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Characterization of FGFRL1, a Novel Fibroblast Growth Factor (FGF) Receptor Preferentially Expressed in Skeletal Tissues

2003; Elsevier BV; Volume: 278; Issue: 36 Linguagem: Inglês

10.1074/jbc.m300281200

1083-351X

Beat Trüeb, Lei Zhuang, Sara Taeschler, Markus Wiedemann,

Craniofacial Disorders and Treatments

Clones for a novel transmembrane receptor termed FGFRL1 were isolated from a subtracted, cartilage-specific cDNA library prepared from chicken sterna. Homologous sequences were identified in other vertebrates, including man, mouse, rat and fish, but not in invertebrates such as Caenorhabditis elegans and Drosophila. FGFRL1 was expressed preferentially in skeletal tissues as demonstrated by Northern blotting and in situ hybridization. Small amounts of the FGFRL1 mRNA were also detected in other tissues such as skeletal muscle and heart. The novel protein contained three extracellular Ig-like domains that were related to the members of the fibroblast growth factor (FGF) receptor family. However, it lacked the intracellular protein tyrosine kinase domain required for signal transduction by transphosphorylation. When expressed in cultured cells as a fusion protein with green fluorescent protein, FGFRL1 was specifically localized to the plasma membrane where it might interact with FGF ligands. Recombinant FGFRL1 protein was produced in a baculovirus system with intact disulfide bonds. Similar to FGF receptors, the expressed protein interacted specifically with heparin and with FGF2. When overexpressed in MG-63 osteosarcoma cells, the novel receptor had a negative effect on cell proliferation. Taken together our data are consistent with the view that FGFRL1 acts as a decoy receptor for FGF ligands. Clones for a novel transmembrane receptor termed FGFRL1 were isolated from a subtracted, cartilage-specific cDNA library prepared from chicken sterna. Homologous sequences were identified in other vertebrates, including man, mouse, rat and fish, but not in invertebrates such as Caenorhabditis elegans and Drosophila. FGFRL1 was expressed preferentially in skeletal tissues as demonstrated by Northern blotting and in situ hybridization. Small amounts of the FGFRL1 mRNA were also detected in other tissues such as skeletal muscle and heart. The novel protein contained three extracellular Ig-like domains that were related to the members of the fibroblast growth factor (FGF) receptor family. However, it lacked the intracellular protein tyrosine kinase domain required for signal transduction by transphosphorylation. When expressed in cultured cells as a fusion protein with green fluorescent protein, FGFRL1 was specifically localized to the plasma membrane where it might interact with FGF ligands. Recombinant FGFRL1 protein was produced in a baculovirus system with intact disulfide bonds. Similar to FGF receptors, the expressed protein interacted specifically with heparin and with FGF2. When overexpressed in MG-63 osteosarcoma cells, the novel receptor had a negative effect on cell proliferation. Taken together our data are consistent with the view that FGFRL1 acts as a decoy receptor for FGF ligands. Most bones of the vertebrate skeleton are formed by a complex process termed endochondral ossification which involves a cartilage intermediate (1Bianco P. Descalzi Cancedda F. Riminucci M. Cancedda R. Matrix Biol. 1998; 17: 185-192Crossref PubMed Scopus (158) Google Scholar). This intermediate represents a highly specialized connective tissue. It consists of a single cell type, the chondrocytes, which are embedded in a rich extracellular matrix (2Heinegard D. Oldberg A. FASEB J. 1989; 3: 2042-2051Crossref PubMed Scopus (460) Google Scholar). Typically, this matrix makes up more than 90% of the cartilage volume and consists of collagens (types II, IX, X, and XI), proteoglycans (aggrecan, small leucine-rich proteins), and glycoproteins (matrilins, COMP). During the first step of endochondral ossification, mesenchymal cells condense and differentiate into chondrocytes (3Cancedda R. Descalzi Cancedda F. Castagnola P. Int. Rev. Cytol. 1995; 159: 265-358Crossref PubMed Scopus (351) Google Scholar). These chondrocytes proliferate rapidly and lay down the cartilaginous model of the future bones. The chondrocytes undergo a complex series of distinct developmental stages, including proliferation, maturation, and hypertrophy. The hypertrophic cartilage is calcified and becomes vascularized. Finally, the calcified cartilage is invaded by osteoclasts and osteoblasts, which replace the cartilaginous tissue by bone. Cartilage has become a popular tissue to study cell proliferation and differentiation in vitro (3Cancedda R. Descalzi Cancedda F. Castagnola P. Int. Rev. Cytol. 1995; 159: 265-358Crossref PubMed Scopus (351) Google Scholar). When cultivated on plastic dishes, chondrocytes rapidly dedifferentiate into fibroblast-like cells. In three-dimensional lattices, however, the chondrocytes undergo the ordered sequence of events observed during differentiation and maturation of cartilage in vivo. Three stages of chondrocyte differentiation have been defined in vitro: proliferative chondrocytes producing mainly collagen II, hypertrophic chondrocytes producing collagen X, and osteoblast-like cells producing collagen I and alkaline phosphatase. Many vitamins, hormones, and growth factors are involved in the regulation of chondrocyte proliferation and differentiation (4Vortkamp A. Osteoarthritis Cartilage. 2001; 9: S109-S117PubMed Google Scholar). The mechanisms, how these substances act on gene expression, however, are not yet understood in detail. Vitamin D3, ascorbic acid, and retinoic acid as well as the growth factors FGF, 1The abbreviations used are: FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; FGFRL, fibroblast growth factor receptor-like; GFP, green fluorescent protein; IL-1, interleukin-1; TGF-β, transforming growth factor β. TGF-β, bone morphogenetic protein (BMP), and insulin-like growth factor (IGF) play critical roles during endochondral ossification. Indian hedgehog (Ihh) and the parathyroid-hormone related peptide (PTHrP) constitute a paracrine feedback loop that regulates differentiation of proliferative chondrocytes into hypertrophic cells in the growth plate of long bones. At the level of gene expression, the transcription factors Sox9, L-Sox5, and Sox6 play an important role in the determination of the cartilage cell lineage (5de Crombrugghe B. Lefebvre V. Nikashima K. Curr. Opin. Cell Biol. 2001; 13: 721-727Crossref PubMed Scopus (404) Google Scholar). In particular Sox9 appears to act as a key differentiation factor for chondrocytes analogous to the way that MyoD acts as a master gene during muscle differentiation. We have recently set out to identify and characterize novel cartilage proteins by a subtractive cDNA cloning approach (6Belluoccio D. Trueb B. FEBS Lett. 1997; 415: 212-216Crossref PubMed Scopus (32) Google Scholar, 7Belluoccio D. Schenker T. Baici A. Trueb B. Genomics. 1998; 53: 391-394Crossref PubMed Scopus (37) Google Scholar). Special emphasis was put on regulatory proteins that might play a role during chondrocyte proliferation and differentiation. Two cDNA libraries were constructed with mRNA from cartilage and subtracted with mRNA from skin or skeletal muscle. These libraries comprised many clones for known cartilage-specific proteins. In addition, our libraries contained several novel clones whose sequences have not yet been stored in public data banks. Some of the novel clones coded for a structural protein that was highly related to the family of the matrilins (6Belluoccio D. Trueb B. FEBS Lett. 1997; 415: 212-216Crossref PubMed Scopus (32) Google Scholar, 7Belluoccio D. Schenker T. Baici A. Trueb B. Genomics. 1998; 53: 391-394Crossref PubMed Scopus (37) Google Scholar). Another set of clones was found to code for a novel transmembrane protein related to members of the fibroblast growth factor receptor family. Here we describe the characterization of this novel membrane protein and demonstrate that it represents a new player in the FGF signaling system. RNA Extraction and Northern Blotting—Total RNA was isolated from various sources by the guanidinium thiocyanate method (8Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar) utilizing the RNeasy kit from Qiagen (Hilden, Germany). When the RNA was prepared from cultured cells, the cell lysate was passed through a Qiashredder as suggested by the supplier (Qiagen); when the RNA was prepared from embryonic or adult tissues, the samples were homogenized in lysis buffer and extracted once with an equal volume of phenol/chloroform. Following isolation on RNeasy columns, the purified RNA was resolved on 1% agarose gels in the presence of 1% formaldehyde and transferred to nylon membranes by vacuum blotting. Radioactively labeled cDNA probes were hybridized at 42 °C to the membranes in a buffer containing 50% formamide as described (9Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing Associates, New York1987Google Scholar, 10Schenker T. Trueb B. Exp. Cell Res. 1998; 239: 161-168Crossref PubMed Scopus (43) Google Scholar). Following stringent washing, the membranes were exposed to x-ray film. Subtracted cDNA Libraries—Poly(A) RNA was purified from total RNA by chromatography on oligo(dT)-Sepharose (Amersham Biosciences). Two subtracted cDNA libraries were constructed by the biotin/streptavidin/phenol method (10Schenker T. Trueb B. Exp. Cell Res. 1998; 239: 161-168Crossref PubMed Scopus (43) Google Scholar, 11Duguid J.R. Dinauer M.C. Nucleic Acids Res. 1990; 18: 2789-2792Crossref PubMed Scopus (177) Google Scholar). In brief, the poly(A) RNA isolated from embryonic chicken sterna and from embryonic chicken control tissues (skin and skeletal muscle) was transcribed separately into double stranded cDNA and provided with specific adapters. An adapter with an internal SalI restriction site was used for the cDNA from sterna, whereas a biotinylated adapter without restriction site was used for the cDNA from the control tissues. The cDNAs were amplified by PCR with primers designed according to the adapter sequences. The cDNA preparation from sterna was then hybridized overnight at 68 °C with a 10-fold excess of biotinylated cDNAs from the control tissues. All hybrids containing at least one biotinylated strand were removed by extraction with phenol in the presence of streptavidin. After a second round of hybridization and extraction, the material remaining in the aqueous phase was amplified by PCR to replenish the cDNAs from cartilage. The entire procedure was repeated a total of three times and then the products were ligated into the SalI restriction site of the cloning vector pUC13. The plasmids were transfected into competent bacteria and plated onto selective agar plates. The inserts from clones of interest were radioactively labeled and used as probes to screen a conventional cDNA library, which had been generated from the cartilage of embryonic chicken sterna (6Belluoccio D. Trueb B. FEBS Lett. 1997; 415: 212-216Crossref PubMed Scopus (32) Google Scholar, 9Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing Associates, New York1987Google Scholar). The DNA sequences of the inserts were determined by the dideoxy chain termination method. All sequences were analyzed with the software computer package of the genetics computer group at the University of Wisconsin. In Situ Hybridization—In situ hybridization studies were performed essentially as described (9Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing Associates, New York1987Google Scholar, 12Wälchli C. Koch M. Chiquet M. Odermatt B.F. Trueb B. J. Cell Sci. 1994; 107: 669-681Crossref PubMed Google Scholar) utilizing labeled RNA probes. Samples from 17-day-old mouse embryos were embedded in paraffin and cut into serial sections. Riboprobes were transcribed from the mouse FGFRL1 cDNA sequence (nucleotides 661–1417), which had been cloned into the vector pSK+, with RNA polymerase T7 (antisense) or T3 (sense) in the presence of 35S-uridine 5′-triphosphate. The probes were purified by gel filtration and separated on a 5% polyacrylamide gel to confirm size and purity. The tissue sections were digested with proteinase K and hybridized with the probes at a concentration of ∼5 × 107 dpm/ml for 18 h at 60 °C. Following hybridization, the slides were treated with RNase A and washed in 0.1 × standard saline citrate at 65 °C for 2 h. The slides were then coated with NTB-2 emulsion, exposed for 3 days at 4 °C, and developed with d-19 developer and fixer (Eastman Kodak Co.). Following staining with hematoxilin and eosin, the slides were inspected under a Nikon Eclipse E1000 microscope equipped with dark field optics. Green Fluorescent Protein (GFP) Fusion Protein Expression—MG-63 and COS-1 cells were obtained from the American Type Culture Collection (Manassas, VA). The cells were grown under an atmosphere of 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 9% fetal bovine serum. The cDNA sequence of mouse FGFRL1 corresponding to amino acid residues 1–468 was subcloned into the SacI/BamHI restriction site of the GFP expression vector pEGFP-N1 (Clontech). Likewise, the sequence for the intracellular domain (residues 493–end) was ligated into the HindIII/KpnI site of the expression vector pEGFP-C3. The reading frame of the resulting constructs was verified by DNA sequencing. The plasmids (1 μg/well) were mixed with Opti-MEM 1 (Invitrogen) containing 3 μl of FuGENE 6 reagent (Roche Diagnostics) and added to cultivated cells that were growing on circular cover slips placed into 6 well plates (13Li B. Trueb B. J. Biol. Chem. 2001; 276: 33328-33335Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). One and 2 days after transfection, the slides were inspected under a Zeiss LSM 410 confocal microscope. Analogous experiments were also performed with the human FGFRL1 sequence. Protein Expression in Insect Cells—The extracellular portion of the human and the chicken FGFRL1 protein was expressed in insect cells utilizing the BacVector transfection kit from Novagen. The cDNA sequences corresponding to amino acid residues 25–355 of the chicken or 36–368 of the human protein were ligated into the EcoRI/HindIII or BamHI/SacI restriction site, respectively, of the transfer plasmid pBAC-3. This transfer plasmid harbors the polyhedrin promoter and the sequence for the signal peptide of the baculovirus envelope protein gp64 to direct expression of proteins into the secretory pathway of infected insect cells. Furthermore, the fusion protein is expressed together with a His-tag that allows the purification of the recombinant protein on nickel affinity columns. Authenticity and reading frame of the resulting constructs were verified by DNA sequencing. The transfer plasmids (500 ng) were transfected together with the BacVector-3000 Triple Cut virus DNA (100 ng) into Sf9 cells that had been cultivated in Grace medium (Invitrogen) supplemented with 9% fetal bovine serum. Three days after transfection, viral particles of the supernatant were purified to homogeneity by the agarose overlay technique and amplified to a viral titer >107/ml. Recombinant protein was produced by infection of fresh Sf9 cells growing in 75-cm2 flasks with this viral stock utilizing a multiplicity of 1–4 plaque forming units/cell. Two days after infection, the supernatant of the insect cells was harvested, and the expressed proteins were purified by chromatography on nickel agarose (HIS-select HC nickel affinity gel, Sigma) and/or heparin-Sepharose (Amersham Biosciences) as suggested by the supplier. Interactions of the Recombinant Protein—Recombinant protein (50 μg) was loaded onto a small heparin-Sepharose column (bed volume: 0.5 ml, Amersham Biosciences) that had been equilibrated with 150 mm NaCl, 0.2% Triton X-100, 50 mm sodium phosphate, pH 8.0. The column was extensively washed with the same buffer, and bound protein was eluted with a linear gradient of 0.15–1.35 m NaCl in a total volume of 22 ml. Fractions of 0.5 ml were collected and checked for their NaCl concentration by measuring the conductivity with a WTW LF330 conductivity meter (Weilheim, Germany). Aliquots (32 μl) from every third fraction were separated on a 10% SDS-polyacrylamide gel, transferred to a nylon membrane, and detected by immunoblotting with an antibody directed against the His-tag of the expressed protein. To test for a potential interaction with FGF, human recombinant basic fibroblast growth factor was radiolabeled to a specific activity of 900 Ci/mmol (100 μCi/ml, Amersham Biosciences) using 125I and chloramine T. Aliquots of the expressed protein (5 μg) were incubated for 2 h at room temperature with 2.5 μl of labeled FGF2 and 25 μl of nickelagarose (Sigma) in 1 ml of 300 mm NaCl, 0.2% Triton X-100, 2 mg/ml bovine serum albumin, 50 mm sodium phosphate, pH 8.0. In some experiments, the binding reaction was competed with 5 μg of recombinant basic fibroblast growth factor (Roche Applied Science) or with 10 μl of fetal bovine serum. The beads were collected by centrifugation and washed three times with 1 ml of 300 mm NaCl, 0.2% Triton X-100, 50 mm sodium phosphate, pH 8.0. Bound proteins were eluted with 40 μl of hot SDS sample buffer containing 2% β-mercaptoethanol and analyzed on a 15% SDS-polyacrylamide gel, followed by autoradiography. Cell Proliferation Assay—A cell proliferation assay kit from Roche Applied Science was used to analyze the effect of FGFRL1 on cell growth. MG-63 cells were grown on cover slips to 30% confluence. The cDNA sequences for chicken and mouse FGFRL1 were ligated into the eukaryotic expression vectors pcDNA3.1(+) and pcDNA3.1(–) and transfected into the cells as described above. Following transfection, the cells were synchronized by starvation in medium lacking any fetal bovine serum. After 24 h the cells were stimulated to proliferate again by the addition of insulin (0.5 μg/ml), FGF2 (5 ng/ml), and heparin (300 ng/ml) in Dulbecco's modified Eagle's medium. After 18 h, the cells were labeled with bromodeoxyuridine and fixed with cold glycine-ethanol buffer as described by the supplier of the kit. Proliferating cells were visualized by indirect immunofluorescence utilizing a monoclonal anti-bromodeoxyuridine antibody and a secondary, fluorescein-labeled anti-mouse Ig antibody. Cell growth was determined by counting immunoreactive cells in relation to the total cell number. Cloning of Chicken FGFRL1—Our cDNA libraries that had been constructed with mRNA from chicken cartilage and subtracted with mRNA from chicken skin and muscle comprised more than 500 clones. About 300 individual clones were analyzed with respect to their insert size (200–600 bp), their DNA sequence, and their hybridization pattern. One-third of these clones showed the expected behavior on a Northern blot: they were expressed in sternal cartilage but not in skeletal muscle (Fig. 1). A high redundancy was observed as the majority of the latter clones coded for type II collagen, the predominant collagen of cartilage. The remaining cartilage-specific clones coded for type IX collagen, type XI collagen, aggrecan, link protein, chondromodulin, and matrilin-3 (Fig. 1). Finally, seven clones were found to code for short fragments of a novel protein. The novel clones were utilized as probes to identify overlapping clones in a conventional cDNA library prepared from chicken cartilage. Our efforts led to the isolation of 14 cDNA clones that altogether spanned a cDNA of 6175 nucleotides (GenBank™ accession number AJ535114). This cDNA contained an open reading frame of 1461 nucleotides that could be translated into a novel protein of 487 amino acids with a molecular mass of 54,000 Da. Utilizing the information of the chicken sequence and various expressed sequence tags, we were able to clone the homologous proteins from man (GenBank™ accession number AJ277437), mouse (GenBank™ accession number AJ293947), and rat (GenBank™ accession number AJ536020). The sequences for the human and the mouse protein have already been published as short sequence papers (14Wiedemann M. Trueb B. Genomics. 2000; 69: 275-279Crossref PubMed Scopus (115) Google Scholar, 15Wiedemann M. Trueb B. Biochim. Biophys. Acta. 2001; 1520: 247-250Crossref PubMed Scopus (29) Google Scholar). A strong conservation of the amino acids was observed when the four protein sequences were compared (Fig. 2). The chicken amino acid sequence shared 74% sequence identity (81% sequence similarity if conservative amino acid replacements were included) with the human and 72% identity (78% similarity) with the rat sequence. The mouse sequence differed from the rat sequence only at 11 positions (not shown). Computer predictions revealed that the novel protein represented a typical membrane protein that was highly related to the members of the FGF receptor family. We therefore termed the novel protein fibroblast growth factor receptor-like protein (approved gene symbol FGFRL1). Similar to the four members of the FGF receptor family (16Szebenyi G. Fallon J.F. Int. Rev. Cytol. 1999; 185: 45-106Crossref PubMed Google Scholar), the novel chicken protein contained a signal peptide and a single membrane-spanning domain as well as three extracellular Ig-like repeats. Each of these repeats possessed two conserved cysteine residues that may be involved in the formation of a disulfide bridge. The three Ig-loops of the chicken FGFRL1 protein shared 39–48% sequence similarity with the extracellular domain of chicken FGFR3 (CEK2). At the intracellular side, the novel protein differed completely from all members of the FGF receptor family. FGFR1–4 are known to contain a cytoplasmic protein tyrosine kinase domain that plays an important role in signal transduction (16Szebenyi G. Fallon J.F. Int. Rev. Cytol. 1999; 185: 45-106Crossref PubMed Google Scholar, 17Ornitz D.M. Bioessays. 2000; 22: 108-112Crossref PubMed Scopus (627) Google Scholar). This kinase domain was completely missing in the novel protein. Instead, FGFRL1 contained a short cytoplasmic tail of about 100 amino acids. This cytoplasmic tail showed a much lower degree of conservation among the four species examined than the extracellular domain with the exception of a histidinerich stretch at the C terminus (Fig. 2). This stretch revealed some similarity to the histidine-rich region of homeodomain proteins. FGFRL1 during Evolution—A peculiar observation was made with the murine FGFRL1 sequences. Compared with the chicken and the human sequences, the rat sequence diverged in the intracellular, histidine-rich region at residue 475 and stopped after 54 unrelated residues (Fig. 2). If a single nucleotide would be deleted at this position, the reading frame would change to a highly similar sequence that would end after 21 residues with the motif Y-Q-C as found in the chicken and the human protein. Nevertheless, the frameshift is real, since it was confirmed in the mouse sequence. It is therefore likely that the murine FGFRL1 genes have sustained a frameshift mutation relatively late during evolution. The complete genomic sequences of several organisms have recently been elucidated and deposited in public data banks. We therefore checked whether other species may also contain a receptor similar to FGFRL1. Neither the fruit fly Drosophila melanogaster nor the roundworm Caenorhabditis elegans possessed any gene that would give rise to a transmembrane protein with three related Ig-like repeats. A similar sequence, however, was identified in the genome of the pufferfish Fugu rubripes. This fish contained a gene with six exons that could be transcribed into a mRNA of ∼3000 nucleotides and translated into a protein of ∼500 residues (Fig. 2). The putative fish protein shared 67–73% sequence identity with FGFRL1 from chicken, rat and man. It should be noted that the fish sequence also ended with the peculiar histidine-rich region and the motif Y-Q-C as the human and the chicken sequence. Thus, vertebrates from fish to man contain a novel, homologous protein that belongs to the FGFR family. Lower animals, including insects and nematodes, do not appear to possess this protein. Expression of FGFRL1—The expression of FGFRL1 was analyzed on Northern blots containing RNA from various chicken tissues (Fig. 3). Two bands of similar intensities corresponding to mRNA species of 7 and 4 kb were detected. The size of the larger band is consistent with the total length of our cDNA sequence. The shorter band might represent a mRNA species that was generated by utilization of an alternative polyadenylation site at position 3416 of our cDNA sequence. This notion is consistent with the fact that a radioactively labeled probe derived from the very 3′ end of the total cDNA sequence hybridized with the 7-kb mRNA species but not with the 5-kb species (not shown). The two mRNA species were detected in RNA preparations from the cartilaginous sterna of 16-day-old chicken embryos (Fig. 3, left). Very faint bands that became clearly visible after prolonged exposure were also noticed with RNA preparations from embryonic femur, skeletal muscle, and heart. In contrast, RNA preparations from skin, gizzard, liver, and brain did not reveal any signal. Fairly strong bands were also detected with RNA preparations from adult chicken sterna (Fig. 3, right). In this case, the signal obtained with the cranial portion of the sternum, which is known to contain many hypertrophic chondrocytes in a mineralized matrix, barely differed from that of the caudal portion, which contains proliferative and resting chondrocytes in a non-mineralized matrix. All the other tissues investigated from the adult animal (brain, gizzard, skeletal muscle, calvaria) revealed faint bands that became clearly visible after prolonged exposure. Thus, the FGFRL1 gene is expressed at fairly high level in cartilage and at very low level in many other tissues. Tissue Distribution—The tissue distribution of the FGFRL1 mRNA was further investigated by in situ hybridization on whole body sections of 17-day-old mouse embryos (Fig. 4). Our antisense probe hybridized specifically with a mRNA in all cartilaginous structures. A relatively strong signal was observed in the nasal cartilage, the ribs, and the sternum as well as in the cartilaginous rudiments of developing bones such as the vertebrae and the pelvic bone. Strong expression was also observed in some muscular tissues, including the muscles of the tongue and the diaphragm. In contrast, no signal was detected in the eye, the brain, and the spinal cord. Moreover, the lung and most of the inner organs, including liver, stomach, intestine, and colon, showed very low signal. Hybridization of a consecutive tissue section with our sense probe showed very low background signal, demonstrating the specificity of our probe (Fig. 4). The cartilaginous vertebrae of a 17-day-old embryo was investigated in greater detail (Fig. 5). Relatively strong expression of FGFRL1 mRNA was observed in the developing vertebral bodies. No differences were noted between the cranial and the caudal portion of the vertebrae. Regions of mineralizing cartilage containing hypertrophic cells showed substantially reduced signal. Likewise, the nucleus pulposus that would later give rise to the intervertebral disc revealed only a weak signal. All the tissues adjacent to the vertebrae, including the spinal cord at the dorsal part and the inner organs at the ventral part, reacted only weakly with our probe. Nevertheless, the signal at these locations appeared to be slightly stronger than the background observed with the sense probe (Fig. 5). These results are consistent with the view that FGFRL1 is expressed at fairly high level in all cartilaginous tissues of the skeleton as well as in a few specialized muscles and at very low level in several other tissues. Subcellular Localization—As a receptor for growth factors, the FGFRL1 protein should be located at the plasma membrane. To study the subcellular distribution, we fused the cDNA sequence for mouse and human FGFRL1 to the sequence for GFP and transfected the resulting constructs into human (MG-63) and monkey (COS-1) cells. When inspected by confocal microscopy, the majority of the signal emitted from GFP was found to be distributed along the plasma membrane (Fig. 6). Some signal could also be detected at compartments of the secretory pathway (Golgi, secretory vesicles), but virtually no signal was detected in the nucleus or the cytoplasm. Thus, the novel receptor is faithfully expressed from our constructs and inserted into the plasma membrane where it could theoretically interact with ligands. A similar experiment was performed with the cytoplasmic portion of the FGFRL1 protein fused to GFP. After transfection of the corresponding construct into MG-63 or COS-1 cells, the fusion protein was found to be distributed all over the cytoplasm and the nucleus in a very diffuse fashion (not shown). The distribution could not be distinguished from that obtained with cells that had been transfected with a construct for GFP alone. Thus, the cytoplasmic tail of the FGFRL1 protein does not appear to interact with proteins of any specific subcellular site. Interactions of the FGFRL1 Protein—To investigate a possible interaction of FGFRL1 with putative ligands, chemical amounts of the FGFRL1 protein were required. We therefore placed the chicken and the human cDNA sequence into a prokaryotic expression vector and expressed the extracellular domain of FGFRL1 as a fusion protein in Escherichia coli. Although we were able to isolate high amounts of fusion proteins from inclusion bodies, the purified proteins did not fold in a correct way as demonstrated by SDS-polyacrylamide gel electrophoresis. In the absence of reducing agents, the proteins formed large aggregates linked by disulfide bon