The Journal of Biological Chemistry - The …tain binding sites for specific macromolecules...

THE JOURNAL OF BIOLOGICAL CHEMISTRY

The Interaction of Plasma Suspension*

Vol. 260, No. 7, Issue of April 10, pp. 4492-4500, 1985 Printed in U.S.A.

Fibronectin with Fibroblastic Cells in

(Received for publication, March 20, 1984)

Steven K. AkiyamaS and Kenneth M. Yamada From the Membrane Biochemistrv Section, Laboratow of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 202&

We have examined the interaction of soluble plasma [‘Hlfibronectin with fibroblastic cells in suspension. Fibronectin labeled by reductive methylation binds to baby hamster kidney cells in serum-free medium in a time-dependent manner at 4,22, and 37 “C, with half- maximal binding occurring in 12-15 min at 22 “C. The binding is saturable and reversible. At least 90% of the cell-associated fibronectin is external to the plasma membrane, as judged by trypsin susceptibility of the bound radioactivity. Scatchard analysis of the concentration dependence of binding indicates the presence of a single class of binding sites, even at low input concentrations of fibronectin. There are approximately 5 f 1 X lo6 sites/cell with an apparent dissociation constant of 8.0 f 0.5 X 10” M; thus, the binding of soluble fibronectin to these cells is of moderate affinity. This putative fibroblast fibronectin receptor is resistant to trypsin in the presence of physiological concentrations of divalent cations but is susceptible to trypsin in the presence of 5 mM EDTA. Binding of 0.1 mg/ml [‘Hlfibronectin is 60440% inhibited by 8 mg/ml unlabeled fibronectin and 95% inhibited by 1 mg/ml purified 75-kDa fibronectin cell-binding domain, but is unaffected by 1 mg/ml44-kDa collagen-binding domain or 5 mg/ml ovalbumin. The binding parameters determined in this study further define the fibroblast cell-surface fibronectin receptor.

Fibronectins are adhesive glycoproteins found in blood, on cell surfaces, and in extracellular matrices (reviewed in Refs. 1-10). Fibronectins have been found in all vertebrates and in most invertebrates examined by radioimmunoassay (11). The fibroblast cellular and plasma forms of fibronectin are very similar but are not identical (12). Plasma fibronectin is di- vided into a series of protease-resistant domains which con- tain binding sites for specific macromolecules including fibrin, gelatin, heparin, and actin. These functional domains have been isolated and purified (13-21). The interactions of fibronectin with biological macromolecules are, thus, relatively well-characterized.

A crucial but poorly understood aspect of fibronectin is its interaction with the cell surface. For example, substrate- adsorbed fibronectin mediates fibroblast cell attachment and spreading on plastic (22), collagen (23-25), and fibrin (26)

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adoertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Partially supported by Public Health Service Fellowship CA 06782 awarded by the National Cancer Institute, National Institutes of Health, Department of Health and Human Services.

substrata. Fibronectin promotes fibroblast cell-cell adhesion (27) and cell motility (28). When exogenous fibronectin is added to transformed cells in culture, they can regain a more normal, flattened phenotype with less overlapping and bleb- bing (29, 30). Fibronectin also appears to promote the phag- ocytic function of macrophages (31-34).

Fibronectin, therefore, appears to interact directly with a wide variety of cells, but especially with fibroblasts. Quanti- tation of the fibroblast-fibronectin interaction is most complete for plasma fibronectin that has been adsorbed onto polystyrene. In a biological assay, fibronectin-mediated cell spreading on plastic is concentration-dependent and exhibits a threshold effect with half-maximal spreading occurring if the initial solution concentration is approximately 1.3 pg/ml. Maximal spreading occurs if the initial fibronectin solutions are above approximately 8-10 pglml, representing about 45,000 polystyrene-adsorbed fibronectin molecules under each cell (35).

Polystyrene beads containing adsorbed fibronectin can bind to fibroblastic cells in suspension (36), probably as the first step toward phagocytosis of the beads (37). This binding is specific for fibronectin, is time- and temperature-dependent, and appears to occur uniformly over the entire surface of cells in suspension (36). Adsorption of fibronectin to a substrate, however, presumably results in a substantial increase in its local concentration in a thin layer adjacent to, and on, the substrate. The analysis of the interaction of fibronectin with cultured fibroblasts would be considerably simplified if it were possible to measure directly its binding to the cells.

While the binding of fibronectin to activated platelets has been examined (38), the direct interaction of soluble fibronectin with fibroblasts in suspension has been extremely difficult to characterize. In one study, the binding of fibronectin to fibroblasts in suspension was found to occur at 4 “C but not at 22 or 37 “C (39). However, the binding assay was performed using a simple salt solution and binding was extremely slow.

In this report, we demonstrate that tritiated plasma fibronectin interacts directly with the fibroblast cell surface under physiological conditions and with a time course consistent with biological processes. The binding of fibronectin to BHK’ fibroblasts is saturable, restricted to the cell surface, and occurs at 4-37 “C. These provide a direct characterization of the fibroblast receptor for fibronectin.

EXPERIMENTAL PROCEDURES

pared from fresh-frozen human plasma (National Institutes of Health Preparation of Fibronectins a d Fragments-Fibronectin was pre-

The abbreviations used are: BHK, baby hamster kidney; DMEM/ HEPES, Dulbecco’s modified Eagle’s medium supplemented with 25 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid; TPCK- trypsin, trypsin treated with L-1-tosylamido-2-phenylethyl chloro- methyl ketone; HBSS, Hank‘s balanced salt solution without calcium and magnesium.

4492

Fibronectin Binding to Fibroblastic Cells 4493

Blood Bank) using a combination of published procedures at 23 "C

with 87.5 ml of 0.2 M EDTA (BDH), 17.5 ml of 0.2 M phenylmeth- (40,41). Freshly thawed human plasma (3.5 liters) was supplemented

anesulfonyl fluoride (Calbiochem-Behring) freshly prepared in 95% ethanol and 22.9 g of c-aminocaproic acid (Calbiochem-Behring), and then centrifuged at 9000 X g for 10 min to remove any aggregated material. The supernatant plasma was applied to a 700-ml precolumn of Sepharose CL-4B which had been prewashed with 0.05 M Tris- HCl, 0.05 M e-aminocaproic acid, 0.02 M sodium citrate, 0.02% sodium azide, pH 7.5 (buffer C). The flow-through fraction was applied immediately to a 700-ml gelatin-Sepharose column (40) which had been previously washed with 3 liters of 4 M urea, 10 mM Tris-HC1, pH 7.0, and then equilibrated with buffer C. The gelatin-bound material was washed with 2 liters of 1.0 M NaCl in buffer C, followed by 1.2 liters of buffer C with no additional salt. The fibronectin was eluted with 0.1 M NaCl, 0.05 M citric acid, pH 5.5. The fractions containing protein as judged by the absorbance at 280 nm were pooled and neutralized by adding 1/20 volume 1.0 M Na2HP04. The fibronectin was precipitated with 50% saturated ammonium sulfate, suspended in 0.2 M 3-cyclohexylamino-1-propanesulfonic acid (Calbi- ochem), pH 11.0, and dialyzed exhaustively at 4 "C against DMEM/ HEPES.

[3H]Fibronectin was prepared by reductive methylation using NaB[3H]H4 (60 Ci/mmol, New England Nuclear) and formaldehyde exactly as described (42) except that the procedure was scaled up to radiolabel 10 mg of plasma fibronectin with 500 mCi of NaB[3H]H4. The [3H]fibronectin was concentrated by precipitation with 50% ammonium sulfate and resuspension in 0.15 M NaCl, 0.01 M Tris- HCI, pH 7.0, followed by exhaustive dialysis against the same buffer at 4 "C.

The 75-kDa cell-binding domain was generated from human plasma fibronectin as follows. Purified fibronectin in 0.15 M NaCl, 0.01 M Tris-HC1, pH 7.0, was diluted to a concentration of 3 mg/ml and a final volume of 110 ml with 0.03 M NaCl, 0.05 M Tris-HC1, 1 mM CaC12, pH 7.0, and digested with 6.6 mg of TPCK-trypsin (Worthington) for 30 min at 30 "C. The reaction was terminated with two 0.55-ml additions of 0.2 M phenylmethanesulfonyl fluoride freshly prepared in 95% ethanol. The cell-binding domain was purified from the crude digest by sequential chromatography on DE52 cellulose (Whatman), gelatin-Sepharose, and heparin-Sepharose exactly as described (13).

The 44-kDa gelatin-binding domain from human plasma fibronectin was generated by thermolysin digestion and was purified by affinity chromatography on gelatin-Sepharose exactly as described for chicken fibronectin (15).

Cells-The BHK cells were a gift from Prof. Frederick Grinnell, University of Texas Health Science Center at Dallas, and were cultured as described (431, either in suspension for not greater than 48 h prior to use in experiments or in monolayer culture for longer term maintenance.

Binding Assay-The BHK cells were harvested from the suspension culture by centrifugation at 750 X g and washed twice with DMEM/HEPES supplemented with 2% (w/v) bovine serum albumin (binding medium). The cells were suspended to a concentration of 1- 10 X lo7 cells/ml in binding medium. Where indicated, [3H]fibronectin was diluted with unlabeled fibronectin in binding medium. The cells, [3H]fibronectin, and either binding medium alone or with excess unlabeled fibronectin, or other competing material was mixed in microfuge tubes (Sarstedt) to a final volume of 150 ~l at approximately 0.5-4 X lo7 cells/ml and the final concentrations of fibronectin specified. The cells and fibronectin were continuously mixed by rotating the tubes end over end at approximately 6 rpm. To terminate the binding reaction, 100 p1 of the mixture were diluted in 1.0 ml binding medium and the cells were collected by centrifugation for 1 min in a Beckman Microfuge 12 equipped with a Type 13.2 fixed- angle rotor. The sedimented cells were resuspended in 1.0 ml of binding medium and centrifuged again. The pellet was then resuspended in 200 p1 of binding medium and layered on 1.0 ml of binding medium containing 10% bovine serum albumin and sedimented. The bound radioactivity was quantitated by suspending the cells in two aliquots of 200 pl of deionized water, mixing with 10 ml of Ultrafluor liquid scintillation fluid (National Diagnostics), and counting in a LKB Rack-Beta 1215 counter. In separate controls further washing was found to have no further effect on the amount of radioactivity in the washes. To quantitate radioactivity nonspecifically trapped in the pellet, control tubes contained [~arboxy-'~C]inulin (New England Nuclear) substituted for the fibronectin. In all cases, the fraction of

trapped radioactivity measured in this manner was less than 10" of the total. The cell count in individual experiments was determined with a Coulter counter.

Other Methods-The cell-spreading assays were performed as described (43) except that the adhesion buffer was replaced by DMEM/ HEPES and spreading was quantitated (12). The fibronectins and cell- and collagen-binding domains were analyzed for purity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described (44). Protein concentrations were measured by the method of Lowry et al. (45) using crystalline bovine serum albumin (Calbiochem- Behring) as a standard. All reagents were purchased from the com- pany specified or were the best grade available. All fibronectins and fragments were stored in small aliquots at -80 "C or in liquid nitro- gen. Before all experiments, the fibronectin was centrifuged at max- imum speed in a Beckman Microfuge 12 for 5 min to remove any aggregates.

RESULTS

Analysis of 3H-labeled Fibronectin-Human plasma fibronectin was tritiated by reductive methylation to a specific activity of 1.2 x lo9 cpm/mg with a recovery of 77% of the initial fibronectin. The labeled fibronectin migrated as a clear doublet species when analyzed by sodium dodecyl sulfate-gel electrophoresis (not shown). When assayed for cell-spreading activity, the [3H]fibronectin mediated half-maximal spreading of BHK cells when incubated on plastic tissue culture dishes at a concentration of 1.7 f 0.5 pg/ml (n = 5, f S.E.). In a parallel assay, unmodified fibronectin had half-maximal activity at a concentration of 1.1 k 0.3 pg/ml (n = 5, f S.E.). Over 90% of the radioactivity was precipitable with 10% trichloroacetic acid.

Time Course of Binding-As shown in Fig. 1, 0.1 mg/ml [3H]fibronectin binds to BHK cells in suspension in a time- dependent manner at 4, 22, and 37 "C. At 22 "C, binding is half-maximal in approximately 12 min and is essentially complete after 50-60 min. Furthermore, 60-80% of the binding can be inhibited by 8 mg/ml unlabeled fibronectin. There were no major differences in the time courses or competability of binding at 4 "C compared to 37 "C (Fig. 1).

Characterization of Bound PHIFibronectin-It was con- ceivable that the observed binding of [3H]fibronectin to the BHK cells was due to a minor subpopulation of the labeled material. To help exclude this possibility and to examine whether radioactively labeled and unlabeled fibronectins interact with the cells with similar affinities, tritiated and unmodified fibronectins were mixed in varying proportions, maintaining the same total concentration of fibronectin (0.05 mg/ml) for addition to the cells at 22 "C. As shown in Fig. 2, a plot of [3H]fibronectin bound to cells uersus the per cent of [3H]fibronectin added was linear (correlation coefficient, 0.998). This result indicates that the labeled and unlabeled fibronectins interact with the cells with similar affinities, and suggests that the cell-associated radioactivity we observe is not due only to a minor subpopulation of [3H]fibronectin modified by the labeling procedure.

In another control, the unbound fraction of [3H]fibronectin which had been incubated with cells was recovered and subsequently was found to bind to fresh cells with efficiencies identical to the original material (not shown). This result also suggests that the observed binding to cells is not due to only a small subpopulation of the labeled fibronectin.

Fig. 3 shows that [3H]fibronectin binds to the surface of the cells and that the binding is reversible. In Fig. 3A, 0.1 mg/ ml [3H]fibronectin was incubated with BHK cells for 60 min at 22 "C. After separation from unbound fibronectin, the cells were suspended in 2.5 mg/ml TPCK-trypsin in HBSS for 20 min with constant mixing at 22 "C. The cells were then sedimented by centrifugation at 750 X g and washed in binding

4494 Fibronectin Binding to Fibroblastic Cells I , , I , , , , ,

A. 4°C

c. P C

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 TIME (Minutes)

FIG. 1. Time course of binding of ['Hlfibronectin to cells in suspension. Cell bound radioactivity in counts/min is plotted as a function of incubation time in minutes. BHK cells, 0.1 mg/ml [3H] fibronectin, and binding medium with (0) or without (0) 8 mg/ml unlabeled plasma fibronectin in a total volume of 800 gl were mixed in microfuge tubes at 4 (panel A ) , 22 (panel B) , and 37 "C (panel C). At the indicated times, 100 p1 of the mixture was removed, the cells were washed as described under "Experimental Procedures," and the cell bound radioactivity was counted. A, the specific activity of the [3H]fibronectin was 9.3 X lo6 cpm/mg and the cell concentration was 4.7 X lo7 cells/ml. B, the specific activity of the [3H]fibronectin was 1.1 x lo7 cpm/mg and the cell concentration was 5.0 X lo7 cells/ml. C, the specific activity of the [3H]fibronectin was 9.3 X 10' cpm/mg and the cell concentration was 2.6 X lo7 cells/ml. The amounts of specific binding are also indicated (O), as the difference between the total binding (0) and the nonspecific binding (0).

medium as usual to remove any released radioactivity. As shown in Fig. 3A, more than 90% of the bound radioactivity is lost after trypsin treatment, compared with cells which had been treated in an identical manner without trypsin in the incubation in HBSS. This result is consistent with the binding and retention of fibronectin almost exclusively on the external surface of the plasma membrane; at most only 10% of the cell-associated radioactivity had been internalized during the initial 60-min incubation.

The reversibility of binding was tested by binding 0.1 mg/ ml [3H]fibronectin to cells for 60 min, washing the cells free of unbound fibronectin, and then suspending them in fresh binding medium with 10 mg/ml unlabeled fibronectin (Fig. 3B) or fresh binding medium alone (Fig. 3C). In both cases, the total radioactivity bound to the cells decreased with time, while the nonspecific component of binding remained essentially constant for the duration of this experiment (90-120 min). When the cells containing bound [3H]fibronectin were incubated with excess unlabeled fibronectin, over 90% of the specifically bound radioactivity dissociated from the cells in

80000 -

60000 - n Z

0 20 40 60 PERCENT 'HH-FIBRONECTIN

FIG. 2. Relative binding of ['Hlfibronectin and unlabeled fibronectin to BHK cells in suspension. The proportion of labeled and unlabeled fihronectin was varied, while the concentration of total fibronectin (0.05 mg/ml) and the number of cells (1.6 X lO'/ml) were kept constant. Binding was measured after 60 min at 22 "C. The linear coefficient of regression of the fit line is 0.998.

less than 90 min (Fig. 3B). Even under these conditions, the nonspecifically bound [3H]fibronectin appears to be essentially irreversibly bound. In separate controls, the number of cells recoverable from the incubation tubes remained constant over this time period. Therefore, the specific binding of [3H] fibronectin to the BHK cells in suspension is reversible and is restricted to the cell surface.

The [3H]fihronectin bound to cells was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. [3H]Fibro- nectin was incubated with cells for 30 or 60 min, then the unbound fibronectin was washed away. The cells were then extracted by boiling in 3% sodium dodecyl sulfate in 10 mM sodium phosphate, 2 mM phenylmethanesulfonyl fluoride, 5 mM EDTA, pH 7.0. The extracted material was chemically reduced with 0.1 M dithiothreitol and analyzed (Fig. 4). The starting [3H]fibronectin yielded one major band (lane 1) at the same mobility as unlabeled fibronectin. In the cell pellet after both 30- and 60-min incubations, the major and minor bands had the same mobilities as unlabeled fibronectin (lanes 2 and 3) .

Binding Saturation and Analysis of Binding Parameters- To examine for saturation of binding, cells were incubated at 22 "C with increasing amounts of [3H]fibronectin ranging from 2 rg/ml to 1.8 mg/ml, with or without excess (8 mg/ml) unlabeled fibronectin. The amounts of total and nonspecific binding are shown in Fig. 5A. The data are the results of averaging three to seven determinations. Specific binding is calculated as the difference between the total and nonspecific binding at each point, and it amounts to 40-50% of the total binding as shown in Fig. 5B. When the data in Fig. 5B are analyzed by the Scatchard method (46), these points could be fit to a straight line by linear regression with a correlation coefficient (r) of 0.936, indicating only a single class of sites (Fig. 6). From the intercept and the slope of the fitted line, there are 5.3 f 0.6 X lo5 sites/cell with an apparent dissociation constant of 8.0 f 0.5 X M, respectively.

Calcium Sensitivity of the Fibronectin Receptor-There have been reports (47) that fibronectin receptor function is resistant to proteolysis as measured in a bioassay, but only in the presence of divalent cations (48). To test this model, BHK cells were washed twice in HBSS, then suspended in HBSS alone or in 1 mg/ml TPCK-trypsin in HBSS supplemented either with 1.3 mM ca2+ and 0.9 mM M$+ or with 5 mM


( A B 1 +

200 - 160 - 150-

0 I

2 93-

TIME [Minuted

FIG. 3. Restriction of [3H]fibronectin to the cell surface and reversibility of binding. A, trypsin removal of bound [3H]fibronectin. [3H]Fibronectin (0.1 mg/ml, 1.4 X lO'cpm/mg) was incubated with 1.8 X lo7 cells/ml in a total volume of 1.2 ml. After incubating for 60 min at 22 "C, 100-pl aliquots were removed and washed as described under "Experimental Procedures," except that the centrifugation steps were performed at low speed for 5 min. Three aliquots of washed cells were counted immediately for bound radioactivity (control). Four aliquots each were suspended in 1 ml of HBSS either with (+ trypsin) or without (-trypsin) 2.5 mg/ml TPCK-trypsin for 20 min at 22 "C. The cells were sedimented by centrifugation, washed as described, and counted for cell bound radioactivity. B, reversibility of [3H]fibronectin binding to cells in the presence of unlabeled fibronectin. [3H]Fibronectin (5.6 X lo7 cpm/mg) and cells (2.6 X 107/ml) were mixed in the presence (0) and absence (0) of 8 mg/ml unlabeled fibronectin. After 60 min, the cells were washed as described under "Experimental Procedures," except the centrifugations were at 750 X g for 5 min. The cells were then suspended in 10 mg/ml unlabeled fibronectin at 22 "C. At the indicated times, the cells were washed and assayed for bound radioactivity. C , reversibility of [3H]fibronectin binding to cells. [3H]Fibronectin (0.1 mg/ml, 5.7 X lo6 cpm/mg) was mixed with 1.4 X lo7 cells/ml in 1.4 ml with (0) and without (0) 8 mg/ml of unlabeled plasma fibronectin. After 60 min, the cells were washed as described in B. The cells were then suspended in fresh binding medium without fibronectin at 22 "C. At the indicated times, the cells were washed and assayed for bound radioactivity.

EDTA, and incubated with constant mixing at 22 "C for 10 min. After quenching the reaction with 1 mg/ml soybean trypsin inhibitor, the cells were collected by centrifugation, washed, and assayed for binding of 0.1 mg/ml [3H]fibronectin with and without 8 mg/ml unlabeled fibronectin. In separate controls, incubating the cells in HBSS with the divalent cations, or EDTA, but without trypsin, had no effect on either the total or nonspecifically bound radioactivity. As shown in Table I, the level of specific fibronectin binding to cells treated with trypsin in the presence of Ca2+/Mg+ was essentially the same as that for the control cells. The cells treated with trypsin in the presence of EDTA had only 35% of the specific fibronectin binding of the control cells. Although the trypsin- treated cells in the presence of divalent cations had less total bound fibronectin than the control cells, the entire difference can be attributed to a decrease in the nonspecific, rather than the specific, component of binding. The cells treated with trypsin in EDTA had reduced levels of both specific and nonspecific fibronectin binding when compared with the control cells, and had approximately the same level of nonspecific binding as cells treated with trypsin in the presence of calcium/magnesium.

43 - 30 -

D-

1 2 3

69 -

FIG. 4. Autoradiograms of plasma fibronectin and cell- bound fibronectin. [3H]Fibronectin (0.1 mg/ml, 4.8 X 10' cpm/mg) was incubated with 6.5 X lo7 cells/ml for 30 min (lane 2) or 60 min (lane 3) at 22 "C. The cells were then washed and the bound fibronectin was extracted by boiling in 300 pl of sodium dodecyl sulfate 3%, 10 mM sodium phosphate, 2 mM phenylmethanesulfonyl fluoride, 5 mM EDTA, pH 7.0. The samples were then reduced in 0.1 M dithiothreitol and analyzed by sodium dodecyl sulfate-gel electrophoresis on a 7.5% polyacrylamide gel. Lane 1 shows [3H]fibronectin before incubating with cells. The arrow indicates the position of unlabeled fibronectin. The position of the dye front is indicated by D.

A trivial explanation for these results would be that TPCK- trypsin is more active in the presence of EDTA than with Ca2+/Mg+. To test this possibility, we assayed TPCK-trypsin at 22 "c in either 1.3 mM Ca" and 0.9 mM M e , or in 5 mM EDTA by the method of Hummel (49) using p-toluenesul- fonyl-L-arginine methyl ester (Sigma) as a substrate. The specific activity of TPCK-trypsin in C a 2 + / M e was 83.7 f 3.6 units/mg (n = 6, f S.E.), while in EDTA the specific activity was only 5.9 f 0.9 units/mg (n = 6, f S.E.). Thus, the results in Table I are actually more striking than indicated, because the cells treated with trypsin in the presence of divalent cations have been exposed to 10 times more units of tryptic activity than the cells treated in the presence of EDTA.

The apparent protective effect of divalent cations on fibronectin-binding activity appears to be quantitative rather than qualitative, i.e. if the cells are treated with enzyme for sub- stantially longer periods or with higher concentrations of trypsin, the binding capacity of the cells treated in the presence of divalent cations eventually decreased to that of the cells treated in the presence of EDTA (data not shown).

Spreading of Cells Mediated by Receptor-bound Fibronec- tin-We investigated whether receptor-bound fibronectin was biologically active in a subsequent assay for cell spreading. BHK cells were pre-incubated with 1.8 mg/ml plasma fibronectin at 22 "C for 1 h and the unbound fibronectin was removed by the standard washing procedure except that the centrifugation steps were performed at 750 x g. The cells were then suspended at 5 X 106/ml in DMEM/HEPES, and lo6 cells were assayed for cell-spreading activity either on uncoated dishes or on dishes pre-coated with 20 pg/ml plasma

4496 Fibronectin Binding to Fibroblastic Cells

I 1 I 1 I 1

300 600 900 1200 1500 1800

TOTAL FIBRONECTIN (pglrnl)

FIG. 5. Saturation of binding of ['Hlfibronectin to cells in suspension. Cell bound fibronectin is plotted as a function of concentration of total fihronectin. A, increasing concentrations of [3H] fibronectin (2000-6000 cpm/pg specific activity) as indicated were mixed with 2-5 X lo7 cells/ml in a total of 150 pl of binding medium with (0) or without (D) 8 mg/ml unlabeled fibronectin. Each point represents the mean of three to seven independent determinations which were normalized to the same cell concentration and specific radioactivity. 8, the specific binding calculated by subtracting the nonspecific binding (0) from the total binding (D) in panel A . The ordinate scale in B is %fold expanded relative to that in A.

12. t a -

6 -

4 -

2 -

10 l h 6 -

4 -

2 -

BOUND (wg/107 Cells) FIG. 6. Scatchard analysis of the binding of ['Hlfibronectin

to cells in suspension. The data in Fig. 5B were analyzed by plotting the ratio of the bound concentration of fihronectin to the free concentration as a function of the bound concentration of fihronectin. The data were fit by linear regression analysis to a straight line with a correlation coefficient ( r ) of 0.936. The slope indicates an apparent dissociation constant ( K d ) of 8.0 f 0.5 X M, and the x-intercept indicates 530,000 f 60,000 sites/cell.

fibronectin. The results are shown in Table 11. The cells were fully competent to spread on immobilized fibronectin whether or not the cells had been pre-incubated with fibronectin, i.e. the washing procedure did not damage the cells, and auto- inhibition of attachment and spreading (50) by receptor- bound fibronectin is not significant in these experiments. Cells that were not pre-incubated in fibronectin did not spread

TABLE I Binding of PHlfibronectin to trypsin-treated cells

BHK cells (2 X lo') were incubated for 10 min at 22 "C in a 20-ml volume of either HBSS in the absence of TPCK-trypsin (control) or in HBSS plus 1 mg/ml TPCK-trypsin either with 1.3 mM CaC12, 0.5 mM MgC12/6H20, 0.4 mM MgS0,/7H20, or with 5 mM EDTA at 22 "C. After quenching with 20 ml of 2 mg/ml soybean trypsin inhibitor in binding medium, the cells were washed in soybean trypsin inhibitor solution and once in binding medium. The cells were then suspended to a concentration of 4 X lo7 cells/ml in binding medium and mixed with 0.1 mg/ml [3H]fibronectin (8.8 X lo6 cpm/ml) with or without 8 mg/ml unlabeled fibronectin in a total volume of 150 pl. After incubating for 60 min at 22 "C, the cells were washed, and the cell-associated radioactivity (f S.E., n = 3) was determined as described under "Experimental Procedures."

Radioactivity bound Specific

NO unlabeled +8 mg/ml binding fibronectin fibronectin

cpm Control 6002 f 27 3168 f 7 2834 f 34 Trypsin/Ca/Mg 4039 f 30 1391 f 6 2648 f 36 Trypsin/EDTA 2281 f 5 1327 f 17 954 f 22

TABLE I1 Spreading of BHK cells mediated by receptor-bound fibronectin

Fibronectin Fibronectin Per cent cells concentration" coating spread'

w l m l d m 1 % 1.8 20 89 f 3 1.8 0 26 f 2 0 20 0 0

91 f 2 I f 1

"The concentration of fibronectin pre-incubated with 4.6 X lo6 cells at 22 "C for 60 min is given. The cells were subsequently washed as described under "Experimental Procedures" and allowed to attach and spread on plastic tissue culture dishes for 90 min at 37 "C.

The concentration of fihronectin used to coat the 35-mm tissue culture dishes is given.

'The per cent of cells spread (+ S.E., n = 6) is indicated. Cell spreading was quantified as described in Ref. 12.

in the absence of immobilized fibronectin. Thus, the cells probably do not retain appreciable amounts of fibronectin from the culture medium. When cells were pre-incubated in 1.8 mg/ml soluble fibronectin and added to plastic dishes in the absence of immobilized fibronectin, a significant fraction (26 f 5%, n = 6, k S.E.) of the cells were able to attach and spread. These results indicate that the receptor-bound fibronectin is available to mediate cell attachment and spreading, although only to a limited degree.

Inhibition of Binding by Purified Cell-binding Domain- The 75-kDa cell-binding domain isolated from tryptic digests of human plasma fibronectin retains a biological molar activity equal to that of intact plasma fibronectin, as judged by the concentrations required for half-maximal cell spreading (13). This domain was also found to inhibit the binding of [3H] fibronectin to the BHK cells. Cells were incubated for 60 min at 22 "C with 0.1 mg/ml [3H]fibronectin either alone, with 5 mg/ml unlabeled fibronectin, or with varying amounts of purified cell-binding domain. As shown in Fig. 7, the purified cell-binding domain can competitively inhibit the binding of intact fibronectin, consistent with the interaction of these two species with the same binding site. At a concentration of 1 mg/ml, the purified cell-binding domain inhibits approximately 95% of the binding of intact fibronectin, and 80 pg/ ml of cell-binding domain has the same apparent inhibitory activity as 5 mg/ml intact fibronectin. In parallel control experiments, purified 44-kDa gelatin binding domain isolated from thermolysin digests of human plasma fibronectin and


”4’ CELL-BINDING DOMAIN (pg/ml)

FIG. 7. Inhibition of binding of [SH]fibronectin to cells by purified cell-binding domain. Per cent inhibition of [3H]fibronectin binding to cells is plotted as a function of the concentration of cell-binding domain (0) or a reference concentration of intact unlabeled fibronectin (0). [3H]Fibronectin (0.1 mg/ml, 2.8 X lo7 cpm/mg specific activity) was incubated with 1.5 X lo6 cells with and without 5 mg/ml unlabeled fibronectin or with the indicated concentrations of purified fibronectin cell-binding domain in a total volume of 150 pl at 22 “C. After 60 min, bound fibronectin was determined as described under “Experimental Procedures.”

TABLE 111 Inhibition of binding of PHIfibronectin to cells Inhibitow protein or peptide Relative binding“

Controlb 1.00 5 mg/ml unlabeled fibronectin 0.50 k 0.02 1 mg/ml cell-binding domain 0.04 k 0.02 1 mg/ml collagen-binding domain 1.01 f 0.04 5 mdml ovalbumin 1.02 f 0.21

“Relative binding is the amount of radioactivity bound to cells when incubated with 0.1 mg/ml [3H]fibronectin at 22 “C in the presence of the protein or fragment indicated, normalized to the control value in parallel controls (+ S.E., n = 3).

* Control binding is the amount of [3H]fibronectin bound in the absence of any other additive as described under “Experimental Procedures,” normalized to 1.00.

the glycoprotein ovalbumin lacked any inhibitory activity. The relative inhibitory activities of the fragments, fibronectin, and ovalbumin controls are listed in Table 111.

DISCUSSION

We have developed an assay which quantitates the binding of soluble [3H]fibronectin to BHK cells suspended in physiological serum-free medium. We are thus able to define quan- titatively the parameters describing the interaction between these cells and soluble human plasma [3H]fibronectin. The assay involves the simple mixing of cells with fibronectin which has been tritiated by reductive methylation, followed by removal of unbound fibronectin with a procedure that involves multiple, rapid centrifugations of the cells through a series of wash buffers. The assay does not require the activation of the fibronectin receptor (as with platelets), nor any form of pretreatment of the labeled fibronectin such as immobilization on beads as with other binding assays (36, 37), or the use of nonphysiological temperatures (39). The [3H] fibronectin labeled by the reductive methylation procedure is equally active as unmodified fibronectin in a biological assay measuring fibronectin-dependent spreading of fibroblastic

cells on plastic. There is no evidence of a small subpopulation of [3H]fibronectin being responsible for the binding we observe, and both labeled and unlabeled fibronectins appear to have similar binding affinities for the cells (Fig. 2).

As measured by our assay, the binding of soluble [3H] fibronectin to fibroblastic cells in suspension in physiological buffer is time-dependent and is usually at least 60-80% specific (Fig. 1). The binding of [3H]fibronectin observed in the presence of 8 mg/ml unlabeled fibronectin is most likely true nonspecific binding because: 1) it is not competable with unlabeled fibronectin (Fig. 1); 2) it is essentially irreversible (Fig. 3); and 3) in contrast to the specific binding, it is abolished to the same extent by trypsin whether divalent cations or EDTA are present (see below and Table I).

Binding occurs at 4 “C (Fig. lA) and also at the more biologically relevant temperatures of 22 and 37 “C (Fig. 1B and C). At all temperatures, binding is essentially complete after approximately 60 min, which is within the time frame of biological processes such as fibronectin-mediated cell attachment or spreading. At all temperatures, there is a nonspecific component of binding as judged by the amount of cell-associated radioactivity remaining in the presence of an 80-fold excess of unlabeled fibronectin. At 22 “C this nonspecific binding occurs relatively rapidly and is essentially complete after only 10-15 min (Fig. 1B). The half-time required for the specific component of binding is 12-15 min when the fibronectin concentration is 0.1 mg/ml.

At least 90% of the cell-associated radioactivity is released by subsequent trypsin treatment of the cell-bound fibronectin (Fig. 3A). This protease sensitivity is consistent with the localization of bound [3H]fibronectin to the outside of the cells, and it demonstrates that not more than 10% of the cell- associated fibronectin is being accumulated via an internali- zation mechanism. In fact, this figure of 10% may be an overestimation, since the cell-binding region of human plasma fibronectin has been shown in earlier studies to be relatively resistant to proteolysis (13, 14). It is therefore possible that even after trypsin treatment, a subpopulation of labeled fibronectin fragments could still remain bound on the outer surface of cells.

The specific component of [3H]fibr~nectin binding to cells is reversible. Over 90% of the specifically bound fibronectin dissociates from the cells in 90 min when the cells are incubated in excess unlabeled fibronectin (Fig. 3B). In contrast, the [3H]fibronectin which is bound to the cells in the presence of excess unlabeled fibronectin (assumed to be nonspecific binding) appears to be essentially irreversibly bound, even in the presence of excess unlabeled fibronectin (Fig. 3, B and C) indicating that nearly all of the dissociation of fibronectin from the cells can be attributed to the loss of specifically bound fibronectin.

The specific component of binding is saturable and hyper- bolic (Fig. 5). In a Scatchard analysis of the data (Fig. 6), the points could be fit to a straight line by linear regression with a correlation coefficient of 0.936, consistent with the presence of only a single class of binding sites, and simple, noncoop- erative binding. An apparent dissociation constant (Kd) of 8.0 f 0.5 X M was calculated assuming a molecular size of 440 kDa. A K d of 0.80 p~ indicates that the binding of [3H] fibronectin to cells is only moderately tight. This dissociation constant is similar to that predicted by biological autoinhibi- tion studies (50). The binding is several orders of magnitude lower in affinity than might be predicted by biological assay (35,43) if the concentrating effect of adsorption to a substrate is ignored. This result may explain why other investigators


have been previously unsuccessful in characterizing this interaction.

For comparison, the concentration of fibronectin in normal plasma is 4.5-9 X M or 0.2-0.4 mg/ml (51), which is very similar to the apparent &, and suggests a possible biological relevance for this type of receptor. If the affinity was higher, fibronectin receptors in the presence of plasma would always be maximally occupied, and cells could not respond to changes in the concentration of fibronectin. In general, receptor occupancy is most sensitive to changes in ligand concentration near the K d .

Finally, the Scatchard analysis indicates the presence of approximately 5.3 & 0.6 X 10' specific binding sites/cell. This number suggests that the possible candidates for this receptor are limited to major membrane components.

The total concentration of fibronectin used to generate the binding isotherms in Figs. 5 and 6 range from 2 to 1800 pg/ ml. Even at the lowest fibronectin concentrations, there is no suggestion of the presence of a class of higher affinity binding sites. We believe that there is only a single class of binding sites for the following reasons. 1) Analysis of points in a higher concentration range (21-1800 pg/ml) indicates the presence of a single class of sites (Kd = 8.4 X 500,000 sites/cell) with a linear correlation coefficient of 0.990, sug- gesting that the model with only a single class of sites is appropriate. If points in the range of 2-21 pg/ml fibronectin are included, the binding parameters are essentially un- changed, and the correlation coefficient changes only to 0.936 (Fig. 5). Although the inclusion of points at low input concentrations of fibronectin marginally reduces the quality of the fit of the one-site model, there is no good evidence in favor of a two-site model. 2) It could be argued that there is an "invisible" class of very tight binding sites undetected by this assay, which comprises 0.5-1% of the total number of receptors. For this entire hypothetical class of sites to remain undetected, essentially all of these sites (>95%) would have to be occupied, even at the lowest ligand concentration, i.e. the tighter class of sites would require a dissociation constant of 2 x 10"' M. This value is a extremely high affinity. Furthermore, our results indicate that even if any such a high affinity class of sites does exist, they can have little apparent biological significance. These sites would be 99% occupied at the concentrations of fibronectin in 10% serum-containing medium. Yet, cells removed from serum-containing medium and plated onto plastic culture dishes do not show any appreciable fibronectin-mediated attachment and spreading2 (see Table 11). High affinity binding sites (& = 10"' M) would still be 75% occupied even at 0.33 pg/ml fibronectin, a concentration at which only 5-10% cell attachment and spreading occurs' (35).

As shown in Table I, the putative fibronectin receptors are both trypsin and divalent cation-sensitive. Trypsin sensitivity suggests that the receptor contains a glycoprotein component (52-54) rather than only a glycolipid component (55-57). This finding cannot entirely exclude heparan sulfate as a possible cell-surface receptor (58, 59), although the cell-binding fragments do not bind to immobilized heparin at physiological salt concentrations (13, 14).

The putative fibronectin receptor appears to be sensitive to the presence of physiological concentrations of calcium and magnesium. In the presence of these divalent cations, the receptor is resistant to trypsin as judged by our assay, in agreement with biological assays (47, 48). The apparent divalent cation sensitivity of the fibronectin receptor suggests

S. K. Akiyama, and K. M. Yamada, unpublished data.

that conformational alterations may occur in the presence of physiological concentrations of calcium and magnesium. The significance of this finding is not yet clear, because the binding of [3H]fibronectin is not affected by 5 mM EDTA (data not shown and Ref. 48).

The receptor-bound fibronectin is available for mediating cell attachment and spreading, although only to a limited degree (Table 11). Whereas 90% of the cells can attach and spread on fibronectin immobilized on plastic, only 26% of the cells pre-incubated with 1.8 mg/ml soluble fibronectin can subsequently attach and spread on uncoated plastic. There are several plausible explanations for this discrepancy. A likely hypothesis is that the cells require multivalent binding to fibronectin for maximal biological response (6, 23, 37, 60, 61). Fibronectin immobilized on plastic would be much more amenable for such a multivalent interaction. Soluble fibronectin prebound to cells may not participate in multivalent binding to its receptors. If released from the cell surface, the prebound fibronectin would be present at only -0.37 pg/ml (see below), a concentration too low to coat the plastic at a density sufficiently high to allow a multivalent interaction. Multivalent interactions also greatly increase the apparent affinity of receptors, the affinity increasing correspondingly as larger amounts of a ligand such as fibronectin becomes available for binding (62).

Another possible explanation and point to emphasize is that the immobilization of fibronectin on plastic has the effect of concentrating the protein in a thin layer between the cells and the plastic. If a 10 pg/ml solution of fibronectin is used to coat a dish, a monomolecular layer of 0.2-0.4 pg/cm' of fibronectin will result3 (63). In a layer 200 nm above the plastic, the effective concentration of fibronectin will be approximately 13-20 mg/ml, or enough to yield 97-98% receptor occupancy for cells within this layer (assuming K d = M) and greater than 90% fibronectin-mediated cell spreading.'

In our cell-spreading experiments, when lo6 cells are added to a dish in a volume of 1 ml, the actual concentration of fibronectin is only 0.37 pg/ml. If 0.37 pg/ml of fibronectin is immobilized on plastic, it can mediate the spreading of only approximately 10-20% of the added cells? For the total amount of fibronectin bound to the cells in the experiments in Table 11, we might also expect to observe only partial cell spreading in the absence of immobilized fibronectin.

The purified 75-kDa cell-binding domain from tryptic digests of human plasma fibronectin competes effectively with [3H]fibronectin for cell-surface binding sites (Fig. 7). A 50- fold molar excess of cell-binding domain can inhibit 95% of the total [3H]fibronectin binding. This inhibitory activity is specific for the cell-binding domain; the purified gelatin- binding domain of fibronectin did not inhibit the binding of [3H]fibronectin to the cells (Table 111). The simplest inter- pretation of these data is that the cell-binding domain is a more effective inhibitor than intact unlabeled fibronectin because the smaller fragment can diffuse more rapidly to the fibronectin receptor. Another possible explanation is that the proteolytic cleavage of fibronectin results in conformational alterations within the cell-binding domain which enables it to compete more effectively for the fibronectin receptor. The exposure of altered binding and inhibitory activities in proteolytic fragments of fibronectin is surprising, but examples have been observed previously (16,64,65).

Our results are similar to those of Plow and Ginsburg (38) for activated (but not resting) platelets, which have an apparent dissociation constant of 3 X M for a single class of

S. K. Akiyama, unpublished result.


120,000 sites/cell. The lower number of sites is not surprising because platelets are much smaller than fibroblasts. The case of fibronectin binding to platelets may be special, however, because binding requires pre-activation of the receptor by thrombin; no binding was observed with normal, unactivated platelets (38).

It was reported previously that soluble fibronectin bound to cells in suspension but only at 4 “C; there was very little or no specific binding at 22 and 37 “C (39). Moreover, at all temperatures, the binding continued to increase with time, even after 4 h. The exact reasons for the discrepancies between these data and ours are not totally clear. The Dulbecco’s medium we used in the present study is more physiological than the phosphate-buffered salt solution used previously. We also used a considerably higher concentration of cells, [3H] fibronectin labeled to a higher specific activity, and more rapid washing steps. Moreover, it is possible that “binding” at 4 “C was simply a cell-catalyzed aggregation of plasma fibronectin at low temperatures. Our data also demonstrate the need to use high concentrations of fibronectin in saturation and competition experiments; the high apparent dissociation constant (8 X M) requires that 5-10 mg/ml fibronectin be used to demonstrate competition and 1.8 mg/ ml of [3H]fibronectin ( 5 x Kd) to demonstrate saturation of specific binding.

The binding of fibronectin to confluent monolayers of human fibroblasts has been examined using lZ5I-labeled fibronectin (66). These authors observed a single class of 128,000 binding sites/cell with an apparent dissociation constant of 3.6 X IO-*, indicating tight binding. However, binding was not measured at equilibrium. In addition, these cells have a considerable extracellular matrix, and it appears likely that at least part of the binding observed in this case was incor- poration of fibronectin into the matrix as reported for exogenous cellular fibronectin (67). In fact, after 1 h, 46% of the binding was found to be to the cell matrix rather than directly to the cells in the above study (66). In our study, the fibronectin binding occurred even after trypsin treatment, sug- gesting that binding is not occurring to endogenous fibronectin. Furthermore, we can rule out binding to collagen because the purified gelatin-binding domain does not inhibit binding of intact fibronectin.

The fibronectin receptor can be compared to the laminin receptor, which has been recently characterized and isolated (68-70). Laminin binds to cells and its receptor with an apparent dissociation constant of 2 X IO-’ M. There are only approximately 50,000-110,000 sites/cell (68, 69). The binding of laminin to its receptor is therefore very tight; laminin has a 400-fold higher affinity for its receptor than does fibronectin for its receptor. This difference may account for the relative ease of isolation of the laminin receptor by conventional affinity chromatographic techniques, whereas the fibronectin receptor has yet to be isolated. The direct binding assay for fibronectin and the determination of binding parameters in this study should facilitate further in vitro analysis and the eventual isolation of the fibronectin receptor.

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