Jun 11, 1984 - Pearlstein, E. (1978) Znt. J. Cancer 22.32-35. Johansson, S., and HGiik, M. (1984) J. Cell BWl. 98,810-817. Vuento, M., and Vaheri, A. (1979) ...
Vol. 260, No. 3, Issue of February 10, pp. 1557-1561,1985 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Societyof Biological Chemists,Inc.
Demonstration of HighAffinity Fibronectin Receptors on Rat Hepatocytes in Suspension* (Received for publication, June 11,1984)
Staffan Johansson From the Demrtment of Medical and Physiological Chemistry, University of Uppsala, The Biomedical Center, S-75123 Uppsala, Sweden
A cell-binding peptide(M. = 86,000)which lacks the cellular matrices or by coating onto plastic surfaces (11). gelatin- andheparin-bindingdomains, was purified Recently, support for the latter theory was presented (12). from trypsin-digested fibronectin. Preincubation ofThe ratability of soluble fibronectin to compete with immobilized hepatocytes in suspension with the peptide, inhibited fibronectin for binding to cellular receptors and thereby ininitial attachment of the cells to immobilized fibronec- hibit initial attachment of rat hepatocytes to fibronectintin while attachment to immobilized laminin andcol- coated plastic dishes, was markedly increased by a limited lagen was unaffected.'2"I-iabeled 86-kDa peptide trypsin digestion of the soluble fibronectin. The interaction boundtothe cells insuspension at 4 "C inatimeof soluble fibronectin with hepatocytes in suspension could dependent,saturable,andpartiallyreversiblereacalso be stimulated by addition of collagen or heparan sulfate, tion. Scatchard analysis of the binding data indicated as measured as increased inhibition of initial attachment to a single class of receptors with a K d of 1.7 X lo-* M. The number of binding-sites was -2.8 X lO"/cell. Un- fibronectin dishes. These results suggested that tryptic fibronectin fragments labeled 86-kDa peptide inhibited the binding of laaIlabeled 86-kDa peptide 30-fold more effectively than could be useful in studies of fibronectin receptors. In this intact fibronectin. These results provide direct evi- investigation an 85-kDa fibronectin fragment, which lacks dence for the presence of a domain in the fibronectinaffinity for collagen and heparin, has been used to demonmolecule which has or may acquire a high affinity for strate the presence of a specific fibronectin receptor on rat receptors involved in adhesion of hepatocytes to im- hepatocytes. The binding of the fibronectin fragment to the cells in suspension has been characterized and compared with mobilized fibronectin. that of intact fibronectin. The glycoprotein fibronectin is a mediator of cell-matrix interactions (1).It has affinity for matrix components such as collagens (2), glycosaminoglycans (31, and fibrin (4). These interactions have been studied in some detail and domains in the fibronectin molecule, responsible for the interactions, have been identified and isolated after proteolytic fragmentation of fibronectin. A large number of different cell types, including fibroblasts (5), hepatocytes (6), and platelets (7) have been shown to adhere to and spread out on substrates of immobilized fibronectin. A cell-binding tetrapeptide in the fibronectin molecule has been identified by use of a monoclonal antibody which inhibits cell attachment to fibronectin substrates (8,9).However, the cellular receptor for fibronectin has not been identified. A problem in studiesof fibronectin receptors is thatsoluble fibronectin binds very poorly to cells, making classical receptor-ligandstudies difficult to perform. Furthermore, since fibronectin has binding sites for a number of different components it may be difficult to interpret binding data, particularly if binding to collagen- and proteoglycan-containing cell layers is studied. The explanations suggested for the lack of binding of soluble fibronectin to cells have been either (a) that cooperative interactions between immobilized fibronectin molecules and many cell surface receptors are required for stable binding (10) or (b) that fibronectin becomes activated by conformational changes during incorporation into extra* This work was supported by Swedish Medical Research Council Grant 06525. The costa 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 18U.S.C. Section 1734 solely to indicate this fact.
MATERIALS ANDMETHODS
Trypsin (TPCW-treated, type XIII), bovine serum albumin, phenylmethylsulfonyl fluoride, EDTA, dithiothreitok Bis-Tris, and guanidinium chloride were purchased from Sigma. DEAE-Sephacel, heparin-Sepharose, gelatin-Sepharose, and Percoll were obtained from Pharmacia, Uppsala, Sweden. TSK 3000 columns were from LKB, Bromma, Sweden. Na'=I (carrier-free) was purchased from the Radiochemical Centre, Amersham, England and Iodo-Beads from Pierce Chemical Co. Fibronectin was purified from human plasma according to the method of Vuento and Vaheri (13). Neutral salt-soluble collagen from rat skin (type I and 111) and laminin were kind gifts from Dr. K. Rubin, University of Uppsala, Sweden, and Dr. R. Timpl, Max Planck Institut, West Germany, respectively. Purifiqatwn of the Cell-binding 85-kDa Fibronectin FragmentFibronectin (3 mg/ml) in 10 mM Tris-HC1 buffer, pH 7.5, containing 0.14 M NaCl and 0.02% NaNs was digested with trypsin (7Fg/ml) for 90 min at 37 "C. The digestion was terminated by addition of phenylmethylsulfonyl fluoride to a final concentration of 0.4 mM. The digest was dialyzed against 50 mM Tris-HC1buffer, pH 7.0, containing 0.1 M NaCl, 10 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, and 0.02% NaN3, and sequentially applied to columns of heparinSepharose (1 mlof gel/lO mgof fibronectin digest) and gelatinSepharose (1ml of ge1/2 mg of fibronectin digest) in the same buffer. During a second passage of the unbound fraction through the columns no further material was retained, demonstrating that no heparin- or gelatin-binding peptides remained in this fraction. The unbound material was dialyzed against 10 mM Bis-Tris buffer, pH 6.0, containing 50 mM NaCl and 0.02% NaN3, and applied to a DEAE-Sephacel column (50 mgof protein/lO mlof gel). The DEAE column was eluted with a linear gradient (100 ml) of NaCl from 50 to 300 mM in this buffer (Fig. 1). The fractions which contained peptides that The abbreviations used are: TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; Bis-Tris, bis(2-hydroxyethyl)iminotris(hydroxymethy1)methane; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; SDS, sodium dodecyl sulfate.
1557
Increased Cell Binding of Fibronectin Fragmentation after
1558
A
B
FN-
top of gel 0 OD c\1
a 94
-
\
67-
20
40 60 Fraction number FIG. 1. DEAE ion-exchange chromatography of tryptic fibronectin peptides. A trypsin digestof fibronectin, which had been passed throughgelatin-andheparin-Sepharose two times,asdescribed under“MaterialsandMethods,” wasapplied to a 10-ml column of DEAE-Sephacel in 10 mM Bis-Tris buffer, p H 6.0, containing 50 mM NaCI and 0.02% NaN3. The arrow indicates thestart of washing of the column after the sample had been applied, and the dotted line depicts the salt gradient. Fractions of 2.5 mlwere collected and analyzed for absorbance at 280 nm (0)and ability to promote attachmentandspreading of rathepatocytes(not shown inthe figure), as described under “Materials and Methods.” Fractions were pooled as indicated (hatched area).
0 Q)
N
U
30
20 E f f l u e n t volume (ml)
FIG. 2. Gel chromatography of partially purified cell-binding fragment. The pooled fihronectinpeptides from Fig. 1 were concentrated and dialyzed as described under “Materials and Methods.” Samples of 1 mg were applied to a TSK 3000 column, eluted at a flow rate of 0.2 ml/min with 20 mM Tris-HCI buffer, pH 7.0, containing 4 M guanidinium chloride, and 2 mM dithothreitol. Absorbance a t 280 nm was measured and fractionsof 0.2 ml were analyzed for ability to promote attachment and spreading of rat hepatocytes (not shown in thefigure). Fractions were pooled as indicated (hatched area). induced attachment and spreading of rat hepatocytes (see below for assayconditions) were pooled andconcentrated to 20 mg/ml by vacuum dialysis. After dialysis against 4 M guanidinium chloride, 2 mM dithiothreitol, in 20 mM Tris-HCI buffer, pH 7.0, the peptides were chromatographed ona TSK 3000 column (volume, 40 ml; length, 100 cm) in the same buffer (Fig. 2). The first peak contained the adhesion promoting material which in SDS electrophoresismigrated as a single peptide with an apparent M, of 85,000 (Fig. 3). The 85kDa fraction was pooled and dialyzed back to phosphate-buffered saline (10 mM PO,, 0.14 M NaCI, pH 7.4) by stepwise reducing the concentration of guanidinium chloride from 4, 3, 2, 1, 0.5 to 0 M in the presence of 2 mM dithiothreitol. Lastly, dithiothreitol was dialyzed away. This treatment was necessary for the peptide to refold into a soluble, monomeric, trypsin resistant, and thus presumably native
43
-
30-
-
20
14 -
i4
v
-.-
FIG. 3. SDS-polyacrylamide gel electrophoresis of cell binding fibronectin fragment. A, intact fibronectin (lane I ) , M , standards (lane 2) and purified cell-binding fibronectin fragment (85 a 10-15% kDa)(lane 3) were reduced andalkylatedandrunon polyacrylamide gradient gel as described under “Materials and Methods.” After electrophoresis, the gel was stained with Coomassie Brilliant Blue. B, fibronectin (1 mg/ml in buffer 3) was digested with trypsin (2 pg/ml) a t 37 “C for various times, prepared for electrophoresis as in A and run ona 7-15% polyacrylamide gel; lane I , M, standards (the same as in A); lune 2, fibronectin digested for 40 min; lune 3, fibronectin digested for 90 min; lane 4, fihronectin digested for 180 min. form. The overall yield of the 85-kDa peptide was 11 mg from 100 mg of fibronectin, equivalent to 28% if one copy of 85-kDa peptide/ 220 kDa is assumed. Electrophoresis-Electrophoresis was performed on polyacrylamide gradient gels in SDS according to the method of Blobel and Dobberstein (14). Iodination of the 85-kDa Fragment-Labeling with [’251]iodinewas performed by the chloramine-T method (15) using Iodo-Beads. The 85-kDa fragment was labeled to a specific activity of lo’ cpm/pg. The integrity of the labeled peptide was ascertained by SDS electrophoresis followed by autoradiography. Preparation of Cell Attachment Substrates-Microtiter wells (Nunc, Denmark) were coated with 50 pl of fibronectin, fibronectin peptides, laminin (50 pg/ml), or collagen (100 pg/ml) in phosphatebuffered saline by adsorption a t 37 “C for 2 h. The protein solution was removed and the wells were incubated with 50 pl of phosphatebuffered saline containing 1.5% bovine serum albumin for 30 min a t 22 “C toblock remaining protein binding sites on the plastic surface. This solution was removed before seeding of the cells. CellAttachment Assay-Hepatocytes were isolated from male Sprague-Dawley rats after perfusion of the liver in situ with collagenase as described (16). Cells (5 X IO‘) in 50pl of buffer 3 (see Ref 16; 0.137 M NaCI, 4.7 mM KCI, 0.65 mM MgSO, X 7 H20, 1.2 mM CaCI2 X 2 H20, 10 mM HEPES) containing 1.5% bovine serum albumin, were seeded in each well and incubated a t 37 “C in humidified air. After incubations for indicated times the microtiter plates were washed and the number of cells attached determined as described (17). In short, the cells were lysed in a solution of Triton X-100, and the activity in the lysateof the enzyme hexosaminidasewas determined. Cell spreading was studied in the presence of cycloheximide (25 pg/ml) as described (18). Assay for Binding of lZI-lubeled 85-kDa Peptide to HepatocytesCells (5 X 106/ml) in buffer 3 containing 1.5% bovine serum albumin were incubated end over end with ’*’I-labeled 85-kDa peptide (IO6
Increased Cell Binding of Fibronectin after Fragmentation
I
I
20 85K fragmentconc.(ug/ml>
1559
60 Time ( m i d
I 00 Protein conc. (ug/ml)
FIG. 4 (left). Inhibition of cell attachment by the 85-kDa fragment. Rat hepatocytes (1 X 106/ml) were preincubated at 4 "C for 20 min in the presence of the indicated amounts of purified 85-kDa fragment, and then After incubation seeded into fibronectin-coated wells under conditions described under "Materials and Methods." for 6 min at 37 "C the wells were washed and the number of cells attached determined. Per cent inhibitionwas calculated as: 100-100 X (number of cells attachedin the presence of 85-kDa fragment/numberof cells attached in the absence of 85-kDa fragment). The values shown represent averages of two parallel incubations. FIG. 5 (center). Time course of binding of "'1-labeled 85-kDa fragment to rat hepatocytes in suspension. Cells were incubated with '251-labeled 85-kDa fragment at 4 "C and specific binding of radioactivity determined (0).In a parallel incubation,unlabeled 85-kDa at the indicated timesas described under "Materials and Methods" fragment was added to give a concentration of 10 pg/ml at the time point indicatedby the arrow (0). FIG. 6 (right). Inhibition of binding of 1a61-labeled85-kDa fragment to hepatocyt- by fibronectin and the 85-kDa fragment. Cells were incubated with'9-labeled 85-kDa fragment at 4 "C for 2 h in the presence of indicatedamounts of fibronectin (0)and 85-kDafragment (0),respectively, and the bound radioactivity determined as described under "Materialsand Methods." cpm/ml; lo7 cpm/pg unless otherwise indicated)at 4 "C for indicated times. The incubations were terminated by removing samples of 100 pl which were centrifuged at 1500 X g through 10 ml of 20% Percoll in buffer 3. The supernatant was aspirated off until 300 pl remained over the pellet. After freezing at -20 "C, the bottom tip of the tubes were cut off and counted in a Packard model 5260 Auto-Gamma Scintillation spectrometer. Unspecific binding (-0.9% of added radioactivity) was determined in the presence of 50 pg unlabeled 85kDa fragment/mL The values shown in the figures represent averages of duplicate determinations aftersubtraction of unspecific binding.
The effect of the 85-kDa peptide in solution on cell attachment was specific in that only attachment to immobilized fibronectin was inhibited while attachment of hepatocytes to immobilized laminin or collagenwas unaffected (Table I). The 85-kDa peptide immobilized on plastic dishes was as effective as intact fibronectin in promoting both attachment and spreading of hepatocytes (data not shown). This is in accordance with the results of Hayashi et al. (22) who studied the ability of fibroblasts to attach and spread out on dishes coated with the partly purified 85-kDa peptide. RESULTS '261-labeled85-kDa fragment incubated with hepatocytes in Trypsin-digested fibronectin added to themedium has pre- suspension at 4 "C bound to the cells in a time-dependent viously been shown to inhibit initial cell attachment to fibro- reaction which was completed after 1-2 h (Fig. 5). Previously nectin-coated dishes more efficiently than intact fibronectin bound lZ5I-labeled85-kDa fragment was partially displaced (12). Such a digest was fractionated as described under "Ma- (45%) by addition of excess unlabeled 85-kDa fragment (10 terials and Methods" to obtain a cell-binding peptide devoid pg/ml) (Fig. 5 ) . However, complete reversal of the binding of of gelatin- and heparin-binding domains. Cell-binding activity radioactivity could not be achieved even if the amount of was associated with an 85-kDa peptide (not shown) which unlabeled 85-kDa fragment was further increased (data not was accumulating during the incubation of fibronectin with shown). The presence of unlabeled 85-kDa fragment or intact trypsin (Fig. 3, A and B ) . Since the 85-kDa peptide does not fibronectin from the start of the incubation, inhibited the bind to gelatin or heparin it must originate from the central binding of '251-labeled 85-kDa peptide in a dose-dependent region of the fibronectin arms (-70-160 kDa from the N- manner. On a molar basis -30-fold more fibronectin (counted terminal) (19-21). Tryptic fibronectin fragments apparently on number of monomers of M, = 220,000) than 85-kDa identical to this 85-kDa fragment have been described by fragment was required to achieve 50% inhibition of the bimlothers (22,23), buthave not been obtained free from contam- ing of '251-labeled85-kDa fragment to the hepatocytes (Fig. inating peptides. In the present study, denaturing conditions 6). When the cells were incubated with increasing amounts of '251-labeled85-kDa fragment a saturation curve of binding were used in order to purify the 85-kDa peptide? was obtained (Fig. 7A). Half-maximal binding occurred at 15Preincubation of hepatocytes with the purified 85-kDa frag20 nM (1.3-1.7 pg/ml) and saturation was achieved at 60-100 ment in suspension, inhibited initial attachment to dishes nM. A Scatchard plot of the binding data, indicated a single coated with intact fibronectin. Almost complete inhibition class of receptors. The apparent K d was 1.7 X lo-' M and the (95%) of attachment to fibronectin dishes was obtained at a average number of binding sites was 2.8 X 105/cell. concentration of 50 pg/ml of the 85-kDa fragment (Fig. 4). When unfractionated peptides of trypsin-digested fibronecThese data indicate that the 85-kDa fragment contains all tin was '251-labeledand incubated with the hepatocytes, the structures in fibronectin to which hepatocytes can attach. 85-kDa peptide was selectively enriched in the cell-associated fraction (Fig. 8). This suggests that the 85-kDa peptide has Under nondenaturing conditionspeptides of molecular mass the highest affinity for the fibronectin receptors among the 65,000 and 20,000 co-purify with the 85-kDa peptide. These peptides trypsin-generated fibronectin fragments. are slowly formed during trypsin digestion of purified 85-kDa peptide or fibronectin (S. Johansson, unpublished results). Preparations of DISCUSSION partly cleaved 85-kDa peptide purified under nondenaturing con&tions gave essentially identical results as shown in Figs. 4-7 for intact, Recently several reports have indicated that the shape of renatured 85-kDa peptide. fibronectin molecules may vary from globular to extended
Increased Cell Binding of Fibronectin after Fragmentation
1560
forms depending on ionic strength and pHof the solute (2427). Alsoimmobilized fibronectin attains different conformations, depending on the nature of the surface to which it is coated (24). Changes in absorption spectra induced by binding of collagen (27) or heparin (28) to fibronectin have previously been reported, and interpreted to reflect conformational changes in fibronectin. The increased binding of soluble fibronectin to hepatocytes in thepresence of collagen or heparan sulfate/heparin or after limited trypsin digestion of the fibronectin suggests that such conformational changes in fibronectin are functionally important (12). Fibrillogenesis of fibronectin provides another example, since a conformar tional change must precede the disulfide bridging between thiol groups, as these are not exposed in native soluble fibronectin (29, 30). In this study the increased affinity of fibronectin for cell surface receptors, induced by uactivation" of fibronectin, has been investigated by a more direct approach than previously. The major cell-binding peptide of trypsin-digested fibronectin was identified and purified. It had an apparent M , of 85,000 and did not contain binding sites for gelatin or heparin. In contrastto soluble '251-labeled fibronectin, which did not detectably bind to hepatocytes (12), the 85-kDa tryptic cellbinding fragment of fibronectin bound to the cells in suspension at 4 "C with high affinity (1.7 X lo-' M) inatimedependent, saturable,and specific manner. Furthermore, preincubation of the cells with the 85-kDa fragment in suspension inhibited the initial attachmentof the cells to dishes coated with fibronectin. This result strongly suggests that the binding sites recognizing the 85-kDa peptide are identical
with the receptors used by hepatocytes for adhesion to fibronectin matrices. In contrast, thecell-binding peptide isolated by Piersbacher et al. (31) did not detectably bind to cells in suspension. Neither did it affect cell attachment tofibronectin dishes at concentrations where the 85-kDa fragment is completely inhibitory (31). However, at millimolar concentrations of the tetra-peptide, cell attachment tofibronectin dishes was inhibited. From these data a K d of 5 X M for the interaction of the tetra-peptide and itsreceptor was calculated (9). The difference in affinity between the tetra-peptide and the 85-kDa fragment, respectively, for the fibronectin receptor (>104-fold) may indicate that the 85-kDa fragment contains additional structures which are required for optimal binding strength. The tetra-peptide is also present in collagen and a few other proteins (9). However, cell attachment to collagen (as well as to laminin) was not affected by the presence of the 85-kDa peptide in the medium indicating that also collagen contains additional cell-binding structures, which are recognized by specific collagen receptors. Intact fibronectin at high concentrations did compete with lZ5I-labeled85-kDa peptide for binding to the hepatocytes although 30-fold less effectively than the 85-kDa peptide. However, it is not clear whether this competition is due to a lower affinity of intact fibronectin for the receptor or to a small fraction of the fibronectin molecules being "activated" to thehigh-affinity form. A recent paper by McKeown-Longo and Mosher (32) can be interpreted in favor of the idea that activation of fibronectin is a cellular event that precedes the actual binding of fibronectin to itsreceptor. They could detect a binding of '%I-labeled fibronectin at 37 "C to cell layers of growing fibroblasts. A fraction of the bound fibronectin was found in a saturable, detergent-soluble pool with a calculated TABLE I Kd of 3.6 X lo-' M. Thus it quite is possible that thehepatocyte Specificity of inhibition of cell attachment by the 85-kDa fragment receptor for the 85-kDa fragment and the detergent-soluble Rat hepatocytes (1 X 10B/ml) were preincubated a t 4 "C for 20 min fibronectin receptor on the fibroblasts are homologous. In the in the absence and presence of purified 85-kDa fragment (50 pg/ml), and then seeded into wells coated with the indicated proteins as study of McKeown-Longo and Mosher (32), transformed cells described under "Materials and Methods." After incubation for 12 were shown to attach to fibronectin-coated dishes but failed min at 37 "C the wells were washed and thenumber of cells attached to bind soluble '251-labeledfibronectin. The transformed cells determined. The number of cells attached in the absence of 85-kDa apparently had functional fibronectin receptors but may have fragment to wells coated with fibronectin is set to 100%. The values been defective in the activation of fibronectin to a form which shown represent averages of two parallel incubations. can bind to its receptor. A possible candidate as cellular Number of cells attached to wells "activator" of fibronectin would bemembrane-bound heparan Amount 85-kDa fragment coated with sulfate proteoglycan (33, 34). A close co-distribution of cell added to the medium Fibronectin Laminin Collagen surface heparan sulfate proteoglycan, fibronectin, and actin has been demonstrated on fibroblasts early in their spreading dm1 76 process (35). Furthermore, exogenously added heparan sulfate 0 100 50.6 69.0 72.4 50 20.750.2 has been shown to stimulate the binding of fibronectin to
l
I
L
c
0
L
e
m
85K fragment conc. @g/ml)
u l
c
FIG. 7. Concentration dependence of binding of 1z61-labeled86-kDa fragment to hepatocytes in suspension. A, the cells were incubated with increasing amounts of '%I-labeled85-kDa fragment (specific activity 2 X 10' cpm/pg) and thebound radioactivity determined as described under "Materials and Methods." The ranges obtained from duplicate incubations are shown. Specific binding (0)was calculated by subtracting nonspecific binding (A) from total binding (0).Nonspecific binding was determined in thepresence of 100 pgof unlabeled 85kDa fragment/ml (the dotted line denotes extrapolated values). B, Scatchard plot of the data presented in A . The curve was fitted by the least square method (regression coefficient = 0.97).
Increased Cell Binding ofFibronectin after Fragmentation
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2. Engvall, E., Ruoslahti, E., and Miller, E. J. (1978)J. Exp. Med. 147,1584-1595 3. Yamada, K. M., Kennedy, D.W., Kimata, K., and Pratt, R.M. (1980)J. BWL Chem. 255,6055-6063 4. Sekiguchi, K., Fukuda, M., and Hakomori, S. (1981) J. BWL Chem. 256,6452-6462 5. Klebe, R.J. (1974)Nature (Lord.)250,248-251 6. Rubin, K., Johansson, S., HGiik, M., and bbrink, B. (1981)Exp. Cell Res. 135,127-135 7. Kotoliansky, V. E., Leytin, V. L., Svirdov, D. D., Repin, V. S., 94- ” and Smirnov, V. N. (1981)FEBS Lett. 123,59-62 8. Piersbacher, M. D., Hayman, E. G., and Ruoslahti, E. (1981)Cell 67 26,259-267 9. Piersbacher, M. D., and Ruoslahti, E. (1984)Nature ( L o r d ) 309, 30-33 43 10. Grinnell, F. (1980)J. Cell BWL 86,104-112 11. Pearlstein, E. (1978)Znt. J. Cancer 22.32-35 12. Johansson, S.,and HGiik, M. (1984)J. Cell BWl. 98,810-817 30 13. Vuento, M., and Vaheri, A. (1979)Biochem. J. 183,331-337 14. Blobel, G., and Dobberstein, B. (1975)J. Cell Biol. 67,835-851 20 15. Hunter, W. M. (1973)in Handbook of Experimental Immunology (Weis, D.M., ed) pp. 17. 1-17.36, Blackwell Scientific Publi14 cations, London FIG. 8. SDS-polyacrylamide gel electrophoresis of “%la16. Rubin, K., Kjellh, L , and Obrink, B. (1977)Exp. Cell Res. 109, beled fibronectin fragments. Fibronectin was digested with tryp413-422 sin for 180 min as described in the legend to Fig. 3,and labeled with 17. Landegren, U. (1984)J. ZmrnunoL Methods 67,379-388 lZ5I as described under “Materialsand Methods” ( l a n e I). The labeled 18. Johansson, S.,Kjellin, L., Hiiiik, M., and Timpl, R. (1981)J. CeU peptide mixture was incubated with the hepatocytes for 3 h a t 4 “C BWL 90,260-264 and cell bound material separated from unbound material as described 19. Ruoslahti, E., Hayman, E. G., Engvall, E., Cothran, W. C., and under “Materials and Methods.” The cell bound radioactivity was Butler, W. T. (1981)J. BWL Chem. 256,7277-7281 solubilized for 15 min at 22 “C in 10 mM Tris-HC1 buffer, pH 8.0, 20. Ehrismann, R., Roth, D. E., Eppenberger, H. E., and Turner, D. containing 0.5% Triton X-100,1 mM phenyimethylsulfonyl fluoride, C. (1982)J. BWl. Chem. 257, 7381-7387 2 m M EDTA, 1 mM N-ethylmaleimide, and pepstatin (10 rg/ml) ( l a n e 21. Vartio, T., Barlati, S., De Petri, G., Miggiano, V., Strihli, C., 2).The samples were prepared for electrophoresis as described in the Tacacs, B., and Vaheri, A. (1983)Eur. J. Biochern 135, 203legend to Fig. 3 and run on a 7-15% polyacrylamide gel. After drying, 207 the gel was subjected to autoradiography using Kodak X-Omat x-ray 22. Hayashi, M., and Yamada, K.M. (1982) J. BWL Chem. 258, film. The migration distance of molecular mass standards (kDa) are 3332-3340 indicated. 23. Smith, D. E., Mosher, D. F.; Johnson, R. B., and Furcht, L. T. (1982)J. BWl. Chem. 257,5831-5838 hepatocytes (12). In agreement with this hypothesis, tumor 24. Tooney, N.M., Mosesson, M.W., Amrani, D. C.,Hainfeldt, J. cells generally synthesize heparan sulfate with a low sulfate F., and Wall, J. S. (1983)J. CellBWL 97,1686-1692 content (36-38) and reduced affinity for fibronectin (37). Cell 25. Erickson, H. P., and Carrell, N.A. (1983)J. BWl. Chem. 258, 14539-14544 attachment andspreading to a performed “matrix” would not be affected by reduced sulfation of heparan sulfate since a 26. Rocco, M., Carson, M., Hantgan, R., McDonagh, J., and Hermans, J. (1983)J. BWL Chem. 258, 14545-14549 fibronectin-heparan sulfate interaction apparently is not required for these events. This was demonstrated in this study 27. Williams, E. C., Janmey, P. A., Ferry, J. D., and Mosher, D. F. (1982)J. BioL Chem. 257,14973-14978 to be the casefor hepatocytes whichin the presence of cycloheximide spread out on dishes coated with the 85-kDa 28. Frangou, S. A., Moms, E. R, Rees, D. A., Welsh, E. J., and Chavin, S. I. (1983)Biopolymers 22,821-831 peptide. 29. Williams, E. C., Janmey, D. A., Johnson, R. B., and Mosher, D. In conclusion, the results presented here support the conF. (1982)J. Biol. Chem. 258,5911-5914 cept that activation of fibronectin by conformational changes 30. McConnell, M.R., Blumberg, P. M., Roesow, P. W. (1978)J. is important for cell binding. The purified cell-binding fragBWl. Chem 253,7522-7530 ment of trypsin-digested fibronectin should be useful instud- 31. Piersbacher, M.D.. Hayman, E.G., and Ruoslahti, E. (1983) ies of fibronectin receptor function on different cell types and Proc. Natl. Acad. Sci. U.S. A. 80,1224-1227 possibly in purification of fibronectin receptors. The advan- 32. McKeown-Longo, P., and Mosher, D. F. (1983)J. Celt BWL 97, 466-472 tages of the 85-kDa fragment over intact fibronectin is (a) increased affinity for the receptor and ( b ) lack of affinity for 33. KjellBn, L.,Pettersson, I., and HMk, M. (1981)Proc. NatL A d . Sci. U.S.A. 78,5371-5375 gelatin and sulfated glycosaminoglycans, which makes the interaction with cell membrane components more specificand 34. Rapraeger, A.C., and Bernfield, M. (1983)J. BioL Chern. 258, 3632-3636 interpretable. 35. Woods, A., Hiiiik, M., Kjellin, L., Smith, C. G., and Rees, D. A. (1984)J. Cell BioL 99,1743-1753 Acknowledgments-I would like to thank Ake Engstriim for constructive help with the purification of fibronectin peptides and Lena 36. Winterbourne, D. J., and Mora, P. T. (1981)J. Biol. Chem. 256, 4310-4320 KjellBn for valuable discussions. 37. Robinson, J., Viti, M., and Hook, M. (1984)J. CeU BioL 98,946REFERENCES 953 1. Hynes, R. O., and Yamada, K. M. (1982)J. Cell Biol. 95, 369- 38. Hook, M.,KjellBn, L., Johansson, S., and Robinson, J. (1984) 377 Annu. Rev. Biochem. 53,847-869 top o f gel -
