[CANCER RESEARCH 52, 746-748, February I, 1992] Advancesin Brief

FLT4) a Novel Class III in

Olga Aprelikova, Katri Pajusola, Juha Partanen, Elina Armstrong, Rutta Alitalo, Stacie K. Bailey, Jeannette McMahon, John Wasmuth, Kay Huebner, and Kari Alitalo Cancer Biology Laboratory, Departments of Virology and Pathology, and Transplantation Laboratory, University of Helsinki, Haartmaninkatu 3, 00290 Helsinki 29, Finlandia. A., K. P., J. P., E. A., R. A., K. A.]; Department of Biological Chemistry, University of California, Irvine, California 92717 fS. K. B., J. M., J. W.]; and Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107-5541 [K. H.]

Abstract Materials and Methods

The receptors for at least two hematopoietic growth factors, namely Identification of Tyrosine Kinases from a HEL Cell cDNA Library. the stem cell factor and colony-stimulating factor 1, belong to class III The polymerase chain reaction technique was used to amplify tyrosine receptor tyrosine kinases. Here we describe cloning of a partial comple kinase sequences from phage aliquots of an oligodeoxythymid> late- mentary DNA for U 14. an additional member of this family from primed human HEL cell cDNA library in bacteriophage Xgtl 1 (a kind human leukemia cells. The 1114 tyrosine kinase domain is 79% homol gift from Dr. Mortimer Poncz, Childrens Hospital of Philadelphia, ogous with the previously cloned FLT1 (M. Shibuya et al.. Oncogene, 5: PA) (9). Briefly, guessmers based on the amino acid sequence of a 519-524, 1990) tyrosine kinase and maps to the chromosomal region conserved domain of tyrosine kinases (10) and aliquots of about IO7 5q33-qter. We have found I-I.T4 expression in human placenta, lung, phages were used for amplification. This resulted in the identification heart, and kidney, whereas the pancreas and brain appeared to contain of four novel kinases, OTK-1-OTK-4.1 The OTK-1 clone was subjected very little if any FLT4 RNA. The results suggest that lì14 functions in to further analysis. multiple adult tissues. The HEL cDNA library was screened with the 210- OTK- 1 cDNA fragment. Among 1.5 x IO6 independent clones, 27 plaques were positive; 7 of them were purified as described (11) and about 1.2 Introduction kilobases of the cDNA were sequenced. We have been interested in RTKs2 expressed in megakaryo- Analysis of Somatic Cell Hybrids. Isolation, propagation and char acterization of the mouse-human somatic cell hybrids containing de blastic leukemia cell lines because the factors regulating the fined human has been described earlier (12, 13). DNA proliferation and differentiation of megakaryoblasts are poorly from human and mouse cell lines and from mouse-human hybrid cell known and knowledge of their receptors could eventually lead clones was extracted and analyzed by standard methods (11). to their identification (1). Two known hematopoietic growth factors, the stem cell factor and colony-stimulating factor 1, are Results and Discussion known to exert their effects on target cells through binding and activating specific protooncogene-encoded receptor tyrosine ki Polymerase chain reaction amplification of tyrosine kinase- nases, named c- and c-fms, respectively (see Refs. 2 and 3). related sequences from the HEL cell cDNA library resulted in These receptors, together with the platelet-derived growth fac the identification of clone OTK-1, which did not correspond to tor receptors a and ß,belongto class III RTKs (4, 5). Members any reported sequence in the available databases. The subdo- of this class are characterized by extracellular domains com main VIII (14) WMAPE amino acid sequence motif deduced posed of five so-called immunoglobulin-like loops and an intra- from the amplified nucleotide sequence of OTK-1 contained cellular tyrosine kinase domain interrupted by a kinase insert the methionine residue indicative of transmembrane tyrosine sequence (6). As our approach to studies of the relevant RTKs, kinases. The HEL cell cDNA library was therefore screened we have used polymerase chain reaction cloning of novel tyro with the probe, positive clones were isolated, and the region of sine kinases expressed by leukemia cells with megakaryoblastic cDNA corresponding to the putative tyrosine kinase and trans- differentiation potential. Earlier we reported the cloning of membrane domains was sequenced. The deduced amino acid several RTKs from the K562 leukemia cell line which retains sequence of the long open reading frame shows extensive ho- the potential for erythroid/megakaryoblastic differentiation (1, mology with sequences of class III RTKs (Fig. 1). Furthermore, the FLT1 and FLT3/FLK2 RTK cDNAs (14-17), encoded by 7). However, none of the we cloned previously was specific for the megakaryoblastic cell lineage. We have therefore loci in human chromosome band 13ql2 (18, 19), appeared as searched for additional RTKs in the HEL erythroleukemia cell its closest homologues. The FLT1 cDNA was originally cloned line, which also has a dual erythroid/megakaryoblastic pheno- by Shibuya et al. (15) as a c-//«i-related RTK from a human type and is inducible to further expression of megakaryoblastic placental cDNA library. The FLT3 and FLK2 mouse cDNAs markers by treatment with the tumor promoter 12-O-tetrade- were cloned independently by two groups (16, 19), but sequence canoylphorbol-13-acetate(8). Here we report the identification, comparisons showed that they represent the same gene, the chromosomal mapping, and partial characterization of a novel human homologue of which was isolated and chromosomally member of RTK class III from this cell line. mapped. As also the name FLT2, referring to the FGFR1 (flg) gene, has appeared in the literature (albeit transiently; see Received 10/22/91; accepted 12/4/91. Rosnet et al., 1991 (19), we have named our novel tyrosine The costs of publication of this article were defrayed in part by the payment kinase FLT4 to avoid any confusion. of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. As can be seen from Fig. 1, the deduced FLT4 polypeptide 1This study was supported by the Finnish Cancer Organizations, The Finnish has a putative transmembrane region followed by a sequence Academy, The Sigrid Juselius and Finnish Cultural Foundations. Orion Corpo ration Research Fund, and the Research and Science Foundation of Farms, and homologous to the tyrosine kinase domains of other members byNIHGrantCA21124. of this receptor family. This domain is divided into two distinct 1The abbreviations used are: RTK, ; cDNA, comple mentary DNA. 3O. Aprelikova et ai, unpublished data. 746

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1992 American Association for Cancer Research. NOVEL RECEPTOR TYROSINE KINASE

FLT4 FLT1 743-.YLT.Q.TS. .SNL.LIT.TC.C.A.TL. .L. .T.LIRK.K.-SSSK. ..D D....D....R.P....K...A.E..K..KS FLT3 529-FLNSPGPFPFIODNISFYA-TI.LCLP.IV. .IV. .CHKYKKOFRYESOLOMIOVTGPLDNEYFYVDFRDYE. .L-K N.EF.K.

FLT1 ..R Q K.SPT.R YK. ..T Y. ..H QG Y Y .KS .. FLT3 ..S....R.MN.T.Y. .S.TGVSIQ K.DSC.KE MMT.L. H.D.I LS-. .VYL. F.Y.C. .D. L. Y. .S. .

FLT4 DAFSPCAEKSPEQRGRFRAMVELARLDRRRPGSSDRVLFARFSKTEGGARRASPDQEAEDLWLSP.... FLT1 DLFFLNKDAALHMEPKKEKMEPGLEQGKKPRLDSVTSSESFASSGFQEDKSLSDVEEEEDSDGFYKEP FLT3

FLT4 FLT1 I IS...... S NN. . . .N DT I.S.K Y. FLT3 ..F...L.FAY. ..EFKS.V. .V.VTHGK. .LS.SS..VR.NA...V.. ..L.EGI..IK.. ..Y.

FLT1 G MD.D..S...E.M YS..E.YQ...D..HR...E..R.A....K ANVQ. DGKDYIPINAIL-1176 FLT3 I VN. . . .IPVDAN.YKLIQS.FK.EQ.FY. .EG.YFV.QS. .AF.SRK. .S.PN.TSF. .CQ.AEAEEACIRTSIHL.KQA-968 Fig. 1. Deduced amino acid sequence of the cytoplasmic domain of FLT4 and its comparison with the corresponding FLTI and FLT3/FLK2 receptor tyrosine kinase sequences (IS. 16, 19) (only differing residues are marked). The amino acid sequences were aligned and a few gaps (dashes) were introduced for optimal homology. The putative transmembrane domains have been underlined and the characteristic residues for binding of ATP and autophosphorylation as well as the kinase insert are marked with boldface letters. regions, TKl and TK2, by a 65-amino acid kinase insert se The degree of homology is less with other members of RTK quence (boldface letters in Fig. 1). The putative ATP binding class III. site in the TKl domain and autophosphorylation site (Y) in the It was of great interest to determine the chromosomal local TK2 domain are also marked with boldface letters in Fig. 1. ization of FLT4 because some clustering of class III receptor The TKl and TK2 domains of FLT4 are 78 and 80% homolo genes has been found to take place (19). Thus rodent-human gous with the corresponding domains of FLT1 and 59 and 55% cell hybrids were analyzed, indicating linkage QÎFLT4tohuman homologous with the corresponding domains of FLT3/FLK2. (data not shown). Then regional assignment on Chromosome 5 was determined using hybrids carrying partial chromosome 5s as shown in Fig. 2. The presence of human S m ce .H .H chromosome 5q33-qter in the hybrids correlated with the pres ence OÕFLT4sequences. The regional mapping results indicated co 1-H that the FLT4 locus is telomeric to the CSFlR/platelet-derived M PO O Oí

15.3 U 15.1 [ 05 14 13.3 [ to S 12 a 11.2 I « sa« 12 2 .2a~ .2 ilav .S CS 13.1l p* 13.s[ kb 14 15 6.2 21 5.8 22 4.5 23.31 31.1 31.3 [ 32 33.3 [ 34 35.3 [

FLT4 + + + + + - + - + Fig. 2. Localization of the /"¿TVgenein the region 5q33 —¿Â»Sqter.The portions Fig. 3. /•'//•/mRNAexpression in adult human tissues. Two pg of polyaden- of chromosome 5 retained in the different hybrid cell lines indicated on the top abscissa are sketched to the right of the chromosome 5 ideogram (12, 13, 20). ylated RNA from the indicated tissues (Multiple Tissue Northern Blot; Clontech These hybrids were tested for presence of the FLT4 locus by filter hybridization Inc.) were analyzed by Northern blotting and hybridization with FLT4 cDNA and the results are shown below the sketches. The region of chromosome 5 probe. The estimated sizes of the transcripts are shown on the left in kilobases common to fZ. TV-positive hybrids and absent from the Fi. TV-negative hybrids is (kb). Control hybridizations with probes for constitutively expressed genes showed 5q33.1-qter. an even loading of the lanes (data not shown). 747

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1992 American Association for Cancer Research. NOVEL RECEPTOR TYROSINE RÕÑASE ß(PDGFRB) locus as well as to the ß- 2. Witte, O. N. Steel locus defines new multipotent growth factor. Cell, 63- 5- 6, 1990. adrenergic receptor (ADRBR) locus since these loci are all 3. Sherr, C. Colony-stimulating factor-1 receptor. Blood, 75: 1-12, 1990. present in the hybrid GB 13, which was negative for FLT4. 4. Claesson-Welsh, L., Eriksson, A., Westermark, B.. and Heldin. C-H. cDNA cloning and expression of the human A-type platelet-derived growth factor Hybridization of polyadenylated RNA from various human (PDGF) receptor establishes structural similarity to the B-type PDGF recep tissues with the FLT4 cDNA fragment showed mRNA bands tor. Proc. Nati. Acad. Sci. USA, «6:4917-4921, 1989. of 5.8- and 4.5-kilobase mobility and a weakly labeled band of 5. Matsui, T.. Heideran, M., Miki, T., Popescu, N., La Rochelle, W., Kraus, M., Pierce, J., and Aaronson, S. A. Isolation of a novel receptor cDNA 6.2 kilobases in placenta, lung, heart, and kidney. Faint mRNA establishes the existence of two PDGF receptor genes. Science (Washington bands were seen in the liver and skeletal muscle, whereas the DC), 243: 800-803, 1989. 6. Ullrich, A., and Schlessinger, J. Signal transduction by receptors with tyro- pancreas and brain appeared to contain very little if any FLT4 sine kinase activity. Cell, 61: 203-212, 1990. RNA (Fig. 3). Although high amounts of FLT1 and FLT3 7. Alitalo, R. Induced differentiation of K-562 leukemia cells: a model for studies of gene expression in early megakaryoblasts. Leuk. Res. It: 501-514, mRNAs have also been reported in the placenta, differences in 1990. the expression patterns o(FLT4 and the previously cloned FLT1 8. Papayannopoulou, T., Raines, E., Collins, S., Nakamoto, B., Tweeddale, M., and FLT3/FLK2 genes are exemplified by the absence of FLT4 and Ross, R. Constitutive and inducible secretion of platelet derived growth factor analogs by human leukemic cell lines coexpressing erythroid and mRNA signals in the brain sample, where abundant FLT3 megakaryocytic markers. J. Clin. Invest., 79: 859-866, 1987. mRNA and some FLT1 mRNA have been reported (lf>, 17). 9. Poncz. M., Surrey, S., LaRocco, P., Weiss, M., Rappaport, E. F., Conway, T. M., and Schwartz, E. Cloning and characterization of platelet factor 4 Also, no FLTÃŒmRNA has been seen in the heart, where FLT4 cDNA derived from a human erythroleukemic cell line. Blood, 69: 219-223, expression was present. Interestingly, FLT3/FLK2 mRNA has 1987. also been detected in hematopoietic progenitor-enriched cell 10. Wilks, A. F., Kurban, R. R., Hovens, C. M., and Ralf, S. J. The application of the polymerase chain reaction to cloning members of the tyrosine populations from fetal mouse liver (16). kinase family. Gene, «5:67-74, 1989. The FLT4 sequence is interesting, because it is homologous 11. Sambrook, J., Fritsch, E. F., and Maniatis, T. Molecular Cloning: A Labo ratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratories. to two hematopoietic receptor tyrosine kinases, because the 1989. FLT4 mRNA is expressed in a restricted set of leukemia cells,4 12. Armstrong, E., Partanen, J.. Cannizzaro. L.. Huebner, K., and Alitalo, K. Localization of fibroblast growth factor receptor-4 gene to chromosome and because the FLT4 gene maps to the distal long arm of region 5q33-qter. Genes, Chromosomes Cancer, 4: 1-5, 1991. chromosome 5, near a region where a variety of growth factors 13. Warrington, J. A., Hall, L. V., Hinton, L. M., Miller, J. N., Wasmuth, J. J. and Lovett, M. Radiation hybrid map of 13 loci on the long arm of chro and growth factor receptors have previously been located (20). mosome 5. Genomics, //: 701-708, 1991. We are currently analyzing possible lesions of this locus in 14. Hanks, S. Quinn, A., and Hunter, T. The family: conserved hematopoietic dyscrasias and malignancies with deletions and features and deduced phytogeny of the catalytic domains. Science (Washing ton DC), 241:42-52, 1988. translocations of this region of chromosome 5. 15. Shibuya, M., Yamaguchi, S., Yamane, A., Ikeda, T., Tojo, A., Matsushime, II.. and Sato, M. Nucleotide sequence and expression of a novel human receptor type tyrosine kinase gene (fit) closely related to the fms family. Acknowledgments Oncogene, 5: 519-524, 1990. 16. Matthews, W., Jordan, C. T., Weigand, G. W., Pardo»,D., and Lemischka, We thank Dr. Mortimer Poncz (Childrens Hospital, Philadelphia, I. R. A receptor tyrosine kinase specific to hematopoietic stem and progenitor ceU-enriched population. Cell, 65: 1143-1152, 1991. PA) for kindly giving us the HEL cell cDNA library. We also thank 17. Rosnet, O., Marchetto, S., deLapeyriere, O., and Birnbaum, D. Murine Fltì, Elina Roimaa, Kirsi Pylkkänen,Hilkka Toivonen, Teresa Druck, and a gene encoding a novel tyrosine kinase receptor of the PDGFR/CSFIR Tapio Tainola for expert technical assistance. family. Oncogene, 6: 1641-1650. 1991. 18. Satoh, H., Yoshida, M. C., Matsushime, H., Shibuya, M., and Sasaki, M. Regional localization of the human c-ros-I on 6q22 ami //r on 13ql2. Jpn. J. Cancer Res.. 78: 772-775, 1987. References 19. Rosnet, O., Mallei, M <... Marchetto, S., and Birnbaum, D. Isolation and chromosomal localization of a novel FMS-like tyrosine kinase gene. Gen 1. Partanen, J., Mäkelä,T.P.. Alitalo, R„Lehväslaiho,H., and Alitalo, K. omics, 9: 380-385, 1991. Putative tyrosine kinases expressed in K-562 human leukemia cells. Proc. 20. Huebner, K., Natarajan, L., Besä,E., Angert, E., Lange, B. J., Cannizzaro, Nati. Acad. Sci. USA, 87; 8913-8917, 1990. L. A., van den Berghe, H., Santoli, D., Finan, J., Croce, C. M., and Nowell, P. C. Order of genes on human chromosome 5q with respect to 5q interstitial 4 Unpublished observations. deletions. Am. J. Hum. Genet., 46: 26-36, 1990.

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Olga Aprelikova, Katri Pajusola, Juha Partanen, et al.

Cancer Res 1992;52:746-748.

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