Proc. Natl. Acad. Sci. USAVol. 90, pp. 9355-9358, October 1993Biochemistry

tie-i and tie-2 define another class of putative receptor tyrosinekinase genes expressed in early embryonic vascular systemTHOMAS N. SATO*t, YING QIN*, CHRISTINE A. KOZAKt, AND KENNETH L. AUDUS§*Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110; and tViral Biology Section, Laboratory of Molecular Microbiology,National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892; and §The University of Kansas, Department of Pharmaceutical Chemistry,Lawrence, KS 66047

Communicated by Herbert Weissbach, June 14, 1993

ABSTRACT We report the molecular cloning and char-acterization of two structurally related putative receptor tyro-sine kinases, encoded by distinct genes (tie-i and tie-2) onmouse chromosome 4. Both tie-i and tie-2 encode receptorproteins possessing unique multiple extracellular domains: twoimmunoglobulin-like loop domains flanking three epidermalgrowth factor repeats followed by three fibronectin-type IIIrepeats. Both genes are expressed in early embryonic vascularsystem and in maternal decidual vascular endothelial cells,where the vasculature undergoes an active angiogenesis. tie-2,but not tie-i, expression was also detected in extraembryonicmesoderm of the amnion. tie-i, but not tie-2, is expressed in anacute myelogenic cell line in vitro. tie-i and tie-2 may formanother class within the receptor tyrosine kinase gene family,and further characterization of these genes and identification oftheir putative ligands should define the nature of the signal-transduction cascades underlying early vascular system devel-opment, as well as their differential roles in mesodermal cellsof the amniotic and myeloid lineages.

Receptor tyrosine kinases play key roles in signal transduc-tion across cell surfaces in many biological systems (1, 2),including the vascular system. Induction of interleukin 6expression in endothelial cells has been shown to correlatewith angiogenesis (3) and to be regulated by oncostatinM-receptor-mediated tyrosine kinase activities (4). Recently,one of the putative receptors for the vascular endothelialgrowth factor, the only known endothelial-cell-specificgrowth factor, was identified and found to be a member of thereceptor tyrosine kinase gene family (5). The importance oftyrosine kinase activities in development of cardiac tissueexplants has been suggested (6).To identify additional tyrosine kinase genes expressed in

endothelial cells and study their biological functions, a PCR-based approach was used to amplify putative tyrosine kinasegenes from a brain microvessel endothelial-cell cDNA li-brary. Two cDNA clones were further characterized becauseof their sequence similarities to the recently reported humanreceptor tyrosine kinase gene tie., which was also shown to beexpressed in endothelial cells (7). Here we report the com-plete deduced amino acid sequences, the chromosomal lo-cation, and the in vitro and in vivo expression patterns ofthese genes.

MATERIALS AND METHODSCell Culture. Bovine brain microvessel endothelial cells

(BME) were isolated from the gray matter of cerebral corti-ces, as detailed by Audus and Borchardt (8, 9). The suspen-sion cultures of KG-1 and Jurkat cells were provided byWanda Depinto (Hoffmann-La Roche).

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

cDNA Libraries. The oligo(dT)-primed cDNA libraryBBE-dT was constructed with Uni-ZAP XR vector (Strata-gene) from the poly(A)+ RNA purified from bovine brainmicrovessel endothelial cells. The random-primed bovinebrain endothelial cDNA library BBE-RP was also con-structed in the A ZAP II (Stratagene) vector. The size-selected library ML-RP was constructed from the poly(A)+RNA purified from adult mouse lung (BALB/c, 4 weeks old)in the A ZAP II vector.

Oligonucleotide Primers. TK1-N was 5'-GAGGATCCRT-NCAYMGNGAYYT-3', and TK2-N was 5'-TCGAATTC-CCNWASWCCANACRTC-3.

Cloning of Bovine and Mouse tie-2. The vector-ready dou-ble-stranded bovine brain microvessel endothelial-cell-derived cDNA (50 ng per reaction) was used for the ampli-fication of tyrosine kinase-related genes with the TK1-N andTK2-N primers. One PCR clone was found to differ, thesize-selected BBE-dT library was screened, and a cDNAclone TKFS.9 with an insert of 4.4 kb was obtained. Bothstrands were then completely sequenced. The mouse ho-molog was cloned from the ML-RP library by using therandom-primer-labeled HincII-Pvu II DNA fragment ofTKFS.9.

Cloning of Bovine and Mouse tie-i. Another PCR-amplifiedclone, PDTKF. 15, was found to be identical to human TIE (7)and was used to isolate a full-length cDNA clone from theBBE-RP library. The mouse homologcDNA was cloned fromthe ML-RP library by using the full-length bovine clone(PDTKF15-lfu11.6).Plasmid Subclones. The 3'-untranslated region of tie-2

(pmhTKFS9.3'UT), the complete second and third fibronec-tin (FN) repeats of tie-2 (pmhTKFN.1), the 3' untranslatedregion of tie-i (pmhPD3UT.5), and the complete second andthird FN repeats of tie-i (pmhPDFN.5) were all subclonedinto the Bluescript SKII(+) vector (Stratagene).

In Situ Hybridization Histochemistry. In situ hybridizationwith paraffin-embedded sections (10) was done as described.The tie-i and tie-2 cRNA probes were transcribed frompmhPDFN.5 and pmhTKFN.5, respectively, and the speci-ficities of each probe were confirmed by genomic Southernblot and RNA blot analyses.

Genetic Mapping. DNAs of the progeny of two multilocuscrosses were typed for restriction enzyme polymorphisms oftie-i and tie-2, using as probes inserts of clones pmhPDFN.5and pmhTKFS9.3'UT, respectively. The first cross, (NFS/Nor C58/J x Mus musculus)F1 x M. musculus; ref. 11, wasalso typed for the markers Jun (c-jun oncogene) and Mtv-13(mammary tumor virus 13), as described (12). Ccnbl-rs4(cyclin p-1-related sequence 4) was typed as described (13) inthe M. musculus cross and in a second cross (NFS/S x Musspretus)Fl x C58/J or M. spretus (14). Ifa (interferon a) wastyped by using a probe from T. Mariano (Johnson Medical

Abbreviations: EGF, epidermal growth factor; FN, fibronectin.tTo whom reprint requests should be addressed.

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School, University ofMedicine and Dentistry ofNew Jersey,Piscataway) after digestion with EcoRI in the M. musculuscross and Sac I in the M. spretus cross. The probes for Lck(lymphocyte tyrosine kinase) and Lmyc-i (L-myc oncogene)were obtained from Oncor. Lmyc-1 was typed in the M.musculus cross after digestion with Bgl II and in the M.spretus cross after digestion with Sac I. The M. spretus crosswas typed for the chromosome 14 marker Ccnbl-rsS, asdescribed (13), and np-1, as described (15). Glud-i was typedin these mice after Apa I digestion, using the probe describedby Tzimagiorgis et al. (16), and Cchlla2 was typed afterEcoRI digestion using the probe described by Chin et al. (17).Recombination and SEs were calculated according to Green(18). Data were stored and analyzed by the computer pro-gram LOTUS designed by C. E. Buckler (National Institutes ofHealth).

RESULTS AND DISCUSSIONStructure of tie-i and tie-2. The EMBL accession numbers

of tie cDNA sequences are X71423 (bovine Tie-i), X71424(bovine Tie-2), X71425 (mouse tie-i), and X71426 (mousetie-2). The deduced amino acid sequences of mouse tie-i andtie-2 contain 1134-aa and 1122-aa proteins, respectively. Thefirst methionine is followed by the stretches of hydrophobicresidues of 22 aa and 18 aa in tie-i and tie-2, respectively,which complies with the prediction of the signal peptidesequence (19, 20). The extracellular regions contain five(tie-i) and nine (tie-2) putative N-linked glycosylation sitesfollowed by single hydrophobic regions for both tie-i andtie-2, which may constitute transmembrane domain. Thecytoplasmic region consists of the split-type tyrosine kinasedomains. tie-i shows strong similarity, 92% overall aminoacid identity, with recently reported human TIE, anotherendothelial receptor tyrosine kinase (7), indicating that tie-iis the mouse homolog of the human TIE. In a search of thecurrent data banks, GenBank (Version 73) and EMBL (Ver-sion 31), tie-2 was found to be novel.The extracellular portions of both tie-i and tie-2 consist of

multiple domains (Fig. 1): two immunoglobulin-like domains

Identity (%) Domains

(21) flanking three epidermal growth factor (EGF)-like repeat(22). These domains are followed by three ,3sheet FN typeIII repeats. This particular combination of all these extracel-lular domains can be found only for the tie genes. Thesequence similarities of the two immunoglobulin-like loopsare low (20-30%); however, all the three EGF-like repeatsare relatively conserved (50%). The amino acid spacingbetween cysteine residues of each EGF-like repeat is well-conserved, and cysteine spacing pattern is most closelyrelated to that of the Drosophila neurogenic genes delta (23)and serrate (24). In addition to the cysteine spacing, there areconserved glycine residues in each repeat. The FN-type IIIrepeat is most closely related to the repeats found in therecently cloned putative receptor phosphatase (25). The firstFN-type III repeat has the highest homology (40% identity),and the second and the third repeats are less conserved (14%and 13% identity, respectively) between tie-i and tie-2.The cytoplasmic domains consist of two tyrosine kinase

domains, and both domains are highly conserved (80% iden-tity) between tie-i and tie-2. There is no tyrosine residue inthe kinase insert domain, the ligand-dependent autophospho-rylation of which is implicated for the association withphosphatidylinositol 3-kinase (26).

A

a -4.4

-2.4-1.4

41044'

-9.5

**-2.4-1.4

-0.24-0.24

" go G3PDH

B

22% Ig

49%55%50%

EGF

30% IgI

-4.4

-4.4

-2.4-1 .4

-9.5-7.5_-4.4-2.4-1.4

40% -0.24

-0.2414% FNIII

0 0 ~~13%40% TM

0 0 84% TKI

0 0 78% TKI

Tie-1 Tie-2

FIG. 1. Domain structure of tie-i and tie-2. Amino acid identitiesbetween mouse tie-1 and tie-2 within each domain are indicated atright by percentages. Ig, immunoglobulin; TM, transmembrane; TK,tyrosine kinase.

* * G3PDH

FIG. 2. RNA blot analyses of tie-i and tie-2 gene expression. (A)pmhPDFN.5 insert DNA was used as a probe for tie-l. (B) pmh-TKFN.1 insert DNA was used as a probe for tie-2. The in vitro-synthesized tie-1 and tie-2 (0.75 ng each), poly(A)+ RNA (2 mg each)from Jurkat cells, KG-1 cells, and poly(A)+ RNA (1 ug) fromcultured adult bovine brain endothelial cells (ED), and adult (6 to 8weeks old) mouse brain, heart, kidney, and mouse embryos [em-bryonic day (E) 7.5] were separated by 1% agarose-formaldehyde gelelectrophoresis. The RNA from embryonic day 7.5 was purified fromembryos with the corresponding maternal decidua (D). The humanRNA blot (Jurkat and KG-1 cell RNA) was rehybridized with humanglyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA probeto indicate equal loading of samples.

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In Vivo and in Vitro Expression of tie Genes. Tissue distri-bution of tie-i and tie-2 gene expression was examined byRNA blot analyses (Fig. 2). The specificity ofeach probe wasverified by hybridization to the in vitro synthesized RNAfrom the cDNA clones encoding the complete coding regions.tie-1 and tie-2 probes hybridized to a single band, 4.3 kb and4.6 kb, respectively, in all mouse tissues examined and inprimary cultured bovine brain microvessel endothelial cells.Human leukemia cell lines were examined because the hu-man TIE gene was reported to be expressed in severalmyelogenic lymphoma cell lines (7). As expected, the tie-1transcript was detected in KG-1 cells but was not detected inJurkat cells. No signals of tie-2 expression were detected ineither cell line.

In vivo endothelial cell expression of tie-i and tie-2 expres-sion was examined by in situ hybridization. In embryonic day8 mouse embryos, where RNA blot analysis indicated rela-

tively abundant tie-i and tie-2 expression, both tie-i and tie-2expression was detected in cells associated with vascularsystem, such as in the endocardium and dorsal aortae (Fig. 3A and C). Furthermore, abundant expression of both geneswas detected in maternal decidual blood vessel (Fig. 3 B andD). This observation may indicate a role for tie-i and tie-2 inangiogenesis of the deciduum during pregnancy. It would beinteresting to examine tie expression in other angiogenicadult tissues and also in tumors. Expression of both tie genesin yolk sac blood islands was also detectable (data notshown). Study of early mammalian vascular system devel-opment has been hindered by the lack of specific molecularmarkers (27); tie-i and tie-2 may now provide such markersfor study of the molecular processes involved in early mam-malian vascular system development. Any correlation of tiegene expression to angiogenesis and vasculogenesis (28)would clearly be of interest. In this regard, use of in vitrodifferentiation system of vasculatures (29, 30) would beextremely useful to study tie gene expression. Besides vas-cular system localization, the tie-2 transcript, but not the tie-1transcript, was detected in the extraembryonic mesoderm ofthe amnion (Fig. 3 A and C). Both tie-i and tie-2 geneexpressions in adult vascular system were less abundant (Fig.4). Because tie-i and tie-2 expressions were detectable byRNA blot analysis and particularly strong hybridizationsignal with RNA from adult heart was detected (Fig. 2), celltypes other than vascular endothelial cells could express tie-iand/or tie-2. It is also possible that different amounts ofbloodvessels in different tissues and in embryonic tissues may bereflected on the RNA blot-analysis result.

> t ~~~~~~~~~~~~~~'....10<Ss,,

cz~~~4

FIG. 3. Expression of tie-i and tie-2 genes in embryonic day 8mouse embryo and in extraembryonic structures. (A-D) Antisenseprobes. Under dark-field illumination, the silver grains are over cellsassociated with embryonic vascular system in endocardium anddorsal aorta with the tie-i antisense cRNA probe (A) and the tie-2antisense cRNA probe (C). tie-2 antisense cRNA probe gave hy-bridization signals in cells located in the outer amniotic layer,indicated by arrow in C; no signals were detected with tie-i antisensecRNA probe in the amnion (A). Both tie-i (B) and tie-2 (D) antisenseprobes hybridized in maternal decidual blood vessels. (A'-D') Senseprobes. Neither tie-i nor tie-2 control sense cRNA probe hybridizedspecifically. Under dark-field illumination, nucleated blood cells atthe yolk sac and other vascular system reflected artificial light.Dorsal aorta (arrowhead) and endocardium (*) are indicated in A'and C'.

,~~- '- 0su''' J s .,4

FIG. 4. Expression of tie-i and tie-2 genes in adult vascularsystem. The bright-field photomicrographs show barely detectablesilver grains over the blood vessels, indicated by arrows and arrow-heads. (A) Heart, tie-i antisense probe. (B) Brain, tie-i antisenseprobe. (C) Heart, tie-2 antisense probe. (D) Brain, tie-2 antisenseprobe. Hybridization with sense control probes shows only back-ground signals. (A') Heart, tie-i sense probe. (B') Brain, tie-i senseprobe. (C') Heart, tie-2 sense probe. (D') Brain, tie-2 sense probe.

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Table 1. Linkage of tie with markers on chromosome 4

Recombinants/ RecombinationGenes total t SE, %

First cross:(NFS/N or C58/J x M. musculus)Fl x M. musculus

Ifa, tie-2 3/120 2.5 t 1.4tie-2, Jun 1/88 1.1 ± 1.1Jun, Mtv-13 4/39 10.3 t 4.9Mtv-13, tie-i 3/42 7.1 t 4.0tie-I, Ccnbl-rs4 2/68 2.9 ± 2.0Ccnbl-rs4, Lmyc-J 2/42 4.7 ± 3.2Lmyc-1, Lck 4/59 6.8 ± 3.3

Second cross:(NFS/N x M. spretus)Fl x C58/J or M. spretus

Ifa, tie-2 0/86 3.4*tie-2, tie-i 15/112 13.3 ± 3.2tie-i, Ccnbi-rs4 0/84 3.5*Ccnbl-rs4, Lck 6/80 7.5 ± 2.9*Upper limit of 95% confidence interval.

tie-i and tie-2 Are Encoded by Distinct Genes on MouseChromosome 4. DNAs ofparental mice oftwo genetic crosseswere typed for restriction enzyme polymorphisms for tie-iand tie-2. For the analysis of the M. musculus cross, theprobe for tie-2 identified 7.8- and 6.5-kb HindlIl bands inparental NFS/N and M. musculus DNAs, respectively, andthe probe for tie-i identified 6.4- and 3.0-kb Pst I bands inthese same mice. In the M. spretus cross, the probe for tie-2identified a 7.8-kb HindIII fragment in NFS/N and 9.2- and12.2-kb fragments in M. spretus; the probe for tie-i identified9.7- and 10.4-kb HindIII fragments in these mice. In bothcrosses, polymorphic fragments of tie-i were inherited assingle genes, as was tie-2 in the M. musculus cross. The twotie-2 fragments in M. spretus represented unlinked loci,"tie2" (9.2 kb) and "tie2ps" (12.2 kb). The pattern ofinheritance of these three genes was compared with theinheritance of over 350 previously mapped markers that hadbeen typed in both crosses; "tie] " and "tie2" were found tobe linked to markers on chromosome 4 (Table 1) but wereseparated from one another by 12.2 centimorgans (recombi-nation = 31/246 = 0. 122 + 0.021). The gene order establishedfor chromosome 4 is as follows: Ifa-"tie-2"-Jun-Mtv-13-"tie-i"-Ccnbl-rs4-Lmyc-i-Lck. This order places both tie-iand tie-2 in a region of chromosome 4 homologous to humanchromosome lp consistent with the previous assignment ofhuman TIE to 1p33-1p34 (7). Analysis of the cross (NFS/Nx M. spretus)F1 x C58/J for "tie2ps" showed this gene tobe on proximal chromosome 14 with the following gene orderand recombination fractions: Ccnb-rs5-(6/82)-Cchlla2-(2/88)-"tie2ps"-(1/90)-Glud-(5/90)-Np-1. Because"tie2ps" was detected only in the M. spretus cross, this genemay be a species-specific pseudogene.

tie Genes Form Another Class of Tyrosine Kinase GeneFamily. On the basis of the structural relatedness, the samechromosomal localization, and the identical expression pat-tern in the early embryonic vascular system, tie genes appearto form another class of tyrosine kinase gene family that havean important function in vascular system development. Thediscovery that tie genes are differentially expressed in me-sodermal cells of the amnion and a cell line of myeloid lineagealso suggests that tie-i and tie-2 may play distinct roles inthese cell types.

Recently, partial characterization of the tyrosine kinasegene tek, located on mouse chromosome 4 and expressed in

embryonic endothelial cells, was reported (31). AlthoughRNA blots indicated the existence of the multiple tek tran-scripts, including a major 2.7-kb transcript as opposed to thesingle 4.6-kb transcript of tie-2 gene, partial sequence of thetek kinase domain is identical to the kinase domain sequenceof the tie-2 gene. The relation of the tek gene to the tie genefamily deserves further study.

We thank Drs. Tom Gridley and Andy McMahon for comments onthe manuscript, and Ms. Enid Alston for manuscript preparation.

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