TROY Newly Identified Member of the Tumor Necrosis Factor

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TROY, A Newly Identified Member of the Tumor Necrosis Factor Receptor Superfamily Exhibits a

homology with Edar and is Expressed in Embryonic Skin and Hair Follicles

§ † ‡ ‡ ‡
Tetsuo Kojima *, Yoshihiro Morikawa , Neal G. Copeland , Debra J. Gilbert , Nancy A. Jenkins , Emiko

† §1
Senba and Toshio Kitamura

*Cytokine Research Program, Chugai Research Institute for Molecular Medicine, Inc., Niihari, Ibaraki,

300-4101, Japan.

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†
Department of Anatomy and Neurobiology, Wakayama Medical School, Kimiidera, Wakayama, 641-8509,

Japan

‡
Mouse Cancer Genetics Program, National Cancer Institute, Frederick Cancer Research and Development

Center, Frederick, MD 21702, USA.

§
Department of Hematopoietic Factors, The Institute of Medical Science, The University of Tokyo,

Minato-ku, Tokyo 108-8639, Japan.

1
Corresponding author: Toshio Kitamura (tel:+81-35449-5758, fax:+81-35449-5453, e-mail:

kitamura@ims.u-tokyo.ac.jp).

Running title: TROY, a new member of TNFRSF exhibits a homology with Edar
SUMMARY

In a signal sequence trap screening of the murine brain, we identified a new member of the TNF receptor

superfamily designated TROY. TROY is a type-I membrane protein of 416 amino acids with characteristic

cysteine-rich motifs in the extracellular domain and a TRAF2 binding sequence in the cytoplasmic domain of

223 amino acids. In fact, activation of NF-κB was induced by the overexpression of TROY and was

inhibited by dominant negative forms of TRAF2, 5 and 6, indicating that TRAFs and NF-κB are involved in

signal transduction of TROY. We also cloned a cDNA for a human counterpart, which showed 75%

homology with mouse TROY at the amino acid level. The extracellular domain of TROY exhibits an

extensive homology with that of Edar, a receptor that specifies hair follicle fate. TROY mRNA is strongly

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expressed in brain and embryo, moderately in heart, lung and liver, but not in spleen. In the embryo, the

expression level is particularly strong in the skin. Interestingly, in situ hybridization analysis of the embryo

showed that TROY mRNA was exclusively expressed in the epithelium of many tissues. On the other hand,

in neonatal mice, TROY is expressed in hair follicles like Edar as well as in the neuron, suggesting pleiotropic

functions of TROY in the development as well as in the adult mice. The Troy gene locates near the waved

coat (Wc) locus, a mutant that relates to abnormalities in skin and hair.
INTRODUCTION

Tumor necrosis factor (TNF)-related cytokines form a large family of pleiotropic mediators of host

defense and immune systems, which act either locally as membrane proteins, or on distant target cells as

secreted proteins. Members of the TNF receptor superfamily (TNFRSF) mediate the action of TNF-related

cytokines leading to cell death or to cell proliferation and differentiation (1-3). Most of the genes for

TNFRSF encode type I transmembrane glycoproteins with an extracellular ligand-binding domain, a single

membrane-spanning region and a cytoplasmic region that activates cell functions. The characteristic pattern

is found within the extracellular domain formed by cysteine-rich 40 residues repeats with a 25-30% homology.

The majority of conserved positions are cysteine residues. The cysteine-rich repeats reflect the characteristic

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structural domain of the TNFRSF (4-6). Overall this family of glycoproteins shows a relatively low level of

sequence conservation, despite sharing a common fundamental structure. Comparison of the cytoplasmic

sequences of the receptors shows considerably more diversity than do extracellular regions in sequence and

size. Although there is no common motif found in all members of TNFRSF, some elements are shared

between subsets of family members. For instance, TNF (4,7,8), CD95/FAS (9) and TRAIL receptor

(TRAILR) (10-12) share a domain of approximately 80 amino acids near their C-terminal called the ''death

domain''(13-15) which is required for induction of apoptosis by these receptors. On the other hand, the

majority of TNFRSF recruit the TNFR-associated factor (TRAF) family of intracellular adaptor molecules

through cytoplasmic tails, to promote cell survival by activation of downstream protein kinase cascades and,

ultimately, transcription factors of the NF-κB and AP-1 family (16,17). A major TRAF2-binding consensus

sequence, (P/S/A/T)x(Q/E)E, and a minor consensus motif, PxQxxD, were defined, based on structural

analyses (18).

TNFRSFs are not only involved in the immune system but also in many other biological systems. For

example, OPG/RANK has been shown to play critical roles in osteoclast differentiation and function (19,20).

The downless (dl) gene, isolated by positional cloning, encodes Edar, a new member of TNFRSF (21,22).

Mice with mutations in this gene have defects in hair follicle induction, lack sweat glands and have

malformed teeth. Based on the mutant phenotype and Edar expression pattern, it was proposed that Edar
specify the fate of hair follicles.

We have now identified a new member of TNFRSF expressed on the mouse embryo (TROY), using

a newly established signal sequence trap method SST-REX (23). TROY shows a unique tissue distribution

in the embryo as well as after the birth; in the embryo, TROY is exclusively expressed in the epithelium

including neuroepithelium, skin, bronchiolar epithelium, conjunctiva and so forth, while after the birth TROY

is strongly expressed in hair follicles like Edar, as well as in neurons.

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MATERIALS AND METHODS

Reagents and Cell Line-----Recombinant murine IL-3 was produced in silk worms (24). Ba/F3 cells

(25) were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and 1ng/ml

mIL-3. Human embryonic kidney (HEK) 293T cells (26) were cultured in DMEM supplemented with

10%FCS. An ecotropic retrovirus packaging cell line BOSC23 (26) was maintained in DMEM containing

10% FCS and a guanine phosphoribosyl transferase (GPT) selection reagent (Specialty Media, Lavallette, NJ).

Two days prior to transfection, cells were transferred to DMEM/10%FCS not containing GPT selection

reagents. A nullipotent embryonal carcinoma NF-1 was obtained from ATCC, and human glioma U251 and

gliosarcoma GI-1 were from RIKEN (Japan).

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Cloning of a cDNA for TROY-----Total RNA was prepared from mouse brain, and poly(A)+RNA was

further purified using oligo(dT)-cellulose chromatography. Complementary DNA was synthesized by

random hexamers primed with SuperScript Choice System (GIBCO-BRL), separated through SizeSep 400

Spun Column (Pharmacia), and inserted into the BstXI site of pMX SST, using BstXI adapters (Invitrogen).

The ligated DNA was introduced into DH10B cells (Electromax, GIBCO-BRL) by electroporation. Plasmid

DNA was prepared using standard methods. Infection of Ba/F3 cells with retroviruses carrying a cDNA

library for SST, and isolation of cDNA fragments by PCR from factor-independent clones was performed as

described (23). Full-length cDNA clones for TROY were obtained from a mouse brain and embryonic day

17.5 skin cDNA library synthesized by oligo dT and random priming, respectively. A biotinylated probe of

460bp was obtained by PCR from TROY cDNA using primers 5’-CAAGGTCCTACCTCTACACA and

5’-AAGGTTCACCTTGCTGGTAC, using 50 microM biotin-21-dUTP (Clontech), 200µM dATP, dGTP,

dCTP and 10uM dTTP. Mouse brain and embryonic skin cDNA libraries were screened using biotinylated

linear DNA probes coated with RecA proteins, as described (27-29). To isolate a cDNA clone encoding a

human counterpart of TROY, a human gliosarcoma cell line GI-1 cDNA library synthesized by random

priming was screened using mouse TROY cDNA coding region as a probe.

RNA Blot Hybridization-----Total RNAs of cell lines were extracted with TRIZOL (GIBCO BRL), and

poly(A)+RNA of mouse embryo was purified with FastTrack2.0 (Invitrogen). Thirty micogram of total
RNA and 3µg of poly(A)+RNA were fractionated in a 0.7% agarose gel containing formaldehyde and

transferred to a Hybond N+ nylon filter (Amersham). Mouse Multiple Tissue Northern blot was obtained

from Clontech. The Northern blot was hybridized with radiolabeled TROY cDNA.

In situ Hybridization-----A 456 bp BamHI-HindIII cDNA fragment (coding region 151-607) of mouse

TROY was inserted into a pBluescriptII KS vector. Digoxigenin-labeled riboprobes for TROY were

transcribed in vitro from linearized, gel-purified plasmids, using T7 polymerase (antisense cRNA probe) and

T3 polymerase (sense cRNA probes). Embryos were embedded in OCT compound (Miles, Elkhart, IN),

frozen in n-hexane, and then cut and analyzed by in situ hybridization, using a modification of reported

methods (30,31). When control sections were hybridized with identical quantities of sense cRNA, signals

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were nil (data not shown).

NF-κB-dependent Reporter Assays-----HEK293T cells were plated in 6-well plates at the concentrations

of 106/well. On the following day, using FuGENE6 (Roche), the cells were transfected with 100ng of

pTK-lacZ, a thymidine kinase promoter-driven beta-galactosidase expression plasmid to normalize for

transfection efficiency, together with 100ng of a reporter plasmid and various amounts of each expression

vector. Total DNA (3µg) was kept constant by supplementation with DNA derived from the pCOSI vector.

A reporter plasmid, κB-luc, has five repeats of the NF-κB site upstream of a promoter derived from HTLV-1

and a luciferase reporter gene (32). Twenty four hours after the transfection, the cells were harvested in

phosphate-buffered saline and lysed in the luciferase lyses buffer. The lysates were assayed for luciferase

and beta -galactosidase activities (33).

Interspecific Mouse Backcross Mapping-----Interspecific backcross progenies were generated by mating

(C57BL/6J x M.spretus) F1 females and C57BL/6J males, as described (34,35). A total of 205 N2 mice

were used to map the Troy locus (see text for details). The probe, a 1690 bp fragment of mouse cDNA was

32
labeled with [α- P] dCTP, using nick translation labeling kits (Boehringer Mannheim). A fragment of 9.6

kb was detected in PstI digested C57BL/6J DNA and a fragment of 15.5 kb was detected in Pst I digested M.

spretus DNA. The presence or absence of the 15.5 kb Pst I M. spretus-specific fragment was monitored in

the backcross mice.
A description of the probes and FRLPs for the loci linked to Troy including Ctsg, Gjb2 and Fzd3 has

been reported (36,37). One locus for our interspecific backcross has not been reported. The Blk probe, an

854 bp EcoRI fragment of mouse cDNA, detected KpnI fragments co-segregated. Recombination distances

were calculated using Map Manager, version 2.6.5. Gene order was determined by minimizing the number

of recombination events required to explain the allele distribution patterns.

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RESULTS

Isolation of the Mouse TROY cDNA Clone Encoding a Newly Identified Member of the TNFR

Superfamily-----In the SST-REX method (23), Ba/F3 clones transduced with cDNAs containing signal

sequences showed factor-independent growth through surface expression of a constitutively active MPL, as a

fusion protein. Among cDNA clones isolated by SST-REX from a mouse brain cDNA library, the LTI9

clone showed homology with members of the TNFR superfamily, at the ami no acid sequence level.

Positions of cysteine residues in the cysteine rich repeat were perfectly conserved. Full-length cDNAs

obtained from oligo-dT primed cDNA libraries derived from mouse brain, using the RecA screening method

(27-29), were confirmed to contain a sequence that was identical to LTI9.

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These cDNAs contain an open reading frame of 1251 nucleotides. The putative initiation codon is

preceded by a sequence (AGAGCC), in good agreement with the Kozak's consensus sequence for initiation of

translation in eukaryotes (38). The termination codon is followed by a 3' untranslated region of 2590 bp.

A canonical polyadenylylation signal is present 36 bp upstream of the poly(A) tail (39). The protein

putatively encoded by the TROY mRNA is a cysteine-rich protein of 416 amino acids with a calculated

molecular mass of 45kDa (Fig. 1). Two hydrophobic regions are present in the protein, representing the

signal sequence and the transmembrane region. A hydrophobic stretch of 25 amino acids toward the C

terminus (amino acids 169-193) was assigned as a transmembrane domain, because it had a potentially single

helical span. A cleavage site for the signal peptide was found between Cys (at position 29) and Glu (at

position 30) .

Like other members of the TNFRSF, TROY contains the characteristic cysteine-rich motifs (C - x(4,6) -

[FYH] - x(5,10) - C - x(0,2) - C - x(2,3) - C - x(7,11) - C - x(4,6) - [DNEQSKP] - x(2) - C) that have been

shown by x-ray crystallography to represent a repetitive structural unit (40,41) (Fig. 1). TROY contains two

perfect TNFR motifs and one imperfect motif in which C2 and C6 are not present. TROY exhibited a

significant and extensive homology (33%) with all of three motifs of Edar, dl gene product (Fig. 2) (21,22)

(Fig. 2).

The cytoplasmic tail of TROY spans amino acids 194-416 of the precursor protein and does not harbor
the death domain descovered in the intracellular domains of CD95 (9), TNFR (4,7,8), TRAILR (10-12) and

Edar, however it does contain a major TRAF2-binding consensus sequence, TLQE (276-279).

A shorter clone, dTROY was also identified from a cDNA library of mouse E17.5 skin. The

extracellular and transmembrane domains of the dTROY were identical with those of TROY. However, the

cytoplasmic tail of dTROY has only 21 amino acids, and does not contain a TRAF2-binding consensus

sequence (Fig. 3). A consensus sequence of the 5’ boundary of the intron was present on the transition point

from the common sequence to the unique sequence of dTROY, suggesting that dTROY contains same

sequence from the intron at 3’ end. To confirm that dTROY was not an artifact, a dTROY-specific RT-PCR

was done, using a sense primer of the common sequence and an antisense primer of the unique sequence of

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dTROY. A band of dTROY was amplified only from total RNA of E17.5 skin but not from mouse brain or

liver (data not shown).

Isolation of a cDNA Clone Encoding a Human Counterpart of TROY-----A human counterpart of TROY

expression in the human gliosarcoma cell line GI-1 was detected in the Northern hybridization (Fig. 4). A

human counterpart was obtained from a cDNA library derived from the GI-1 cell line using RecA screening.

This cDNA contains an open reading frame of 1269 nucleotides and encoded a cysteine-rich protein of 423

amino acids with a calculated molecular mass of 46kDa. The overall identity between hTROY and mTROY

at the amino acid level is 75% and with the identity being 92% in the extracellular and transmembrane

domain. The cytoplasmic tail of hTROY was 234 amino acids long, and had 57% homology with mTROY

(Fig. 1).

Expression of TROY RNA -----Expression of TROY mRNA was examined by Northern blot analysis,

using a cDNA for the ORF of TROY as a probe (Fig. 4). TROY mRNA was strongly expressed in the brain,

with an approximate molecular size of 4.5 kilo bases, which was close to the length of the TROY cDNA clone

(3970 nucleotides) identified. TROY mRNA was detected in most tissues but not in the spleen. In the

embryo, the expression level was periodically increased and was particularly strong in the skin. TROY

mRNA was also detected in human glioma U251, GI-1, the embryonic stem cell A3-1 and nullipotent

embryonal carcinoma NF-1 (data not shown). Cellular localization of TROY mRNA was investigated by in
situ hybridization in mouse embryo day 13.5. Interestingly, TROY mRNA was detected exclusively in

epithelium i.e. neuroepithelium in frontal and lateral lobes, epidermis of skin, bronchiolar epithelium,

epithelium of tongue, gastric epithelium, conjunctiva, and cochlea, while in neonatal mice TROY mRNA was

mainly detected in hair follicles like Edar, as well as in neuron but not skin epidermis (Fig. 5).

TROY Induces NF-κB Activation-----Because the TRAF2-binding consensus sequence TLQE was found

in the cytoplasmic domain of TROY, we asked if TROY activates NF-κB through TRAF2. HEK293T cells

were transiently transfected with an IL-2 promoter-derived NF-κB-luciferase reporter gene (32) and a TROY

expression vector (Fig. 7). Co-transfection of TROY induced over a 3.5-fold higher luciferase activity, as

compared with the vector control. To determine if TRAF2, 5 and 6 are involved in the NF-κB activation by

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TROY, we tested whether TROY-induced NF-κB activation was inhibited by dominant negative mutants of

TRAF2, 5 and 6 lacking N-terminal zinc binding domain required for NF-κB activation (42-48).

Overexpression of dominant negative TRAF2, 5 or 6 blocked NF-κB activation in the reporter assay. These

results demonstrated that TRAFs were involved in TROY-mediated NF-κB activation. The dTROY with a

small cytoplasmic domain did not activate NF-κB.

Troy Locates Near Wc-----The mouse chromosomal location of Troy was determined by interspecific

backcross analysis, using progeny derived from matings of [(C57BL/6J x M.spretus) F1 X C57BL/6J] mice.

This interspecific backcross mapping panel has been typed for over 2900 loci that are well distributed among

all the autosomes as well as the X chromosome (34). C57BL/6J and M.spretus DNAs were digested with

several enzymes and analyzed by Southern blot hybridization for informative restriction fragment length

polymorphisms (RFLPs), using a mouse cDNA TROY probe. The 15.5 kb PstI M.spretus RFLPs (see

Materials and Methods) was used to follow the segregation of the Troy locus in backcross mice. Mapping

showed that Troy is located in the central region of mouse chromosome 14 linked to Ctsg, Gjb2, Blk and Fzd3.

Although 161 mice were analyzed for every marker (shown in the segregation analysis) (Figure 7), up to 191

mice were typed for some pairs of markers. Each locus was analyzed in pairwise combinations for

recombination frequencies, using additional data. Ratios of the total number of mice exhibiting recombinant

chromosomes to the total number of mice analyzed for each pair of loci and the most likely gene order are:
centromere - Ctsg - 1/191 - Gjb2 - 2/180 - Troy - 2/171 - Blk - 4/175 - Fzd3. The recombination frequencies

[expressed as genetic distances in centiMorgans (cM) +/ - the standard error] are: certromere - Ctsg - 0.5 +/-

0.5 - Gjb2 - 1.1 +/- 0.8 - Troy - 1.2 +/- 0.8 - Blk - 2.3 +/- 1.1 - Fzd3.

We compared our interspecific map of chromosome 14 with a composite mouse linkage map that shows

the location of many uncloned mouse mutations (provided from Mouse Genome Database, a computerized

database maintained at The Jackson Laboratory, Bar Harbor, ME), and found that Troy localized in the

vicinity of the waved coat (Wc) locus, a mutant that presents abnormality in skin and hair (49-51).

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DISCUSSION

We isolated and characterized a newly identified member of TNFRSF, TROY, from a cDNA library of

the murine brain, using SST-REX. In structural analysis of the cDNA, the translation product of TROY was

shown to be a type-I transmembrane protein of 416 amino acid residues. While the extracellular domain of

TROY retains the characteristic structure of TNFRSF, the intracellular domain does not contain the death

domain.

During our characterization of TROY, Hu et al. reported a novel member of TNFRSF (TNFRSF19),

which was identical to TROY except that the cytoplasmic tail was shorter than that of TROY by 68 amino

acids (52). In a search of the expressed sequence tag (EST) database, three clones (GenBank Accession No.

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AI551729, AA445805 and AI48211) encoding the C-terminus region of TROY were identified, all of which

were identical to the C- terminal region of TROY but not to that of TNFRSF19. Moreover, the cytoplasmic

tail of hTROY identified in this study was similar to mTROY but not to TNFRSF19 in the size and the

sequence, indicating that TROY is a major and complete cDNA clone.

When compared to other members of TNFRSF, the closest known relative of TROY is a receptor called

Edar (21,22) that specifies hair follicle fate and is 33% identical to TROY in the extracellular domain. Mice

with mutations in the Edar gene not only have defects in hair follicle induction but also lack sweat glands and

have malformed teeth (21,22). The expression of TROY is strong in embryonic skin and hair follicles, as is

the case for Edar. This high homology in the extracellular domain and the similarity of distribution between

TROY and Edar strongly suggests that TROY also plays some role in development of embryonic skin and

induction of hair follicles. While Edar harbors a death domain, TROY dose not contain the one, and

activates NF-κB through TRAF proteins. Therefore, it would be interesting to know how TROY and Edar

functionally interact with each other.

In this context, of interest is the fact that the gene for Troy locates near the waved coat (Wc) locus, a

mutant of which presents abnormality in skin and hair. Heterozygotes of Wc locus have a wavy coat and

whiskers, and have a dry and scaly skin, and homozygotes die in utero (49-51). The central region of mouse

chromosome 14 shares regions of homology with human chromosomes 7q, 8p, 13q and 14q. The draft
sequence of human chromosome13q12.11-3 from the Sanger center has been registered in the EMBL

database, and includes the sequence of hTROY (624-1586). It would be interesting to examine if there is a

similar genetic disease in the human.

TROY exhibits a major TRAF2-binding consensus sequence in the cytoplasmic tail, and overexpression

of TROY induced NF-κB activation, as noted in the reporter assay using luciferase. This activation was

blocked by overexpression of dominant negative TRAF2, 5 and 6. Like most members of the TNFRSF,

TROY will be homotrimerized by stimulation of its ligands and recruit TRAF2, 5 and 6 to the cytoplasmic

tail which will promote cell survival and proliferation by activating downstream protein kinase cascades and,

eventually, transcription factors in the NF-κB and AP-1 family. Hu et al. reported that NF-κB activation is

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not induced by TNFRSF19, a truncated version of TROY lacking the C-terminal 77 amino acid residues of

the cytoplasmic tail, indicating that the C-terminus region of TROY is essential for activation of NF-κB and

for signal transduction.

We also found a truncated form of TROY, dTROY that harbors a smaller cytoplasmic tail than that of

TROY. The consensus sequence of the 5’ boundary of the intron was found on the transition point from the

TROYs common nucleotide sequence to dTROY unique sequence, indicating that dTROY is an alternative

splicing form containing an unspliced intron. Unlike TROY, dTROY can not induce NF-κB activation, and

may play a role as a decoy receptor like TRAIL-R3 (53) and DcR3 (54) for the CD90 ligand, to negate a

signal from a ligand for TROY. The expression of dTROY mRNA, detected using dTROY-specific

RT-PCR, was found only in the embryonic skin but not in the brain or liver. In the embryo, the expression

level of TROY appears to be significantly increased during the late stages of embryogenesis and fluctuated

from E11.5 to E15.5. Moreover, TROY sequence was found as an EST from the mouse four-cell-embryo

(Accession No. AU041881). These results suggest that TROY may play pleiotropic roles in embryogenesis.

Of particular interest is the fact that homozygotes for the Wc locus result in embryonic lethality.

In summary, TROY is a novel member of TNFRSF which exhibits a similarity with Edar in the structure

and the expression patterns, and the gene of which locates in the vicinity of Wc. The present data also

suggests that TROY is involved in embryonic development and development of skin and hair follicles. For
further study, it is important to identify the cognate ligand, which is now under investigation.

Acknowledgements

This research was supported by the Ministry of Education, Science, Sports and Culture of Japan, and in part

by the National Cancer Institute, DHHS. We thank Dedorah B. Householder for excellent technical

assistance, Dr. Hiroyoshi Nakano, Dept. Immunology, Juntendo University School of Medicine for the

TRAFs expression vector, and Ms.Mari Ohara for editing the English. The Department of Hematopoietic

Factors is supported in part by Chugai Pharmaceutical Ltd.

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FOOTNOTES

The nucleotide sequences for the mouse TROY, dTROY and human TROY have been deposited in the

DNA Data Bank of Japan database under DDBJ/EMBL/GenBankAccession Number AB040432, AB040433

and AB040434, respectively.

The abbreviations used are: TNF, tumor necrosis factor; TNFR, TNF receptor; TNFRSF, TNFR

superfamily; EST, expressed sequence tag; TRAF, TNFR-associated factor.

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FIGURE LEGENDS

Figure 1. Comparison between the protein sequence of mouse and huma n TROY. The signal sequence and

transmembrane domain are underlined, and common amino acid residues are shaded. Two perfect TNFR

cystaine-rich motifs are dot underlined.

Figure 2. Alignment of the extracellular domain of mouse TROY with that of mouse Edar. Identical

residues are indicated with asterisk and similar residues are indicated by a dot.

Figure 3. Structure of dTROY, a putative decoy receptor. The region of dTROY, which is structurally

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different from TROY, is boxed. The consensus sequence of 5' boundary of the intron is shaded. The

dTROY specific antisense primer region is underlined.

Figure 4. Northern blot analysis for detection of TROY transcripts. Blotted membranes were probed with

a 32P-dCTP-labeled TROY cDNA fragment in a PerfectHyb solution (TOYOBO, Japan) and then washed

with 0.2xSSPE at 65°C. Hybridized signals were identified using BAS2000 or X-ray films (FUJI Film,

Japan).

Figure 5. in situ hybridization of TROY. The sections of embryo at ED13.5 (A, lung; B, oculus; C, skin;

D, frontal lobe) and neonatal (E, skin; F, ventricular zone) were incubated with 0.1 ml of hybridization buffer

containing 40 ng of the cRNA, then washed for 1 hr in 0.1X SSC at 65°C. The sections were then stained

with an alkaline phosphatase-conjugated anti-digoxigenin Fab fragment (Boehringer Mannheim), a nitro blue

tetrazolium (Boehringer Mannheim) a 5-bromo-4-chloro-3-indolyl phosphate (Boehringer Mannheim), and

levamisole. TROY mRNA is seen in epithelium i.e. bronchiolar epithelium (arrowhead), conjunctiva

(arrowhead), epidermis of skin (arrowhead), neuroepithelium (arrowhead), while in neonatal mice TROY

mRNA is detected in hair follicles (arrowhead), as well as in neuron (arrowhead) but not skin epidermis.

Bar = 100 micrometer.
Figure 6. TROY mediates NF-κB activation. 293T cells were transiently transfected with 100ng of luc

reporter plasmid, 100ng of beta-galactosidase plasmid and indicated expression plasmids. Twenty four

hours later, the cells were collected, and the luciferase activity in each sample was determined. The values

were normalized to the expression of beta-galactosidase. The level of induction in the luciferase activity is

indicated as compared with cells transfected with the control vector. Data are shown as the average ± SD of

triplicate samples and represent one of three independent experiments all with similar results.

Figure 7. Troy maps in the central region of mouse chromosome 14. Troy was placed on mouse

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chromosome 14 by interspecific backcross analysis. The segregation patterns of Troy and flanking genes in

161 backcross animals that were typed for all loci are shown at the top of the figure. For individual pairs of

loci, over 161 animals were typed (see text). Each column represents the chromosome identified in the

backcross progeny inherited from the (C57BL/6J x M.spretus) F1 parent. The shaded boxes represent the

presence of a C57BL/6J allele and white boxes represent the presence of a M.spretus allele. The number of

offspring inheriting each type of chromosome is listed at the bottom of each column. A partial chromosome

14 linkage map showing the location of Troy in relation to linked genes is shown at the bottom of the figure.

Recombination distances between loci in centimorgans are shown to the left of the chromosome and the

positions of loci in human chromosomes, (where known) are shown to the right. References for the human

map positions of loci cited in this study can be obtained from GDB (Genome Data Base), a computerized

database of human li nkage information maintained by The William H. Welch Medical Library of The Johns

Hopkins University (Baltimore, MD).
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mTROY MALKVLPLHRTVLFAAILFLLHLACKVSCETGDCRQQEFKDRSGNCVLCKQCGPGMELSKECGFGYGEDAQCVPCRPHRFKEDWGFQKCKPCA
hTROY MALKVLLEQEKTFFTLLVLLGYLSCKVTCETGDCRQQEFRDRSGNCVPCNQCGPGMELSKECGFGYGEDAQCVTCRLHRFKEDWGFQKCKPCL

mTROY DCALVNRFQRANCSHTSDAVCGDCLPGFYRKTKLVGFQDMECVPCGDPPPPYEPHCTSKVNLVKISSTVSSPRDTALAAVICSALATVLLALL
hTROY DCAVVNRFQKANCSATSDAICGDCLPGFYRKTKLVGFQDMECVPCGDPPPPYEPHCASKVNLVKIASTASSPRDTALAAVICSALATVLLALL

mTROY ILCVIYCKRQFMEKKPSWSLRSQDIQYNGSELSCFDQPRLRHCAHRACCQYHRDSAPMYGPVHLIPSLCCEEARSSARAVLGCGLRSPTTLQE
hTROY ILCVIYCKRQFMEKKPSWSLRSQDIQYNGSELSCLDRPQLHEYAHRACCQCRRDSVQTCGPVRLLPSMCCEEACSPNPATLGCGVHSAASLQA

mTROY RNPASVGDTMPAFFGSVSRSICAEFSDAWPLMQNPLGGD-SSLCDSYPELTGEDTNSLNPENESAASLDSSGGQDLAGTA--ALESSGNVSES
hTROY RNAGPAGEMVPTFFGSLTQSICGEFSDAWPLMQNPMGGDNISFCDSYPELTGEDIHSLNPELESSTSLDSNSSQDLVGGAVPVQSHSENFTAA

mTROY TDSPRHGDTGTVWEQTLAQDAQRTPSQGGWEDRENLNLAMPTAFQDA
hTROY TDLSRYNN--TLVESASTQDALTMRSQLDQESGAIIHPATQTSLQVRQRLGSL
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mTroy ETGDCRQQEFK-DRSGNCVLCKQCGPGMELSKECGFGYG-EDAQCVPCRPHRFKEDWGFQKCKPCADCALVNRFQRA
* .:* ::*:: : :* * * * ** * .**:* :* **** ..:*.: *:* *: ** : * **
mEdar EDSNCGENEYHNQTTGLCQQCPPCRPGEEPYMSCGYGTKDDDYGCVPCPAEKFSK-GGYQICRRHKDC---EGFFRA

mTroy N----CSHTSDAVCGDCLPGFYRKT-KLVGFQDMECVPCGDPPPPYEPHCTSKVNLVK--ISSTVS----SPRDT
. . .** ** ****:* : .: .* * .* .** : .*.. .. *. *** . ** :
mEdar TVLTPGDMENDAECGPCLPGYYMLENRPRNIYGMVCYSCLLAPPNTK-ECVGATSGVSAHSSSTSGGSTLSPFQH
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Troy K R Q F M E K K P S W S L R S Q D I Q Y N G S E L S C F D Q
AAGAGGCAGTTCATGGAGAAGAAACCCAGCTGGTCTCTGCGGTCACAGGACATTCAGTACAATGGCTCTGAGCTGTCATGCTTTGACCAGC
AAGAGGCAGTTCATGGAGAAGAAACCCAGCTGTAAGCTCCCATCCCTCTGTCTCACTGTGAAGTGAGCTTGTTAGCATTGTCACCCAAGAG
DTroy K R Q F M E K K P S C K L P S L C L T V K *
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Skeletal Muscle
Mouse Embryo

Kidney
Spleen

Testis

Brain
Heart

Brain

Lung

Liver

Skin
10.5 11.5 12.5 13.5 14.5 15.5 16.5 17.5 U251 GI-1
7.5kb
4.4kb

G3PDH
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D

F
B

E
A

C
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Mock

TROY