyn. 1992; 194: 63-70Crossref PubMed Scopus (34) Google Scholar). Cultured cells and granulation tissue of healing wounds have co-linear transmembranous associations of fibronectin-containing extracellular matrix fibers and bundles of actin microfilaments localized at dense submembranous plaques (26Singer I.I. Mosher D.F. Fibronectin. Academic Press, New York1989: 139-162Google Scholar). Somehow, therefore, the intracellular cytoskeleton determines the pattern of assembly of extracellular matrix and/or the extracellular matrix determines the pattern of assembly of intracellular cytoskeleton.We had studied previously the effects of activated factor XIII (factor XIIIa, plasma transglutaminase) on the incorporation of fibronectin and its N-terminal fragments into extracellular matrix by cultured fibroblasts (16Barry E.L. Mosher D.F. J. Biol. Chem. 1988; 263: 10464-10469Abstract Full Text PDF PubMed Google Scholar, 27Barry E.L.R. Mosher D.F. J. Biol. Chem. 1989; 264: 4179-4185Abstract Full Text PDF PubMed Google Scholar). When factor XIIIa was included in the binding medium, accumulation of 125I-fibronectin in the deoxycholate-insoluble matrix was increased in the form of cross-linked nonreducible high molecular weight aggregates. Factor XIIIa also cross-linked 125I-labeled 27- and 70-kDa N-terminal fragments into high molecular weight aggregates that could not be extracted from cell layers with deoxycholate. The characteristics of the cross-linked partners were similar to those reported when bound 27-kDa fragment was cross-linked with the membrane-impermeable reagent bis(sulfosuccinimidyl)suberate (28Limper A.H. Quade B.J. LaChance R.M. Birkenmeier T.M. Rangwala T.S. McDonald J.A. J. Biol. Chem. 1991; 266: 9697-9702Abstract Full Text PDF PubMed Google Scholar) or cellular fibronectin was derivatized, bound to cells, and cross-linked with photoactivable agents (29Perkins M.E. Ji T.H. Hynes R.O. Cell. 1979; 16: 941-952Abstract Full Text PDF PubMed Scopus (138) Google Scholar). These previous cross-linking studies are clouded by several unknowns: presence of pre-existing matrix, long incubation times, and, when photoactivable cross-linkers were not used, possibility of cross-linking of receptors to other molecules or to multiple labeled ligand during the exposure to cross-linkers. Another shortcoming inherent to this analytical approach is the absence of an unequivocal way to distinguish between cross-linking events with cell surface molecules critical for fibrillogenesis (“productive”) and events that are specific yet irrelevant to matrix assembly (“nonproductive”). We reasoned that we could deal with these problems using cells stimulated with lysophosphatidic acid (LPA), 1The abbreviations used are: LPAlysophosphatidic acidLAMMslarge apparent molecular mass moleculesPAGEpolyacrylamide gelsSASDsulfosuccinimidyl 2-(p-azidosalicylamido)-1,3′-dithiopropionateFITCfluorescein isothiocyanate. a specific up-regulator of fibronectin binding and assembly (30Zhang Q. Checovich W.J. Peters D.M. Albrecht R.M. Mosher D.F. J. Cell Biol. 1994; 127: 1447-1459Crossref PubMed Scopus (93) Google Scholar). Because fibronectin binding is as responsive to stimulation by LPA when cells spread shortly after dense seeding as when cells become confluent days after sparse seeding, we could compare cross-linking in monolayers of cells with little or no matrix shortly after seeding and abundant matrix upon reaching confluence. Furthermore, the addition and withdrawal of LPA cycles cells so rapidly between assembly-competent and assembly-deficient phenotypes (30Zhang Q. Checovich W.J. Peters D.M. Albrecht R.M. Mosher D.F. J. Cell Biol. 1994; 127: 1447-1459Crossref PubMed Scopus (93) Google Scholar) that one would expect minimal changes in the composition of molecules on the surface of cells and in the extracellular matrix. Thus, binding to molecules not at assembly sites, i.e. to molecules at sites irrelevant to and nonproductive of assembly, should be the same in control and LPA-treated cultures, and a comparison of the cross-linked complexes following the binding in the absence and presence of LPA should identify the labile receptor molecules.RESULTSThe purpose of the present studies was to characterize the cell surface molecules responsible for binding and assembly of fibronectin. Fibronectin matrix assembly is highly regulated, such that addition or withdrawal of LPA rapidly causes a 2-4-fold stimulation or less, respectively, of the binding of fibronectin or the 70-kDa fragment to cell layers of MG63 osteosarcoma cells or human foreskin fibroblasts (30Zhang Q. Checovich W.J. Peters D.M. Albrecht R.M. Mosher D.F. J. Cell Biol. 1994; 127: 1447-1459Crossref PubMed Scopus (93) Google Scholar, 33Checovich W.J. Mosher D.F. Arterioscler. Thromb. 1993; 13: 1662-1667Crossref PubMed Google Scholar). Binding of the 70-kDa fragment is to a single class of binding sites on unstimulated and LPA-stimulated cells, with increases of both avidity and number of binding sites upon stimulation (30Zhang Q. Checovich W.J. Peters D.M. Albrecht R.M. Mosher D.F. J. Cell Biol. 1994; 127: 1447-1459Crossref PubMed Scopus (93) Google Scholar). Protein kinase C inhibitors, in contrast, cause a 5-10-fold decrease in binding (42Somers C.E. Mosher D.F. J. Biol. Chem. 1993; 268: 22277-22280Abstract Full Text PDF PubMed Google Scholar). Cross-linking studies of cells treated to be assembly-competent or assembly-deficient, therefore, will yield information about the molecules that comprise the labile binding sites. The N-terminal 70-kDa fragment of fibronectin binds reversibly to cells in monolayer culture with the same avidity as the initial reversible binding of intact fibronectin (20McKeown-Longo P.J. Mosher D.F. J. Cell Biol. 1985; 100: 364-374Crossref PubMed Scopus (247) Google Scholar). The fragment consists of an N-terminal domain of 27 kDa and a 40-kDa gelatin-binding domain (2Hynes R.O. Fibronectins. Springer-Verlag Inc., New York1990Crossref Google Scholar, 3Mosher, D. F., (ed) (1989) Fibronectin, Academic Press, San Diego.Google Scholar). Near the N terminus is a glutamine residue that is attacked by factor XIIIa (43McDonagh R.P. McDonagh J. Petersen T.E. Thøgersen H.C. Skorstengaard K. Sottrup-Jensen L. Magnusson S. FEBS Lett. 1981; 127: 174-178Crossref PubMed Scopus (76) Google Scholar). The five type I modules of the 27-kDa domain are most important for binding (19Sottile J. Schwarzbauer J. Selegue J. Mosher D.F. J. Biol. Chem. 1991; 266: 12840-12843Abstract Full Text PDF PubMed Google Scholar, 20McKeown-Longo P.J. Mosher D.F. J. Cell Biol. 1985; 100: 364-374Crossref PubMed Scopus (247) Google Scholar, 21Quade B.J. McDonald J.A. J. Biol. Chem. 1988; 263: 19602-19609Abstract Full Text PDF PubMed Google Scholar), whereas the adjacent gelatin-binding domain does not bind specifically to cell layers (44Chernousov M.A. Fogerty F.J. Koteliansky V.E. Mosher D.F. J. Biol. Chem. 1991; 266: 10851-10858Abstract Full Text PDF PubMed Google Scholar). Nevertheless, the gelatin-binding domain must contribute to the binding, because the whole 70-kDa fragment has approximately 10-fold higher avidity for the binding site (Kd is 3 nM on LPA-treated cells (30Zhang Q. Checovich W.J. Peters D.M. Albrecht R.M. Mosher D.F. J. Cell Biol. 1994; 127: 1447-1459Crossref PubMed Scopus (93) Google Scholar)) than the isolated 27-kDa domain (20McKeown-Longo P.J. Mosher D.F. J. Cell Biol. 1985; 100: 364-374Crossref PubMed Scopus (247) Google Scholar, 44Chernousov M.A. Fogerty F.J. Koteliansky V.E. Mosher D.F. J. Biol. Chem. 1991; 266: 10851-10858Abstract Full Text PDF PubMed Google Scholar). Our cross-linking studies, therefore, were done with the 70-kDa fragment used at concentrations in which binding sites would be 5-20% saturated with ligand.Fig. 1 shows PhosphorImager scans of discontinuous SDS gels of 125I-70-kDa fragment bound to MG63 osteosarcoma cells for 60 min, cross-linked with factor XIIIa for 5 min, and reduced prior to electrophoresis. The fragment was cross-linked into complexes that migrated at the top of separating gel or failed to enter the stacking gel. In this and other experiments, there was diffuse radioactivity below the top of the running gel and in the stacking gel of lanes containing the cross-linked samples. Cross-linking into both types of complexes was enhanced 3-4-fold in cell layers treated with LPA and diminished in cell layers incubated with unlabeled 70-kDa fragment or H7 kinase inhibitor. In control studies, no cross-linking was seen if thrombin-activated factor XIII or Ca2+ was omitted from the 5-min cross-linking step (data not shown). Similar cross-linking profiles were observed with NIH 3T3 mouse fibroblasts (data not shown) and human foreskin fibroblasts (see below).After factor XIIIa cross-linking, approximately 60% of bound 70-kDa fragment was associated irreversibly with cell layers and not released back to the medium during a 6-h chase (Fig. 2A). Without cross-linking, most of the bound 70-kDa fragment re-appeared in the medium in the same period of time (Fig. 2A). To characterize the cross-linked complex, solubility in Triton and urea was examined. Cross-linked 70-kDa fragment at the tops of the stacking gel or the separating gel could not be extracted with either 1% Triton X-100 or 2 M urea. The combination of 1% Triton X-100 and 2 M urea extracted the bands almost completely (Fig. 2B).Fig. 2Association of 125I-70-kDa fragment of fibronectin with osteosarcoma cells after cross-linking. Cells were incubated for 60 min with 125I-70-kDa fragment and 200 nM LPA, washed, and cross-linked with factor XIIIa, 10 μg/ml, for 5 min. A, cell layers with (XL) or without (No XL) factor XIIIa cross-linking were incubated for 6 h with Tyrode's buffer containing 0.2% fatty acid-free bovine albumin. At the designated time points, small portions of medium were sampled to quantify dissociation of specifically bound 70-kDa fragment. Each point represents the average of duplicate values that varied <5%. B, the factor XIIIa cross-linked cell layers were extracted with 1% Triton X-100 or 2 M urea or 1% Triton X-100 plus 2 M urea for 10 min. The remaining pellets were extracted with 2% SDS. The fractions were analyzed by SDS-PAGE and autoradiography. Int, interface of the 8% running and 3% stacking gel; Top, top of the stacking gel.View Large Image Figure ViewerDownload Hi-res image Download (PPT)When increasing amounts of unlabeled 70-kDa fragment were incubated with LPA-treated cell layers, the dose responses for inhibition of binding of 125I-70-kDa fragment to cell layers and for inhibition of formation of cross-linked complexes at the tops of the stacking and separating gels were identical (Fig. 3). At the highest concentration of unlabeled 70-kDa fragment, binding and cross-linking were 18-20% of control. Cross-linking was also inhibited by monodansylcadaverine, which competes for protein-protein cross-linking and incorporates into factor XIIIa-reactive glutamines (Fig. 4). Quantification with a PhosphorImager indicated that the radioactivity missing from the top could be accounted for by radioactivity at the position of the monomer. Cross-linking to form the labeled band at the top of the separating gel, however, was not inhibited at the same rate as cross-linking to form the labeled band at the top of the stacking gel.Fig. 3Dose responses of inhibition of binding and factor XIIIa-mediated cross-linking of 125I-70-kDa fragment by unlabeled fragment. Foreskin fibroblasts were incubated 60 min with 125I-70-kDa fragment and LPA followed by a wash and 5-min incubation with factor XIIIa. Cell layers were extracted with 2% SDS sample buffer and analyzed by SDS-PAGE and autoradiography. Variable amounts of unlabeled 70-kDa fragment were present in the 60-min incubation. Binding of labeled fragment (70-kDa) and amounts of cross-linked fragment at the top of the stacker (Top) and near the top of the separating gel (Int) were quantified by phosphorimaging and expressed as % of values determined when no unlabeled fragment was present. Points represent the mean ratios ± S.D. (n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Competition for cross-linking of 125I-70-kDa fragment by monovalent amine. Osteosarcoma cells were incubated with 125I-70-kDa fragment and LPA for 60 min followed by a wash and 5-min incubation with factor XIIIa except that variable amounts of dansylcadaverine were present during the 5-min incubation with factor XIIIa. Actual counts from PhosphorImager scans are plotted versus dansylcadaverine concentration. Radioactivity lost from the top of the stacking gel (Top) and from near the top of the separating gel (Int) was found at the position of the monomer (70-kDa). The reaction schemes show how incorporation of dansylcadaverine (CadD) into a reactive glutamine (Q) of protein P1 competes for ε(γ-glutamyl)lysine (EK) cross-links between proteins P1 and P2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In order to study the influence of preformed fibronectin-containing extracellular matrix, osteosarcoma cells and foreskin fibroblasts were studied shortly after plating on vitronectin-coated plates or at confluence without or with LPA stimulation. Immunofluorescence studies demonstrated scant organized fibronectin in freshly seeded cells (see Fig. 8B) and copious fibronectin matrices in confluent cultures (data not shown). Under all conditions, >60% of 125I-labeled 70-kDa fragment or fibronectin remained insoluble in 1% Triton X-100 when cell layers were extracted after cross-linking; the Triton-insoluble fractions are shown in Fig. 5. Treatment with LPA resulted in increased cross-linking of the 70-kDa fragment to both cell types under both culture conditions (Fig. 5A). Similarly, 125I-fibronectin was cross-linked to bands at the top of the stacker and at the top of the separating gel in all four types of samples (Fig. 5B). The major difference in cross-linking patterns was a band somewhat larger than monomeric fibronectin (300 kDa) that was seen when the 70-kDa fragment was cross-linked. This band was most prominent in extracts of foreskin fibroblasts after 3 days in culture. Occasionally, several bands with sizes less than 200 kDa were observed, but this was not a reproducible finding.Fig. 8Localization of bound 70-kDa fragment and pre-existing fibronectin on cell surface. MG63 cells were cultured for 4 h on a vitronectin-coated coverslip and then incubated for 1 h with FITC-70-kDa fragment, 13 μg/ml, and 200 nM LPA in Tyrode's solution containing 0.2% albumin. The coverslip was washed, and cells were fixed with 3% paraformaldehyde. Fibronectin was stained with IST-2 followed by rhodamine-labeled goat anti-mouse IgG. A, FITC-70-kDa fragment; B, IST-2; C, phase. The open arrow points to a linear array of 70-kDa fragment on the lateral cell surface where there is little IST-2 epitope. The arrowhead points to a linear array of 70-kDa fragment where four cells come together. In controls in which the 40-kDa gelatin-binding domain of fibronectin was substituted for the 70-kDa fragment, no specific fluorescence was seen.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Cross-linking of 125I-labeled 70-kDa fragment (A) or fibronectin (B) to osteosarcoma cells (MG63) or human foreskin fibroblasts (HFSF). Cell layers were studied 4 h after dense seeding in vitronectin-coated wells in the absence of serum or 3 days after sparse seeding and culture in fetal bovine serum until confluent. Layers were incubated for 60 min with labeled protein without (−) or with (+) 200 nM LPA, washed, and cross-linked with factor XIIIa, 10 μg/ml, for 5 min. Cell layers were first extracted by 1% Triton X-100, and the Triton-insoluble materials were collected with 2% SDS sample buffer and analyzed. Shown are phosphorimages of dried mini-gels, with a 3% stacker, and 10% running gel.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The fact that the 70-kDa fragment did not form a dimer > trimer > tetramer … ladder upon cross-linking indicates that the formation of large complexes is not due to the 70-kDa fragment cross-linking to itself. Cellular targets of the cross-linking, however, potentially could be cross-linked to one another or to other molecules as well as to the labeled ligand during the exposure to factor XIIIa. Such cross-linking could account for the extraordinarily large apparent size of the complexes. Therefore, we compared results obtained with factor XIIIa with those obtained with SASD, a heterobifunctional, iodinatable, cleavable, photoreactive cross-linking agent that has been proven useful in a variety of situations, such as identification of the receptor for interleukin-3 (35Sorensen P. Farber N.M. Krystal G. J. Biol. Chem. 1986; 261: 9094-9097Abstract Full Text PDF PubMed Google Scholar). The advantages of SASD are that the only cross-linking events should be between the ligand and the molecules that bind the ligand and that the label is in part transferred (trans-labeling) to the binding molecules after reduction.125I-SASD·;70-kDa fragment was bound to confluent fibroblasts, and cross-linking was initiated by photolysis. With increasing time of irradiation, photoaffinity labeling was initially to the band at the top of the separating gel and, later, at the top of stacking gel when products were analyzed under nonreducing condition (Fig. 6). In control experiments, no high molecular weight complexes were generated if fibronectin or 70-kDa fragment labeled with 125I by the chloramine-T method was treated by photolysis in solution or bound to cell surface (data not shown). Cross-linked 125I-SASD-ligand was not solubilized with 1% Triton (data not shown). Upon reduction, part of the radioactive signal was transferred to the molecules that migrated near the top of the separating gel, with an apparent size similar to that obtained with factor XIIIa-mediated cross-linking of 70-kDa fragment. The major difference between factor XIIIa- and SASD-mediated cross-linking was the persistence of label at the top of the stacking gel in the factor XIIIa cross-linked samples after reduction. In order to learn whether this was an artifact of factor XIIIa cross-linking of the cellular targets to each other or other large molecules, the cell surface molecules were first exposed to factor XIIIa and then probed with 125I-SASD·;70-kDa fragment (Fig. 7). Upon reduction, the radioactive signal present at the top of the stacker became diminished, and increased label was detected at the top of the separating gel and at the position of the 70-kDa fragment, just as in the sample generated without the pretreatment with factor XIIIa. This observation suggests that the redistribution of the label upon reduction of the samples probably represents the dissociation of multiple 70-kDa fragments from the targets. The cell surface molecules to which 125I-70-kDa fragment cross-linked after specific binding to assembly sites, therefore, have an extraordinary Large Apparent Molecular Mass, which, in the absence of further characterization, we shall refer to as “LAMMs.”Fig. 6Cross-linking with a bifunctional photoactivable reagent, 125I-SASD·;70-kDa. Confluent human foreskin fibroblasts were incubated for 1 h with 125I-SASD·;70-kDa fragment and 200 nM LPA in Tyrode's buffer containing 0.2% albumin. After washing, cells were photolyzed for increasing amounts of time (0-10 min) and then extracted with SDS sample buffer. Cell lysates were analyzed on a 3.3/8% SDS-PAGE with or without reduction followed by scanning of radioactive bands. The 70-kDa fragment in some lanes ran off the gel. Phosphorimaging quantitation of the 10-min photolysis showed that the radioactive signal at the top of the gel decreased from 35,000 to 11,000, while signal from the interface band increased from 9,000 to 22,000 upon reduction.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 7Photoaffinity labeling of factor XIIIa cross-linked cell surface molecules with 125I-SASD·;70-kDa fragment. Freshly seeded" @default.
• W2000201832 created "2016-06-24" @default.
• W2000201832 creator A5037893640 @default.
• W2000201832 creator A5039135526 @default.
• W2000201832 date "1996-12-01" @default.
• W2000201832 modified "2023-09-27" @default.
• W2000201832 title "Cross-linking of the NH2-terminal Region of Fibronectin to Molecules of Large Apparent Molecular Mass:" @default.
• W2000201832 cites W1483633222 @default.
• W2000201832 cites W1494049520 @default.
• W2000201832 cites W1504442241 @default.
• W2000201832 cites W1506082716 @default.
• W2000201832 cites W1510915684 @default.
• W2000201832 cites W1511824111 @default.
• W2000201832 cites W1515455066 @default.
• W2000201832 cites W1523064851 @default.
• W2000201832 cites W1529402516 @default.
• W2000201832 cites W1539168089 @default.
• W2000201832 cites W1540200437 @default.
• W2000201832 cites W1549463507 @default.
• W2000201832 cites W1568784633 @default.
• W2000201832 cites W1586370846 @default.
• W2000201832 cites W1587369907 @default.
• W2000201832 cites W1588599526 @default.
• W2000201832 cites W1597229078 @default.
• W2000201832 cites W1601295859 @default.
• W2000201832 cites W1605270049 @default.
• W2000201832 cites W1775749144 @default.
• W2000201832 cites W1965576964 @default.
• W2000201832 cites W1972270878 @default.
• W2000201832 cites W1977378217 @default.
• W2000201832 cites W1978300767 @default.
• W2000201832 cites W1981254630 @default.
• W2000201832 cites W1986783451 @default.
• W2000201832 cites W1989792558 @default.
• W2000201832 cites W2008615479 @default.
• W2000201832 cites W2013079236 @default.
• W2000201832 cites W2018620812 @default.
• W2000201832 cites W2020645507 @default.
• W2000201832 cites W2020683610 @default.
• W2000201832 cites W2023692836 @default.
• W2000201832 cites W2023989365 @default.
• W2000201832 cites W2035873802 @default.
• W2000201832 cites W2035978536 @default.
• W2000201832 cites W2041984131 @default.
• W2000201832 cites W2044499728 @default.
• W2000201832 cites W2045684496 @default.
• W2000201832 cites W2048442712 @default.
• W2000201832 cites W2052371064 @default.
• W2000201832 cites W2057516353 @default.
• W2000201832 cites W2059082715 @default.
• W2000201832 cites W2066665315 @default.
• W2000201832 cites W2067910961 @default.
• W2000201832 cites W2075151357 @default.
• W2000201832 cites W2083216138 @default.
• W2000201832 cites W2085804760 @default.
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