they may all interact with the same receptor(s), and the two closest members of the family, bFGF and acidic FGF, have been shown to compete for receptor ...
Proc. Nad. Acad. Sci. USA Vol. 87, pp. 4378-4382, June 1990 Biochemistry
A murine fibroblast growth factor (FGF) receptor expressed in CHO cells is activated by basic FGF and Kaposi FGF (fibroblast growth factor receptor phosphorylation/growth factors/mitogenic activity)
ALKA MANSUKHANI, DAVID MOSCATELLI, DANIELA TALARICO, VERA LEVYTSKA, AND CLAUDIO BASILICO Departments of Microbiology and Cell Biology, New York University School of Medicine, New York, NY 10016
Communicated by David D. Sabatini, March 26, 1990
ter cell exposure to both FGFs, and a significant proliferative response is obtained.
We have cloned a murine cDNA encoding a ABSTRACT tyrosine kinase receptor with about 90% similarity to the chicken fibroblast growth factor (FGF) receptor and the humanfms-like gene (FLG) tyrosine kinase. This mouse receptor lacks 88 amino acids in the extracellular portion, leaving only two immunoglobulin-like domains compared to three in the chicken FGF receptor. The cDNA was cloned into an expression vector and transfected into receptor-negative CHO cells. We show that cells expressing the receptor can bind both basic FGF and Kaposi FGF. Although the receptor binds basic FGF with a 15- to 20-fold higher affinity, Kaposi FGF is able to induce down-regulation of the receptor to the same extent as basic FGF. The receptor is phosphorylated upon stimulation with both FGFs, DNA synthesis is stimulated, and a proliferative response is produced in cells expressing the receptor, whereas cells expressing the cDNA in the antisense orientation show none of these responses to basic FGF or Kaposi FGF. Thus this receptor can functionally interact with two growth factors of the FGF family.
MATERIALS AND METHODS Library Screening and Sequencing. A Agt1O cDNA library was screened using an 800-base-pair fragment from the tyrosine kinase portion of the bek gene as a probe (8). Filters were hybridized overnight at 650C in 5x SSC/10x Denhardt's solution/0.1% SDS containing denatured calf thymus DNA (100 ,ug/ml). (1 x SSC = 0.15 M NaCl/0.015 M sodium citrate, pH 7.0; lx Denhardt's solution = 0.02% polyvinylpyrrolidone/0.02% Ficoll/0.02% bovine serum albumin.) The filters were then washed at 650C with 2x SSC/0.1% SDS for 30 min and with lx SSC/0.1% SDS for 45 min. The cDNAs were subcloned into pBluescript (Stratagene) and sequenced by the dideoxynucleotide chain-termination method (9). Cell Lines and Transfections. CHO-DG44 cells, which are dihydrofolate reductase-negative (DHFR-), were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (vol/vol) fetal calf serum, 5 mM hypoxanthine, 2 mM thymidine, and 10 mM proline. Cells were transfected by calcium phosphate precipitation with cDNA 4A in p91023B vector (10), which carried DHFR as a selectable marker. Positive clones were selected in medium lacking hypoxanthine and thymidine. Scatchard Analysis. Recombinant bFGF was iodinated as described (11) (specific activity = 552 cpm/fmol). CHO clone 3-3 and clone 4-1 cells were incubated at 40C with DMEM containing 0.15% gelatin, 25 mM Hepes (pH 7.4), and various concentrations of '2"I-labeled bFGF from 0.3 to 7 ng/ml. After 2 hr, the medium was removed, the cells were washed once with ice-cold isotonic phosphate-buffered saline (PBS), and 1251I-labeled bFGF bound to the extracellular matrix was removed by washing twice with 2 M NaCl/20 mM Hepes, pH 7.4 (11). Radioactive bFGF bound to high-affinity receptors was released with two subsequent washes with 2 M NaCl/20 mM sodium acetate, pH 4.0. Radioactivity in the combined medium and PBS wash (free fraction) and in the combined 2 M NaCl washes buffered at pH 4 (bound fraction) was quantitated on a Beckman y scintillation counter. The cells of parallel cultures were treated with trypsin and quantitated using a Coulter particle counter. Competition Assays. To investigate the ability of K-FGF to displace 1251-labeled bFGF, CHO cells were incubated at 40C with DMEM containing 0.15% gelatin, 25 mM Hepes (pH 7.4), 1251I-labeled bFGF (3 ng/ml), and the indicated concentrations of unlabeled bFGF or K-FGF. To decrease nonspecific binding, heparin (10 ,ug/ml) was included in all assays (11). After 2 hr, the medium was removed, and 1251-labeled
The mitogenic effect ofgrowth factors requires binding to and activating specific cell surface receptors, many ofwhich have tyrosine kinase activity (1, 2). Basic fibroblast growth factor (bFGF) and Kaposi fibroblast growth factor (K-FGF) belong to a family of heparin-binding polypeptides that are mitogenic for a variety of mesoderm- and neuroectoderm-derived cell lineages, are angiogenic, and are also thought to play a role in development (3). The protein sequences of bFGF and K-FGF share a conserved region of about 40% homology, but the N-terminal 80 amino acids of K-FGF are unique (4). Furthermore, whereas bFGF lacks a signal peptide and is not efficiently secreted, K-FGF has a hydrophobic leader sequence directing cleavage and secretion into the extracellular compartment (5). In spite of these differences, bFGF and K-FGF, and indeed most fibroblast growth factors (FGFs), appear to exhibit a similar spectrum of action, suggesting that they may all interact with the same receptor(s), and the two closest members of the family, bFGF and acidic FGF, have been shown to compete for receptor binding (6). Crosslinking studies with radiolabeled FGFs have identified two highaffinity receptors with molecular masses ranging between 125 and 210 kDa on a variety of cell types (3, 6). The cloning of a chicken cDNA encoding a putative bFGF tyrosine kinase receptor was reported (7). Here we describe that the protein encoded in a murine cDNA clone that we isolated and sequenced* can bind both bFGF and K-FGF when expressed in receptor-negative CHO cells, although with different affinities. The receptor molecule, which is about 90% homologous to the chicken bFGF receptor but is shortened in the extracellular portion, is internalized and phosphorylated af-
Abbreviations: FGF, fibroblast growth factor; bFGF and K-FGF, basic FGF and Kaposi FGF, respectively; DHFR, dihydrofolate reductase; FLG, fms-like gene. *The sequence reported in this paper has been deposited in the GenBank data base (accession no. AA05374).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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Biochemistry: Mansukhani et al. bFGF bound to high-affinity receptors was determined as described above. The ability of bFGF and K-FGF to displace 125I-labeled K-FGF was also compared. Recombinant K-FGF was iodinated with "25I-labeled Bolton-Hunter reagent. The radioactive K-FGF was repurified on heparin-Sepharose with 0.15% gelatin added to all buffers as a carrier protein and had a specific activity of 63 cpm/fmol. After 2 hr of incubation, the medium was removed, the cells were washed twice with ice-cold PBS and were extracted in 0.5% Triton X-100/0.1 M sodium phosphate, pH 8.1. Radioactivity in the extracts was quantitated in a y scintillation counter. Down-Regulation. CHO clone 3-3 cells were incubated for 5 hr at 370C in DMEM containing 0.15% gelatin, heparin (10 ttg/ml), and the indicated concentrations of bFGF or K-FGF. The cells were washed twice with ice-cold 2 M NaCl/20 mM sodium acetate, pH 4.0, and twice with ice-cold PBS to remove bound growth factors. The cells were then incubated at 40C in DMEM containing 0.15% gelatin, 25 mM Hepes (pH 7.4), heparin (10 tzg/ml), and 125I-labeled bFGF (5 ng/ml). After 2 hr, the cells were washed as above, and 125I-labeled bFGF bound to high-affinity receptors was quantitated. Immunoblot Analysis. Clones 3-3, 4-1, and A-1 were exposed to bFGF or K-FGF at 20 ng/ml for 15 min at 37°C after serum deprivation for 24 hr. Then 60-mm plates with 1 x 106 cells were rinsed with PBS/5 mM sodium orthovanadate and immediately lysed with 100 ,ul of boiling lysis buffer [2.5% (wt/vol) SDS/125 mM Tris-HCl, pH 6.8]. Protein lysates were sonicated, electrophoresed on an 8% polyacrylamide/ SDS gel, and transferred to nitrocellulose. The blot was incubated with affinity-purified anti-phosphotyrosine antibodies (gift from H. Hanafusa, Rockefeller University) and was developed using 125I-labeled protein A (Amersham). DNA Synthesis Assay. Approximately 2 x 104 cells were plated in DMEM/10% fetal calf serum on 24-well plates. The next day the cells were washed and transferred to DMEM/ F-12 containing 0.25% bovine serum albumin fraction V (Sigma). Forty-eight hours later, bFGF (10 ng/ml) or K-FGF (10 ng/ml) was added alone or with transferrin (5 ,ug/ml). Twelve hours later cells were labeled with [3H]thymidine (5 ,uCi/ml; 6.7 Ci/mmol; 1 Ci = 37 GBq) for 6 hr. Cells were washed with PBS and harvested in 0.05 mM Tris HCl, pH 7.5/5 mM EDTA/0.15 M NaCl/0.6% SDS, and the amount of [3H]thymidine incorporated into trichloroacetic acidprecipitated DNA was determined in a Beckman liquid scintillation counter. Growth Response. Approximately 5 x 104 cells were plated in 35-mm dishes in DMEM/10% fetal calf serum. Cells were transferred to DMEM/F-12 containing 0.25% bovine serum albumin. Forty-eight hours later (time 0), cells on two plates per clone for clones 4-1, 3-3, and A-1 were counted and transferrin (5 ,ug/ml) was added alone or along with K-FGF or bFGF at 10 ng/ml. The medium was changed every two days.
RESULTS AND DISCUSSION Several lines of evidence indicated that the FGF receptor(s) had tyrosine kinase activity (12-15), and the sequence of the chicken FGF receptor reported by Lee et al. (7) showed homology to two putative tyrosine kinases. Those were the human fms-like gene (FLG) (16) and the mouse bek (8) proteins, whose sequence had been deduced from partial cDNAs. Conserved regions within the kinase domains of identified genes have been used to search for related genes using cross-hybridization techniques (16). We used this approach to isolate murine cDNA clones encoding functional FGF receptors. Cloning of a Murine cDNA Encoding an FGF Receptor. A cDNA library constructed in the AgtlO vector from A-15 cells
Proc. Natl. Acad. Sci. USA 87 (1990)
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(4) (NIH 3T3 cells transformed by the K-fgf cDNA was screened with the tyrosine kinase portion of the mouse bek cDNA (8). Eighteen positive clones were isolated from 5 x 105 plaques. Upon subcloning and dideoxynucleotide sequencing 8 of these clones proved to be shorter versions of a long (3.0 kilobase) cDNA clone named 4A. Clone 4A contained a long open reading frame (2.2 kilobase) that could encode a protein with 93-94% similarity with the partial sequence of the FLG protein and the chicken bFGF receptor, respectively. It also showed significant but lower homology (91%) to the bek-encoded protein. The 2.2-kilobase open reading frame has a methionine codon 43 base pairs downstream from the 5' end of the cDNA, and the first 20 amino acids encode a putative hydrophobic signal sequence, suggesting that this clone does contain the N terminus of the receptor. The 3' end of the cDNA has a 741-nucleotide untranslated region that contains a polyadenylylation signal (ATTAAA) 13 bases upstream of the poly(A) tail. The cDNA encodes a protein of 733 amino acids with a deduced mass of 80.6 kDa (Fig. 1). Using the cDNA as a probe, a single band of =3.4 kilobases is seen on Northern blots of RNA extracted from NIH 3T3 cells and various mouse tissues (data not shown). Like the chicken FGF receptor, the mouse protein has the features of a membrane-spanning glycoprotein (Fig. 1). A hydrophobic stretch of 21 amino acids is seen (residues 287-307), characteristic of a transmembrane region, and eight consensus sequences for N-linked glycosylation are found mostly in the presumptive extracellular portion of the protein. The intracellular kinase domain has %-97% similarity to the chicken and human (FLG) FGF receptors. This sequence of 425 amino acids is a split kinase domain with an unusually short kinase-insertion region of 14 amino acids, and a long juxtamembrane region (residues 308-396) as in the chicken FGF receptor. It maintains all of the features of tyrosine kinases including the Gly-Xaa-Gly-Xaa-Xaa-Gly motif im-
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sented by straight lines. Boxes indicate coding regions. Crosshatched box designates the transmembrane region. Solid box represents the acidic region. The predicted amino acid sequence of the longest open reading frame is shown using the single-letter code. The cysteine residues are marked by solid squares and the potential N-linked glycosylation sites by ovals. The arrow marks the site of the deletion of 88 amino acids with respect to the chicken receptor (7). The transmembrane region is underlined, and the acidic segment in the extracellular domain and the conserved motifs of the tyrosine kinase domain are highlighted. bp, Base pairs.
Biochemistry: Mansukhani et al.
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portant for ATP binding, and the conserved His-Arg-AspLeu-Ala-Ala-Arg-Asn-Val-Leu and Asp-Phe-Gly-Leu-AlaArg motifs (17). The extracellular domain of the mouse FGF receptor is 90% similar to the chicken FGF receptor. The cysteine residues in this region are perfectly conserved, suggesting that the immunoglobulin-like domain structure is maintained. However, the mouse receptor clone lacks 88 amino acids (between residues 30 and 31) comprising the first immunoglobulin domain of the chicken receptor (7). This mouse FGF receptor, therefore, has only two such domains in the extracellular portion whereas the chicken has three. The unusual acidic region ofeight consecutive acidic residues [Glu-(Asp)7] is maintained. Expression in Receptor-Negative Cells. The 4A cDNA was cloned into the p91023B vector (10) under the control of the adenovirus major late promoter and transfected into DHFRCHO cells (clone DG44) that lack FGF receptors and do not express any RNA hybridizing to a 4A cDNA probe (unpublished observations). DHFR' clones were selected and tested for their ability to bind 1251-labeled bFGF. Cells transfected with the cDNA inserted in the vector in the antisense orientation were used as negative controls. CHO parental cells or cells expressing the antisense cDNA (clone A-1) bound equal amounts of '251-labeled bFGF, representing background levels (1000-2000 high-affinity binding sites), whereas positive clones ranged from 10,000 to 140,000 sites per cell (data not shown). Two positive clones were used for further analysis. When cells from the positive clones (clones 3-3 and 4-1) were incubated with 125I-labeled bFGF and a homobifunctional cross-linking reagent, a cross-linked 125[labeled bFGF-receptor complex with a molecular mass of 150 kDa was obtained (Fig. 2). Subtraction of the molecular mass of bFGF yields an estimated molecular mass of 130 kDa for the expressed receptor. The difference between this molecular mass and that of the primary translation product is probably due to glycosylation. Binding of bFGF and K-FGF. The concentration dependence of the binding of 125I-labeled bFGF to CHO clones 3-3 and 4-1 was investigated. Scatchard analysis of binding to high-affinity receptors gave a straight line for both clones, indicating a single class of binding sites (Fig. 3). From this data CHO clone 3-3 was calculated to have 21,000 receptors per cell with a Kd of 1.7 x 10-11 M. The Kd is similar to the Kd calculated for the bFGF receptor of BHK cells (11). CHO clone 4-1 was calculated to have 100,000 receptors per cell with a Kd of 7.4 x 10-11 M. The reason for the difference in 1
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FIG. 2. Cross-linking of 125Ilabeled bFGF to CHO cells expressing the FGF receptor. CHO cells transfected with the FGF receptor in the antisense orientation (clone A-1, lanes 1 and 2) or with the FGF receptor in the sense orientation (clone 4-1, lanes 3 and 4) were incubated at 4°C with 1251-labeled bFGF (10 ng/ml) in DMEM containing 0.15% gelatin and 25 mM Hepes (pH 7.4) (lanes 2 and 4) or in the same medium containing unlabeled bFGF (1 ,ug/ml) (lanes 1 and 3). The cross-linking reagent, bis(sulfosuccinimidyl)suberate, dissolved in dimethyl sulfoxide was added to the medium to a final concentration of 1 mM, and the cells were incubated for a further 15 min at room temperature. The cells were then extracted in electrophoresis sample buffer, and the extracts were analyzed on a 7% polyacrylamide gel containing SDS, followed by auto-
radiography.
Proc. Natl. Acad Sci. USA 87 (1990)
Kd is unknown. There was no difference in 125I-labeled bFGF binding to extracellular matrix in clones A-1, 3-3, or 4-1. To determine whether the mouse FGF receptor also bound K-FGF, the ability of K-FGF to displace 125I-labeled bFGF was investigated. The curves in Fig. 4A show that about 20 times more K-FGF than bFGF is needed to compete for the binding of 125I-labeled bFGF to CHO clone 3-3. The reverse experiment was performed using clone 4-1 cells, which express a higher number of receptors, because of the lower specific activity of 1251I-labeled K-FGF. Also in this case, bFGF was more efficient than K-FGF in competing for the binding of 125I-labeled K-FGF (Fig. 4B). These experiments demonstrate that this mouse FGF receptor binds both bFGF and K-FGF but has a greater affinity for bFGF. Despite this difference in affinity, bFGF and K-FGF had similar abilities to activate the receptor. When CHO clone 3-3 cells were incubated for 5 hr at 37°C with various concentrations of bFGF or K-FGF, the number of cell surface receptors was down-regulated to a similar extent by both growth factors (Fig. 5). K-FGF could interact with the receptor in a functionally more efficient manner than bFGF. Alternatively, K-FGF may bind to other cellular proteins that are involved in presenting the growth factor to the receptor, an interaction that may not be detected in the binding assays that are performed at 4°C. Receptor Activation. The action of FGFs on target cells results in a number of cellular responses that lead to cell proliferation. Although the precise steps in the transduction pathway remain to be elucidated, the effect of growth factor action can be assessed by morphological changes, receptor phosphorylation, stimulation of DNA synthesis, and proliferation in the target cells. To test whether our mouse cDNA A
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functional receptor, we analyzed the level of receptor phosphorylation on tyrosine residues in transfected CHO cells treated with bFGF or K-FGF. Western blots of clones 3-3, 4-1, and A-1 stimulated with bFGF and K-FGF were incubated with anti-phosphotyrosine antibodies (Fig. 6). A band of 130 kDa is seen in clones 3-3 and 4-1 when they are exposed to bFGF or K-FGF at 20 ng/ml. The band is not seen in untreated 3-3 or 4-1 cells (data not shown) or in similarly treated A-1 cells. Thus, FGF stimulation leads to phosphorylation of a protein whose molecular mass is similar to that of the protein detected by bFGF crosslinking and that is likely to correspond to the activated receptor. The additional 90-kDa band seen in anti-phosphotyrosine immunoblots of Swiss 3T3 or NIH 3T3 cells treated with bFGF or K-FGF is not detectable in the CHO clones (14, 18). Although receptor kinase activation is one of the initial steps in the signal transduction pathways, this step can be uncoupled from the mitogenic response in certain mutant receptors (19). We thus studied the effect of bFGF and K-FGF on the morphology, rate of DNA synthesis, and a
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FIG. 5. Down-regulation of high-affinity bFGF receptors by bFGF and K-FGF. Clone 3-3 cells were incubated at 370C for 5 hr in serum-free medium containing heparin (10 ,ug/ml) and the indicated concentrations of bFGF (solid circles) or K-FGF (open circles). The cells were washed with 2 M NaCl buffered at pH 4.0 to remove bound growth factors and were incubated for 2 hr at 40C with 125I-labeled bFGF (5 ng/ml). The amount of 1251-labeled bFGF bound to highaffinity receptors was quantitated.
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growth of CHO cells expressing the mouse FGF receptor. Cells of clones 4-1 and 3-3 treated with the growth factors show distinct morphological changes compared to untreated cells or treated A-1 cells. The cells appear strikingly more spindle shaped and refractile (data not shown). The transfected cells expressing the receptor showed an increase in the rate of DNA synthesis in response to K-FGF or bFGF (Fig. 7A), as well as in cell number (Fig. 7B and data not shown). Thus K-FGF and bFGF can utilize the same receptor. By using antibodies against human FLG, Ruta et al. (20) have shown that acidic FGF stimulates tyrosine kinase activity of the endogenous FLG protein, suggesting that this receptor can also bind acidic FGF. Whether other members of this family interact with this receptor remains to be seen. Although the incorporation of [3H]thymidine in DNA in clone 3-3 and 4-1 cells is stimulated by bFGF and K-FGF, the 3-3 LU.
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Biochemistry: Mansukhani et al.
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weak response to the growth factor may be due to the fact that the cDNA clone encodes an altered receptor species lacking an immunoglobulin-like domain. Although this possibility cannot be totally excluded, we think it is unlikely, since this domain lies in the extracellular ligand-binding portion of the receptor and the binding capacity of the receptor is unaffected. It is likely that the cDNA described in this paper represent an alternatively spliced form of the mouse FGF receptor RNA. Polymerase chain reaction amplification of NIH 3T3 RNA using primers flanking the presumably deleted region of the mouse 4A cDNA produces multiple products, one of which corresponded in length to the fragment obtained with the 4A cDNA, indicating that several alternate forms of the mRNA exist (data not shown). Additionally, this FGF receptor may well belong to a family of receptors with various affinities for the FGF family. The bek gene (8) is probably one such closely related gene.
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Note. After this manuscript was submitted for review, a paper by Reid et al. (22) appeared describing the sequence of two murine cDNAs encoding proteins homologous to the chicken bFGF receptor. The deduced sequence of the shorter of these proteins, apart from some minor differences, is identical to the sequence of our receptor, whereas the largest one includes an additional extracellular immunoglobulin-like domain. As we suggested, the two cDNAs are probably the result of alternative splicing. We thank S. Kornbluth and H. Hanafusa for providing us with the bek probe. This work was supported by Grants CA 42568, CA 42229, and CA 34282 from the National Cancer Institute.
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Yarden, Y. & Ullrich, A. (1988) Biochemistry 27, 3113-3118. Schlessinger, J. (1988) Biochemistry 27, 3119-3123. Burgess, W. & Maciag, T. (1989) Annu. Rev. Biochem. 58,575-606. Delli-Bovi, P., Curatola, A. M., Kern, F. G., Greco, A., Ittmann, M. & Basilico, C. (1987) Cell 50, 729-737. 5. Delli-Bovi, P., Curatola, A. M., Newman, K. M., Sato, Y., Moscatelli, D., Hewick, R. M., Rifkin, D. B. & Basilico, C. (1988) Mol. Cell. Biol. 8, 2933-2941. 6. Neufeld, G. & Gospodarawicz, D. J. (1986) J. Biol. Chem. 261,
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FIG. 7. Proliferative response to K-FGF and bFGF of CHO cells expressing the mouse FGF receptor. (A) [3H]Thymidine incorporation into DNA. The results shown are the average of two independent experiments done in duplicate. The maximal stimulation seen with 10%o fetal calf serum was 100,000, 240,000, and 200,000 cpm for clones A-1, 3-3, and 4-1, respectively. (B) Growth curve. Clone 3-3 (ri, in), clone 4-1 (o, *), and clone A-1 (n, *) were used. Solid symbols represents K-FGF/transferrin, and open symbols are transferrin alone. Similar results were obtained with bFGF. trans, Transferrin; -, control with no growth factors added. rate is only 12% of the maximal stimulation induced by 10% calf serum. This somewhat weak but reproducible effect is also reflected in the proliferative response of these cells to the growth factor. It may be that CHO cells are lacking some component of the signal transduction pathway utilized- by the FGF receptor (such as the 90-kDa protein substrate). In a similar study of CHO cells reconstituted with the plateletderived growth factor receptor, transfectant clones showed 50% of the rate of DNA synthesis obtained with 10%o serum in response to platelet-derived growth factor (21), but the stimulation of DNA synthesis produced by serum was much
lower than in
our
experiments. It could be argued that the
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