FLT4 Receptor Tyrosine Kinase Contains Seven Immunoglobulin-Like Loops and Is Expressed in Multiple Human Tissues and Cell Lines1

[CANCER RESEARCH 52. 5738-574.1. October 15, 1992| FLT4 Receptor Tyrosine Kinase Contains Seven Immunoglobulin-like Loops and Is Expressed in Multiple Human Tissues and Cell Lines1

Katri Pajusola, Olga Aprelikova, Jaana Korhonen, Arja Kaipainen, Liisa Pertovaara, Rutta Alitalo, and Kari Alitalo2

Cancer Biology Laboratory, Departments of Virology [L. P.I and Pathology [K. P., O. A., J. K., A. K., K. A.], and Transplantation Laboratory [R. A.], University of Helsinki, Haartmaninkatu .?,00290 Helsinki 29, Finland

ABSTRACT Ig-like loops. Although the predicted FLT3/FLK2 proteins also contain five Ig-like domains, the FLT1 and KDR/FLK1 genes The /ms-like tyrosine kinase 4 (FLT4) complementary DNA was encode seven Ig loops (6). cloned from a human lili erythroleukemia cell library by polymerase chain reaction-amplification. We previously reported a partial sequence The eight human class III RTK genes are clustered in three different chromosomes. Chromosome 4 contains the c-kit, of FLT4 and showed that the 11 14 gene maps to chromosomal region Sq33-qter (O. Aprelikova, K. Pajusola, J. Partanen, E. Armstrong, R. PDGFR-a, and KDR genes (7-9). The FLT1 and FLT3 genes Alitalo, S. Bailey, J. McMahon, J. Wasmuth, K. Huebner, and K. are located in chromosome 13ql2 (10, 11), while FLT4 was Alitalo, Cancer Res., 52: 746-748, 1992). Here we present the full- found to be localized in chromosome 5q33-qter (1) close to the length sequence of the predicted FLT4 protein. The extracellular do fms and PDGFR-ft genes (12). main of 11 14 consists of 7 immunoglobulin-like loops, including 12 Here we report the entire amino acid sequence of FLT4 and potential glycosylation sites. On the basis of structural similarities its expression pattern in different tissues and cell lines. FLT4 and the previously known FLT1 and kinase insert domain-con taining receptor tyrosine kinase/fetal liver kinase l (KDR/FLK1) re MATERIALS AND METHODS ceptors constitute a subfamily of class III tyrosine kinases. 1114 was expressed as 5.8- and 4.5-kilobase mRNAs which were found to differ in Isolation and Characterization of cDNA Clones. An oligodeoxythy- their 3' sequences and to be differentially expressed in the HEL and midine-primed HEL cell cDNA library in bacteriophage Xgtl 1 (kindly DAMI leukemia cells. Interestingly, a Wilms' tumor cell line, a retin- provided by Dr. Mortimer Poncz, Childrens Hospital of Philadelphia, oblastoma cell line, and a nondifferentiated teratocarcinoma cell line Philadelphia, PA) was screened with the previously cloned FLT4 cDNA expressed 11 14. whereas differentiated teratocarcinoma cells were neg fragment (1). Positive plaques were identified and purified as described ative. Most fetal tissues also expressed the i Â¡14mRNA, with spleen, (13), and cDNA inserts were isolated from a low-melting-point agarose brain intermediate zone, and lung showing the highest levels. In in situ gel, ligated into Â£coRl-cleaved GEM3Zf(+) plasmid (Promega), and hybridization the ELT4 autoradiographic grains decorated bronchial used to transform Escherichia coli. Both single- and double-stranded epithelial cells of fetal lung. No evidence was obtained for the expres templates were used for sequencing by the dideoxynucleotide chain sion of 1114 in the endothelial cells of blood vessels. termination method (14). New sequencing primers were designed ac cording to the sequences obtained. All portions of the cDNAs were INTRODUCTION sequenced on both strands. Computer analysis of the sequences was done with the GCG package using the Fasta and Wordsearch programs We described previously the identification and partial de duced amino acid sequence of a novel RTK3 initially cloned (15). Inverse Polymerase Chain Reaction. Since cDNA libraries used for from a human erythroleukemia cell cDNA library (1). The pro the screening of FLT4 cDNAs did not contain its most 5' protein- tein, designated FLT4, deduced from the nucleotide sequence coding sequences, inverse PCR was used for the amplification of the 5' of this cDNA belongs to class III RTKs according to the clas end of FLT4. Polyadenylated RNA was isolated from the HEL cells, sification of Ullrich and Schlessinger (2). This class of RTKs and the double-stranded cDNA copy was synthesized using the Amer- includes two protooncogenes (c-fms and c-kit) encoding recep sham cDNA Synthesis System Plus kit and a gene-specific primer, tors for colony-stimulating factor 1 and stem cell factor, respec 5'-TGTCCTCGCTGTCCTTGTCT-3', which was located 195 base pairs downstream of the 5' end of clone S2.5. The cDNA was treated tively, and two PDGFRs (Â«and ÃŸ). Additional members of class III RTKs, the human FLT1 and with T4 DNA polymerase to blunt the ends and purified with Centricon FLT3 cDNAs were cloned using the \-ros and c-fms sequences, 100 (Amicon). Circularization was made in a total volume of 150 n\ in a reaction mixture containing ligation buffer, 5% PEG-8000, 1 mivi respectively, under low-stringency hybridization conditions dithiothreitol, and 8 units of T4 DNA ligase. Ligation was carried out (3, 4). The mouse FLKl cDNA, as well as FLK2, which is the at 16Â°Cfor16 h. Fifteen n\ of this reaction mix were used in a standard mouse homologue of FLT3, and human FLT4 were identified 100-MlPCR reaction. Primers used in PCR were oriented in opposite by PCR amplification using degenerate oligonucleotides de directions and contained Sad and Pstl restriction sites, present in this signed according to conserved regions of tyrosine kinase cata region of the FLT4 cDNA. The sequences of the primers were 5'- lytic domains (1, 5, 6). A typical feature originally reported for TCCCCTGCAGGAGATGGACA-3' and 5 -CTTGGCCAGGAGCT- CAGGAGG-3'. Two rounds of PCR were performed using 33 cycles class III RTKs is a long extracellular domain organized into five (denaturation at 95Â°Cfor 1 min, annealing at 55Â°Cfor 2 min, and elongation at 72Â°Cfor 4 min). The PCR mixture was treated sequen Received 5/4/92; accepted 8/6/92. The costs of publication of this article were defrayed in part by the payment of tially with the Sad and Pstl restriction enzymes, and after purification page charges. This article must therefore be hereby marked advertisement in accord with MagicPCR Preps (Promega) DNA fragments were subcloned in ance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by the Finnish Cancer Organizations, the Finnish Academy, and the to the pGEM3Zf(+) vector for sequencing. The sequences obtained Sigrid Juselius and Finnish Cultural Foundations. were confirmed by cloning and analysis of the corresponding genomic 2 To whom requests for reprints should be addressed. sequence. -'The abbreviations used are: RTK, receptor tyrosine kinase; FLT1,/mi-like mRNA Expression Analyses. The tumor cell lines used in this study tyrosine kinase; FLKl, fetal liver kinase: TK, tyrosine kinase; cDNA, complemen have been reported in several previous publications: JEG-3, a chorio- tary DNA; PDGFR, platelet-derived growth factor receptor; PCR, polymerase chain reaction; VEGF/VPF. vascular endothelial growth factor/vascular permeabil carcinoma; A204, a rhabdomyosarcoma; SK-NEP-1, a nephroblas- ity factor; KDR, kinase insert domain-containing receptor tyrosine kinase. toma; BT-474, a breast carcinoma; Y79, a retinoblastoma; and 5738

MOLT-4, a T-cell leukemia, all from the American Type Culture Col lection; JOK-1, a hairy cell leukemia (16); HEL erythroleukemia (17); and DAMI megakaryoblastic leukemia (18). The cells were grown in , Extracellular Domain TM TK1 TK2 CT RPMI containing 10% fetal calf serum and antibiotics. All specimens â€¢¿.S 1.9 from fetal abortuses were obtained with permission of the ethical com 1.2 0.6 mittees of the Helsinki University Central Hospital and the University of Turku. II.l Total RNA was extracted using guanidinium thiocyanate as de scribed (13), and 20 Mgwere electrophoresed in agarose gels containing Jl.l formaldehyde and blotted using standard conditions. The inserts of EE cDNA clones were labeled by the random priming method and hybrid S2.5 ized to the blots. Hybridization was carried out in 50% formamide, 5x Denhardt's solution (lOOx Denhardt's solution is 2% each of Ficoll, B polyvinylpyrrolidone, and bovine serum albumin), 5x saline-sodium phosphate-EDTA (3 MNaCl, 200 HIMNaH2PO4 â€¢¿H2O,20 IHMEDTA, pH 7.0), 0.1% sodium dodecyl sulfate, and 0.1 mg/ml of sonicated salmon sperm DNA at 42Â°Cfor 18-24 h. The filter was washed at 65Â°C in Ix standard saline citrate (150 mivi NaCl, 15 HIMsodium citrate, kb pH 7.0) and 0.1% sodium dodecyl sulfate and exposed to Kodak XAR-5 film. 5.8 in Situ Hybridization. Lung tissue from a 15-week-old human fetus 4.5 was obtained with the permission of the joint ethical committee of the University Central Hospital and the University of Turku, Finland. The sample was fixed in 10% formalin for 18 h at 4Â°C,dehydrated, embed ded in wax, and cut into 6-Mmsections. The cRNA probes of 206 and 157 bases (antisense and sense) were generated from linearized plasmid using SP6 and T7 polymerases and 15S-UTP. In situ hybridization of 0.5kb 1.9kb 1.2kb 0.6kb sections was performed according to the method of Wilkinson et al. (19, 20) with the following modifications: (a) instead of toluene, xylene Hybridization probes was used before embedding in paraffin wax; (b) 6-Mmsections were cut, Fig. 1. A, schematic structure of the FLT4 cDNA clones analyzed. Arrows, placed on a layer of diethylpyrocarbonate-treated water on the surface subcloned restriction fragments (whose sizes are shown in kilobases) used for of glass slides pretreated with 2% 3-triethoxysilylpropylamine (Sigma); probing Northern blots in B. E, EcoRl site: S. Spk\ site. B. Northern hybridization analysis of DAMI and HEL leukemia cell RNAs with the probes shown in A. (c) alkaline hydrolysis of the probes was omitted; (d) the hybridization mixture contained 60% deionized formamide; (e) the high-stringency wash was for 80 min at 65Â°Cin a solution containing 50 HIMdithio- threitol and 1 x standard saline citrate; and (/) the sections were cov ered with NTB-2 emulsion (Kodak) and stored at 4Â°C.After an expo pÃetesignal sequence for polypeptide translocation into the en- sure time of 14 days the slides were developed for 2.5 min in a Kodak doplasmic reticulum. We therefore extended the 5' end of the D-19 developer and fixed for 5 min with Unifix (Kodak). The sections FLT4 cDNA sequence from polyadenylated RNA of the HEL were stained with hematoxylin in water. Control hybridizations with cells using a specific oligonucleotide for cDNA priming and sense strand and RNAse A-treated sections did not give a signal above ligation of blunt-ended cDNA into circles followed by inverse background. PCR. This resulted in the identification of 12 additional amino acids including the translational initiator codon. RESULTS Predicted Amino Acid Sequence and Protein Structure of FLT4. The complete sequence of the FLT4 cDNA clones ex Isolation of Full-length FLT4 cDNA. We previously ampli tends for 4416 nucleotides and contains an open reading frame fied a FLTV-specific 210-base pair fragment from the HEL cell of 1298 amino acids. The predicted FLT4 polypeptide is shown cDNA library using PCR and degenerate oligonucleotides cor in Fig. 2. A putative signal peptide sequence of mostly hydro- responding to conserved motifs of RTK amino acids (21). This phobic amino acids follows the initiator methionine. The se fragment was used for screening the HEL library, and a partial quence surrounding the corresponding ATG codon is in agree 1.2-kilobase FLT4 cDNA sequence was reported previously (1). ment with the consensus translation initiation sequence Fig. \A shows schematic structures of overlapping A clones, reported by Kozak (22). The predicted extracellular portion of from which the full-length FLT4 cDNA sequence was obtained, the FLT4 polypeptide is 775 amino acids long and contains 12 and the corresponding functional domains of the receptor se potential sites for asparagine-linked glycosylation (NXS/T). It quence. Several subcloned fragments were used in the Northern also contains several amino acid residues exhibiting a pattern of blot hybridization of DAMI and HEL leukemia cell RNAs spacing described for members of the immunoglobulin super- (Fig. \B). All probes revealed two major transcripts of 4.5 and family of proteins (23). It has 12 cysteine residues, and it can be 5.8 kilobases, except that a 1.0-kilobase probe, located in the 3' organized into seven immunoglobulin-like domains (Ig I-VII in end of the 11.1 cDNA, hybridized only to the 5.8-kilobase tran Fig. 2). The predicted Ig-like domain IV lacks cysteine residues. script in addition to some shorter bands of unknown nature Fig. 2 also shows the amino acid sequence of FLT1 (3, 24), (data not shown). In HEL cells the two RNAs were present in which is the closest human homologue of FLT4. From this roughly equal amounts, whereas in DAMI cells the shorter one figure one can see the alignment of cysteine residues of the was more prevalent. The most 5' end of the 2.5-kilobase sub- extracellular domain and the very similar composition of the clone did not encode a translational initiation codon or a com- Ig-like regions. 5739

FLT4 1 [HO. . RGAALCLRLMLCLGLLDGt.VSGYSMTPPTLHlIEESHVIDTGDSI.S 48 FLT4 IBI IGTGVIAVFFMVLLLI.IFCNMRRPAHADIKTGYLSIIMDPGEVPLEEQCEY830 TM I ::.::! : I : I I . I II .:..!.I.:.:. I:::.I:.I SS .1.1 l. : l l : l l l :: .1:1.. .: l l l : l l l l l l l l : l l l l : l l l l FLT1 1 |MVSYHDTGVLLCALLSCLLLTG^SSGSKLKDPELSLKGTQHIHQAGQTI,H 50 FLT1 764 ICTCVAATLFHLLLTLLI^KMKRSS.SEIKTDYLSIIHDPDEVPLDEQCER 812 FLT4 831 LSYDASQHEFPRERLHLGRVLG'YG'AFG'KWEASAFGIHKGSSCDTVAVI?Â«880 ~ FLT4 49 ISbRGQHPLEWAWPGAQEAPATGDKDSEDTGWRDCEGTDARPYCKVLLL 98 :III::.I.:I: ..1:11 ::.:.. I : : : . : I .. I I 1.1111.111:1111.11: II lllllll:llllll.I:..I lililÃ FLT1 51 LQÃ‡JÃŽGEAAHKWSLPE MVSKESERLSITKSACGRNGKQFCSTLTL 94 FLT1 813 LPYDASKWEFARERLKLGKSLGRGAFGKWQASAFGIKKSPTCRTVAVKM 862 â€¢¿â€¢¿n IKi FLT4 99 HEVHAHEIGSYvttYKYIKARIEGTTAASSYVFVRDFEQPFINK. . . .PD Iti FLT4 881 LKEGATASEHRALMSELKILIHIGNHLNWNLLGACTKPQGPLMVIVEFC 930 TK1 :..:!! Il I IIII : I... I:I:.I :.II::. I: lllllllll.:lll.llM l.lll:lllllllllllll..llllllll:l FLT1 95 NTAQANHTGFYSCKYLAVPTSKKKETESAIYIFISDTGRPFVEMYSEIPE 144 FLT1 863 LKEGATASEYKALMTELKILTHIGHHLNWNLLGACTKQGGPLHVIVEYC 912

FLT4 145 TLLVNRKDAMWVpELvSIPGLNÃ•ILRS.QSSVLWPDGQEVVWDDRRGMLV 193 FLT4 931 KYGNLSNFLRAKRDAF...SPCAEKSPEQRGRFRAMVELARLDRRRPGSS 977 _ .: :. .: Hill. I.:.III:. . ..I III. ::II.I:I::: lllllll:l:.lll l ...l . ...:::: : :l :: .1 ...l FLT1 145 IIHMTEGRELVIPckvTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFII 194 FLT1 913 KYGNLSNYLKSKRDLFFLNKDAALHMEPKKEKMEPGLEQGKKPRLDSVTS 962 KI ign FLT4 978 DRVLFARFSKTEGGARRASPDQEAEDLWLSPLTMEDLVCYSFQVARGMEF 1027~ FLT4 194 STPLLHDALYLQCETTWGDQDFLSNPFLVHITGNELYDIQLLPRKSLELI. 243 I.: ..: .1 lit. .:: : .1 :I.I .I.:.I:I: ..:.:.!! FLT1 195 SNATYKEIGLLTgpATVNGHLYKTN.YLTHRQTNTIIDVQISTPRPVKLL 243 FLT1 963 SESFASSGFQEDKSLSDVEEEEDSDGFYKEPITMEDLISYSFQVARGMEF 1012

FLT4 244 VGEKLVLÃ•JcicVWAEFNSGVTFDWDYPGKQAERGKWVPERR....SÃ›QTHT289 FLT4 1028 LASRKCIHRDLAARNILLSESDWKICDFGLARDIYKDPDYVRKGSARLP 1077 I..IIIIIII. ..:I. I :.I.II: I:.I:.. II I. l.llllllllllllllllll.:lllllllllllllll:lllllll..lll FLT1 244 RGHTLVLNyrATTPLNTRVQMTWSYPD...EKNKRASVRRRIDQSNSHAN 290 FLT1 1013 LSSRKCIHRDLAARNILLSENNWKICDFGLARDIYKNPDYVRKGDTRLP 1062 â€¢¿tâ€¢¿n ' ... igin TK2 FLT4 290 ELSSILTImaSOHDLGSYVbKANNGIQRFRESTEVIVHENPFISVEWLK 339 FLT4 1078 LKWMAPESIFDKVYTTQSDVWSFGVLLWEIFSLGASPYPGVQINEEFCQR 1127 : 1:111..: . . I I .lit ... I . .1.1 : . : . : I I . I . I FLT1 291 IFYSVLTIDKMQNKDKGLYTÂ£JtVRSGPSFKSVNTSVHIYDKAFITVKHRK 340 FLT1 1063 LKWMAPESIFDKIYSTKSDVWSYGVLLWEIFSLGGSPYPGVQMDEDFCSR 1112

FLT4 340 GPILEATAGDELVKLPVKLAAYPPPEFQWYKDGKALSGRHS PHAL 384 FLT4 1128 LRDGTRMRAPELATPAIRRIMLNCWSGDPKARPAFSELVEILGDLLQGRG 1177_ ..: I I.. I I. :!.:!: 1:1.11. I . I I I : .:: . ... I 11:1 llllll..ll.l .111:11 lll.ll l.llll MINI:.. FLT1 341 OOVLETVAGKRSYRLSKKVKAFPSPEWWLKDGLPATEKSARYLTRGYSL 390 Ig IV FLT1 1113 LREGMRMRAPEYSTPEIYQIMLDCWHRDPKERPRFAELVEKLGDLLQANV 1162 CT

FLT4 385 VLKEVTEASTGTYTLALWNSAAGLRRtUSLELWNVPPQIHEKEASS... 431 FLT4 1178 LQEEEEVCMAPRSSQSSEEGSFSQVSTMALHIAQADAEDSPPSLQRHSLA 1227 : : I : I I I ... I . I I : I : : I :. .I : I I I . I I I . I I . . I I FLT1 391 IIKDVTEEDAGNYT1LLSIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPD 440 FLT1 1163 QQDGKDYIPINAILTGNSGFTYSTPAFSEDFFKESISAPKFNSGSSDDVR 1212 FLT4 432 PSIYSRHSRQALT0TAYGVPLPLSIQWHWRPWTPCKMFAQRSLRRRQQQD 481 ~ FLT4 1228 ARYYNWVSFPGCLARGAETRGSSRMKTFEEFPMTPTTYKGSVDNQTDSGM 1277 I.:I. Ill 1111111:1 I .1.1 1:1 .. . I. .: ...::. FLT1 441 PALYPLGSROILTJrJTAYGIPQP.TIKHFWHPCNHNHSEARCDFCSNNEES 489 FLT1 1213 YVNAFKFMSLERIKTFEELLPNATSHFDDYQGDSSTLLASPMLKRFTWTD 1262

FLT4 482 LMPQCRDWRAVTTQDAVNPIESLDTWTEFVEGKtUSlVSKLVIQNAiÃ¼ÃSAM531 FLT4 1278 VLASEEFEOIESRHRQESGFR 1298 :: : .. : .1.111:. : .::IIII1 .1.11:.:..:!:: Ig V FLT1 490 FILD ADSNMGNRIESITQRMAIIEGKNKMASTLWADSRISGI 532 FLT1 1263 SKPKASLKIEV 1273

FLT4 532 YKcWsNKVGODERI,IYFYVTTIPDGFTIESKPSEELLEGOPVLLsEpAD 581 I It.Hill :I I II:I.:I:II ::.... II:.: IIl[.: FLT1 533 YIJTJIASNKVGTVGRNISFYITDVPNGFHVNLEKMPT..EGEDLKLSgJTVN 580 â€¢¿ ... FLT4 582 SYKYEHLRWYRLH1STLHDAHGNPLLLDCKNVHLFATPLAASLEEVAPGA 631 .: I .: I. I :.I: .... I :.:I . Ig VI FLT1 581 KFLYRDVTWILL RTVNNRTMHYSISKQKMAITK 613 â€¢¿â€¢¿H FLT4 632 RHA.TLSLSIPRVAPEHEGHYVCEVQDRRSHDKHCHKKYLSVQALEAPRL 680 I. II.I.I .I. :..I I.I..: . :. :II :.:.. Ill I FLT1 614 EHSITLNLTIMNVSLQDSGTYAj^ARNVYTGEEILQKKEITIRDQEAPYL 663

FLT4FLT1 TQtUiDLLVmSDSLEMO664 730jjiANGVPEPQITWFKNNHKIQQEPGIILGPGS

LRNLSDHTVAISSSTTLD' 713Â¡VCNAKGCVMÂ£Â£ASVAVEGSEDKGSMEJiyÂ£Ly] r731 â€¢¿â€¢¿â€¢¿ Ig VII FLT4FLT1681 QKLSIQRVREEDAGRYLC 780 1714 1 I'll 1 1 1 . 1 1 .IATNQKGSVESSAYLTVQGTSDKSNLEJLITLT]..1.11:1:111 :.1:1..11:.:1::.1 STLFIERVTEEDEGVYHCrLvAGAHAPSIVWYKDERLLEEKSGVDLADSN 763 Fig. 2. Deduced amino acid sequence of the FLT4 and its comparison with the FLT1 t>rosine kinase sequence (3, 24). I, identical residues: :, conservative changes: , nonconservative substitutions. The signal sequence and the transmembrane sequence as well as conserved cysteine residues potentially participating in the formation of Ig-like loops have been boxed. â€¢¿,residuesconforming to the consensus motifs for Ig-like loops (Ig I through VII). The IV Ig-like loop does not contain cysteine residues but has instead several residues typical for an Ig-like loop. Potential glycosylation sites have been underlined. Arrows, TK domains. Also, marked are the ATP-binding lysine residue (aa 879) 21 residues downstream from the consensus ATP recognition sequence GXGXXG (â€¢)andthe potential autophosphorylation site (D). The amino acid sequence of the short version of FLT1 (24) was aligned with FLT4 using the Wisconsin University GCG package progam (15) and manual alignment. The FLT4 nucleotide sequence has been deposited in the EMBL database under the accession number X 68203.

The cytoplasmic domain of FLT4 is separated from the ex evolution we hybridized a 2.5-kilobase cDNA fragment (S2.5 in tracellular part by a putative transmembrane region of 23 hy- Fig. 1) to genomic DNAs purified from different animals and drophobic amino acid residues. This sequence is flanked on the from yeast (Fig. 3). Specific bands were found in all animal cytoplasmic side by basic amino acid residues typical for the species tested, but yeast DNA did not give a signal. junction between the transmembrane and cytoplasmic domains. FLT4 Is Expressed in Multiple Fetal Tissues. In our previ The tyrosine kinase homologous domain begins at residue 843 ous report we published the results of an RNA analysis of and includes an ATP-binding pocket and a putative autophos multiple human adult tissues using the 1.9-kilobase FLT4 phorylation site homologous to Y416ofc-srcat Y1068 (Fig. 2). cDNA probe (see Fig. 1), which covers part of the extracellular The tyrosine kinase catalytic domain of FLT4 is divided into domain of FLT4. In adult human tissues FLT4 was expressed in two subdomains by a 65-amino acid sequence (aa 944-1008), placenta, lung, kidney, heart, and liver in a decreasing order (1). which is mostly hydrophilic and does not show homology to Analysis of human fetal tissues showed that all except the thy FLT1. Unlike FLT1, FLT4 does not contain tyrosine residues mus and small intestine contain FLT4 transcripts (Table 1). in its kinase insert. The highest expression levels were found in lung and spleen. To FLT4-Homologous Sequences in Other Animal Species. In determine which cells in lung tissue possess FLT4 transcripts, order to reveal how well the FLT4 gene has been conserved in in situ hybridization of human fetal lung was carried out. From 5740

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1992 American Association for Cancer Research. FLT4 RECEPTOR TYROSINE K1NASE tÃ Table l FLT4 mRNA expression in human fetal tissues 0) o A Northern blot containing total RNA from the listed tissues of 16-19-week -* V) human fetuses was hybridized with the 1.9-kilobase FLT4 cDNA fragment, and tÃ 3 o the resulting autoradiograph was scanned with a densitometer. The results were o normalized for the amount of RNA estimated from an UV picture of the corre Kb sponding cthidium bromide-stained gel. Fetal tissue mRNA" Brain Meninges 23.1 Cortical plate Intermediate zone 9.4 Ependymal zone 6.6 Cerebellum Choroid plexus Liver Pancreas 4.4 Small intestine Heart Lung Kidney Adrenal Skin 2.3 Spleen Thymus 2.0 The following symbols denote mRNA levels in an increasing order: â€”¿,+,++,

Fig. 3. Analysis of ÃŽÃ•.7Vsequencesin DNA samples from different species. A "Zooblot" (Clontech) containing DNA digested with EcoRl was used for hybrid ization with the S2.5 cDNA fragment encoding part of the extracellular domain cording to the deduced amino acid sequence FLT4 belongs to of FLT4 (see Fig. 1). class III RTKs. More specifically, FLT4 belongs to a subfamily of RTKs, which contain seven Ig loops in their extracellular the results shown in Fig. 4 we conclude that epithelial cells of domain and thus differ from other members of class III RTKs small bronchi are mainly responsible for FLT4 expression in the which contain five Ig-loops. lung. FLT4 is most closely homologous with the prototype recep FLT4 Is Down-regulated during Teratocarcinoma Cell Differ tor of the FLT family, FLT1, which was cloned as a \-ros- entiation. We also analyzed the expression of FLT4 in different related DNA from a human genomic DNA library (3) and with tumor cell lines. Fig. 5A shows the presence of FLT4 transcripts the mouse FLK1 receptor, which was cloned from hematopoi- in a Wilms' tumor cell line (SK-NEP-1 ) and in a retinoblastoma etic stem cell-enriched fractions of mouse liver (5, 6). Fig. 6 cell line (Y79). The association between FLT4 expression and shows a comparison of the class III RTKs in a schematic form, cell differentiation was examined using the Tera-2 human ter- including the percentage of identity of selected domains. The atocarcinoma cells. FLT4 was highly expressed in undifferenti- extracellular domain of FLT4 shows 33% and 37% of amino ated Tera-2 cells, but not in cells differentiated for 10 days with acid sequence identity with human FLT1 and mouse FLK1, retinoic acid (Fig. 5B). respectively. FLT1 and FLK1, like FLT4, are widely expressed in various normal tissues, such as lung, heart, kidney, and brain. DISCUSSION In addition, a recently identified human endothelial cell recep tor tyrosine kinase KDR (9) shows considerable homology with We have characterized a novel receptor tyrosine kinase FLT4 and the other FLT family members. From the available cDNA which is expressed in several human fetal tissues. Ac- sequence data one can calculate that KDR is 81% identical with

Fig. 4. Localization of FLT4 mRNA in epithelial cells of small bronchioli of a 15-week-old human fetus. Hybridization with theantisense RNA is shown in lightfield (A) and darkfield (B) photography. Hybridization with sense RNA gives a faint unspecific background (C). Bar. 0.1 mm. 5741

FLT4IIIIIIIVVVIVIIFUTIO30Q34O3Â« . vf "f tÂ» M O Â® O H "S t ti kb "5 5 < CO E SI 3303.03

5.8 FLT4 4.5 36TM

U TM58

T"'80KI NTKI::i

IIIIIIjlIITK2 -.57c-fms61- -59C-*ll6159PDGFRo/ÃŸ00000IIIIIIIVVUKI60

8]KDWFLK1(37)802381FLT3IFLK258_ TÃo

2.2 ÃŸ-actin Fig. 6. Comparison of the FLT4 receptor with other class III receptor tyrosine kinases. The percentage of amino acid sequence identity is given for the individual Ig-like loops of FLT4 versus FLTI as well as for the extracellular domains and kinase inserts of FLT4 versus FLTI and KDR/FLK1. All class III receptors are compared with FLT4 for identity in the TK1 and TK2 domains. Number in parentheses, comparison of human and mouse receptors: all other values represent comparisons between human sequences.

kb Tera2 In order to reveal possible cells and tissues where FLT4 is important we analyzed its expression pattern in several tissues. We found FLT4 transcripts in most human fetal tissues studied. While FLT4 is highly expressed in human fetal brain, we did 5.8 FLT4 not detect its expression in the brain tissue of adults (1). We 4.5 also analyzed the presence of FLT4 transcripts in different tu mor cell lines. FLT4 expression in undifferentiated teratocarci noma cell line Tera-2 and its down-regulation upon retinoic acid-induced differentiation are interesting. Two other tumor cell lines, a Wilms' tumor cell line and a retinoblastoma cell RA - + line, gave FLT4 mRNA signals. Our in situ hybridization also revealed FLT4 expression in bronchial epithelial cells of human Fig. 5. FLT4 mRNA expression in tumor cell lines. In A, polyadenylated RNA from the indicated cell lines was analyzed by Northern blotting and hybridization fetal lung tissue. with the S2.5 FLT4 cDNA probe. Hybridization with rf-actin probe was used as an The most important question which may provide a valuable internal control for the loading of even amounts of RNA to the analysis. B, Tera-2 teratocarcinoma cells were analyzed after a 10-day treatment with vehicle (â€”)or insight into the function of the FLT genes is the identification retinole acid (+) to induce neuronal differentiation (30). of the ligands for these receptors. So far, only the ligand for FLTI has been published (24). This ligand, the VEGF/VPF, FLT4 in the tyrosine kinase domain (Fig. 6). In addition, the stimulates vascular permeability and endothelial cell prolifera extracellular domain of KDR also has a 7-Ig-loop structure,4 tion in vivo (14, 27, 28). Recent unpublished evidence indicates that KDR also binds VEGF/VPF.4 Both of these receptors are and its TK1 and TK2 domains are 95% and 97% identical with the corresponding domains of mouse FLK1 receptor. This sug expressed in endothelial cells, whereas the present experiments gests that KDR is the human homologue of mouse FLK1. show that FLT4 is mainly expressed in epithelial cells of the While the FLT4 TK domain is about 80% identical with the developing lung. Thus one could speculate that the FLT4 re TK domains of FLTI and KDR/FLK1, it is only about 60% ceptor is more likely to function in the regulation of growth and identical with the TK domains of other receptors of RTK class permeability of bronchial epithelial cells. The recently de III. Since these other receptors also have only five Ig-like do scribed placenta! growth factor (29) is another VEGF/VPF- mains in the extracellular region, one can group FLT4, FLTI, like factor, which needs to be tested for binding to the FLT and KDR/FLK1 in a separate FLT subfamily within class III receptors. RTKs. The tyrosine residue located in the sequence D/E-D/E-Y-M/ ACKNOWLEDGMENTS V-P/D/E-M (25) in kinase inserts of PDGFRs, c-fms, and c-kit We thank Dr. Harri Hirvonen for the human fetal RNAs and Dr. is an autophosphorylation site, which, when phosphorylated, Minna Sandberg for human fetal lung in situ samples and Elina binds the SH2 domain of phosphatidylinositol 3-kinase (26). Roimaa, Kirsi PylkkÃ¤nen,Hilkka Toivonen, Kaarina Kronqvist, and Interestingly, unlike these class III RTKs, members of the FLT Tapio Tainola for expert technical assistance. subfamily or the FLT3/FLK2 receptor do not contain such consensus motifs. REFERENCES I. Aprelikova. <>..Pajusola. K . Partanen, J.. Armstrong, 1 .. Alitalo. R., Bailey, ' B. Terman. personal communication. S.. McMahon. J.. Wasmuth. J., Huebner. K.. and Alitalo, K. FLT4. a novel 5742

class III receptor tyrosine kinase in chromosome 5q33-qter. Cancer Res.. 52: mia cell lines. In: R. F. Revoltella (ed.). Expression of Differentiated Func 746-748, 1992. tions in Cancer Cells, pp. 239-245. New York: Raven Press, 1982. 2. Ullrich. A., and Schlessinger, J. Signal transduction by receptors with 17. Martin. P., and Papayannopuolou, T. H. HEL cells: a new human erythro- tyrosine kinase activity. Cell, 61: 203-212, 1990. leukemia cell line with spontaneous and induced globin expression. Science 3. Shibuya. M., Yamaguchi, S., Yamane, A., Ikeda, T.. Tojo, A., Matsushime, (Washington DC). 216: 1233-1235, 1982. H.. and Sato, M. Nucleotide sequence and expression of a novel human 18. Greenberg, S. M., Rosenthal, D. S., Greeley, T. A., Tantravahi, R., and receptor type tyrosine kinase gene (fit) closely related to the fins family. Handin. R. I. Characterization of a new megacaryocytic cell line: the Dami Oncogene. 5: 519-524, 1990. cell. Blood, 72: 1968-1977, 1988. 4. Rosnet, O., Marchetto, S., deLapeyriere, O., and Birnbaum, D. Murine Fll3, 19. Wilkinson, D. G., Bailes, J. A., and McMahon, A. P. Expression of the a gene encoding a novel tyrosine kinase receptor of the PDGFR/CSFÃŒR protooncogene Â¡nt-iis restricted to specific neural cells in the developing family. Oncogene. 6: 1641-1650. 1991. mouse embryo. Cell, 50: 79-88, 1987. 5. Matthews, W., Jordan, C. T., Weigand. G. W., Pardoll. D., and Lemischka, 20. Wilkinson. D. G., Bailes. J. A., Champion. J. E., and McMahon, A. P. A I. R. A receptor tyrosine kinase specific to hematopoietic stem and progenitor molecular analysis of mouse development from 8 to 10 days post coitum cell-enriched population. Cell, 65: 1143-1152, 1991. detects changes only in embryonic globin expression. Development (Camb.), 6. Matthews, W., Jordan, C. T., Gavin. M., Jenkins. N., Copeland, N. G., and 99:493-500. 1987. Lemischka, I. A receptor tyrosine kinase cDNA isolated from a population of 21. Wilks, A. F.. Kurban. R. R.. Hovens, C. M.. and Ralf, S. J. The application enriched primitive hematopoietic cells and excibiting close genetic linkage to of the polymerase chain reaction to cloning members of the protein tyrosine c-kit. Proc. Nati. Acad. Sci. USA, 88: 9026-9030, 1991. kinase family. Gene (Amst.), 85: 67-74, 1989. 7. Yarden. Y.. Kuang. W. J., Yang-Feng, T., Coussens, L., Munemitsu, T. J., 22. Kozak, M. An analysis of 5'-noncoding sequences from 699 vertebrate mes Dull, T. J., Chen, E., Schlessinger. J., Francke, U., and Ulrich, A. Human senger RNA. Nucleic Acids Res., 15: 8125-8135, 1987. proto-oncogene c-kit: a new cell surface receptor tyrosine kinase for an uni 23. Williams, A., and Barclay, A. N. The immunoglobulin superfamilyâ€” dentified ligand. EMBO J., 6: 3341-3351, 1987. domains for cell surface recognition. Annu. Rev. Immunol., 6: 381-405, 8. Stenman. G., Eriksson, A., and Claesson-Welsh, L. Human PDGFA receptor 1988. gene maps to the same region on chromosome 4 as the KIT oncogene. Genes 24. de Vries, C., Escobedo, J. A., Ueno, H., Houck, K., Ferrara, N., and Wil Chromosomes Cancer, /: 155-158, 1989. liams. L. T. The/ms-like tyrosine kinase, a receptor for vascular endothelial 9. Terman, B. I.. Carrion, M. E., Kovacs. E.. Rasmussen. B. A., Eddy. R. L., and growth factor. Science (Washington DC), 255: 989-991. 1992. Shows. T. B. Identification of a new endothelial cell growth factor receptor 25. Cantley, L. C., Auger, K. R., Carpenter, C, Kapeller, R., and Soltoff, S. tyrosine kinase. Oncogene, 6: 1677-1683, 1991. Oncogenes and signal transduction. Cell, 64: 281-302. 1991. 10. Satoh, H.. Yoshida, M. C., Matsushime, H.. Shibuya, M.. and Sasaki, M. 26. Reedijk, M., Liu, X., van der Geer. P.. Lctwin, K., Waterfield, M. D., Hunter. Regional localization of the human c-rcw-l on 6q22 anaÃŸton 13ql2. Jpn. J. T., and Pawson, T. Tyr721 regulates specific binding of the CSF-1 receptor Cancer Res., 78: 772-775, 1987. kinase insert to PI 3'-kinase SH2 domains: a model for SH2-mediated re 11. Rosnet, O., Mattei, M-G., Marchetto, S., and Birnbaum, D. Isolation and ceptor-target interactions. EMBO J.. //: 1365-1372, 1992. chromosomal localization of a novel FMS-like tyrosine kinase gene. Genom- 27. Ferrara, N., and Henzel, W. Pituitary follicular cells secrete a novel heparin- ics, 9:380-385, 1991. binding growth factor spesific for vascular endothelial cells. Biochem. Bio- 12. Warrington, J. A., Hall, L. V., Hinton, L. M., Miller, J. N., Wasmuth. J. J., phys. Res. Commun., 161: 851-858, 1989. and Lovett, M. Radiation hybrid map of 13 loci on the long arm of chromo 28. Gospodarowicz, D.. Abraham. J. A., and Schilling. J. Isolation and charac some 5. Genomics. //: 701-708, 1991. terization of a vascular endothelial cell mitogcn produced by pituitary-derived 13. Sambrook, J., Fritsch, E. F., and Maniatis, T. Molecular Cloning: A Labo folliculo stellate cells. Proc. Nati. Acad. Sci. USA, 86: 7311-7315. 1989. ratory Manual. Cold Spring Harbor. NY: Cold Spring Harbor Laboratory. 29. Maglione, D.. Guerriero. V., Viglietto, G., Delli-Bovi, P., and Persico, M. G. 1989. Isolation of a human placenta cDNA coding for a protein related to the 14. Sanger. F., Niklen, S., and Coulson, A. R. DNA sequencing with chain vascular permeability factor. Proc. Nati. Acad. Sci. USA, 88: 9267-9271, terminating inhibitors. Proc. Nati. Acad. Sci. USA. 74: 5463-5467, 1977. 1991. 15. Devereux, J.. Haeherly, P.. and Smithies, O. Nucleic Acids Res., 12: 387- 30. Thompson. S.. Stern, P. L., Webb, M.. Walsh, F. S., EngstrÃ¶m.W., Evans, 395, 1983. E. P., Shi, W-K., Hopkins. B.. and Graham, C. F. Cloned human tcratoma 16. Andcrsson, L. C.. SÃ»mes,M. A., Lehtonen, E., and Gahmberg, C. G. Struc cells differentiate into neuron-like cells and other cell types in retinoic acid. tural and functional markers during induced differentiation in human leuke J. Cell Sci., 72:37-64, 1984.

5743

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1992 American Association for Cancer Research. FLT4 Receptor Tyrosine Kinase Contains Seven Immunoglobulin-like Loops and Is Expressed in Multiple Human Tissues and Cell Lines

Katri Pajusola, Olga Aprelikova, Jaana Korhonen, et al.

Cancer Res 1992;52:5738-5743.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/52/20/5738

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/52/20/5738. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.