Proc. Natl. Acad. Sci. USA Vol. 88, pp. 1095-1099, February 1991 Cell Biology

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Isolation of an additional member of the fibroblast growth factor receptor family, FGFR-3 (tyrosine kinase growth factor receptor/activation by acidic and basic fibroblast growth factors/K-562 cells/cDNA)

KATHLEEN KEEGAN*, DANIEL E. JOHNSONt, LEWIS T. WILLIAMSt, AND MICHAEL J. HAYMAN*t *Department of Microbiology, State University of New York at Stony Brook, Stony Brook, NY 11794; and tDepartment of Medicine, Howard Hughes Medical Institute, Program of Excellence in Molecular Biology, Cardiovascular Research Institute, University of California, San Francisco, CA 95143

Communicated by William J. Lennarz, November 5, 1990

ABSTRACT The fibroblast growth factors are a family of polypeptide growth factors involved in a variety of activities including mitogenesis, angiogenesis, and wound healing. Fibroblast growth factor receptors (FGFRs) have previously been identified in chicken, mouse, and human and have been shown to contain an extracellular domain with either two or three immunoglobulin-like domains, a transmembrane domain, and a cytoplasmic tyrosine kinase domain. We have isolated a human cDNA for another tyrosine kinase receptor that is highly homologous to the previously described FGFR. Expression of this receptor cDNA in COS cells directs the expression of a 125-kDa glycoprotein. We demonstrate that this cDNA encodes a biologically active receptor by showing that human acidic and basic fibroblast growth factors activate this receptor as measured by 45Ca2 efflux assays. These data establish the existence of an additional member of the FGFR family that we have named FGFR-3.

A

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FIG. 1. (A) Northern blot of K-562 RNA hybridized with clone 17B. A major mRNA of 4.5 kb and a minor mRNA of 7 kb are seen in these cells. Sizes of molecular size standards, in kb, are indicated at left. (B) Primer extension of clone 17B. An oligonucleotide that hybridized near the 5' end of clone 17B was hybridized to K-562 RNA, extended with reverse transcriptase, and electrophoresed on 5% sequencing gel next to a sequencing ladder (lanes G, A, T, and C). The 175-nucleotide extension product is indicated in lane 1 by the arrow.

Acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) are members of a family of multifunctional polypeptide growth factors that have been shown to stimulate proliferation of cells of mesenchymal, epithelial, and neuroectodermal origin. They also play a role in other processes, including angiogenesis, promotion of differentiation of adipocytes and neurons, and would healing (1-3). In addition to aFGF and bFGF, five other members of the fibroblast growth factor (FGF) family have been identified. These include the oncogenes int-2 (4) and hst/Kaposi FGF (5), and three others-FGF-5 (6), FGF-6 (7), and keratinocyte growth factor (8), which may play roles in normal growth and development. Recently, the human, chicken, and mouse FGF receptors (FGFRs) have been shown to be members of the tyrosine kinase receptor family (9-15). The FGFRs are predicted to encode proteins with an extracellular domain containing either two or three immunoglobulin-like domains, a transmembrane domain, and a cytoplasmic tyrosine kinase domain. The FGFR was initially purified from chicken embryo as a receptor that bound bFGF (9). The receptor cDNA was cloned by using oligonucleotide probes based on this protein sequence. The extracellular region of the chicken FGFR was predicted to encode three immunoglobulin-like domains (9). The gene for human FGFR was shown to encode multiple forms of the receptor, including one in which the extracellular region contains the coding potential for two immunoglobulinlike domains and one with the coding potential for three immunoglobulin-like domains (12). These different forms appear to be generated by alternatively spliced mRNAs (12). Two similar forms of the receptor have been isolated from mouse cDNA libraries (13-15). Subsequent characterization of the protein products of the human and chicken receptor

genes has demonstrated that both aFGF and bFGF bind (12) and stimulate tyrosine kinase activity of these receptors (14, 16). The mouse FGFR tyrosine kinase activity is stimulated by Kaposi FGF in addition to bFGF (15). These results raise the question of whether all members of the FGF family share a common cell-surface receptor or interact with distinct receptors. In this report, we describe the isolation and characterization of a gene§ that is very similar to, yet distinct from, the previously described FGFR-encoding gene. Furthermore, we demonstrate that the protein product of this gene can be activated by both aFGF and bFGF, thus establishing the existence of an additional member of the FGFR

family.

MATERIALS AND METHODS cDNA Cloning. A human K-562 cDNA library (from Owen Witte, University of California, Los Angeles; ref. 17) was hybridized under low-stringency conditions [50% (vol/vol) formamide/5 x SSC (1 x SSC is 0.15 M sodium chloride/0.015 M sodium citrate, pH 7)/25 mM sodium phosphate, pH 6.5/1 x Denhardt's solution (lx Denhardt's solution is 0.02% polyvinylpyrrolidone/0.02% Ficoll/0.02% bovine serum albumin) and salmon sperm DNA at 250 ,ug/ml/10% (wt/vol) dextran sulfate for 18 hr at 38°C]. The entire v-sea gene was [a-32P]dCTP-labeled with a random primer kit (Boehringer Mannheim) and used as probe. Filters were washed once for 15 min in 1 x SSC/0.1% SDS at 42°C and twice for 20 min in Abbreviations: FGF, fibroblast growth factor; aFGF, acidic FGF; bFGF, basic FGF; FGFR, FGF receptor. iTo whom reprint requests should be addressed. §The sequence reported in this paper has been deposited in the GenBank data base (accession no. M58051).

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.

1095

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Proc. Natl. Acad. Sci. USA 88 (1991)

Cell Biology: Keegan et al.

phate, pH 6.5/1x Denhardt's solution/salmon sperm DNA at 250 AgIml/l0o (wt/vol) dextran sulfate, 42°C] for 18 hr and washed at O.1x SSC/0.1% SDS at 65°C for 2 hr. An RNA ladder (BRL) was used for molecular size standards. Primer Extension. An oligonucleotide, 5'-GACGGGCAGCGTGCCTGC3' beginning 80 base pairs (bp) downstream from the 5' end of clone 17B was labeled with [,y-32P]ATP and hybridized to 40 ,ug of RNA at 30°C in 50% formamide/20 mM Pipes, pH 6.5/0.9 M NaCI/1 mM EDTA. Primer extension was done as described (20), and the products were electrophoresed on a 5% sequencing gel next to a sequencing ladder. Transient Expression and Immunoprecipitation. Clone 17B was subcloned into the simian virus 40- based expression vector pMT2 (22, 23). Electroporation into COS cells was done at 675 V/cm. Sixteen hours later, cells were treated with increased amounts of tunicamycin (0, 2.5, and 5.0 ,g/ml) for

0.2x SSC/0.1% SDS at 42°C. Full-length clones were then isolated by screening at high stringency (using the hybridization buffer described above at 42°C for 18 hr) with a [a-32P]dCTP-labeled partial cDNA clone of FGFR-3 as probe. The cDNAs were subcloned into pBSKS+ (Stratagene) and sequenced using the dideoxynucleotide chaintermination method (18). The putative hydrophobic signal sequence and transmembrane sequence were identified by Kyte and Doolittle hydropathy analysis (19). Northern (RNA) Blot Analysis. mRNA from K-562 cells was prepared by using the guanidinium hydrochloride/CsCl method (20). Ten micrograms of total RNA was loaded on a formaldehyde gel, and a Northern blot was done as described (21). The blot was hybridized with a [a-32P]dCTP-labeled probe derived from the kinase domain of FGFR-3 at high stringency [50% formamide/5 x SSC/25 mM sodium phos-

1 CGCGCGCTGCCTGAGGACGCCGCGGCCCGCCCGCCATGGGCGCCCCTGCCTGCGCCTCGCGCTCTGCGTGGccGTGGccArcGTGGCCGGCGCCTCCTCGGAGTCCTTGGGGACG M G A P A C A L A L C V A V A I V A G A S S E S L G T

120

121 GAGCAGCGCGTCGTGGGGCGAGCGGCAGAAGTCCCGGGCCCAGAGCCCGGCCAGCAGGAGCAGTTGGTCTTCGGCAGCGGGGATGCTGTGGAGCTGAGCTGTCCCCGCCGGGGGTGGT 28 E Q R V V G R A A E V P G P E P G 0 Q E 0 L V F G S G D A V E L S C P P P G G G

240 67

241 68

27

CCCATGGGGCCCACTGTCTGGGTC3GGATGGCACAGGGCTGGTGCCCTCGGAGCGTGTCCTGGTGGGGCCCCAGCGGCTGCAGGTGCTGATGCCTCCCACGAGGACTCCGGGGCCTAC360 S G A Y

107

361 AGCTGCCGGCAGCGGCTCACGCAGCGCGTACTGTGCCACTTCAGTGTGCGGGTGACAGACGCTCCATCCTCGGGAGATGACGAAGACGGGGAGGACGAGGCTGAGGACACAGGTGTGGAC 108 S C R Q R L T 0 R V L C H F S V R V T D A P S S G D D E D G E D E A E D T G V D

480 147

M G P T V W V K D G T

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481 ACAGGGGCCCCTTACTGGACACGGCCCGAGCGGATGGACAAGAAGCTGCTGGCCGTGCCGGCCGCCAACACCGTCCGCTTCCGCTGCCCAGCCGCTGGCMCCCCACTCCCTCCATCTCC 148 T G A P Y W T R P E R M D K K L L A V P A A N T V R F R C P A A G N P T P S I S

600 187

601 TGGCTGAGACGGCAGGGAGTTCCGCGGCGAGCACCGCATTGGAGGCATCAGCTGCGGCATCAGCAGTGGAGCCTGGTCATGGAAGCGTGGTGCCCTCGGACCGCGGCAACTACACC 188 W L K N G R E F R G E H R I G G I K L R H Q Q W S L V M E S V V P S D R G N Y T

720 227

721 228

840 TGCGTCGTGGAG4C0GTTTGGCAGCATCCGGCAGACGTACACGCTGGACGTGCTGGAGCGCTCCCCGCACCGGCCCATCCTGCAGGCGGGGCTGCCGGCCACCAGACGGCGGTGCTG V V E

C

N

K

F G S

I

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Y

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841 GGCAGCGACGTGGAGTTCCACTGCAGGTGTACAGTGACGCACAGCCCCACATCCAGTGGCTCAAGCACGTGGAGGTGAACGGCAGCAAGGTGGGCCCGGACGGCACACCCTACGTTACC 268 G S D V E F H C K V Y S D A 0 P H I Q W L K H V E V N G S K V G P D G T P Y V T 961 308

267

960 307

1080 GTGCTCAAGACGGCGGGCGCT1CACCACCGACAAGGAGCTAGAGGTTCTCTCCT0TGCACMCGTCACCTTTGAGGACGCCGGGGAGTACACCTGCCTGGCGGGCAtTCTATTGGGTTT F

347

TCTCATCACTCTGCGTGGCTGGTGGTGCTGCCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAGGCGGGCAGTGTGTATGCAGGCATCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATC S H H S A W L V V L P A E E E L V E A D E A G S V Y A G I L S Y G V G F F L F I

1200 387

1201 CTGGTGGTGGCGGCTGTGACGCTCTGCCGCCTGCGCAGCCCCCCCAAGAAGGCCTGGGCTCCCCCACCGTGCACAGATCTCCCGCTTCCCGCTCAGCGACAGGTGTCCCTGGAGTCC 388 L V V A A V T L C R L R S P. P K K G L G S P T V H K I S R F P L K R Q V S L E S

1320 427

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1321 428 1441 468 1561 508

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1440 4CGCGTCCATGAGCTCC4CACACCACTGGTGCGCATCGCAAGGCTGTCCTCAGGGGAGGGCCCCACGCTGGCCATGTCTCCGAGCTCGAGCTGCCTGCCGACCCCAATGGGAGCTG A

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1680 18GATGC0GAGACGATGCCACTGACAAGGACCTGTCGGACCTGGTGTCT0GAGATGGAGATGATGAAGATGATCGGGAACACAAAACATCATCAACCTGCTGGGCGCCTGCACGCAG M

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1681 GGCGGGCCCCTGTACGTGCTGGTGGAGTACGCGGCCAAGGGTAACCTGCGGGAGTTTCTGCGGGCGCGGCGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCMGCCGCCCGAGGAG 548 G G P L Y V L V E Y A A K G N L R E F L R A R R P P G L D Y S F D T C K P P E E

1800 587

1801 CAGCTCACCTTCAAGGACCTGGTGTCCTGTGCCTACCAGGTGGCCCGGGGCATGGAGTACTTGGCCTCCCAGAAGTGCATCCACAGGGACCTGGCTGCCCGCAATGTGCTGGTGACCGAG 588 Q L T F K D L V S C A Y Q V A R G M E Y L A S 0 K C I H R D L A A R N V L V T E

1920 627

1921 GACAACGTGATGAAGATCGCAGACTTCGGGCTGGCCCGGGACGTGCACAACCTCGACTACTACAAGAAGACAACCAACGGCCGGCTGCCCGTGAAGTGGATGGCGCCTGAGGCCTTGTTT 628 D N V M K I A D F G L A R D V H N L D Y Y K K T T N G R L P V K W M A P E A L F

2040 667

2041 GACCGAGTCTACACTCACCAGAGTGACGTCTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTTCACGCTGGGGGGCTCCCCGTACCCCGGCATCCCTGTGGAGGAGCTCTTCMGCTGCTG 668 D R V Y T H 0 S D V U S F G V L L W E I F T L G G S P Y P G I P V E E L F K L L

2160 707

2161 708

2280 8GGAGGGCCACCGCATGGAC0GCCCGCCMCTGCACACACGACCTGTACATGATCATGCGGGAGTGCTGGCATGCCGCGCCCTCCCAGAGGCCCACCTTCMGCAGCTGGTGGAGGAC K 0

L V E D

747

2281 CTGGACCGTGTCCTTACCGTGACGTCCACCGACGAGTACCTGGACCTGTCGGCGCCTTTCGAGCAGTACTCCCCGGGTGGCCAGGACACCCCCAGCTCCAGCTCCTCAGGGGACGACTCC 748 L D R V L T V T S T D E Y L D L S A P F E 0 Y S P G G Q D T P S S S S S G D D S

2400 787

2401 GTGTTTGCCCACGACCTGCTGCCCCCGGCCCCACCCAGCAGTGGGGGCTCGCGGACGTGAGGGCCACTGGTCCCCACATGTGAGGGGTCCCTAGCAGCCCTCCCTGCTGCTGGTGCA 788 V F A H D L L P P A P P S S G G S R T *

2520

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P A N C T

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FIG. 2. Sequence of FGFR-3: nucleotide sequence and predicted amino acid (in single-letter code) sequence ofthe longest open reading frame of clone 17B. Bold underlining, hydrophobic sequences; *, cysteine residues; underlining, potential N-linked glycosylation sites; and +, conserved ATP-binding site motif Gly-Xaa-Gly-Xaa-Xaa-Gly (GXGXXG).

Cell

Biology: Keegan et al.

Proc. Natl. Acad. Sci. USA 88 (1991)

1.5 hr before and throughout a 2-hr [35S]methionine-labeling period. Cells were labeled with [35S]methionine at a concentration of 150 puCi/ml (1 Ci = 37 GBq). The labeled cells were lysed in 1% Triton X-100/150 mM NaCl/10 mM Tris HCI, pH 7.5, on ice, precleared with Staphylococcus aureus, and spun at 10,000 x g for 15 min. Immunoprecipitation was performed as described (24) by using rabbit antiserum generated against a trpE fusion protein expressing amino acids 577-806 of FGFR-3. Immunoprecipitates were electrophoresed on a 7.5% SDS/polyacrylamide gel (25). 45Ca2' Efflux Assays. RNAs were transcribed from linearized cDNA templates by using either T7 RNA polymerase (antisense) or T3 RNA polymerase (sense) in the presence of 500 gM rRNA-encoding NTP (200 ttM rRNA-encoding GTP) and 500 ,uM 5'-GpppG-3', according to published protocols (12). Oocytes were surgically removed and enzymatically dispersed by incubation with type II collagenase (1 mg/ml) for 3 hr at room temperature. Individual oocytes were injected into the vegetal pole with 50 nl of water or RNA solution (1 ,ug/,l in water). After injection, oocytes were incubated at 19°C for 48 hr before performing the 45Ca2' efflux assay; 45Ca2' efflux assays were done as described (12). Briefly, oocytes were incubated with 45CaC12 for 3 hr at 19°C and then washed extensively. Groups of five oocytes were placed in individual wells of a 24-well plate. At 10-min intervals, the medium was removed for counting of radioactivity and fresh medium was added. After 40 min, recombinant human aFGF and bFGF (a gift from Chiron) were added to a final concentration of 0.5 nM, and medium collections were continued at the specific times.

1097

as shown by Northern blot (Fig. 1A). In addition, primer extension using a primer near the 5' end of the cDNA demonstrated only 95 base pairs (bp) of additional sequence in the mRNA not present in the cDNA, suggesting that the clone was nearly complete (Fig. 1B). An open reading frame of 806 amino acids was predicted from the nucleotide sequence of this clone (Fig. 2) and was found to be most similar to FGFRs in overall structure and amino acid homology (see Fig. 3A). The predicted initiating methionine of clone 17B, although not residing within a context favorable for strong translation initiation (29), is followed by a hydrophobic sequence characteristic of a signal sequence (30). In addition, this methionine residue is in an analogous position to the initiating methionine of the FGFR. Also following the same pattern as FGFRs, the extracellular domain of clone 17B contains three immunoglobulin-like domains. Constant-type immunoglobulin domains are characterized by a cysteine residue followed by a tryptophan 11 or 12 residues downstream and a Asp-Xaa-Gly-Xaa-Tyr-XaaCys motif 50-70 residues further downstream (31). Consensus sequences for variable-type immunoglobulin domains consist of a cysteine residue followed by a tryptophan 16 amino acids downstream and a Asp-Xaa-Gly-Xaa-Tyr-XaaCys motif 60-75 residues further downstream (31). Immunoglobulin domains I, II, and III of clone 17B fall into the constant-type immunoglobulin domain category. Between the first and second immunoglobulin domains is an acidic run of amino acids (Asp-Asp-Glu-Asp-Gly-Glu-Asp-Glu-AlaGlu-Asp; DDEDGEDEAED in single-letter code) similar to an acidic region found in other FGFRs (Fig. 2). Seven potential N-linked glycosylation sites reside in the extracellular region. A 25-amino acid hydrophobic sequence characteristic of a transmembrane domain lies at amino acids 372-396 (Fig. 2). The sequence of the cytoplasmic domain of clone 17B is typical of tyrosine kinase receptors (ref. 32; Fig. 2). There is a Gly-Xaa-Gly-Xaa-Xaa-Gly (GXGXXG) motif and a conserved lysine residue, both of which are normally found in ATP-binding domains and an Asp-Phe-Gly-Leu-Ala-Arg (DFGLAR) sequence, which is found in known tyrosine kinase catalytic domains. The tyrosine kinase domain of clone 17B has features characteristic of other tyrosine kinase receptors such as FGFR, platelet-derived growth factor receptor (33, 34), and colony-stimulating factor 1 receptor (35). The predicted protein sequence of clone 17B contains an

RESULTS AND DISCUSSION To isolate tyrosine kinases expressed in hematopoietic cells, we screened a cDNA library prepared from the human chronic myelogenous leukemia (CML) cell line, K-562 (17) with the chicken v-sea gene, a receptor-like tyrosine kinase (26) under low-stringency conditions. Among several cDNAs isolated, the nucleotide sequence of one partial cDNA clone indicated that we had isolated a gene that was only 29o homologous to v-sea but was much more similar to the previously described FGFR (11, 12) and the mouse bek gene (27). A 4.5-kilobase (kb) clone, named 17B, was isolated by screening the library at high stringency. The size ofthis clone corresponded to the size of the major mRNA from K-562 cells

A

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TM rqs &I

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23%

22%

48%

70%

28%

44%

82%

43%

91 %

38%

FGFR-2/bek

144%

21

51%

71%

32%

44%

88%

50%

93P

47%

Cek 2

1 8%

37%

76%

87%

68%

73%

9 1%

93%

99%

66%

FIG. 3. (A) Schematic of the human FGFR-3. Shaded box, putative signal sequence; S, immunoglobulin domains with cysteine residues; black box, acidic region; hatched box, transmembrane domain (TM); vertically striped box, kinase domain; and KI, kinase insert region. (B) Amino acid homology between human FGFR-3 and FGFR-1, FGFR-2/bek (36), and chicken Cek-2 sequences. The putative amino acid sequences are broken down into specific regions including the signal sequence (SS), immunoglobulin domains (I, II, and III), transmembrane domain (TM), juxtamembrane region (JM), tyrosine kinase domain (TK 1 and TK 2), kinase insert (KI), and C-terminal tail (C).

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Tunicamycin (ug! ml ) P

50

25 P

P

200-

976843FIG. 4. Transient expression of FGFR-3 cDNA in COS cells with and without tunicamycin. Cells were labeled with [35S]methionine in the presence of increased amounts of tunicamycin (0, 2.5, and 5 ,ug/ml), lysed, and immunoprecipitated with preimmune sera (P) or antisera recognizing FGFR-3 (I). Molecular mass standards are denoted at left in kDa.

inserted sequence that splits the tyrosine kinase domain into two parts. This kinase insert sequence is 14 amino acids long (residues 576-589), which is the same size as the kinase insert region of the FGFR (residues 580-593) but is much smaller than the kinase insert regions of the colony-stimulating factor 1 and platelet-derived growth factor receptors. The juxtamembrane regions of clone 17B (residues 397-478) and FGFR (residues 396-482) are longer than the corresponding regions of other tyrosine kinases and provide a feature that distinguishes this class of growth factor receptors. FGFR and clone 17B appear to encode receptors with similar structures: they possess three immunoglobulin-like domains, a transmembrane domain, and a tyrosine kinase domain. The amino acid homology between FGFR and clone 17B in the kinase domain is 87% (Fig. 3). However, the kinase insert region, the juxtamembrane domain, and the C terminus are less conserved, showing 43%, 44%, and 38% homology, respectively (Fig. 3). These less conserved sequences are most likely to be involved in the specificity of this receptor.

The extracellular region exhibits an overall amino acid identity of 47% when compared with the FGFR, but when it is broken down into immunoglobulin domains, extracellular domain I is less conserved, whereas domains II and III are highly conserved. Extracellular domain I shows the greatest divergence between the two genes, showing only 22% amino acid identity (Fig. 3). Recently, two other genes, bek (36, 38) and Cek2 (37), have been shown to exhibit a high degree of homology to the FGFR gene and clone 17B (Fig. 3) and are predicted to encode proteins with similar structural characteristics. In addition, bek binds aFGF and bFGF (36), thus placing it in the FGFR family. In summary, the degree of amino acid homology between other members of the FGFR family and protein 17B demonstrates that we have isolated a gene with a predicted structure that has significant similarity to FGFRs. To characterize the protein product of clone 17B, we assayed transient expression in COS cells. Cells were transfected with plasmid containing the 17B cDNA in either the sense or the antisense orientation. Immunoprecipitation was then performed with antiserum that recognizes the kinase domain of the cDNA. A protein of 125 kDa is specifically immunoprecipitated in cells expressing the receptor cDNA in the sense orientation (Fig. 4) but not in the antisense orientation (data not shown). A 97-kDa protein is immunoprecipitated from COS cells transfected with clone 17B when treated with the N-linked glycosylation inhibitor tunicamycin. These data indicate that clone 17B encodes a 125-kDa transmembrane glycoprotein. As stated previously, the degree of amino acid identity between the FGFR and protein 17B is 47% in the extracellular domain. The epidermal growth factor receptor and the c-erbB-2 genes show 43% identity in the extracellular domain but do not share the ability to bind epidermal growth factor (28, 39). Because this would suggest that a high degree of amino acid identity does not necessarily predict an overlap in factor binding, we asked whether aFGF and bFGF would activate our FGFR-related receptor. To address this question, we expressed the 17B protein in Xenopus oocytes and measured receptor activation by using a sensitive Ca2+ efflux assay (40). This is a rapid functional assay that measures the ability of a receptor to mobilize intracellular Ca2+ stores upon receptor stimulation. Previous studies have shown that the chicken and human FGFRs bind aFGF and bFGF and are activated by them in this assay system (12). For our exper-

3000 1

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wate r antisense FGFR 3 * -- FGFR 1

-

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1000

0

Minutes FIG. 5. bFGF induces 45Ca2+ efflux from Xenopus oocytes injected with RNA encoding FGFR-3. The graph shows 45Ca2+ efflux from oocytes injected with sense-strand FGFR-3 RNA (V), antisense strand FGFR-3 RNA (*), FGFR-1 (O), or water (o). Injected oocytes were incubated with 45CaC12 for 3 hr at 19°C and then washed extensively. Groups of five oocytes were placed in individual wells of a 24-well plate. At 10-min intervals, the medium was removed to count radioactivity, and fresh medium was added. After 40 min, recombinant bFGF was added to a final concentration of 0.5 nM, and medium removal was continued at specified times. Each data point represents the average of triplicate wells. In general, triplicate points did not vary >15%.

Cell

Biology: Keegan et al.

iments, oocytes were injected with in vitro-transcribed RNAs corresponding to either the sense or antisense of clone 17B. After 2-day incubation, oocytes were preloaded with 45CaC12, stimulated with bFGF, and assayed for levels of 45Ca2+ released into the medium (Fig. 5). Addition of 0.5 nM bFGF to oocytes injected with RNA from clone 17B resulted in a rapid and large efflux of 45Ca2+. Similar results were obtained upon adding 0.5 nM aFGF (data not shown). Stimulation of oocytes injected with human FGFR-1 caused a slightly higher peak of 45Ca2' release that follows a profile similar to that from protein 17B. In contrast, oocytes injected with either water or antisense RNA from clone 17B did not give strong profiles of efflux. These data indicate that clone 17B encodes a receptor capable of being activated by aFGF and bFGF, thus establishing the receptor as an additional member of the FGFR family. Because of the high degree of homology between protein 17B, FGFR, and bek-encoded protein and because of its ability to be activated by aFGF and bFGF, we designate our protein FGFR-3 and propose that the previously described FGFR/flg protein be named FGFR-1 and bek-encoded protein be named FGFR-2. Both aFGF and bFGF bind and activate the chicken and human FGFRs (12). One form of the human FGFR that binds both factors possesses an extracellular domain consisting of only the acidic region at the N terminus followed by immunoglobulin domains II and III (12). Although the extracellular region of FGFR-3 is only 47% identical to FGFR-1, there is a high degree of conservation within the acidic domain and domains II and III; this would suggest that these sequences are sufficient for binding aFGF and bFGF. We have identified another tyrosine kinase receptor gene and have shown that the protein product of this gene can be stimulated by aFGF and bFGF. Identification of FGFR-3-, FGFR-1-, and the FGFR-2/bek-encoding genes offers additional evidence for a family of FGFRs. Recently, the sequence of the chicken gene Cek2 was published (37), and the protein it encodes has greater amino acid identity to FGFR-3 than to either FGFR-1 or FGFR-2/bek and, thus, may represent the chicken homologue of FGFR-3. Although we have shown that FGFR-3 responds to aFGF and bFGF, we do not know the identity of the in vivo ligand that interacts with this receptor. Further studies will be needed to determine whether different members of the FGFR family display different specificities for the known members of the FGF family. We thank L. Morrison, A. Crowe, and W. Schubach for critical reading of the manuscript, John Lu for technical expertise, J. Lipsick for assistance with sequence analysis, and K. Donnelly for manuscript preparation. This work was supported by National Institutes of Health Grants R01 HL-32989 and P01 HL-43821 (to L.T.W.) and R01 CA42573 and P01 CA-28146 (to M.J.H.). D.J. was supported by American Heart Association Fellowship 94-1219116. 1. Baird, A., Esch, F., Mormede, P., Ueno, N., Ling, N., Bohlen, P., Ying, S.-Y., Wehrenberg, W. B. & Guillemin, R. (1986) Rec. Prog. Horm. Res. 42, 143-205. 2. Burgess, W. H. & Maciag, T. (1989) Annu. Rev. Biochem. 58, 575-606. 3. Rifkin, D. B. & Moscatelli, D. (1989) J. Cell Biol. 109, 1-6. 4. Moore, R., Casey, G., Brookes, S., Dixon, M., Peters, G. & Dickson,C. (1986) EMBO J. 5, 919-924. 5. Delli Bovi, P., Curatola, A. M., Kern, F. G., Greco, A., Ittmann, M. & Basilico, C. (1987) Cell 50, 729-737. 6. Zhan, X., Bates, B., Hu, X. & Goldfarb, M. (1988) Mol. Cell. Biol. 8, 3487-3495. 7. Marics, I., Adelaide, J., Raybaud, F., Mattei, M., Coulier, F., Planche, J., Lapeyriere, 0. & Birnbaum, D. (1989) Oncogene

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Isolation of an additional member of the fibroblast growth factor receptor family, FGFR-3.

The fibroblast growth factors are a family of polypeptide growth factors involved in a variety of activities including mitogenesis, angiogenesis, and ...
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