A comparison of the platelet-derived growth factor-dependent tyrosine kinase activity in sparse and confluent fibroblasts

JOURNAL OF CELLULAR PHYSIOLOGY 126:225-236 (1586)

A Comparison of the Platelet-Derived Growth Factor-Dependent Tyrosine Kinase Activity in

Sparse and Confluent Fibroblasts ANDRIUS KAZLAUSKAS AND PAUL E. DiCORLETO'

Atherosclerosis Section, Department of Cardiovascular Research, The Cleveland C h i c Foundation, Cleveland, Ohio 44106 (A.K., P. E.D.), and Department of Chemistry, Cleveland

State University, Cleveland, Ohio 44115 (A. K.)

Confluent (density-inhibited) human foreskin fibroblasts require a higher concentration of platelet-derived growth factor (PDGF) to elicit a mitogenic response than do sparse (nondensity-inhibited) fibroblasts. The PDGF receptor number and apparent affinity were similar in the two preparations of cells. The intrinsic kinase activity of the PDGF receptor from sparse and confluent fibroblasts was therefore examined in an attempt to explain the differential mitogenic response to PDGF. When membranes from sparse and confluent cells containing equal PDGF binding capacity were incubated with increasing concentrations of PDGF, the putative PDGF receptor (a 180-kD component), was phosphorylated on its tyrosyl residues to a similar extent. The time course of tyrosine phosphorylation of the PDGF receptor from sparse and confluent cell membranes was also found to be similar. To determine whether the phosphorylation of the PDGF receptor from isolated membranes differed from the analogous phosphorylation in intact cells, sparse and confluent fibroblasts were metabolically labeled with [32P]H3P04, stimulated with PDGF, solubilized, and the cell proteins were immunoprecipitated with a phosphotyrosine-specific antibody. The extent of PDGF-dependent tyrosine phosphorylation of the PDGF receptor from sparse vs. confluent fibroblasts was quite similar. The time course of the tyrosine dephosphorylation of the PDGF receptor was also similar in the two populations. Because comparable extents of PDGF-induced tyrosine phosphorylation of the PDGF receptor were observed despite the differential PDGF-induced mitogenic response of sparse and confluent fibroblasts, we tentatively conclude that 1) PDGF-dependent tyrosine phosphorylation of the PDGF receptor is not tightly coupled to the propagation of the mitogenic signal and 2) density-dependent inhibition of growth does not reflect any measurable change in the quantity of kinase activity of t h e PDGF receptor.

Density-dependent inhibition of growth is a character- istic of normal diploid cells and can be defined as the decrease in growth rate of cultured cells as they ap- proach confluence. Initial explanations of this phenomenon include the generation of growth inhibitors as a result of increased cell contact in dense cultures (Stoker, 1964), the increased cell-cell contact itself (Todaro et al., 1965; Dulbecco and Stoker, 19701, as well as a depletion of the culture medium (Todaro et al., 1965). At the present time, there is no universally accepted explanation of density-dependent inhibition of growth; however, cer- tain aspects of the initial theories are still under inves- tigation. Several growth inhibitors that might be responsible for density-dependent inhibition of growth have been identified and purified (Steck et al., 1979; Holley et al., 1980; Hare1 et al., 1983). Lieberman et al. (1982) have shown that increased cell contact induced by the binding of isolated cell membranes to cells inhib- its proliferation. A more recent theory of density-dependent inhibition of growth is based on the observation

0 1586 ALAN R. LISS. INC

that the response of cells to serum growth factors is density-dependent (Mierzejewski and Rozengurt, 1977; Holley et al, 1977). In support of this theory, Vogel et al. (1980) have reported that, as cell density increased, 3T3 cells became less responsive to the mitogenic stimulus of platelet-derived growth factor (PDGF). In that PDGF is the major serum mitogen for cells of mesenchymal origin (Ross et al., 1974; Kohler and Lipton, 1974; Wes- termark and Wasteson, 1976), decreased PDGF-sensitivity could explain decreased cell proliferation.

PDGF and other polypeptide hormones elicit their biological effects only after binding to specific plasma mern- brane receptors. The PDGF receptor has been identified

Received August 7, 1985; accepted September 18, 1985.

*To whom reprint requesWcorrespondence should be addressed.

226 KAZLAUSKAS AND DrCORLETO

on cells of mesenchymal origin by the specific binding of [n51JPDGF (Heldin et al., 1981; Bowen-Pope and Ross, 1982; Huang et al., 1982; Williams et al., 1982); and by affinity cross-linking (Glenn et al., 1982; Williams et al., 1984) to be a plasma membrane glycoprotein. The receptor, which has been pursed recently (Daniel et al., 1985) is an approximately 180-kD glycoprotein that is auto- phosphorylated on tyrosyl residues in response to PDGF binding (Ek and Heldin, 1984; Daniel et al., 1985). The cascade of events triggered by binding of PDGF to its receptor that ultimately leads to mitosis remains poorly characterized. However, there are a few well defined post-receptor binding events that may participate in the relay of the mitogenic signal. One of the early events that has been the focus of considerable research is the PDGF-dependent tyrosine phosphorylation of numerous cellular components including the PDGF receptor (Ek et al., 1982; Nishimura et al., 1982; Pike et al., 1983). Though the importance of the PDGF-dependent tyrosine phosphorylation in the mediation of the PDGF mitogenic signal has not been conclusively demonstrated, tyrosine phosphorylation has been implicated in the regulation of growth control. Viral transformation of cells has been correlated to increased cellular phosphotyrosine (Cooper and Hunter, 1981a,b). Numerous oncogene products have been shown to be tyrosine kinases (re- viewed by Heldin and Westermark, 1984). In addition, orthovanadate, an inhibitor of phosphotyrosyl phospha- tases (Swarup et al., 1982) has been shown to stimulate DNA synthesis in a variety of cell types (Carpenter, 1981; Smith, 1983).

Several recent reports have demonstrated a correlation between tyrosine phosphorylation of a growth factor receptor and the biological action of that growth factor. Hepatocytes from diabetic rats expressing a “post-insulin resistance” have been shown to exhibit decreased insulin receptor autophosphorylation relative to insulin- responsive hepatocytes (Kadowaki et al., 1984). Thus insulin receptor phosphorylation appears to be correlated to the biological action of insulin. Carlin et al. (1983) reported a direct relationship between the epidermal growth factor (EGF) mitogenic response and EGF receptor tyrosine autophosphorylation. When stimulated with EGF, young fibroblasts respond mitogenically to a much greater extent than do senescent fibroblasts. Studies with EGF receptors, prepared from cell membranes, show that EGF receptor tyrosine autophosphorylation occurs to a much greater extent in young fibroblasts than in senescent fibroblasts. Thus the mitogenic response to EGF appears to be directly correlated to EGF receptor tyrosine autophosphorylation. Unlike EGF and insulin, there have been no reports to date illustrating a correlation of a decreased biological response to PDGF with decreased tyrosine phosphorylation of the PDGF receptor. In the present report we have tested the hypothesis that tyrosine phosphorylation of the PDGF receptor mediates the density-dependent response to PDGF stimulation.

MATERIALS AND METHODS Materials

[Y-~~P]ATP (1040 Cilmmole), L-[35S] methionine (800 Cilmmole in 50 mM Tricine and 10 pmol /3-mercaptoethanol, pH 7.4) and [3H]thymidine (6.7 Cilmmole) were purchased from New England Nuclear (Boston, MA).

Carrier-free [32P]H3P04 (HC1-free in H20) was purchased from ICN Radiochemicals (Irvine, CA). Trasylol (aprotinin) was purchased from Mobay Chemical Corp. (New York, NY). Unless indicated otherwise, all other chemicals were purchased from Sigma (St. Louis, MO). Media and its supplements were purchased from Grand Island Biological Co (Grand Island, NY). Calf serum was purchased from Hyclone (Logan, UT).

Human plasma-derived serum (HPDS) was prepared by collecting whole blood from healthy donors into pre- chilled 50-ml syringes containing 5 ml of a 3.8% sodium citrate solution. Collected blood was mixed by inverting syringes 20 times, transferred to siliconized 50-ml centrifuge tubes, and centrifuged for 30 min at 40,OOOg at 4°C (Beckman 52-21 centrifuge, JA-17 rotor) to remove cellular components. Plasma was decanted into a glass container, recalcified by bringing the calcium concentration to 20 mM and allowed to clot for 2 hr at 37°C. Plasma-derived serum was centrifuged (30 min at 400g at 4°C) to remove the clotted material, filter sterilized, and stored at -70°C.

Partially purified (up to and including carboxymethyl- Separose chromatography) as well as pure PDGF (Raines and Ross, 1982) and homogenous tU5I]PDGF were a gift from R. Ross, D.F. Bowen-Pope, and E. Raines a t the University of Washington in Seattle. Unless specified otherwise, all experiments were routinely performed using partially purified PDGF and repeated at least once with pure PDGF to verify that the observed effects were in fact due to PDGF.

Cell culture Human foreskin fibroblasts were obtained from ex-

plants of neonatal foreskins and used between passages 2 and 10. Cells were maintained in a humidified 95% air:5% COZ incubator a t 37°C. Culture medium con- sisted of a 1: l mixture of Dulbecco-Vogt modified Eagle’s medium and Ham’s F12 with the following supplements: sodium bicarbonate (0.24%), penicillin (100 dml), streptomycin (100 pg/ml), nonessential amino acids (0.1 mM), sodium pyruvate (1 mM), and L-glutamine (4 mM). Cells were subcultured with trypsin-EDTA [0.05% trypsin (Worthington, Freehold, NJ), 0.02% EDTA] and maintained in fresh culture medium containing 5% calf serum, which was replaced with fresh culture medium every 2-3 days until the cells attained confluence. In preparation for experiments, trypsindetached cells were plated in 5% calf serum for 8-20 hr, after which time the culture medium was changed to 1% HPDS. After an additional 48 hr, cells at all plating densities were quiescent.

Mitogenesis assay The mitogenic response of fibroblasts to PDGF was

determined by incorporation of r3H]thymidine into TCA- precipitable material. Human foreskin fibroblasts were plated in 24-well multiwell plates (Costar, Cambridge MA) at yarse (5 x lo3 cells/cm2) and confluent (1.5 X lo4 cellslcm ) cell densities in culture medium containing 5% calf serum and were allowed to become quiescent in 1% HPDS, at which time sparse wells contained approximately 3.2 x lo3 cells/cm2 and confluent wells contained approximately 1.1 x lo4 celldcm’. The 1% HPDS- conditioned medium was aspirated and pooled, and 0.5 ml was added back to each well along with various

SPARSE AND CONFLUENT CELL KINASES 227

concentrations of PDGF (0-2 ng/ml). Cells were processed according to published protocols (Bowen-Pope and Ross, 1982; DiCorleto and Bowen-Pope, 1983). Briefly, after an 18-hr incubation at 37"C, the medium was aspirated, 0.2 pCi of [3H]thymidine was added, and the incubation was continued for 4 hr at 37°C. Medium was aspirated and the cells were rinsed with 5% trichloroacetic acid and solubilized in 0.25 N NaOH. Aliquots were neutralized with HC1, combined with 5.0 ml Aqua- sol scintillation cocktail (New England Nuclear) and the trichloroacetic acid-precipitable radioactivity was quantitated via a Packard scintillation spectrophotometer. Standard deviations routinely were less than 8%. Auto- radiographic analysis of [3H]thymidine incorporation into DNA was performed using the mitogenesis assay conditions, but cells were labeled with 2.0 instead of 0.2 pCi [3H]thymidine/well and subsequently processed as described by Glenn and Ross (1981).

Quantitation of PDGF receptors The number of PDGF receptors was quantitated as

previously described (Bowen-Pope and Ross,. 1982; Di- Corleto and Bowen-Pope, 1983). Briefly, fibroblasts were plated in 24-well plates at sparse and confluent cell densities, and quiescence was induced as described for the mitogenesis assay. Cells were cooled on ice and washed twice with 0.5 ml ice-cold binding medium [Dul- becco-Vogt modified Eagle's medium without bicarbonate, containing 25 mM Hepes buffer, pH 7.2, and bovine serum albumin (BSA), 2 mg/ml]. This same medium (0.5 ml) was added to each well along with various concentrations of [1251]PDGF (0-6 ng/ml). Cells were incubated for 2 hr at 4°C with gentle mixing, the medium was aspirated, and the cells were rinsed three times with ice- cold phosphate-buffered saline containing BSA (1 mg/ ml). Cell-bound radioactivity was quantitated by solubilizing the cells in a 1% Triton X-100 solution containing BSA (1 mg/ml) and counting in a Packard gamma counter. Nonspecific binding of [1251]PDGF was determined by incubating cells in appropriate sample wells with 100 ng partially purified PDGF in 0.5 ml binding medium for 2 hr at 4°C with gentle mixing, prior to incubation with [1251]PDGF.

Isolation of membranes Fibroblasts were plated in 150-mm tissue culture

dishes (Nunc, Grand Island, NY) or Lux (Naperville, IL) at sparse and confluent cell densities as described, and quiescence was attained in the usual manner. Mem- branes were isolated essentially as described by Pike et al. (1983). Briefly, quiescent cells were rinsed three times with icecold phosphate-buf€er saline, scraped in the presence of 6-ml of ice-cold scraping buffer (5 mM Hepes, 2 mM MgClz, 5 mM P-mercaptoethanol, pH 7.4), and homogenized a t 0°C with 20 strokes of the tightly fitting pestle in a Dounce homogenizer. Homogenized cells were centrifuged for 60 min at 42,OOOg (18,000 rpm) at 4°C in a Beckman 52-21 centrifuge, JA-21 rotor. The supernatant was discarded and the membrane pellet was rehom- ogenized in the centrifuge tube with a Dounce homogenizer pestle, resuspended in ice-cold 20 mM Hepes, pH 7.4, and stored at -70°C. Protein concentration was determined by the method of Bradford (1976) using BSA as a standard.

Quantitation of specific PDGF binding to isolated membranes

To quantitate the relative amount of PDGF binding to isolated membranes from sparse and confluent cells, the following assay, developed by Drs. R. Seifert and D. Bowen-Pope at the University of Washington, was em- ployed. Increasing quantities of membrane protein (0- 15 pg) were diluted in binding medium and added to a fixed dose of [1251]PDGF (0.7 ng). The final volume was adjusted to 300 p1 with binding medium, and samples were incubated at 37°C for 30 min. Because it was difficult to quantitate the amount of [1251]PDGF bound to membranes because of high nonspecific binding, un- bound [ 1251]PDGF of duplicate aliquots was quantitated via a PDGF radioreceptor assay (DiCorleto and Bowen- Pope, 1983). The relative PDGF binding capacity of membranes from sparse and confluent cells was determined by comparing the amount of membranes required to bind one-half of the added ['251]PDGF. Nonspecific binding cannot be determined in the described assay; therefore, only the relative PDGF binding capacity/pg of isolated membrane was determined.

The membrane phosphorylation assay The membrane phosphorylation assay was performed

essentially as described by Pike et al. (1983). For the standard assay, membranes from sparse and confluent cells containing equal PDGF-binding capacity were combined with 20 mM Hepes, pH 7.4, 100 mM NaC1, 0.2% Triton X-100,lO mM MnC12, 5 mM p-nitrophenyl phosphate, and 40 pM adenosine 5'-(~,y-immune)triphos- phate in a final total volume of 30 pl. An appropriate quantity of PDGF was added and the mixture was preincubated for 10 min at 0°C. [Y-~~P]ATP (10-15 pM) was added and the incubation continued for an additional 10 min. Reactions were terminated by the addition of 35 pl of sodium dodecyl sulfate (SDS) electrophoresis buffer [2.5% (w/v)SDS, 100 mM dithiothreitol, 125 mM Tris HC1, 2.5 mM EDTA, 8 M urea, 0.001% bromophenyl blue, 15% sucrose] followed by boiling for 3 rnin at 100°C. Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli (1970) utilizing either 5-12.5% linear gradient or 7.5% separat- ing gels with a 3% stacking gel. Gels were run at 10 mA overnight (12-15 hr), stained with Coomassie blue, de- stained (10% glacial acetic acid, 50% methanol), incubated in 1 N NaOH at 22°C for 15 min and for 1 hr in fresh 1 N NaOH at 40°C to hydrolyze most phosphoserine phosphoester bonds (Cheng and Chen, 19811, fixed (10% glacial acetic acid, 10% methanol, 1% glycerol), and dried onto thick filter paper. Autoradiography was performed with Kodak XK-1 X-ray films with regular inten- sifying screens at -70°C. To quantitate the radioactivity of the 180-kD component, the gel piece containing this band was excised and counted with or without scintil- lant in a Packard (Downers Grove, IL) scintillation spectrophotometer.

Phosphoamino acid analysis Analysis of the phosphoamino acid content of the

PDGF receptor was performed essentially as described by Cooper and Hunter (1981a). The excised 180-kD gel band was rehydrated in 3 ml of a solution containing 50

228 KAZLAUSKAS AND DrCORLETO

mM NH4HC03 and 1% /?-mercaptoethanol heated to 100°C for 1 min and then digested for 18 hr at 37°C with 250pg pronase. The supernatant from the digestion and several washings of the gel fragments were pooled, clarified by centrifugation, lyophilized in a glass tube, redissolved in 0.5 m16 N HCl and hydrolyzed for 1 hr at 110°C. HC1 was evaporated under reduced pressure at 37"C, and the residue was dissolved in 20 pl of electrophoresis buffer (78:25:897 glacial acetic acid:formic acid:water v/v), pH 1.9, containing unlabeled phosphotyrosine, phosphoserine, and phosphothreonine each at 2 mg/ml. The sample was applied to a 20 x 20-cm cellu- lose thin-layer electrophoresis plate without fluorescence indicator Kodak, (Rochester, NY) and electrophoresed for 1 hr at 4°C at 1,000 V. Ascending chromatography was then performed in the second dimension utilizing a fresh solution of 5:3 isobutyric acid:O.5N NH4OH (v/v). Phos- phoamino acid standards were visualized by spraying with ninhydrin (0.3% in 1-butanol) followed by gentle heating (70°C). Radiolabeled phosphoamino acids were visualized via autoradiography as described above.

Preparation of antiserum to phosphotyrosine An antiserum to phosphotyrosine was prepared essen-

tially as described by Ek and Heldin (1984). Rabbits were injected in the footpads with 200 p1 of a 1:l emulsion of Freund's complete adjuvant and phosphate-buffered saline, which caused the popliteal lymph nodes to become intlamed. Phosphotyrosine (2 mg) was coupled to 10 mg of human immunoglobulin by incubating with 20 mg lethyl-3~3-dimethylaminopropyl)-carbodiimide hydrochloride for 20 hr at 22°C in 1.0 ml phosphate- buffered saline. The resulting antigen was combined with an equal volume of Freunds incomplete adjuvant, and 200 pl of this emulsion was injected directly into the swollen popliteal lymph node every 2 weeks. Anti- serum was collected after four such antigen injections.

Phosphotyrosine antisera characterization Antisera were characterized as described by Ek and

Heldin (1984). Approximately 10 pg of membranes from confluent fibroblasts were phosphorylated in the presence or absence of 10 ng of PDGF using the standard assay procedure except that the reaction was terminated by the addition of a 1,000-fold excess of ATP. Antiserum (15 p1) was added and incubated for 1 hr at 0°C with occasional vigorous mixing after which time 60 pI of a 1:l protein A-Sepharose (Pharmacia, Uppsala, Swe- denkbuffer A (0.15 M NaCl, 20 mM Tris HC1, 0.2% Triton X-100, pH 7.4) slurry was added and the incubation continued for 30 min with continous mixing. The immunoprecipitated and Sepharose-immobilized samples were washed three times with buffer A, once with buffer B (0.5 M NaCl, 20 mM Tris HCl, 0.2% Triton X- 100, pH 7.4), and once with buffer C (20 mM Tris HC1, pH 7.4). Sepharose beads were pelleted between washes by centrifugation at 12,OOOg for 3 min in a Beckman (Palo Alto, CA) microfuge. After the final wash, 70 pl of SDS electrophoresis buffer was added, and the samples were alkylated by the addition of 2 p1 of a 0.5 M solution of iodacetamide followed by incubation at 37°C for 1 hr with mixing. The eluted samples were analyzed by 5- 12.5% SDS-PAGE and autoradiography as described above.

Metabolic labeling of cells Fibroblasts were plated in six-well plates (Costar,

Cambridge, MA) at sparse and confluent densities and allowed to become quiescent in 1% HPDS as described above. Cell monolayers were washed two times with Hank's balanced salt solution lacking phosphate, and 0.75 ml of labeling medium was added [Dulbecco-Vogt modified Eagle's medium containing 10% of the normal phosphate concentration and BSA (1 mg/ml)], and [32P]H3P04 was added to a final concentration of 1 mCi/ ml. After 12-16 hrs, the indicated concentration of PDGF was added, and the cultures were incubated for a prede- termined time interval a t 37°C.

For the experiment in which cells were labeled with both [32P]H3P04 and [35S]methionine, cells were plated, allowed to become quiescent, and rinsed as above. La- beling medium [Dulbecco-Vogt modified Eagle's medium containing 5% of the normal methionine concentration, 10% of the normal phosphate concentration, and BSA (1 mg/ml)] was added (0.8 ml) containing 0.4 mCi/ml [35S]methionine. After 60 hr [32P]H3P04 was added to a final concentration of 1 mCi/ml, and after an additional 12-16 hr the cells were stimulated with a maximal dose of PDGF (50 ng/ml), and the incubation was continued for approximately 7 min at 37°C.

Cell solubilization and imrnunoprecipitation Cells were solubilized and immunoprecipitated essen-

tially as described by Ek and Heldin (1984). Immedi- ately following the appropriate PDGF stimulation, cells were placed on ice and rinsed two times with ice-cold 0.15 M NaC1, 20 mM Tris HCl, pH 7.4, 8 pY103 cells of ice-cold lysis buffer (150mM NaC1, 50 mM Tris HC1, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 10 mM EDTA, 5 pM Na3V04, 30 mM Na4P207, 1 mM phenylmethyl- sulfonyl fluoride, 150 kallikrein-inactivating unitdml Trasylol, pH 7.4) was added, and the cells were solubilized for 15-20 min at 0°C with occasional vigorous mixing. Equal aliquots of the solubilized cells were transferred to siliconized microfuge tubes, and 20 p1 of phosphotyrosine antiserum was added and incubated at 4°C for 2 hr with continuous inversion mixing. Protein A-Sepharose slurry, as described above, was added and the incubation continued for an additional 45 min. The immunoprecipitated and Sepharose-immobilized sample was washed three times with 0.5 ml lysis buffer, once with buffer B, and once with buffer C. Adsorbed samples were eluted from the protein A-Sepharose beads, alkylated, and analyzed by 5-12.5% SDS-PAGE and autoradiography as described above. Gel pieces containing the 180-kD band that were labeled with both [32P] and [35S] were rehydrated with 50 p1 HzO for 30 min, digested in a 6% solution of Protosol in Econofluor (New England Nuclear), incubated over night a t 37°C in a shaking water bath, and counted in a liquid scintillation spectrophotometer.

RESULTS Vogel et al. (1980) have reported that 3T3 cells respond

to crude preparations of PDGF in a density-dependent manner. Using highly purified PDGF, we have also observed that in response to a given dose of PDGF, sparse human foreskin fibroblasts incorporate [3HJthymidine


0.6 1.0 2.0 4.0

CWF COUCCNTRATIOW (nJml)

Fig. 1. PDGF-induced [3H]thymidine incorporation into sparse and confluent fibroblasts. Human fibroblasts were plated into 24-well plates at sparse and confluent cell densities, allowed to become quiescent and stimulated with the indicated concentrations of PDGF, and the r3H]thymidine incorporation was assessed as described in Materials and Methods. H, response of sparse cells; 0,response of confluent cells. Error bars represent the standard deviation of duplicate determinations. The data are representative of eight independent experiments. Sparse wells contain 6.1 x lo3 cells, confluent wells contain 1.93 x 104 cells.

I

a 4 a

Y

I !'@ i' *iil

0 1.0 2.0 3.0 4.0 5.0 6.0

11%1 C W F COWCENlRATlOW h k ~ l t

Fig. 2. Specific binding of [l2'1]PDGF to sparse and confluent fibroblasts. Sparse and confluent quiescent fibroblasts were incubated with increasing concentrations of [1251]PDGF, and the amount bound was quantitated as described in Materials and Methods. Data presented are the specific binding, which were calculated by subtracting nonspecific binding ([1251]PDGF bound after exposure of cells to 100 ng partially purified PDGF from total binding. Nonspecific binding was routinely less than 12% of total binding. H , sparse cells; 0 , confluent cells. Data are representative of eight independent experiments.

TABLE 1. Number of PDGF receptors on sparse and confluent cells'

Cultures ( x 10-~)/celI tndml) Number of receptors Apparent receptor affinity

0.35 f 0.09 Sparse 1.18 f 0.25 0.57 * 0.12 Confluent 1.15 f 0.08

'The binding of ['2511]PDGF to quiescent sparse and confluent cells in 24-well plates was performed as described in Materials and Methods and illustrated in Figure 2. Data are the average i SD of duplicate determinations from three experiments. Similar data were obtained for cells plated in six-well plates. Apparent receptor affinity denotes the concentration of PDGF that half- maximally saturates available PDGF receptors.

into DNA to a greater extent than do confluent cells (Fig. 1). The result was not due to differences in thymidine pools, since this observation was confirmed by autoradiography of [3H]thymidine-labeled cells. When cells were stimulated with 1.0 ng/ml PDGF, the labeling in- dex of sparse cells was 36.0 k 3.9% vs. 10.8 + 0.3% for confluent cells. At a PDGF concentration of 100 ng/ml, 42.8 k 1.7% of the nuclei of sparse cells were labeled vs. 17.3 4.4% for confluent cells. The decreased mitogenic response of confluent cells was not caused by the produc- tion of a growth inhibitor: At the start of the mitogenesis assay, whether the conditioned medium from sparse or confluent cells was pooled and added back only to sparse or confluent cells, respectively, or the conditioned medium from sparse and confluent cells was pooled to- gether and added back to both the sparse and confluent cells, did not alter the enhanced PDGF-dependent mitogenic response of sparse cells relative to confluent cells (data not shown).

The number of PDGF rece tors and their apparent affinity were measured with [E5r]PDGF. As is shown in Figure 2 and Table 1, no difference was observed between the sparse and confluent cells. Thus the amount of PDGF that will specifically bind to a given fibroblast does not appear to explain the enhanced mitogenic response of sparse versus confluent cells to PDGF.

Autophosphorylation of the PDGF receptor in membrane preparations

Membranes from sparse and confluent quiescent fibroblasts were prepared as described above. Although intact sparse and confluent fibroblasts contain the same number of PDGF receptors, the yield of membranes from cells of the two populations was not identical, and therefore the number of PDGF receptors in the resultant membrane preparations was determined in each case. Because an antibody to the PDGF receptor is presently unavailable, the relative number of PDGF binding sites present in membrane preparations was assessed in an indirect assay. The PDGF binding capacities of sparse and confluent cell membranes are compared in Figure 3. The greater binding capacity of confluent than sparse cell membranes that is consistently observed might arise from differences in the extracellular and/or intracellular membrane content of the two populations. On the basis of this assay, the PDGF binding capacity was normal- ized in the phosphorylation assay by adding more of either sparse or confluent membranes. The validity of this membrane assay is based on the fact that the PDGF receptors of intact sparse and confluent fibroblasts have similar binding affinities (Table 1). Because the nonspecific binding of [1251]PDGF in the membrane assay cannot be measured, only the relative number of binding sites can be determined for a given membrane prepara-

230 KAZLAUSKAS AND DICORLETO

' - - - - - - - - -

4

o L ' ' I I I

1 2 5 5 10 15

FIBROBLAST MEMBRANE (re protein)

Fig. 3. Comparison of the PDGF binding capacity of membrane preparations from sparse and confluent fibroblasts. Increasing amounts of membrane were added to a sin le dose of ['251]PDGF (0.7 ng, 7,200

radioreceptor assay as described in Materials and Methods. W , membrane from sparse cells; 0 , membrane from confluent cells. Error bars represent the standard deviation of duplicate determinations.

cpm), and the amount of free [12 B IJPDGF was quantitated via a PDGF

Fig. 4. Phosphoamino analysis of the 180-kD band from alkali-treated gels. G4 bands were localized by autoradiography and excised and the protein extracted and hydrolyzed. Thin layer electrophoresis was performed in the first dimension, and ascending chromatography was performed in the second dimension as described in Materials and Methods. Encircled areas represent the location of added, unlabeled phosphoamino acid standards. P-tyr, phosphotyrosine; P-ser, phosphoserine; P-thr, phosphothreonine. The autoradiogram was exposed for 4 days at -70°C.

tion. For its present application, however, this limita- tion does not compromise the usefulness of the assay.

Membranes containing an equal number of PDGF receptors from sparse and confluent cells were phosphorylated in the absence and presence of PDGF. Samples were separated on the basis of molecular mass by use of SDS-PAGE. The resulting gels were alkali-treated, and the prominently phosphorylated 180-kD band, the putative PDGF receptor, was localized by autoradiography, excised, and quantitated via liquid scintillation counting. Phosphoamino analysis of the alkali-treated PDGF receptor band verified that phosphotyrosine is the only radiolabeled phosphoamino acid (Fig. 4). Phosphoamino acid analysis prior to alkali treatment verified previous reports (Nishimura et al., 1982; Pike et al., 1983) that the 180-kD component, in response to PDGF stimulation, incorporated radiolabeled phosphate into phosphoserine to a small extent relative to phosphotyrosine, whereas threonine was usually unlabeled (data not shown). A comparison of the extent of the PDGF-dependent tyrosine phosphorylation of the 180-kD component from sparse or confluent cell membranes over a large range of PDGF is shown in Figure 5. Sparse cells exhibited slightly greater tyrosine phosphorylation than confluent cells. In five replicate experiments with five different membrane preparations, a small difference was observed between the extent of phosphorylation by the two populations; however, this difference was not consistently sparse greater than confluent or the reverse (com- pare Figs. 5 and 6). The difference between averaged sparse and confluent data points from replicate experiments ranged from 3% to 25%. The extent of the phosphorylation of the serine residues of the 180-kD component was also examined in sparse vs. confluent cell membranes. PDGF-dependent serine phosphorylation of the 180-kD component proceeded to a similar extent in sparse and confluent cell membranes (data not shown).

The rate of phosphorylation of the PDGF receptor might exhibit differences between sparse and confluent cells that were not apparent when the extent of phosphorylation was examined. For this reason, the time course of phosphorylation was assessed. The membrane phosphorylation assay was altered such that the reaction mixture, containing 10 ng of PDGF and the standard amount of radiolabeled ATP, was initiated by the addition of membranes and was terminated after the indicated time interval (Fig. 6). A comparison of the time course of PDGF-dependent phosphorylation on tyrosyl residues of the 180-kD protein from sparse and confluent cell membrane preparations is shown in Figure 6 . The two membrane preparations exhibited a similar time course, and the 180-kD component of Confluent cell membranes was phosphorylated to a slightly greater extent relative to sparse cell membranes. In four independent time-course experiments using four different membrane preparations from both sparse and confluent cells, no consistent difference in the rate of phosphorylation of the PDGF receptor was observed. The time course of PDGF-dependent serine phosphorylation of the 180-kD component from sparse and confluent membranes was also found to be similar (data not shown). Thus it appears that the PDGF receptor from sparse and confluent cell membranes is phosphorylated to a similar extent and with a similar time course in response to PDGF.


I 0 4 0 6 0 8.0 10.0

PDOF h l

Fig. 5. Phosphorylation of isolated sparse and confluent cell membranes. A, membranes from sparse and confluent fibroblasts were phosphorylated in the presence of the indicated amount of PDGF (ng) and analyzed via 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and autoradiography. Molecular mass standards are the same as described in Figure 6. The autoradiogram was exposed for 21 hr. B, the 180-kD component was excised and the [32P] content quantitated by Cerenkov counting. The value of I3*P] incorporated in the absence of PDGF was subtracted from the value incorporated in the presence of PDGF. H, sparse; 0, confluent. B, inset, an independent experiment in which higher concentrations of PDGF were used. Excised samples were counted in the presence of scintillation cocktail, and therefore the cpm values were higher.

1100

900

700

500

300

100

0 1 I I 1

1 3 5 1 9 I 1 13 15

TIME (min)

Fig. 6. Time course of the phosphorylation of isolated membranes from sparse and confluent fibroblasts. A, membranes from sparse and confluent fibroblasts were phosphorylated in the described phosphorylation assay except that the membranes were not preincubated with PDGF and the reaction was initiated by the addition of membranes to the complete reaction mixture and terminated after the indicated time (min). PDGF (long) was present (+) or absent (-1 from the reaction mixture as indicated. Samples were analyzed by 7.5% SDS-PAGE and autoradiography. Molecular mass markers and their molecular mass in Daltons. Myosin, 200,000; phosphorylase B, 97,000; bovine serum albumin, 68,000; ovalbumin, 43,000; achymotrypsinogen, 25,7000. The autoradio am was exposed for 4 hr. B, the 180-kD band was excised

of label in the absence of PDGF was subtracted from the amount of label incorporated in the presence of PDGF. H, sparse; 0, confluent.

and the R PI content quantitated by Cerenkov counting. The amount


Fig. 7. Characterization of phosphotyrosine antiserum. Fibroblast membranes were phosphorylated according to the described phosphorylation assay in the absence (-) or presence (+) of 10 ng of PDGF, immunoprecipitated and analyzed by 5.125% SDS-PAGE and autoradiography as described in Materials and Methods. Antiserum was pretreated with phosphotyrosine Ip-tyr), phosphoserine (P-ser), or phosphothreonine (7-thr) at a ratio of 1 pg phosphoamino acid/pl of antiserum and used for the immunoprecipitation of the indicated samples. Nonimmune serum (NS) was used in the immunoprecipitation of the indicated sample in place of P-tyr antiserum. Molecular mass markers are the same as in Figure 6. the autoradiogram was exposed for 45 hr.

Phosphorylation in intact cells It was possible that during the preparations of isolated

membranes, regulatory molecules were lost that play a role in the control of tyrosine phosphorylation in intact cells. Preliminary experiments in which metabolically labeled cells were stimulated with PDGF, lysed, and analysed by SDS-PAGE indicated that the PDGF receptor would have to be purified from the whole cell lysates because of the high background of phosphorylated cellular components. Immunopurification utilizing a phosphotyrosine antibody has been successfully used for this purpose by several other groups (Frackelton et al., 1984; Ek and Heldin, 1984), so we raised such an antibody to phosphotyrosine.

To provide a source of phosphotyrosine-labeled protein with which to characterize the phosphotyrosine antiserum, fibroblast membranes were phosphorylated in the standard membrane phosphorylation reaction in the presence or absence of 10 ng PDGF. The phosphorylated membranes were immunoprecipitated and analyzed by SDS-PAGE as described in Methods and Materials. Anti- serum preexposed to unlabeled phosphotyrosine did not immunoprecipitate the PDGF receptor, whereas phosphoserine or phosphothreonine-preexposed antiserum as well as the native antiserum did immunoprecipitate the 180-kD component (Fig. 7).

[32P]H3P04-metabolically labeled sparse and confluent fibroblasts were stimulated with various concentrations of PDGF and solubilized in an equivalent volume of lysis buEer/cell. Equal aliquots of this solubilized cell solution containing an equal number of sparse and confluent cells, and therefore an equal number of PDGF receptors, were immunoprecipitated and analyzed by SDS-PAGE and autoradiography as described above. Gels were alkali treated to remove any phosphoserine components, the 180-kD band was excised, and the extent of tyrosine phosphorylation was quantitated by scintillation counting. In response to PDGF stimulation, the intact sparse and confluent fibroblasts phosphorylated the tyrosine residues of the PDGF receptor to similar extents (Fig. 8), which was in agreement with the membrane preparation results. The inset in Figure 8B shows that 50 ng/ml PDGF induced maximal tyrosine phosphorylation of the 180-kD component, whereas 1 ng/ml of PDGF did not induce any detectable phosphorylation. The time course of the PDGF-dependent tyrosine dephosphorylation of the PDGF receptor of intact sparse and confluent cells was examined by stimulating [32P]H3P04-metabolically labeled cells with 50 ng/ml PDGF and solubilizing the cells after the indicated time intervals (Fig. 9). The time course of dephosphorylation for the two populations was similar. The extent of phosphorylation appeared to be slightly greater for confluent cells; however, in replicate experiments no consistent difference was observed.

In a final series of control experiments designed to correlate the mass of PDGF receptor to the extent of its phosphorylation, cells were metabolically labeled with [35S]methionine and [32P]H3P04, stimulated with 50 ng/ ml PDGF, immunoprecipitated, and the amount of each radionuclide in the 180-kD component was quantitated. The ratio of [35S]labeled confluent to sparse 180-kD component was 1.8 f 0.5; the ratio of [32P]labeled confluent to sparse 180-kD band was 1.9 f 0.3 (mean SD of triplicate samples). Thus the ratio of these two numbers is 0.95; we therefore conclude that the PDGF receptor is phosphorylated to a similar extent in intact sparse and confluent fibroblasts. The coefficients of variance for triplicate determinations of the extent of phosphorylation were 14% and 17% for sparse and confluent samples, respectively. Thus, differences in the extent of phosphorylation of less than 17% are not resolvable in this assay.

DISCUSSION We have observed that sparse fibroblasts show a

greater mitogenic response to PDGF than do confluent fibroblasts, which is consistent with the observation of


100 I!

250 5 0 0

PDGF CONCENTRATION fnglrnll

Fig. 8. Dose response of [32P]H3P04-labeled sparse and confluent fibroblasts to PDGF. A, labeled cells were stimulated with the indicated concentration of PDGF (ng/ml), solubilized, immunoprecipitated with native antiserum or antiserum preincubated with P-tyr (phosphotyrosine), and analyzed by 5-12% SDSPAGE and autoradiography as described in Materials and Methods. Molecular weight markers are as in Figure 6. Number of cpm f x loadeuane starting from right to left: 2.0, 1.7, 0.8, 0.4, 0.9, 1.8, 0.5, 0.2, 1.3, 2.6, 0.9, 0.8. The autoradiogram was exposed for 31 hr. SPR, sparse samples; CN, confluent samples. B, the 180-kD band was excised and the incorporated [32P] quantitated by Cerenkov counting. Amount of label in the 180-kD band that was immunoprecipitated with P-tyr-preexposed antiserum was subtracted from the amount immunoprecipitated with native antiserum. ., sparse; @, confluent. B, inset, an analogous but independent experiment in which cells were stimulated with higher concentrations of PDGF.

i I 1 1 1 I

0 1

3 5 10 15 20 16 50

TIME (rnin)

Fig. 9. Time course of PDGF stimulation of [32P]H3P04-labeled sparse and confluent fibroblasts. A, Metabolically labeled fibroblasts were stimulated with 50 ngiml PDGF for the indicated time (mid and immunoprecipitated with antiserum pretreated (+) with P-tyr, or native antiserum 1-) and analyzed by 5-12.5% SDS-PAGE and autoradiography as described in Materials and Methods. Molecular mass markers are the same as in Figure 6. Number of cpm (xlO-? loaded/ lane starting from the right: 1.6,2.4, 0.4, 0.6, 1.5, 1.6, 0.4.0.6, 1.9,2.0, 0.6, 0.3. The autoradiogram was exposed for 31 hr. SPR, sparse samples; CN, confluent samples. B, the 180-kD band was excised and the incorporated 13'P] quantitated by Cerenkov counting. Plotted points represent the ["PI content of the 180-kD band precipitated with native antiserum minus the amount immunoprecipitated with P-tyr-pretreated antiserum. ., sparse; @, confluent.

234 KAZLAUSKAS A N D DrCORLETO

Vogel et al. (1980). In this report, we have examined potential mechanisms for the density-dependent response of cells to PDGF. This phenomenon cannot be explained on the basis of a greater concentration of PDGF per sparse cell relative to confluent cell, because at an equivalent amount of PDGF per cell, the density- dependent mitogenic effect is not diminished (Fig. 1). Perhaps the differential mitogenic response to PDGF results from a more rapid degradation of growth factor in confluent vs. sparse cells, yielding a lower actual amount of PDGF per confluent cell relative to sparse cells. Such an explanation is unlikely for the following reasons: 1) Vogel et al. (1980) reported that cell density, not cell number, determines the response to PGDF and that sparse cells can proliferate as well in fresh medium containing 5% calf serum as in medium containing 5% calf serum in which cells have become confluent; 2) At very high concentrations of PDGF (100 ng/ml), at which a relatively low percentage of the PDGF would be de- graded, the density-dependent effect is maintained.

Another explanation for the density-dependent mitogenic response to PDGF is that the majority of sparse cells is mitogenically similar to confluent cells, whereas a small subpopulation of the sparse cells is hyperrespon- sive to the PDGF mitogenic signal, thereby increasing the apparent proliferative response of the entire sparse population. We have no evidence at the present time that such a subpopulation does exist.

A simple explanation for the enhanced mitogenic response of sparse cells relative to confluent cells is that sparse cells have more PDGF receptors. However, our results demonstrated that sparse and confluent fibroblasts had the same number of PDGF receptors per cell. The apparent affinities of the two populations of receptors were also similar. Not unlike the response of cells to PDGF, it has been reported that epithelial, glial, and 3T3 cells respond to EGF in a density-dependent manner (Holley et al., 1977; Mierzejewski and Rozengurt, 1977; Westermark, 1977) such that, at the same concentration of EGF, sparse cells are more responsive than confluent cells. In epithelial cells, this phenomenon has been reported to be due, a t least in part, to a greater number of EGF receptors per sparse cell relative to confluent cell (Holley et al., 1977). In contrast, for both glial and 3T3 cells, confluent cells were reported to have more EGF receptors than sparse cells (Westermark, 1977; Pratt and Pastan, 1978). The receptor affinity was compared in the epithelial and glial cell studies and was determined to be similar. Thus the density-dependent response of cells to EGF, similar to our results with PDGF, is not satisfactorily explained by growth factor receptor number.

Carlin et al. (1983) reported that the decreased mitogenic response of senescent vs. young human fibroblasts to EGF was directly correlated to decreased tyrosine phosphorylation of the EGF receptor. In light of both this report and the apparent importance of tyrosine phosphorylation in growth control, we examined the possibility that regulation of the tyrosine kinase activity of the PDGF receptor was responsible for the density- dependent PDGF response. We found no striking differences in the time course or the extent of tyrosine or serine phosphorylation of the PDGF receptor in the two populations.

The concentrations of PDGF used in the receptor phosphorylation assays were much higher than those needed to stimulate phosphorylation. This requirement of large amounts of PDGF has also been reported by others in similar studies on PDGF-dependent kmase activity (Pike et al., 1983; Frackelton et al., 1984; Ek and Heldin, 1984). A high concentration of PDGF might be needed to fill quickly a sufficiently large number of receptors so that the early post-receptor binding event can be detectable. Thus this difference might reflect the much lower sensitivity of the phosphorylation assay, in which PDGF is present for minutes, relative to the mitogenesis assay, in which the PDGF is present for many hours.

The use of a phosphotyrosine antiserum in these experiments warrants mention of the following caveat. A quantitative comparison of the extent of phosphorylation is not valid if the PDGF receptor is phosphorylated on different domains and therefore is not similar in terms of immunoprecipitability. This, however, seems unlikely for the following reasons: 1) In the double label experiment, we showed that the mass of immunoprecip- itable PDGF receptor in sparse and confluent cells correlated tightly to the extent of tyrosine phosphorylation; 2) In isolated membranes, in which immunoprecipitability does not affect the results, the extent of tyrosine phosphorylation of the receptor in sparse and confluent samples was similar. Both the isolated membrane and whole cell immunoprecipitation phosphorylation assays are somewhat imprecise, and it is therefore possible that there is a small, consistent difference in the extent of the phosphorylation of the PDGF receptor of sparse and confluent cells that we are not able to detect. However, such a difference would be small relative to the difference in the PDGF-induced mitogenic signal of sparse and confluent fibroblasts.

In that sparse fibroblasts respond mitogenically to a greater extent than do confluent fibroblasts when stimulated with PDGF, it is apparent that tyrosine phosphorylation of the PDGF receptor is not tightly correlated to the PDGF mitogenic signal. Although the present work does not conclusively eliminate a role of tyrosine phosphorylation of the PDGF receptor in the mitogenic response, it suggests that there are other factors that regulate the magnitude of the PDGF signal. It would be interesting to examine other known postrecep- tor binding events such as tyrosine phosphorylation of the 42-kD protein that is phosphorylated in response to PDGF and has been implicated to play a role in growth control (Cooper et al., 1982; Nakamura et al., 1983; Kohno, 1985). These tyrosine phosphoproteins were detectable in our phosphotyrosine immunoprecipitated cells; however, they were present in only very small amounts and could not be quantitated in our present system. Alternatively, the density-dependent mitogenic response to PDGF might be manifested later in the mitogenic cascade, for example, a t the level of PDGF- induced c-myc oncogene expression (Kelly et al., 1983).

In light of the work demonstrating a correlation between tyrosine phophorylation of the EGF receptor and the EGF-induced mitogenic response of senescent vs. young fibroblasts (Carlin et al., 1983), it is curious that tyrosine phosphorylation does not constitute a primary factor in the density-dependent response of cells to PDGF. It is possible that regulation of the mitogenic


signal is different in response to PDGF, or, if cells have similar regulatory mechanisms for response to PDGF and EGF, perhaps growth inhibition resulting from density dependence and senescence does not proceed by the same mechanism.

ACKNOWLEDGMENTS We would like to thank Ms. Muriel Daly and Mr.

Endre Ritly for providing excellent secretarial and pho- tographic assistance. Human foreskin fibroblasts were prepared from tissue provided by the Perinatal Clinical Research Center (NIH USPHS MOlRR00210), Cleve- land Metropolitan General Hospital. This work was sup- ported by a grant from the National Heart, Lung, and Blood Institute, National Institutes of Health CHL- 29582). P.E.D. is the recipient of a Research Career Development Award (HL-01561) from the National Insti- tutes of Health.

LITERATURE CITED Bradford, M. (1976) A rapid and sensitive method for the quantitation

of microgram quantities of protein utilizing the principle of protein- dye binding. Anal. Biochem.,72:248-254.

Bowen-Pope, D.F., and Ross, R. (1982) Platelet-derived growth factor. II. Specific binding to cultured cells. J. Biol. Chem., 257:5161-5171.

Carlin, C.R., Phillips, P.D., Knowles, B.B., and Cristofalo, V.J. (1983) Diminished in vitro tyrosine kinase activity of the EGF receptor of senescent human fibroblasts. Nature, 306617-620.

Carpenter, G. (1981) Vanadate, epidermal growth factor and the stimulation of DNA synthesis. Biochem. Biophys. Res. Commun., 102: 1115-1121.

Cheng, Y.E., and Chen, L.B. (1981) Detection of phosphotyrosinecon- taining 34,000-dalton protein in the framework of cells transformed with Rous sarcoma virus. Proc. Natl. Acad. Sci. USA, 78~2388-2392.

Cooper, J.A., Bowen-Pope, D.F., Raines, E., Ross, R., and Hunter, T. (1982) Similar effects of platelet-derived growth factor and epidermal growth factor on the phosphorylation of tyrosine in cellular proteins. Cell, 31~263-273.

Cooper, J.A., and Hunter, T. (1981a) Changes in protein phosphorylation in Rous sarcoma virus transformed chick embryo cells. Mol. Cell. Biol.,I:165-178.

Cooper, J.A., and Hunter, T. (1981b) Four different classes of retrovi- ruses induce phosphorylation of tyrosine present in similar cellular proteins. Mol. Cell. Biol.,1:394-407.

Cooper, J.A., Sefton, B.M., and Hunter, T. (1983) In: Methods in Enzy- mology. S.P. Colowick and N.O. Kaplan, eds. Academic Press, New York, Vol. 99, pp. 387-405.

Daniel, T.O., Tremble, P.M., Frackelton, A.R. Jr., and Williams, L.T. (1985) Purification of the platelet-derived growth factor receptor by using an anti-phosphotyrosine antibody. Proc. Natl. Acad. Sci. USA, 822684-2687.

DiCorleto, P.E., and Bowen-Pope, D.F. (1983) Cultured endothelial cells produce a platelet-derived growth factor-like protein. Proc. Natl. Acad. Sci. USA, 80t1919-1923.

Dulbecco, R., and Stoker, M. (1970) Conditions determining initiation of DNA synthesis in 3T3 cells. Proc. Natl. Acad Sci. USA, 66~204- 210.

Ek, B., and Heldin C.H. (1982) Characterization of a tyrosine-specific k i n e acitivity in human fibroblast membranes stimulated by platelet-derived growth factor. J. Biol. Chem., 257~10486-10492.

Ek, B. and Heldin, C.H. (1984) Use of an antiserum against phosphotyrosine for the identification of phosphorylated components in human fibroblasts stimulated by platelet-derived growth factor. J. Biol. Chem., 259: 11145-11152.

Ek, B., Westermark, B., Wasteson, A., and Heldin, C.H. (1982) Stimu- lation of tyrosine-specific phosphorylation by platelet-derived growth factor. Nature, 295~419-420.

Frackelton, A.R. Jr., Tremble, P.M., and Williams, L.T. (1984) Evidence for the plateletderived growth factor-stimulated tyrosine phosphorylation of the platelet-derived growth factor receptor in vivo. J. Biol. Chem., 259:7909-7915.

Glenn, K.C., Bowen-Pope, D.F., and Ross, R. (1982) Platelet-derived growth factor. III. Identification of a plateletderived growth factor receptor by affinity labeling. J. Biol. Chem., 2575172-5176.

Glenn, K.C., and Ross, R. (1981) Human monocyte-derived growth factorts) for mesenchymal cells: Activation of secretion by endotoxin and concavalin A. Cell., 25:603-615.

Harel, L., Blat, C., and Chatelain, G . (1983) Density-dependent inhibition of growth: Fractionation of inhibitory diffusible factorb) re- leased by dense cultures of 3T3 cells. Biol. Cell, 48:ll-16.

Heldin, C.H. and Westermark, B. (1984) Growth factors: Mechanism of action and relation to oncogenes. Cell, 37:9-20.

Heldin, C.H., Westermark, B., and Wasteson, A. (1981) Specific receptors for platelet-derived growth factor on cells derived from connec- tive tissue and glia. Proc. Natl. Acad. Sci. USA, 78~3664-3668.

Holley, R.W., Armour, R., Baldwin, J.H., Brown, K.D., and Yeh, Y.C. (1977) Density-dependent regulation of growth of BSC-1 cells in cell culture: Control of growth by serum factors. Proc. Natl. Acad. Sci. USA, 74:5046-5050:

Hollev. R.W.. Bohlen. P.. Fava, R., Baldwin, J.H., Kleeman, G., and A h o u r , R.'(1980) Purification of kidney epithelial cell growth inhibitors. Proc. Natl. Acad. Sci. USA, 77:5989-5992.

Huang, J.S., Huang, S.S., Kennedy, B., and Deuel, T.F. (1982) Platelet- derived growth factor. Specific binding to target cells. J. Biol. Chem., 257:8130-8136.

Hunter, T., and Sefton, B.M. (1980) Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc. Natl. Sci. USA, 77~1311- 1315.

Kadowaki, T., Kasuga, M., Akanuma, Y., Ezaki, O., and Takaku, F. (1984) Decreased autophosphorylation of the insulin receptor-kinase in streptozotocindiabetic rats. J. Biol. Chem., 259:14208-14216.

Kelly, K., Cochran, B.H., Stiles, C.D., and Leder, P. (1983) Cell-specific regulation of the c-myc gene by lymphocyte mitogens and platelet- derived growth factor. Cell, 35~603-610.

Kohler, N., and Lipton, A. (1974) Platelets as a source of fibroblast growth-promoting activity. Exp. Cell Res., 87r297-301.

Kohno, M. (1985) Diverse mitogenic agents induce rapid phosphorylation of a common set of cellular proteins at tyrosine in quiescent mammalian cells. J . Biol. Chem., 260:1771-1779.

Laemmli, U.K. (1970) Cleavage of structural proteins during the as- sembly of the head of bacteriophage T4. Nature, 227~680-685.

Lieberman, M.A., Keller-McGandy, C.E., Woolsey, T.A., and Glaser, L. (1982) Binding of isolated 3T3 surface membranes to growing 3T3 cells and their effect on cell growth. J. Cell. Biochem., 20%-93.

Martin, G.S., Radke, K., Carter, C., Moss, P., Dehazya, P., and Gilmore, T. (1984) The role of protein phosphorylation of tyrosine in transformation and mitogenesis. J. Cell. Physiol. Suppl., 3t139-149.

Mierzejewski, K., and Rozengurt, E. (1977) Density-dependent inhibition of fibroblast growth is overcome by pure mitogenic factors. Nature, 269~155-156.

Nakamura, K.D., Martinez, R., and Weber, M.J. (1983) Tyrosine phosphorylation of specific proteins after mitogen stimulation of chicken embryo fibroblasts. Mol. Cell. Biol., 3~380-390.

Nishimura, J., Huang, J.S., and Deuel, T.F. (1982) Platelet-derived growth factor stimulates tyrosine-specific protein kinase activity in Swiss mouse 3T3 cell membranes. Proc. Natl. Acad. Sci. USA, 79~43034307.

Pike, L.J., Bowen-Pope, D.F., Ross, R., and Krebs, E.G. (1983) Charac- terization of platelet-derived growth factor-stimulated phosphorylation in cell membranes. J . Biol. Chem., 258:9383-9390.

Pratt, R.M., and Pastan, I. (1978) Decreased binding of epidermal growth factor to Balb/c 3T3 mutant cells defective in glycoprotein synthesis. Nature, 272:68-70.

Raines, E.W., and Ross, R. (1982) Platelet-derived growth factor. I. High yield purification and evidence for multiple forms. J. Biol. Chem., 257~5154-5160.

Ross, R., Glomset, J., Kariya, B., and Harker, L. (1974) A platelet- dependent serum factor that stimulates the proliferation of arterial smooth muscle cells in vitro. Proc. Natl. Acad. Sci. USA,71:1207- 1210.

Smith, J.B. (1983) Vanadium ions stimulate DNA synthesis in Swiss mouse 3T3 and 3T6 cells. Proc. Natl. Acad. Sci. USA, 80t6162-6166.

Steck, P.A., Voss, P.G., and Wang, J.L. (1979) Growth control in cultured 3T3 fibroblasts. Assay of cell proliferation and demonstration of a growth inhibitory activity. J. Cell. Bio1.,83:562-575.

Stoker, M.G.P. (1964) Regulation of growth and orientation in hamster cells transformed by polyoma virus. Virology, 24:165-174.

Swarup, G., Speeg, K.V.Jr., Cohen, S., and Garbers, D.L. (1982) Phos- photyrosyl-protein phosphatase of TCRC-2 cells. J . Biol. Chem., 257t7298-7301.

Todaro, G., Lazar, G., and Green, H. (1965) Initiation of cell division in a contact-inhibited mammalian cell line. J. Cell. Comp. Phys- io1.,66:325-334.

Vogel. A,. Ross. R.. and Raines. E. (1980) Role of serum comwnents in Zekityidependent inhibition' of growth of cells in cult&e. J. Cell Biol., 85~377-385.

Westermark, B. (1977) Local starvation for epidermal growth factor cannot explain densitydependent inhibition of normal glial cells.


Roc. Natl. Acad. Sci. USA,74:1619-1621. Westermark, B., and Wasteson, A. (1976) A platelet factor stimulating

human normal glial cells. Exp. Cell Res.,98:170-174. Williams, L.T., Tremble, P., and Antoniades, H.N. (1982) Platelet-

derived growth factor binds specifically to receptors on vascular

smooth muscle cells and the binding becomes nondissociable. Proc. Natl. Acad. Sci. USA,79:5867-5870.

Williams, L.T., Tremble, P., Lavin, M.F., and Sunday, M.E. (1984) Platelet-derived growth factor receptors form a high affinity state in membrane preparations. J. Biol. Chem., 259:5287-5284.

Documents

A comparison of the platelet-derived growth factor-dependent tyrosine kinase activity in sparse and confluent fibroblasts