Growth Factors

ISSN: 0897-7194 (Print) 1029-2292 (Online) Journal homepage: http://www.tandfonline.com/loi/igrf20

cDNA Cloning and Expression of a Human FGF Receptor which Binds Acidic and Basic FGF Stefan Wennström, Charlotte Sandström & Lena Claesson-welsh To cite this article: Stefan Wennström, Charlotte Sandström & Lena Claesson-welsh (1991) cDNA Cloning and Expression of a Human FGF Receptor which Binds Acidic and Basic FGF, Growth Factors, 4:3, 197-208, DOI: 10.3109/08977199109104816 To link to this article: http://dx.doi.org/10.3109/08977199109104816

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.o 1991 Harwood Academic Publishers GmbH

Growth Factors, 1991, Vol. 4, pp. 197-208 Reprints available directly from the publisher Photocopying permitted by license only

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cDNA Cloning and Expression of a Human FGF Receptor which Binds Acidic and Basic FGF STEFAN WENNSTROM, CHARLOTTE SANDSTROM and LENA CLAESSON-WELSH* Ludzuig Institute for Cancer Rescarcli, Bioniedical Ceiiter, B m 595, 5-751 24 Uppsala, SZO&II

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(Received September 3 7 990, Acceprd Septeniher 10 1990) We have isolated and characterized a cDNA clone, phFGFR, encoding a human fibroblast growth factor (FGF) receptor. phFGFR contains an open reading frame which encodes an 820 amino acid polypeptide with three immunoglobulin-likedomains in the extracellular part and an intracellular split tyrosine kinase domain. Transient expression in COS-1 cells and

immunoprecipitation using an antiserum raised against a C-terminal peptide, gave rise to two components, representing mature (130 kDa) and precursor (115 kDa) forms of the phFGFR encoded polypeptide, which was denoted hFGFR-1. Crosslinking of iodinated acidic FGF (aFGF) and basic FGF (bFGF) to transiently expressing COS-1 cells revealed a major band of 95 kDa, which was competed for by both aFGF and bFGF. From Scatchard analyses, the K,:s for binding of aFGF and bFGF to hFGFR-1 were estimated to 25 pM and 41 pM, respectively. Thus, phFGFR encodes a human FGF receptor with high affinity for both aFGF and bFGF.

INTRODUCTION The fibroblast growth factors comprise a growing family of structurally related, heparin-binding proteins. The family currently includes acidic FGF (aFGF) and basic FGF (bFGF) (for reviews, see Burgess and Maciag, 1989; Gospodarowicz et a]., 1987; Rifkin and Moscatelli, 1989), the int-2 gene product (Smith et al., 1988) the hst/KS3 gene product (Delli Bovi et al., 1987; Yoshida et al., 1987), FGF-5 (Zhan et al., 1988), KGF (Finch et al., 1989) and FGF-6 (Marics et al., 1989). The first discovered and most closely related members of this family are aFGF and bFGF. bFGF has been isolated from a variety of tissues and transformed cell lines (Gospodarowicz, 1987; Lobb et al., 1986a) whereas sources of aFGF have been neural tissues, such as brain and retina (Lobb et al., 1986a; Lobb et al., 1986b). Both aFGF and bFGF promote angiogenesis (Folkman and Klagsbrun, 1987). bFGF has been shown to induce mesoderm formation in Xenopus embryos (Kimelman et al., 1988). The amino acid sequences for aFGF (Jaye et al., ‘Correspondence to L. Claesson-Welsh at the address above Phone +46-18-551688. Fax +46-18-106867.

1986) and bFGF (Abraham et al., 1986) lack N-terminal signal sequences, in contrast to those for the other members of the FGF family. While it remains to be determined how aFGF and bFGF are released from the cell, efficient secretion has been demonstrated after fusion at the cDNA level of the immunoglobulin signal sequences with the bFGF coding sequence (Rogelj et al., 1988); this construct caused transformation after introduction into NIH 3T3 cells. Receptors for aFGF and bFGF have been identified on a wide variety of neuroectoderm- and mesoderm-derived cells. The two factors appear to interact with two components with molecular weights of 125000 and 145000 as determined by affinity labeling techniques. In cross-competition experiments bFGF appears to bind stronger to the 145 kDa component, whereas aFGF binds stronger to the 125 kDa component (Neufeld and Gospodarowicz, 1986). It has also been suggested that both factors bind with high affinity to a common receptor (Burrus and Olwin, 1989). Recently, a chicken bFGF receptor was purified and a cDNA clone was isolated through the use of primers, based on partial amino acid sequence information (Lee et al., 1989). The cDNA encodes a poly-

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peptide equipped with a tyrosine kinase domain. This is in agreement with reports which show that the interaction of both aFGF and bFGF with their receptors results in stimulation of tyrosine phosphorylation (Coughlin et al., 1988; Friesel et a]., 1989; Huang and Huang, 1986). We have isolated and determined the sequence of a cDNA clone encoding a human FGF receptor, which we denote hFGFR-1. It is an 820 amino acid glycoprotein with an intracellular tyrosine kinase domain. Crosslinking and ligand-binding assays using iodinated aFGF and bFGF on COS-1 cells, either untransfected or transiently expressing the cDNA, was performed in order to characterize the ligand binding properties of this receptor.

MATERIALS AND METHODS Isolation and Characterization of cDNA Clones Oligo (dT)-primed cDNA was synthesized using the cDNA synthesis kit from Pharmacia LKB and mRNA (kindly provided by Dr. E. J. J. van Zoelen, Dept. of Cell Biol., Katholieke Universiteit, Nijmegen, The Netherlands, and Mrs. A. Feyen, Hubrecht Lab., Utrecht, The Netherlands) isolated from the teratocarcinoma cell line Tera-2 clone 13 (Thompson et al., 1984). The cDNA was cloned into bacteriophage LgtlO. The cDNA library was first screened with a 750 base pair (bp) fragmet covering the tyrosine kinase domain in the human PDGF a-receptor (Claesson-Welsh et al., 1989). Hybridization with the PDGF a-receptor probe to Hybond C Filter (Amersham) duplicates was performed under medium stringency [40% formamide in 5 XSSC (1XSSC = 15 mM sodiumcitrate pH 7.0, 0.15 M NaCI), 10 XDenhardt's solution (Denhardt, 1966), 0.1% sodium dodecyl sulphate (SDS), 0.1 mg/ml salmon sperm DNA, 50mM NaHPO, pH 6.5 at 37"CI. Isolated FGF receptor cDNA fragments were used to rescreen the cDNA library under high stringency (50% formamide in 5 XSSC, 10 XDenhardt's, 0.1% SDS, 0.1 mg/ml salmon sperm DNA, 50mM NaHPO, pH 6.5, at 42°C). The probes were labeled with a-32P]dCTP using; the Multiprime Labelling System Kit (Amersham) and labeled probes were added at 0.5-1.0 xlOhcpm/ml hybridization solution. Filters were washed three times in 0.1 XSSC, 0.1% SDS at 37°C (medium stringency) or at 55°C (high stringency), dried and exposed to Fuji RX Films. Positive plues were picked and purified as described by Maniatis et al. (1982).

Inserts were cloned into the pUC19 vector (Yannisch-Perron et al., 1985) and after restriction enzyme characterization, subcloned into M13 vectors followed by primed DNA synthesis on single stranded DNA templates in the presence of dideoxynucleotide triphosphates, using Sequenase (United States Biochemical Corp.). Sequence determination was done on both strands. The most 5'sequence (150 bp) was isolated using the polymerase chain reaction (PCR) technique. First cDNA strand templates were synthesized from mRNA from the sarcoma cell line B5GT using avian myeloblastosis virus (AMV) reverse transcriptase (Pharmacia LKB). The sense strand PCR primer was a mixture based on the first five amino acids in the chicken FGF receptor signal sequence (Lee et al., 1989); it contained in addition to a 5'-EcoR1 cloning site. The anti-sense primer was based on the sequence of a partial FGF receptor cDNA clone (pl:lb, see Fig. lA), 210 bp 3' of an internal EcoRl site. A low stringency PCR of 25 cycles was performed using Taq polymerase (Perkin ElmedCetus) and amplified fragments were cleaved with EcoRl and cloned into EcoRl cleaved pUC19 vectors. Sequence determination revealed that the sense primer by cross-hybridization had hybridized 5' of the open reading frame of the human cDNA. A new sense primer was synthesized based on the human sequence around the initiating methionine, and a high-stringency PCR of 25 cycles was performed. The PCR fragment was cleaved with EcoRl and fused with the EcoRl cleaved partial cDNA pl:lb, to create phFGFR. Sequence determination was performed on several independent clones of the PCR-fragment to ensure that errors that might have been introduced by the Taq polymerase were avoided. The Genbank accession number of the sequence of phFGFR is M34641. Tissue Culture and Transfection

The Tera-2 clone 13 cell line (a kind gift from Dr. W. Engstrom, Dept. of Pathology, Huddinge Hospital, Sweden, and Dr. C. F. Graham, Dept of Zoology, University of Oxford, Great Britain) were maintained in a-modified minimum essential medium (a-MEM, GibcoBRL) containing 10% fetal calf serum, 100 units/ml of penicillin and 50pg/ml of streptomycin, using gelatinzied [O.l0/o swine skin gelatin (Sigma) in PBS] tissue culture dishes. COS-1 cells (American Type Culture Collection) were cultured in Dulbecco's modified Eagle's

HUMAN RECEPTOR FOR aFGF AND bFGF

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medium, containing 1 O 0 / ~ fetal calf serum, 100 units/ ml of penicillin and 50 pglml of streptomycin. Two EcoRl fragments, one created through PCR, as described above, and the other derived from the partial cDNA pl:lb, were cloned into the simian virus (SV) 40-based expression vector pSV7d (Truett et al., 1985), to yield an insert covering the entire open reading frame (see Fig. 1A). To facilitate cloning, the most 3' EcoRl site in pl:lb was first destroyed using exonuclease Ba131 (International Biotechnologies, Inc.). Transfection of the construct into COS cells was performed by the calcium phosphate precipitation method (Wigler et al., 1979) to yield transient expression and the cells were examined after twothree days.

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4°C. The beads were washed three times with a buffer containing 1% Triton X-100, 1% deoxycholate, O . l o / ~ SDS, 50 mM Tris-HCI pH 7.5, 0.15 M NaCI, 10 mM EDTA, 30 mM pyrophosphate, once with high salt buffer (1%Triton X-100, 20 mM TrisHCI pH 7.5, 0.5 M NaCI) and once in distilled water. The immune-complexes were eluted from the beads by boiling for 3 min in the sample buffer [ 4 O h SDS, 0.2 M Tris-HCI pH 8.0, 0.5 M sucrose, 5 mM EDTA, 0.01% bromophenolblue (Merck), 3% B-mercaptoethanol] and analyzed by gradient (5-1Oo/o) SDSpolyacrylamide gel electrophoresis, as described by Blobel and Dobberstein (1975). Gels were treated for fluorography by soaking in Amplify (Amersham), dried and exposed to Hyperfilm MP (Amersham).

Covalent Cross-linking Antibodies A synthetic peptide based on the 16 C-terminal amino acids of the human FGF receptor sequence (Fig. lB), was coupled to keyhole limpet hernocyanin (KLH) (Calbiochem-Behring). The peptide and KLH were mixed at equal concentrations (0.5 mg/ml) in 0.1 M sodium acetate pH 5.0 for 12 h at 1°C and a covalent crosslinker, ethyldimethyl-carboimide, was added to a final concentration of 20mg/ml and the coupling proceeded for another 12 h at 4°C. The sample was dialyzed against 0.1 M sodium acetate pH 5.0 and used for immunization of rabbits.

Radioactive Labeling and Immunoprecipitation Tera-2, cl. 13 cells, transfected and untransfected COS-1 cells were labeled in cystein- and methionine-free MCDB 104 medium containing 100 pCi/ml each of I"S]cysteine and [ "Slmethionine (Amersham) for 3 h at 37°C. Labeled cells were washed with cold PBS and lysed in a solubilization buffer [0.5% Triton X-100, 0.5% deoxycholate (Merck), 20 mM Tris-HC1 pH 7.5, 0.15 M NaCI, 10 mM EDTA, 1% Trasylol (Bayer) and 1 mM phenyl methylsulfonyl fluoride (Sigma)]. The lysates were centrifuged at 10 000 g for 30 min and the supernatants were passed two times over 1 ml Lens culinaris lectin-Sepharose 4B columns previously equilibrated in solubilization buffer. The glycoprotein fractions were eluted with lO0/o a-methylmannoside (Sigma) in the solubilization buffer and precipitated with 10pl of preimmune or antiserum for 2 h at 4°C. Immune-complexes were bound to 80 pl protein A-Sepharose C1-4B (Pharmacia LKB) for 30min at

Confluent cultures of transfected and control COS-1 cells in 75cm2 tissue culture flasks were washed three times with cold PBS containing 0.2% gelatin and incubated for 2 h at 4°C with either "'I-aFGF (speific activity 135 000 cpm/ng; a kind gift from S. Lewis, Amersham International, Great Britain) or '?'I-bFGF (specific activity 115 000 cpm/ng; Amersham) (25 nglml) in PBS, 0.2% gelatin, in the absence or presence of a ten-fold excess of unlabeled ligands. aFGF purified from a baculovirus expression system, was a kind gift from Drs. Yihai Cao and Ralf Petterson, Stockholm Branch of the Ludwig Institute for Cancer Research. Recombinant human bFGF was from Farm Italia Carlo Erba. The monolayers were washed free of unbound ligand. A 25mM stock solution of the covalent crosslinker DSS (3.60 mg in 0.4 ml DMSO; Pierce) was diluted 500 times with PBS and 2ml was added to each 75 cm2 flask. After 20 min incubation at room temperature, reactions were quenched for 2 min at room temperature with 20 mM Tris-HCI pH 7.5 (final concentration). After washing once with PBS, the cells were pelleted by centrifugation at 10 000 g for 2 rnin and incubated for 30 min on ice with lysis buffer (1%Triton X-100, 20 mM Tris-HCI pH 7.5, 10% glycerol). Unsolubilized material was pelleted by centrifugation at 10 000 g for 15 min and the supernatants were analyzed by gradient (5-10%) SDS-polyacrylamide gel electrophoresis and exposure to Hyperfilm MP (Amersham).

Radio-receptor Assays Confluent cultures of COS-1 cells expressing phFGFR (36 h after transfection) and untransfected

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COS-1 cells, in gelatinized 12-well tissue culture dishes, were serum-starved 16 h before use. The monolayers were washed twice with cold PBS containing 0.2% gelatin and then incubated at 4°C for 2 h in the presence of iodinated aFGF or bFGF and serial dilutions of unlabeled ligands. Cells exposed to 12-iI-aFGFwere washed four times with cold PBS and then lysed for 15 min at room temperature using 0.5 ml/well of 1% Triton-100, 20 mM Tris-HC1 pH 7.5, 10% glycerol. 12'I-bFGF treated cells were washed four times with cold PBS and twice using 20mM Tris-HC1 PH 7.5, 2 M NaCI, in order to remove low affinity binding (Moscatelli, 1987) and then lysed as described above. Cell-associated radioactivity was determined in a gamma-spectrometer and the obtained values were used in the crosscompetition study and analyzed according to Scatchard (1949), both manually and using the RBINDING computer program for the linear subtraction method (van Zoelen, 1989). All series were performed in triplicates.

RESULTS Isolation of an FGF Receptor cDNA A LgtlO cDNA library was prepared from mRNA isolated from a human teratocarinoma cell line, Tera-2, clone 13 (Thompson et a]., 1984) and screened under medium stringency using a probe corresponding to the tyrosine kinase region of the human PDGF a-receptor (Claesson-Welsh et a]., 1989). The deduced amino acid sequence of a partial cDNA clone displayed several characteristics of a growth factor receptor with a tyrosine kinase domain (see below) and it was very similar to the recently described chicken bFGF receptor (Lee et a]., 1989). A full length cDNA was obtained using the polymerase chain reaction (PCR), in which one primer was a mixture of degenerated oligomers corresponding to the five most NH,-terminal amino acids in the chicken bFGF receptor signal sequence. The other, second primer was based on a stretch in our partial cDNA. An 800 basepair (bp) fragment was synthesized and subsequently cloned and sequenced; 650 bp overlapped with and were exactly identical to the corresponding segment in our partial cDNA; 150 bp constituted new sequence. The

sequence of the 150bp stretch was identical in several independent clones of the PCR fragment. A 588bp fragment created by cleavage of the PCR fragment with EcoRl was fused with the EcoRl cleaved partial cDNA to yield phFGFR (Fig. 1A). The nucleotide and deduced amino acid sequences of phFGFR are shown in Fig. 1B. The clone encompasses 3343bp which contains an 820 amino acid open reading frame. The resulting polypeptide can be divided into two parts by a centrally located hydrophobic stretch, probably constituting the transmembrane domain. The most N-terminal part of the polypeptide has the characteristics of a cleavable signal sequence; a preferred cleavage site for the signal peptidase according to von Heijne (1986) would be N-terminal of Ala-21. The proposed extracellular part of the polypeptide contains eight signals for N-linked glycosylation (Asn-X-Ser/Thr, where X may be any amino acid other than Pro; see Creighton, 1984). Six evenly spaced cysteine residues allow the formation of three immunoglobulinlike domains. An unusually long stretch of acidic residues (eight glutamic and aspartic residues in a row) is found between the second and third cysteine residue. The intracellular part of the polypeptide, 86 amino acids from the assumed transmembrane domain, contains a Gly-X-Gly-X-X-Gly motif and a potential nucleotide binding lysine residue at position 512, within a region conserved between tyrosine kinase domains. As for the members of the PDGF receptor family, the tyrosine kinase-region in phFGFR contains an insertion which lacks homology with kinase domains; of the 17 amino acids in the insert sequence, six are acidic. There is a 75 amino acid carboxyterminal tail following the second part of the tyrosine kinase domain. A 450 bp fragment of phFGFR, corresponding to the extracellular domain, was used as a probe in a A 0

1

2

3

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5

I

I

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I

I

I

I

I

v

.......

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mRMA

p1:lb PCR fr phFGFR

FIGURE 1 . (A) Schematic representation of FGF receptor cDNA clones. A schematic outline of the predicted mRNA is shown, with and untranslated (-) regions indicated. The broken line indicates possible extension of a 4.0 kb transcript to yield a 4.8 kb translated (0) transcript, which both were detected in a Northern blot analysis of Tera-2, cl. 13 mRNA, using the 450 bp 5'-fragment of phFGFR as a probe. The partial cDNA p1:lb was fused with the PCR fragment using an internal EcoRl site ( V )to yield phFGFR, which contains the entire open reading frame.

HUMAN RECEPTOR FOR aFGF AND bFGF

GMTTCGGG -

201

Mot T r p S e r T I P L Y S Cya L O U Lou P h e T r p A l a Val Leu Val T h r Ala T h r Leu Cys T h r AlaVA r q P r o S.r P r o T h r Leu ATG T G G A G C T G G M G T G C C T C C T C TTC T G G G C T G T G C T G G T C A C A G C C A C A C T C T G C A C C G C T A G O C C G T C C C C G A C C T T G

U

A r q A s p A s p Val G l n S * r

11. A s n T r p Leu A r q A s p Gly Val G l n Leu A l a Glu S e r Asn A r q - T h r A r q 11. T h r Gly G l u G l u Val G l u CGG G A C G A T G T G C A G A G C A T C M C T O G CTG CGG G A C G G G G T G C A G C T G G C G G M A G C M C C G C ACC C G C A T C A C A GGG G A G G A G GTG G A G

U

Val G l n A s p s e r v a l p r o r l a ASP s e r Gly Leu T y r Ala CYS Val T h r Ser S * r P r o S e r G l y S e r h a p T h r T h r Tyr P h e S e r Val si;;; G T G CAG G A C T C C G T G C C C G C A G A C T C C GGC C T C TAT G C T T G C G T A A C C A G C A G C C C C T C G G G C A G T G A C A C C A C C T A C T T C T C C G T C M T

________---____

_ _ _ _ .

Val S a r A s p A 1 a L e u P r o . S r S e r G l u Asp A s p Asp A s p A s p A s p A s p S O r S e r S a c Glu G l u Lys Glu T h r A s p A s n T h r Lys P r o A s n G T T T C A G A T G C T C T C C C C T C C T C G G A G G A T G A T G A T G A T G A T G A T G A C T C C T C T T C A GAG G A G A M G M A C A G A T M C A C C A M CCA M C

0

87

210 117

360 147 450

P I 0 Val A l a P r o T y r T r p T h r 5.r P r o Glu LYS net G ~ L U y s Lys Leu H i s Ala V41 P r o A l a A l a L y s T h r Val Lys P h e Lys cys P r o C C C G T A G C T CCA TAT T O G A C A T C C CCA G M M G ATG O M M G AAA TTG C A T G C A G T G CCG G C T G C C M G A C A G T G M G T T C AAA T G C C C T

177 540

S e r S e r G l y T h r P r o A s n P r o T h r Leu A r g T r p Leu Lys A s n S e r L y s G l u P h e Lys P r o A s p H i s A r q 11. G l y Gly Tyr L y s Val A r q T C C A G T G G G A C C C C A M C C C C ACA C T G CGC T G G TTG AAA M T AGC AM G M r T c AM CCT GAC C A C AGA ATT GGA G G C TAC M G G T C CGT

201 630

0

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21 90

T y r A l a T h r T r p S e r 11. I l e n e t A s p S.r Val Val P r o S 8 r A s p LyS Gly Asn Tyr T h r CyS I 1 0 Val G l u A s n Glu Tyr G l y 5.1 Ile TAT G C C A C C T O G A G C A T C A T A ATG G A C TCT G T G GTG C C C T C T G A C M G G G C M C T A C A C C T G C A T T GTG G A G M T GAG T A C G G C AGC A T C

231 120

A s n H i s T h r T y r G l n Leu A s p Val Val Glu A r q S e r P r o H ~ s A r q P r o Ile LaU G l n Ala G l y U . L P r o A l a A s n Lyr T h r Val Ala Leu A A C C A C A C A T A C C A G C T G G A T G T C G T G GAG CGG T C C C C T C A C COG C C C A T C CTG C M GCA G G G TTG CCC G C C M C AAA A C A GTG GCC C T G

261

810

G l y S.r A E n Val G l u Oh. M e t Cys Lys Val T y r S.r A s p P r o G l n P r o H i s Ile Gln T r p u Lys H i s 11. G l u Val A s n G l y S . L . r Lys G G T A G C M C G T G G A G TTC ATG T G T M G G T G T A C A G T G A C C C G CAG CCG C A C A T C CAG T O G C T A M G C A C A T C GAG GTG A A T G G G AGC M G

900

I 1 0 G l y P r o A s p Asn Leu P r o T y r Val G l n Il* L*U Lys T h r A l a G l y Val A s n T h r T h r A s p Ly5 G 1 U M e t G l u Val L e u H 1 S LaU A r g ATT G G C C C A G A C AAC CTG C C T TAT G T C CAG A T C T T G M G A C T G C T G G A G T T M T A C C A C C G A C AAA G A G ATG G A G GTG CTT C A C TTA A G A

321 990

U

291

A s n Val S e r P h e G l u A s p A l a G l y Glu T y r T h r Cy6 Leu A l a G l y A s n Ser 11. G l y Leu S e r H I S H i 8 S e r Ala TIP Leu T h r Val Leu G T C T C C TTT G A G G A C G C A G G G GAG TAT ACG T G C T T G G C G GGT M C T c r ATC GGA CTC T C C CAT C A C TCT G C A TGG TTG A C C GTT CTG

1080

G1U Ala L e u G 1 U G 1 U A r q P r o 1\11 Val P4.t T h r S e r P r o G A A G C C CTG G M G A G A G O CCG G C A G T G ATG A C C TCG C C C

387 1170

n e t val G l y s e r v a l 11. val ~ y rLYS net Lys S e r G l y T h r Lys Lys 5.1 A s p P h e His s e r G l n net A l a Val H i s L y s Leu A l a Lys ATG G T G G G G T C G G T C A T C G T C TAC AAG ATG M G A G T G G T A C C M G M G A G T G A C TTC C A C A G C CAG ATG G C T G T G CAC M G CTG GCC M G

1260

S e r A l a A s p S b r S e r Ale S * r Met A s n S O T G l y Val Leu Leu Val A r g P r o S e r A I g L e u T C T G C T G A C T C C A G T G C A T C C ATG M C T C T G G G G T T CTT CTG G T T CGG C C A TCA CGG C T C

441 1350

Val S e r Glu T y r G 1 U Leu P r o G l u A s p P r o A c g T r p GlU Leu P r o A r g A s p A r g Leu V a l G T C T C T G A G TAT G A G C T T C C C G M G A C CCT C G C TGG G A G CTG C C T COG G A C A G A CTG G T C

1440

G l y Gln Val Val Leu A l a G l u Ala Ile Gly Leu A s p Lys A s p Lys P r o A s n A r g Val T h r G G G CAG G T G GTG T T G G C A G A G G C T A T C GGG C T G G A C M G G A C AAA CCC - M C C G T GTG A C C

501 1530

A 1 a T h r Glu LyS A s p L e u S e r A S P Leu I1e S e r G 1 U M e t G 1 U Met M e t L y S l e t 11. Gly G C A A C A GAG AM G A C T T G T C A G A C CTG A T C TCA O M ATG GAG A T G ATG M G A T G ATC G G G

537 1620

A l a Cys T h r Gln A s p G l y P r o Leu T y r Val 11. Val G l u T y r A l a 5 - r Lys G l y A s a L-u G C C T G C ACG CAG G A T G G T C C C T T G TAT G T C A T C GTG G A G TAT G C C T C C M G G G C M C C T G

561 1710

H I S A s n P r o G1U G l u G l n Leu S.r S m r LyS A s p G l y Leu G l u Tyr CyS T y r A s a P r o S.r G G G CTG O M T A C T G C T I C M C C C C A G C CAC M C CCA GAG G A G CAG CTC T C C T C C M G G A C

1800

G l y l4.t G l u Tyr Leu A l a S e r L y a Lys Cys I l e H l s A r q A s p L.Y Alr A l a A r g P.sn V a i G G C ATG G A G TAT CTG G C C T C C M G M G T G C ATA C A C C G A G A C CTG GCA G C C AGG M T G T C

1890

Leu Val T h r G l u A s p Asn Val M e t L y s 11. A l a A s p Ph. G l y Leu A l a A r g A s p 11. H I S H I S 11. A s p T y r Tyr LyS LyS ThC Thr A n n CTG G T G A C A G A G G A C A A T GTG ATG M G A T A G C A G A C TTT G G C C T C G C A COG G A C A T T CAC C A C A T C G A C T A C TAT AAA M G A C A A C C A A C

651 1980

Gly A r g L O U P r o Val Lys T i p net A l a P r o G l u Ala Leu P h e A s p A r g 11. Tyr T h r H i s Gln S e r A s p Val T r p S.r P h e G l y Val Leu G G C C G A C T G C C T G T G M G TGG ATG GCA C C C G A G G C A T T A TTT G A C CGG A T C TIC ACC CAC CAG A G T G A T G T G TGG T C T T T C G G G G T G C T C

2010

Leu T I P G l u 110 P h e T h r Leu G l y Gly s e r P r o T y r P r o Gly Val P r o Val Glu Glu Leu P h e Lys Leu Leu Ly9 Glu Gly H i s A c g n e t CTG T G G G A G A T C T T C A C T CTG G G C G G C T C C CCA T A C C C C G G T G T G C C T GTG GAG G M C T T T T C M G CTG CTG M G GAG G O T C A C C G C ATG

2160

A s p Lys P r o S e r A S n C y s T h r A s n G1u L e u T y r Met n e t net A r q A s p Cys T r p H I S A l a Val P r o Ser Gln A r q P r o T h r P h e Lys G l n G A C M G C C C A G T M C T G C A C C M C GAG CTG T A C A T G ATG ATG CGG G A C T G C T G G CAT GCA G T G C C C T C A CAG A G A C C C A C C T T C M G CAG

2250

Val A l a Leu T h r S e r A s n G l n G l u Tyr Leu A s p Leu S e r net P r o Leu A s p Gln Tyr S e r P r o S e r L*U Val G l u A s p L e u A s p A r q 11. CTG G T G G M G A C C T G G A C CGC A T C GTG G C C T T G A C C T C C M C C A G G A G T A C CTG G A C CTG T C C ATG C C C CTG G A C CAG T A C T C C CCC A G C

2340

P h e P r o A s p T h c A r g S e r Ser T h r Cys S e r S e r Gly Glu Asp S e r Val Ph. S e r H I S Glu P r o Leu P r o Glu Glu P r o Cys Leu P r o A r q TTT C C C G A C A C C C G G A G C T C T ACG T G C T C C T C A G G G GAG G A T T C C G T C T T C T C T C A T GAG CCG CTG C C C GAG G A G C C C T G C C T G C C C C G A

2430

HlS

P r o Alll G l n L e u Ala A s n Gly Gly Leu LyS A r g A r q CAC C C A G C C C A G C T T G C C A A T G G C G G A C T C M A C G C C G C T G A C T G C C A C C C A C A C G C C C T C C C C A G A C T C C A C C G T C A G C T G T M C C C T C A C C C A C A G C C C C T G C T G

820 2531

GGCCCACCACCTGTCCGTCCCTGTCCCCTTTCCTGCTGGCAGGAGCCGGCTGCCTACCAGGGGCCTTCCTGTGTGGCCTGCCTTCACCCCACTCAGCTCACCTCTCCCTCCACCTCCTCT

2651

CCACCTGCTGGTGAGAGGTGCAAAGAGGCAGATCTTTGCTGCCAGCCACTTCATCCCCTCCCAGATGTTGGACCMCACCCCTCCCTGCCACAGCATCGCCTGGAGGGCAGGGAGTGGGA

2177

GCCMTGMCAGGCATGCMGTGAGAGCTTCCTGAGCTTTCTCTGTCGGTTTGGTCTGTTTTGCCTTCACCCATMGCCCCTCGCACTCTGGTGGCAGGTGCCTTGTCCTCAGGGCTACA

2897

GCAGTAGGGAGGTCAGTGCTTCGTGCCTCGATTGMGGTGACCTCTGCCCCAGATAGGTGGTGCAGTGGCTTATTMTTCCGATACTAGTTTGCTTTGCTGACCAAATGCCTGGTACCAG

3011

AGGATGGTGAGGCGAAGGCCAGGTTGGGGGCAGTGTTGTGGCCCTGGGGCCAGCCCCAAACTGGGGGCTCTGTATATAGCTATGMG-CACAAAGTGTATAAATCTGAGTATAT~

3137

AAT

c

_I

351

411

411

591

625

687

717 747

717

801

TACATGTCTTTTTMMGGGTCGTTACCAGAG~CCCATCGGGTMGATGCTCCTGGTGGCTGGGAGGCATCAGTTGCTATATATT~C~G~AGGAAAAT3 G 2T5T7 TTTAAAMGGTCATATATTTTTTGCTACTTTTGCTGTTTTATTTTTTTAAATTATGTTCTAAACTCGTGCCGCTCGTGCCGMTTC

3343

(B) Nucleotide sequence and deduced amino acid sequence of phFGFR. Nucleotides and amino acids are numbered to the right. Amino acids are numbered from the initiating methionine. Arrowhead indicates potential start of mature protein. Potential sites of N-linked glycosylation are overlined and cysteine residues are boxed in the extracellular part of the mature protein. A region containing an unusual row of acidic amino acid residues is indicated by a broken line. The transmembrane region I S boxed and the borders of the two tyrosine kinase segments are indicated by brackets. The position of the nucleotide-binding lysine residue is indicated by a bold line. In the 3' untranslated region two ATTTA motifs, which have been implicated in regulation of mRNA stability (Meijlink et al., 1985; Shaw and Kamen, 1Y8h), are underlined, as well a s primer sequences in the beginning and the end of the cDNA sequence.

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Northern blot analysis. Two transcripts of 4.0 and 4.8 kb were detected in mRNA derived from several sarcoma cell lines and biopsies, as well as from Tera-2, cl. 13 cells (not shown).

a b c d e f

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-200

- 92

- 69

FIGURE 2. lmmunoprecipitation from metabolically labeled COS-1 cells followed by SDS-polyacrylamide gel electrophoresis and fluorography. Metabolically labeled COS-1 cells transfected with phFGFR (lanes a and b), untransfected COS-1 cells (lanes c and d) and Tera-2, cl. 13 cells (lanes e and f) were immunoprecipitated with preimmune serum (lanes a, c and e) and with the FGF receptor peptide antiserum (lanes b, d and f). Migration positions of the precursor (115 kDa) and mature (130 kDa) forms are indicated, as well as those of molecular weight markers run in parallel (myosin, 200 000; phosphorylase b, 92 000; bovine serum albumin, 69 000). The 130 kDa component in transfected COS-I cells (lane b) is readily apparent on the original fluorography, but was difficult to reproduce on the photography.

Transient Expression of phFGFR in COS-1 Cells The insert of phFGFR was cloned into the SV40 based expression vector pSV7d (Truett et al., 1985) and the construction was transfected into COS-1 cells. Two days after transfection, the cells were. metabolically labeled for three hours using [3-'S]methionine and 13'S]cysteine. An antiserum, raised against a synthetic peptide corresponding to the 16 most C-terminal amino acid residues in the open reading frame of phFGFR, was used for immunoprecipitation from a glycoprotein-enriched fraction (Fig. 2). Two bands, one more intense of 115 kDa and one fuzzy, fainter of 130 kDa, were seen upon SDS-polyacrylamide gel electrophoresis, when the peptide antiserum was used on samples from COS-1

cells transfected with the cDNA (Fig. 2, lane b). The preimmune serum (Fig. 2, lane a) did not react with the 115kDa and 130kDa components, and they were not found in samples derived from control COS-1 cells using either preimmune serum (Fig. 2, land c) or the antiserum (Fig. 2, lane d). In samples from metabolically labeled Tera-2 cells, the 115 and 130 kDa bands were seen when using the antiserum (Fig. 2, lane f), but not when using the preimmune serum (Fig. 2, lane e). In a pulse-chase analysis of Tera-2, cl. 13 cells, the 115 kDa band was converted to 130 kDa with time (not shown), indicating that the 115 kDa component is the intracellular precusor form of the mature 130 kDa molecule. The molecular weight of the receptor polypeptide, calculated from the deduced cDNA sequence not including the signal sequence, is 89 185. This is in good agreement with the apparent molecular weight of 115.000 for the immunoprecipitated precursor, considering that eight signal sequences for N-linked glycosylation are found in the extracellular part of the molecule (Fig. 1B).

Crosslinking using lZ5I-aFGFand lZ5I-bFGF Untransfected COS-1 cells and COS-1 cells transiently expressing the phFGFR encoded polypeptide, were incubated with 12sI-aFGFor 12?I-bFGFin the presence or absence of a ten-fold excess of unlabeled ligands. After washing to remove unbound ligand, the cells were treated with the covalent crosslinker disuccinimidyl suberate (DSS). When iodinated aFGF was crosslinked to transiently expressing COS-1 cells, a broad band of approximately 95 kDa (Fig. 3, lane a) was seen. A considerably less intense 145 kDa band could also be detected; the prominence of this band varied between experiments. These bands were not detected when an excess of aFGF (Fig. 3, lane b) or bFGF (Fig. 3, lane c) was included in the binding buffer, or when untransfected COS-1 cells were analyzed (Fig. 3, lanes d-f). I29-aFGF was crosslinked to a separate component in untransfected COS-1 cells, yielding a faint band of 125 kDa. Iodinated bFGF was crosslinked to transfected COS-1 cells, yielding a similar pattern as for iodinated aFGF. Thus, an intense 95 kDa band was seen in the absence of competing unlabeled ligands (Fig. 3, lane g), but not after inclusion of an excess of bFGF (Fig. 3, lane h) or aFGF (Fig. 3, lane i). Iodinated bFGF was not crosslinked to untransfected COS-1 cells to any appreciable extent (Fig. 3, lane j-I).

HUMAN RECEPTOR FOR aFGF AND bFGF

a

b

c

d

e

f

g

h

i

j

k

l

kDa

-200 145 -

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-92

Cross-competitionfor Binding of lZ51-aFGFand lZ5I-bFGFto Transiently Expressing COS-1 cells Iodinated aFGF and bFGF were analyzed with regard to their binding to COS-1 cells 48 h after transfection with phFGFR. As shown in Fig. dA, 12'1aFGF was most efficiently competed for by aFGF, and less well by bFGF. Iodinated bFGF on the other hand was more efficiently competed for by bFGF than aFGF (Fig. 4B). These results indicate that aFGF and bFGF interact with partially non-overlapping sites on the FGF receptor molecule. Binding of iodinated ligands to untransfected COS-1 yielded a low level of specific binding, which was 10-15% of that seen when using transfected cells (not shown). This is in agreement with the weak signal obtained when iodinated aFGF and bFGF were crosslinked to untransfected COS-1 cells (Fig. 3).

Scatchard Analysis of Ligand-binding The affinity of "'1-aFGF and "'I-bFGF to the phFGFR encoded molecule was estimated by performing Scatchard analyses of ligand-binding to transiently expressing COS-1 cells. The K,:s for binding of aFGF (Fig. 5A) and bFCF (Fig. 5B) were estimated to 25 pM anmd 41 pM, respectively. For each ligand there was a straight line in the Scatchard analysis, indicating a single class of binding sites. Thus, both factors bound with very high affinity to the receptor expressed after transfection to a level of about 10 000 molecules/cell.

203

FIGURE 3. Covalent crosslinking of "'I-aFGF and "'I-bFGF to transfected and untransfected COS-I cells followed by SDS-polyacrylamide gel electrophoresis and autoradiography. COS-I cells transfected with phFGFR (lanes a-c, and g-i) and untranfected COS-I cells (lanes d-f and j-I) were crosslinked with "'I-aFGF (lanes a-f) and "'I-bFGF (lanes g-I), in the absence (lanes a, d, g and j ) or presence of a ten-fold excess of unlabeled aFGF (lanes b, e, I and I) or bFGF (lanes c, f , h and k). The apparent molecular weights of crosslinked components are indicated, as well those of molecular weight markers run in parallel (myosin 200 000; phosphorylase b, 92 000).

DISCUSSION In this report, we describe the structure and binding characteristics of a human FGF receptor. A cDNA clone, phFGFR, was isolated from a human teratocarcinoma cell line, Tera-2 c1.13, which was established from a pulmonary metastasis of a testicular carcinoma (Thompson et a]., 1984). phFGFR encodes an 820 amino acid polypeptide with several structural features in common with members of the PDGF receptor family, i.e. the immunoglobulin-like domains in the extracellular domain and the intracellular split tyrosine kinase domain. The amino acid sequence of the tyrosine kinase domain of the cloned receptor is also most similar to that of the PDGF receptors (for a review, see Westermark et al., 1989). The unique features of the FGF receptor include an unusual stretch of aspartic residues in the extracellular domain, and a long juxtamembrane domain (86 amino acids). The number of amino acids between the transmembrane domain and the start of the tyrosine kinase domain is usually very similar (40 amino acids for the members of the PDGF receptor family) between different receptors with tyrosine kinase domains. COS-1 cells transiently expressing the phFGFR cDNA were examined with regard to their ligandbinding characteristics using iodinated aFGF and bFGF. Crosslinking with either iodinated aFGF or bFGF yielded a prominent 95 kDa band upon SDSpolyacrylamide gel electrophoresis. Addition of an excess of unlabeled aFGF and bFGF completely blocked the signal. The 95 kDa band was not seen in

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samples from untransfected cells. Scatchard analyses of the binding of aFGF and bFGF to transiently expressing COS-1 cells showed high affinity binding of both ligands. These data show that the receptor encoded by phFGFR binds aFGF as well as bFGF, probably to a shared but not identical binding-site. To simplify the nomenclature (see below) this receptor will be denoted hFGFR-1 from now on. The results from immunoprecipitations from metabolically labeled, transiently expressing COS-2 cells and Tera-2, cl. 13 cells indicated that the

mature form of hFGFR-1 is 130kDa. A weak 145 kDa component, which could represent the sum of the molecular mass of the ligand bound to the 130 kDa polypeptide, was found in the crosslinking experiments (Fig. 3). The dominating crosslinked component was however 95 kDa, and its relation to the 130 kDa polypeptide is not as apparent. It cannot be excluded that the 95 kDa band arose as a result of partial degradation. There was a low but specific binding of iodinated aFGF and bFGF to untransfected COS-1 cells. A

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A

A

s

Y

U

c

a

0

n

FIGURE 1(A) and (B). Crosscompetition for binding of "'I-aFGF and '?'I-bFGF to transiently expressing COS-7 cells. The binding of "'1-labeled aFGF (A) and bFGF (B) to COS-I cells transfected with phFGFR was determined at o"C, in the presence of increasing concentrations of aFGF (open circles) and bFGF (filled circles). The binding in the absence of unlabeled ligands (1700 c.p.m. "'I-aFGF; 1100 c.p.m. I2'lbFGF) was set to 100%.

10.0

10.0

1

I

0.5

I

I

I

I

2.0

5.0

10.0

50.0

Factor a d d e d (nglml)

HUMAN RECEPTOR FOR aFGF AND bFGF

weak component of 125 kDa was seen as a result of crosslinking of iodinated aFGF to untransfected cells (Fig. 3); this component probably represented binding to an endogenous FGF receptor distinct from hFGFR-1. The level of specific binding of iodinated ligands to untranfected COS-1 cells was too low to permit Scatchard analysis. The amino acid sequence of hFGFR-1 is related to other reported sequences. Lee et al. (1989) isolated a

205

cDNA using sequence information from a receptor purified using binding to biotinylated bFGF-avidin agarose. The binding characteristics of the polypeptide encoded by the chicken cDNA were not examined in the report by Lee et al. The overall amino acid identity between the hFGFR-1 and the chicken bFGF receptor sequences is 92%. The differences are clustered in short stretches in the NH,- and C-terminal part of the polypeptide (Fig. 6). The

A

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0.5

P)

0.1

E

k -0 C

3 0

1

I

I

I

I

I

m

B

0.5

K d = 41 p M

0.1

I

I

I

I

1 .o

I 5.0

Bound fmol/well

I

I F IGURE 5 Scatchard analysis of '?'IaFGF (A) and "'1-bFGF (B) binding to COS-1 cells transiently expressing phFGFR.

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same chicken bFGF receptor cDNA (denoted Cekl) was also isolated using anti-phosphotyrosine antibodies to screen an expression library (Pasquale and Singer, 1989). The Cekl protein was detected in many embryonic tissues, but not in the corresponding adult tissues. The sequence of a human

partial cDNA denoted fms-like gene (flg) (Ruta et al., 1988) is very similar but not identical to the one

reported here (Fig. 6). The flg sequence starts at amino acid 199, according to the numbering in Fig. 1B. The amino acid identity between the sequences is 99%. In a report by Ruta et al. (1989), an anti-

hFGFR- 1 chicken mouse hFGFR- 1 chicken mouse hFGFR- 1 chicken mouse flg hFGFR- 1 chicken mouse flg hFGFR-1 chicken mouse flg hFGFR-1 chicken mouse flg be k

1 MJSWKCLLFWAVLVTATLCTARPSPTLPEQAQPWGAPVEVESFLVHPGDLLQLRCRLRDDVQSINWLRDGVQLAESNRT 1 FT R I L SA A D L -K NI HSA V P N 1 G V L V a0 RITGEEVEVQDSVPADSGLYACVTSSPSGSDTTYFSVNVSDALPSSEDDDDDDDSSSEEKETDNTKPNPVAPYWTSPEK 79 R R E M N E A E A (Q)A Y 80 R (RR) 159 MEKKLHAVPAAKTVKFKCPSSGTPNPTLRWLKNSKEFKPDHRIGGYKVRYA~SIIMDSVVPSDKGNYTCIVENEYGSI 159 G G K 161 G 1 230 NHTYQLDVVERSPHRPILQAGLPANKTVALGSNVEFMCKVYSDPQPHIQWLKHIEVNGSKIGPDNLPYVQILKTAGVNT 238 V 240 HPS 40 317 TDKEMEVLHLRNVSFEDAGEYTCLAGNSIGLSHHSAWLTVLEALEERPAVMTSPLYLEIIIYCTGAFLISCMVGSVIVY 311 I T QS M VT I 319 L I 119 396 KMKSGTKKSDFHSQMAVHKLAKSIPLRRQVTVSADSSASMNSGVLLVRPSRLSSSGTPMLAGVSEYELPEDPRWEL~RD 396 T T N L S M 398 I 198 1 *

hFGFR-1 chicken mouse flg be k hFGFR-1 chicken mouse flg be k hFGFR-1 chicken mouse flg bek hFGFR-1

415 475 417 217 4 554 554 556 356 03 633 633 635 435 162 712

RLVLGKPLGEGCFGQVVLAEAIGLDKDKPNRVTKVAVKMLKSDATEKDLSDLISEMEMMKMIGKHKNIINLLGACTQDG I E

K T M V I KEAVT D V PLYVIVEYASKGNLREYLQARRPPGLEYCYNPSHNPEEQLSSKDLVSCAYQVARGMEYLASKKCIHRDLAARNVLVTED M TRI F R

M STDINRV MTF T L Q N NVMKIADFGLARDIHHIDYYKKTTNGRLPVKWMAPEALFDRIYTHQSDVWSFGVLLWEIFTLGGSPYPGVPVEELFKLL

NN

IYLTG( S ) V

chicken 712

mouse flg bek

714 515 240

-

I

M

KEGHRMDKPSNCTNELYMMMRDCWHAVPSQRPTFKQLVEDLDRIVALTSNQEYLDLSMPLDQYSPSFPDTRSSTCSSGE M

V

T

G

A

I

H

LT

T E

TQ E

Y

-

S

D

hFGFR-1 791 DSVFSHEPLPEEPCLPRHPAQLANGGLKRR chicken 791 D D C PH-SH A H mouse 793 T S flg 594 be k 318 PD M Y QY HINGSVKT---

FIGURE 6. Comparison of the amino acid sequence of hFGFR-I with related sequences. The amino acid sequence of hFGFR-1 (master sequence) was compared with that for the chicken bFGF receptor and Cekl (Lee et al., 1989; Pasquale and Singer, 1989), the mouse bFGF receptor (Reid et al., 1990), the human partial flg sequence (Ruta et al., 1988) and the murine partial bek sequence (Kornbluth et al., 1988). 'Indicates amino acid residue 7 for flg and bek, respectively. Insertions relative to hFGFR-1 are shown in parenthesis for the chicken (Glu-147), mouse (Arg-148 and Arg-148) and flg (Ser-147) sequences. Gaps ( -) were introduced to maximize similarity.

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HUMAN RECEPTOR FOR aFGF AND bFGF

serum raised against a synthetic peptide based on the flg sequence (amino acids 782-806 in Fig. 1B) was used to immunoprecipitate two equally intense components, 150kDa and 130kDa, from NIH 3T3 cells to which iodinated aFGF had been crosslinked. When bFGF was used, only very faint bands of the same sizes were seen. A recent report (Isacchi et al., 1990) shows the sequence of a human cDNA clone identical to phFGFR, but expression and ligandbinding were not examined. A murine partial cDNA denoted bek (bacterially expressed kinase; Kornbluth et al., 1988) displays an amino acid sequence (starting at amino acid 472 according to numbering in Fig. 1B) 85% similar with hFGFR-1 (Fig. 6). Reid et al. (1990) reported the identification of two highly related murine cDNAs. One is apparently more similar to hFGFR-1, the chicken bFGF receptor (Cek 1) and flg whereas the other, although the sequence was not given, is identical to bek. Another recent report (Mansukhani et al., 1990) contained the sequence of a murine cDNA clone very similar to that for phFGFR, but coding for a molecule with a shorter extracellular domain. This receptor was shown to bind bFGF with a K, of 17-74 pM. The influence by members of the FGF family on early embryonic development is currently a focus of interest. It has recently been shown that K-FGF (hst/KS3) and int-2, as well as bFGF can induce mesoderm formation in the Xenopus embryo (Paterno et a]., 1989). The murine int-2 product appears moreover not to be expressed in adult tissues, and there is a strong temporal and spatial restriction of its expression in the mouse embryo (Wilkinson et a]., 1988). Several types of heparinbinding factors are expressed by murine embryonal carcinoma and embryonic stem cells (Heath et a]., 1989; van Veggel et al., 1987), of which some may represent new members of the FGF family. Embryonal carcinoma cells have provided a useful model system for the study of growth factor production during mammalian development. It is obviously relevant to analyze also the pattern of growth factor receptor expression in these cells. The human Tera-2 cells have this far been shown to express receptors for epidermal growth factor (Engstrom, 1986), platelet-derived growth factor receptor-8 (Weima et a]., 1990) and insulin-like growth factor I receptors (Weima et al., 1989). The ability of Tera-2 cells to undergo differentiation in the presence of retinoic acid (Thompson et al., 1984) offers a possibility to analyze also regulatory aspects of receptor expression.

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ACKNOWLEDGMENTS We thank Carl-Henrik Heldin and Bengt Westermark for helpful suggestions, Ulla .Engstrom for peptide synthesis and Ingegard Schiller for preparation of the manuscript.

REFERENCES Abraham, J. A., Whang, J. L., Tumolo, A,, Mergia, A,, Friedman, J., Gospodarowicz, D. and Fiddes, J. C. (1986) Human basic fibroblast growth factor: nucleotide sequence and genomic organization. EMBO 15, 2523-2528. Blobel, G . and Dobberstein, B. (1975) Transfer of proteins across membranes. 11. Reconstitution of functional rough microsomes from heterologous components. 1. Cell. Biol. 67, 835-851. Burgess, W. H. and Maciag, T. (1989) The heparin-binding (fibroblast) growth factor family of proteins. Ann. Rev. Biochem. 58, 575-606. Burrus, L. and Olwin, B. B. (1989) Isolation of a receptor for acidic and basic fibroblast growth factor from embryonic chick. 1. Biol. Chem. 264,18647-18653. Claesson-Welsh, L., Eriksson, A,, Westermark, B. and Heldin, C.H. (1989) cDNA cloning and expression of the human A-type platelet-derived growth factor (PDGF) receptor establishes structural similarity to the B-type PDGF receptor. Proc. Natl. Acad. Sci. U S A 86, 4917-4921. Coughlin, S. R., Barr, P. J., Cousens, L. S., Fretto, L. J. and Williams, L. T. (1988) Acidic and basic fibroblast growth factors stimulate tyrosine kinase activity in vivo. 1. Bid. Chern. 263,988-993. Creighton, T. E. (1984) Proteins (W. H. Freeman and Company, New York), pp. 76-78. Delli Bovi, P., Curatola, A. M., Kern, F. G., Greco, A., Ittman, M. and Basilico, C. (1987) Processing, secretion and biological properties of a novel growth factor of the fibroblast growth factor family with oncogenic potential. Cell 50, 729-737. Denhardt, D. T. (1966) A membrane-filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23, 641-646. Engstrom, W. (1986) Differential effects of epidermal growth factor (EGF) on cell locomotion and cell proliferation in a cloned human embryonal carcinoma-derived cell line in vitro. 1. Cell Sci. 86, 47-55. Finch, P. W., Ruin, J. S., Miki, T., Ron, D. and Aaronson, S. A. (1989) Human KGF is FGF-related with properties of a paracrine effector of epithelial cell growth. Science 245, 752-755. Folkman, J. and Klagsbrun, M. (1987) Angiogenic factors. Science 235, 442-447. Friesel, R., Burgess, W. H. and Maciag, T. (1989) Heparin-binding growth factor 1 stimulates phosphorylation in NIH 3T3 cells. Mol. Cell. B i d . 9, 1857-1865. Gospodarowicz, D. (1987) Isolation and characterization of acidic and basic fibroblast growth factor. Methods Enzymol. 147, 106- 119 .

Gospodarowicz, D., Ferrara, N., Schweigerer, L. and Neufeld, G. (1987) Structural characterization and biological functions of fibroblast growth factor. Endocr. Rev. 8, 95-114. Heath, J. K., Paterno, G. D., Lindon, A. C. and Edwards, D. R. (1989) Expression of multiple heparin-binding growth factor species by murine embryonal carcinoma and embryonic stem cells. Development 107, 113-122. von Heijne. (1986) A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14, 4683-4690. Huang, S. S. and Huang, I. S. (1986) Association of bovine brainderived growth factor receptor with protein tyrosine kinase activity. 1. B i d . Chem. 261, 9568-9571.

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