Cellular Receptors for Type 6 Transforming Growth Factor LIGAND BINDING AND AFFINITY LABELING IN HUMAN AND RODENT CELL LINES*

Type fl transforming growth factor (BTGF) purified from human platelets to homogeneity as judged by NHderminal amino acid sequence analysis has been labeled with 1261 to characterize its interaction with cellular receptors. Binding of lZ6I-gTGF to target cells is temperature- and time-dependent, specific, satura-ble, and reversible. About 1.6-1.9 X lo' binding sites/ cell with high affinity for BTGF (Kd = 5.6-7.8 X lo-" M and 9.1-14 X lo-" M, respectively) are found in NRK-49F and BALB/c 3T3 cells. PTGF receptors do not appear to undergo acute down-regulation by the ligand. Specific binding of '261-@TGF has been ob- served in several human, rat, and mouse fibroblast lines and in some, but not all, tumor-derived cell lines examined. "'I-BTGF has been cross-linked to intact cells and isolated membrane preparations using disuc- cinimidyl suberate. Cells and isolated membranes from human, rat, and mouse origin affinity labeled with "'1- BTGF exhibit a major labeled species of approximately 280 kilodaltons that has the properties of high affinity and specificity expected from a physiologically relevant BTGF receptor. Minor labeled species of 70-90 kilodaltons are also labeled by '261-fiTGF, but they correspond to molecular species with low apparent affinity ( K d - lo-' M) for 12'I-BTGF.

with tyrosine protein kinase activity in vitro (11) and i n uiuo (12). The signal(s) elicited by the EGF/aTGF receptor in NRK-49F cells are necessary but not sufficient for acute transformation of fibroblasts in culture.
pTGF is a polypeptide found in transformed cells that also produce aTGF (5,6) and in normal tissues, including blood platelets (13)(14)(15)(16). It consists of two identically sized 11-12-kDa chains linked by disulfide bonds (13,14,16,17). By analogy to other polypeptide hormone systems, the cellular actions of PTGF are presumably exerted through its interaction with specific cell-surface receptors. This report shows that in various cell types, 'zSI-pTGF binds to a single class of saturable, high-affinity receptors specific for this ligand. Like the receptors for other polypeptide growth factors, receptors for pTGF are found in relatively low numbers in target cells.
To understand the mode of action of pTGF, it is important to gain information on the structural and functional properties of these receptors. However, the low abundance of this receptor type is a limiting factor in efforts to characterize its properties. To obviate this limitation, we have used receptor affinity labeling methodology consisting of cross-linking P G F receptors with receptor-bound ' z61-@TGF using a bifunctional reagent. This methodology has been useful in characterizing receptors for insulin (18,19), insulin-like growth factors I and I1 (20)(21)(22), aTGFs (10,23), platelet-derived growth factor (24), and other polypeptide hormones (25)(26)(27). This report describes the identification and properties of affinity labeled cellular components which have the characteristics of a physiologically relevant receptor for PTGF.

RESULTS
Structural and Biological Integrity of Radwiodinated PTGF-The preparations of platelet PTGF used in these studies contain one major 23-kDa polypeptide consisting of two identically sized chains of 11-12 kDa linked by disulfide bonds, as judged by dodecyl sulfate-polyacrylamide gel electrophoresis with and without reductant (dithiothreitol) and silver staining of the gels (Fig. IA). Two separate microsequencing analyses of reduced and 5'-carboxymethylated PTGF yielded one single N-terminal amino acid sequence, Staining material at the top of lanes b, c, and d is an artifact of the dithiothreitol-containing buffer. kDa, kilodalton. B, dodecyl sulfate-polyacrylamide gel electrophoresis of '*'I-flGF. Aliquots (80,000 cpm) were electrophoresed in the presence of dithiothreitol ( l a n e a) or without reductant ( l a n e b). An autoradiogram (4 h) from the fixed, dried gel is shown. Molecular size markers were run on a parallel lane that was excised and stained for protein after electrophoresis. C, induction of ['Hlthymidine incorporation into DNA by native (0) and '=I-labeled (0) flGF. The indicated concentrations of growth factor were added together with [methyL3H]thymidine (0.5 pCi/well) to sparse monolayers of NRK-49F cells in 16-mm wells that had been growth-arrested by incubation with medium containing 0.1% calf serum. [3H]Thymidine incorporated into trichloroacetic acid-insoluble material was determined 24 h later. ['HJThymidine incorporated into DNA in control cells treated with 5% calf serum was 218 pmol/ IO6 cell. Data are the average of duplicate determinations. D, induction of anchorage-independent proliferation by native flGF (0) and '*'I-PTGF (0). NRK-49F cells were plated in medium containing 0.35% agar, 0.3 nM EGF, and the indicated concentrations of flGF or "'I-flGF. Assays were read 8 days later. The per cent of cells growing into large colonies (>lo0 cells/colony) in each condition is plotted. Data are the average of duplicate plates. sequenators underwent Edman degradation. This sequence is fully consistent with and extends that recently reported for flGF from bovine kidney and human placenta (14). We therefore consider these platelet f l G F preparations to be near-homogeneous. '261-labeling of platelet flGF yielded a radioactive product of 23 kDa that could be reduced by dithiothreitol to ll-12-kDa species (Fig. 1B). Thus, '261-flGF in these preparations has structural properties similar to those of native /3TGF (Fig. 1B).

Lys-Asn-Cys-Cys-Val-X-X-Leu
Radiolabeling of polypeptide hormones has been reported (35) as potentially damaging for their biological activity. We compared the biological competence of native flGF and lZ5I-PTGF in two assays, [3H]thymidine incorporation into DNA in serum-depleted monolayers of NRK-49F cells and induction of anchorage-independent cell proliferation. In both assays, the potency of '251-/3TGF was very close to that of native f l G F (Fig. 1, C and D).
Specificity of lZ5I-j3TGF Binding-When near-confluent monolayers of NRK-49F rat fibroblasts were incubated for 4 h at 4 "C in the presence of 170 PM 1251-flGF, they bound about 2.1% of the input radioactivity. Various concentrations of native flGF and other growth factors were tested for their ability to compete with 1251-/3TGF for binding to NRK-49F cells. Only f l G F inhibited the binding of 1251-flGF in a concentration-dependent manner in NRK-49F cells (Fig. 2) and in BALB/c 3T3 cells (not illustrated). These results indicate that lZ5I-flGF binds specifically to target cells and that binding occurs at receptors that do not recognize other polypeptide growth factors.
BTGF and "'I-flGF bind to nonsiliconized glass and various types of plastic surfaces. Significant adsorption of "'I-PTGF to plastic culture dishes were observed in the absence of cells. However, binding of "'I-flGF to plastic dishes was not decreased by a 100-fold molar excess of PTGF. The presence of a confluent cell monolayer in the dishes markedly prevented nonspecific adsorption of lZ5I-flGF. Thus, the amount of radioactivity nonspecifically bound to culture dishes incubated with 170 PM lZ5I-flGF was decreased from about 4% of the input radioactivity in dishes without cells to 0.5% of the input radioactivity in dishes containing a confluent monolayer of cells (not illustrated).
Time-course and Reversibility of lZ5I-PTGF Binding- Fig. 3 illustrates the kinetics of lZ5I-flGF association to cellular receptors at 4 and at 37 "C. At 4 "C lZ5I-flGF binding was  half-maximal by 30-60 min and reached saturation 4 h after radioligand addition. The binding kinetics at 37 "C were considerably faster, maximal binding being obtained after about 25 min of incubation and remaining constant thereafter for at least 4 h (Fig. 3).
Progressive dissociation of cell-bound '251-flGF was obtained when cells preincubated with radioligand were washed free of unbound hormone and incubated at 37 "C in the presence of binding buffer (Table I). The relative dissociation was approximately the same regardless of whether cells had been preincubated with "'I-flGF for 0.5 or 3.5 h at 37 "C, or for 3.5 h at 4 "C. Very little (12%) dissociation of prebound lZ5I-PTGF was obtained in cells incubated for 3.5 h at 4 "C (Table I).
Affinity lZ5Z-BTGF Binding to Other Cell Lines-Various cell lines in addition to NRK-49F and BALB/c 3T3 cells have been screened for their ability to specifically bind lZ5I-@TGF (Table  11). Skin, kidney, and embryo fibroblasts of human and rodent origin were the cell lines with highest ability to bind lZ5If l G F among those tested. Some tumor-derived cell lines (A431 and HOS) exhibited significant binding of '251-@TGF, whereas others (A875 and H35) did not. Retrovirally transformed rat and mouse fibroblasts known to release @TGF were found to bind 12"I-j3TGF, but at lower levels than nontransformed fibroblasts.
Affinity Labeling of NRK-49F Cells and Membranes with '25Z-/3TGF-We attempted to affinity label cellular receptors for BTGF by cross-linking intact cells and isolated membrane preparations with cell-or membrane-bound "'I-flGF using the homobifunctional agent, disuccinimidyl suberate (DSS) (18). Fig. 6 shows an autoradiogram from a dodecyl sulfatepolyacrylamide electrophoresis gel of NRK-49F cells and isolated NRK-49F membranes cross-linked with '251-@TGF. Cells and membranes were sequentially incubated in the presence of '251-@TGF and DSS. The cross-linked samples were extracted with the nonionic detergent Triton X-100, and the detergent-solubilized material was subjected to electrophoresis and autoradiography. One major labeled species that migrated slowly (M, = 250-300 x lo3) on 7% polyacrylamide gels was observed in samples from affinity labeled intact NRK-49F cells (Fig. 6b). A relatively low (1.5 nM) concentration of @TGF (Fig. 6c) inhibited the labeling of this species by about 70% as determined by excising the corresponding gel segments and counting their radioactivity in a y counter. This labeled species was not observed in cells that were incubated with '261-@TGF but not exposed to DSS (Fig. 6a) or in control experiments in which incubations with '251-@TGF and DSS were performed in the absence of cells (not illustrated). Affinity labeled NRK-49F membranes (Fig. 6, d-f) exhibited a major labeled species with molecular size and labeling properties similar to those of the major species labeled in intact cells. These results are consistent with this labeled species corresponding to a membrane-associated cellular receptor with high affinity for BTGF.
Minor labeled bands were observed in the 70-90-kDa region of the gels ( Fig. 6 and following ones). Their labeling could not be inhibited by 1.5 nM @TGF present during incubation of cells or membranes with '251-@TGF. However, the labeling of some of these bands was effectively inhibited by higher concentrations of BTGF (see below). Thus, these labeled species seem to correspond to cellular components with lower apparent affinity for '251-@TGF. Some of these labeled bands are likely nonspecific since they appeared even in cells and membrane samples that had not been exposed to DSS following incubation with '"I-@TGF (Fig. 6, a and d).
Affinity Labeling and Detergent Solubility of PTGF Receptors in Intact Cell Monolayers-Human WI-38 lung fibroblasts, mouse Swiss 3T3 fibroblasts, and BALB/c 3T3 fibroblasts cross-linked with lZ5I-@TGF showed a major specifically la- Human osteosarcoma 9 f 2 ( n = 2 ) 60 A431 Human epidermoid carcinoma 9 * 1 ( n = 3 ) 54 A875 Human melanoma e 2 (n = 2) e10 beled component similar in size to, or somewhat larger (WI-38 cells, Fig. 7) than, the species labeled in NRK-49F cells (Figs. 7 and 8A). A molecular size of 280 kDa was estimated for these '251-PTGF-labeled species in high-porosity gels (Figs. considered at present an approximate value. Most of the intact cell experiments in these affinity labeling studies were performed with cell suspensions rather than intact cell monolayers to reduce the incubation volume and, consequently, the amounts of homogeneous flGF and lZ5I-PTGF to be used. However, some affinity labeling experiments were done with intact cell monolayers to test whether cell detachment with EDTA-containing buffer might have altered the properties of cellular components labeled with lZ5I-PTGF. The results shown in Fig. 7 correspond to one such experiment. Comparison between the results obtained with BALB/ c 3T3 and NRK-49F cells in this experiment (Fig. 7) and in experiments using cell suspensions (Fig. 8A) shows that apparently the same cellular components were affinity labeled with "'I-PTGF, regardless of whether intact cell monolayers or cell suspensions were used. As in other experiments in this study, the samples electrophoresed on the gel shown in Fig. 8A were extracts from the corresponding affinity labeled cells obtained with the nonionic detergent, Triton X-100. The purpose of this detergent extraction was to optimize electrophoretic resolution by reducing the amount of cellular protein loaded on the gels. Fig. 8B shows an autoradiogram from a gel containing the Tritoninsoluble material corresponding to the samples run on the gel in Fig. 8A. A faint trace of nonspecifically labeled 70-kDa material was the only labeled species detected in these gels (Fig. 8B). In other control experiments (not shown), small samples of '2sI-pTGF-labeled cells that had been directly solubilized with sodium dodecyl sulfate exhibited the same pattern of labeled species as the Triton extracts shown in Figs. 7 and 8. These observations indicate that affinity labeled "TI-PTGF receptors can be quantitatively extracted from intact cells by Triton X-100 and that extraction with this detergent does not alter the electrophoretic properties of "'I-PTGF-labeled cellular components.
Specificity of Cellular Components Affinity Labeled with PTGF-The labeling of the 280-kDa receptor in BALB/c 3T3 cells was detectable in original autoradiograms with as little as 25 PM '2sI-PTGF. The increase in labeling of this receptor species with increasing concentrations of "'I-flGF in the 25-150 PM range (Fig. 9, a-d) roughly paralleled the isotherm of 12'I-PTGF binding to intact cells (Fig. 5). Furthermore, 1.5 nM native D G F added during incubation of cells with 150 PM '2sI-PTGF inhibited almost completely the labeling of the 280-kDa receptor species (Fig. 9e). This observation is consistent with an affinity of the 280-kDa receptor for f l G F close to the affinity of flGF receptors determined in the radioligand binding experiments described above. In contrast, EGF, aTGF, insulin, insulin-like growth factor-11, and nerve growth factor which do not compete with 12sI-PTGF for binding to cellular receptors did not affect the labeling of the 280-kDa receptor (Fig. 9, h-1). Similar results were obtained when isolated membrane preparations were affinity labeled with 12'I-PTGF in the presence of an excess of each of these growth factors (not illustrated). Fig. 9 also shows that a relatively large (45 nM) excess of native PTGF is needed to completely inhibit the labeling of 70-90-kDa components and other minor species by "'I-BTGF in BALB/c 3T3 cells (Fig. 9g). These observations suggest that the 70-90-kDa-labeled bands correspond to cellular components with an average apparent Kd for "'I-flGF of about Effect of Various Cross-linking Concentrations-Various concentrations of DSS over a 10-fold (50-500 p M ) concentration range were assayed for their ability to cross-link "'I-flGF to intact cells. The 280-kDa species was increasingly labeled by increasing concentrations of DSS, but even under the mildest cross-linking condition used (50 p~ DSS), this labeled species exhibited a diffused migration on the electrophoresis gels (Fig. 10). In all the experiments described here, the diffused appearance of the 280-kDa band was in contrast to the sharpness of Coomassie Blue-stained protein bands in the high molecular weight region of the gels (not illustrated). Thus, the anomalous migration of the 280-kDa band on dodecyl sulfate-polyacrylamide gels is not likely due to random cross-linking of the receptor species with other cell and membrane components, as observed with high (>0.5 mM) concentrations of DSS in other receptor affinity labeling studies (36, 37). The inability of N-hydroxysuccinimidyl pazidobenzoate to cause the labeling of the 280-kDa species with '"I-flGF (Fig. lOf) argues that the labeling induced by lo-' M.

_ . C .
_. r " " - DSS is not simply an artifact of derivatization of free amino groups by succinimidyl derivatives.
Effect of Preincubation with PTGF on '2sZ-BTGF Binding and PTGF Receptor Affinity Labeling-An acute decrease, or "down-regulation," of the number of receptors for various growth factors occurs soon after exposure of cells to the respective ligands at 37 "C (38)(39)(40). The acute down-regulation of these receptors often results in biphasic binding kinetics at 37 "C with a peak of maximal ligand binding within the first hour of incubation followed by a marked decrease in binding over the next few hours (38)(39)(40). In contrast to this phenomenology, repeated experiments showed that 12'I-flGF binding at 37 "C does not significantly decrease after reaching apparent equilibrium (Fig. 3). These results suggested that acute down-regulation of pTGF receptors may not occur in the cell lines used in these studies. To further investigate this possibility, monolayers of BALB/c 3T3 cells or NRK-49F cells were incubated in the presence of saturating (0.5-2 nM) concentrations of f l G F for 2 or 6 h. Cells were then washed for 1.5 h at 37 "C, and their ability to specifically bind "' 1-PTGF was measured at 4 "C. It was found that the concentrations of f l G F tested decreased the binding of 150 PM 12' 1-PTGF to 69-84% of the values obtained in untreated cells (not illustrated).
However, control experiments using 1251-flGF instead of native pTGF in the first incubation indicated that a portion of the observed decrease in subsequent '"I-flGF binding was due to residual occupancy of cellular receptors by j3'TGF from the first incubation. Thus, residual receptor occupancy accounted for about half of the measured decrease in '251-flGF binding to cells preincubated with 0.5 nM flGF (data not shown). These results suggest that down-regulation of flGF receptors in BALB/c 3T3 cells occurred to a very limited extent ( 4 5 % of binding to control cells) in these experiments, if at all. Control experiments to monitor degradation of pTGF or '251-pTGF (by radioreceptor assay and trichloroacetic acid precipitability) during incubation in the presence of a cell monolayer at 37 'C indicated that the concentrations of ligand used remained saturating for flGF receptors in these cells throughout the entire incubation period (not shown).
We examined directly the effect of preincubation of NRK-49F cells with f l G F and lBI-flGF on the susceptibility of the cells to affinity labeling by l2'1-flGF. Monolayers of NRK-49F cells were incubated for 20 min or 2 h in the presence of 0.5 nM f l G F or 0.5 nM 1261-flGF. They were then extensively washed at 37 "C and affinity labeled with a subsaturating (150 PM) concentration of lZ5I-flGF in the cold. After electrophoresis and autoradiography of the resulting cell extracts, a progressive decrease in the intensity of labeling of the BTGF receptor could be detected with time of exposure to f l G F (Fig. 11). However, the labeling of this species was increased in cells that had been preincubated with lZ5I-BTGF instead of f l G F (Fig. 11). Thus, the decrease in receptor labeling after exposure to f l G F was most likely due to residual occupancy of receptor sites by f l G F carried over in the dishes from the preincubation step even after extensive washing at 37 "C. DISCUSSION This report demonstrates the presence of high-affinity receptors for f l G F in various cultured cell lines. The binding of lZ5I-flGF to these receptors is time-and temperaturedependent, saturable, reversible, and competed for by P G F but not by five other polypeptide growth factors tested.
The known biological effects of f l G F are half-maximal at 1-10 PM concentration and maximal at about 40 pM concentration ( Fig. 1 and see Refs. 13,14,16,17). Predictably, lZ5I-pTGF interacts with binding sites that exhibit a K d in the picomolar range. According to the K d values obtained here, however, occupancy of only about 20% of the @TGF receptors (by about 40 PM n G F ) is required for maximal biological action. Furthermore, this level of receptor occupancy could be an overestimate, considering that a substantial fraction of the added BTGF is likely to be degraded during the course of bioassays that last for up to several days, as in the case of the soft agar colony formation assay. It is therefore possible that a "spare" number of receptors exists for flGF, as it has been proposed for other polypeptide hormone receptor systems such as the insulin receptor (41,42). However, this possibility is difficult to rigorously assess because of the limitations in accurately determining the affinity of f l G F receptors at the bioassay temperature of 37 "C, a temperature at which complex receptor dynamics and significant degradation of radioligand added at low concentrations could occur.
The bifunctional agent DSS can cause the cross-linking of receptor-bound lZ5I-flGF to a 280-kDa cellular component that has many of the properties expected from physiologically relevant, membrane-associated f l G F receptors. First, this 280-kDa component exhibits an apparent affinity for f l G F high enough to mediate the effects of this growth factor at low PM concentrations. Second, this receptor species is specific for flGF and does not cross-react with other growth factors tested. f l G F purified from feline sarcoma virus-transformed rat cells was also able to inhibit the labeling of the 280-kDa receptor by platelet-derived lZ5I-flGF (not illustrated). Third, this 280-kDa component is labeled in intact cells as well as in isolated membrane preparations. Fourth, it is found in mouse, rat, and human cell types that bind lZ5If l G F specifically and respond to flGF. We have also detected this receptor species in Snyder-Theilen feline sarcoma virus-transformed rat embryo fibroblasts and in Moloney murine sarcoma virus-transformed 3T3 mouse cells (not illustrated), two cell lines that produce f l G F along with aTGF, and which may be stimulated by these factors in an autocrine fashion (1). However, we have been unable to detect the 280-kDa PTGF receptor species in A875 human melanoma and H35 rat hepatoma (not illustrated), two cell lines which do not exhibit high-affinity binding sites for lZ5I-mGF. Finally, the relatively high efficiency (about 13%) of DSS to cross-link lZ5I-flGF with the 280-kDa cellular component is consistent with the hypothesis that this component is the major receptor type for flGF in the cell lines studied here.
Results of experiments using various concentrations of cross-linking agents (Fig. 10) do not support the possibility that the diffused appearance of the 280-kDa-labeled component is due to random cross-linking with other membrane components. In other control experiments (not illustrated), high molecular size receptors for insulin-like growth factors I and 11, insulin, EGF, and aTGF affinity labeled by crosslinking with the respective '261-ligands in BALB/c 3T3 and NRK-49F cells as described before (18-23) migrated on the gels as well-defined labeled bands in parallel with the diffused 280-kDa species labeled by '"I-flGF. This obse'rvation argues that the anomalous migration of the 280-kDa receptor species is not an intrinsic problem of the methodologjl used. Like many membrane-associated proteins, the 280-kDa f l G F receptor identified here may be glycoprotein. In support of this possibility, recent experiments3 show that BTGF-receptors bind specifically to immobilized lectin columns. The migration of certain glycoproteins on dodecyl sulfate-polyacrylamide gels is anomalous due to the heterogeneity of the carbohydrate moiety and variable binding of detergent (43,44). It will be of interest to determine whether the 280-kDa f l G F receptor species identified in the present studies is a heterogeneously glycosylated protein.
A class of 70-90-kDa cellular components with low apparent affinity (&lo-' M) for 1251-flGF has also been detected in the affinity labeling experiments reported here. This class of low affinity binding sites may escape detection in '%If l G F binding studies because its apparent K d is 2 orders of magnitude higher than the K d for high-affinity f l G F binding sites. The significance of this class of binding sites with low affinity for Y -f l G F is unclear. The known cellular effects of f l G F are exerted at concentrations of this growth factor (1-40 PM) which would not effectively bind to this class of sites. It is possible that these lower affinity sites play a role in as yet unidentified cellular events involving flGF. Alternatively, these 70-90-kDa-labeled species may correspond to degradation products of the major 280-kDa receptor species that would pre-exist in the intact cells or occur during incubation with 1251-flGF. The observed heterogeneity and variable labeling of these species in the present studies supports this possibility. The receptor for EGF and aTGF and the PDGF receptor undergo acute down-regulation when cells are exposed to the respective ligands at 37 "C. No evidence has been obtained in the present studies for biphasic binding kinetics of lZ5I-flGF at 37 "C or for a markedly decreased f l G F receptor levels in BALB/c 3T3 or NRK-49F cells pretreated with various concentrations of lZI-flGF. These results suggest that unlike the receptors for EGFIaTGF and PDGF, the receptors for f l G F may not undergo acute down-regulation.
Available information about the target cell selectivity of f l G F is limited because studies have focused on the ability of flGF to induce anchorage-independent cell proliferation. Induction of anchorage-independent proliferation by f l G F is a complex cellular response with specific requirements including the cooperative action other growth factors (5)(6)(7)(8). These requirements may be different for each cell type, making difficult the identification of f l G F target cells. The identification of potential targets for f l G F can therefore be facilitated by structural identification and measurement of cellular receptors for this growth factor. For example, four fibroblast lines that respond differently to PTGF in soft agar assays, rat NRK-49F, human WI-38, mouse BALB/C 3T3, and Swiss 3T3, were all found to exhibit receptors for PTGF.
The studies reported here suggest that the physiologically relevant PTGF receptor consists of a 280-kDa-binding component. Obviously, other PTGF receptor subunits could exist that interact with this binding species and become dissociated upon solubilization and electrophoresis of affinity labeled cells and membranes. Further characterization and isolation of the PTGF receptor is needed to address this and other questions on the structure of BTGF receptors. The information derived from the present studies should help initiate efforts to quantitatively solubilize and isolate this receptor type.
After submission of this paper, Frolik et aE. (45) and Tucke et al. (46) reported specific binding of '251-6TGF to NRK-49F cells. The affinity and binding capacity for '*'1-/3TGF reported by these authors are very similar to the results reported here. These authors, however, report significant down-regulation of '251-PTGF_Pinding to NRK-49F cells after preincubation of cells with unlabeled PTGF (45).