Structure, expression and chromosomal mapping of TKT from man and mouse: a new subdass of receptor tyrosine kinases with a factor VIII-like domain

r

Oncogene (1993), 8, 3433-3440

©

Macmillan Press Ltd, 1993

SHORT REPORT

Structure, expression and chromosomal mapping of TKT from man and mouse: a new subdass of receptor tyrosine kinases with a factor VIII-like domain

Thomas Karn, Uwe Holtrich, Andreas Bräuninger, Beatrix Böhme, Georg Wolf, Helga Rübsamen-Waigmann & Klaus Strebhardt

Chemotherapeutisches Forschungsinstitut, Georg-Speyer-Haus, Paul-Ehrlich-Str. 42-44, 60596 Frankfurt, Germany

Using a polymerase chain reaction-mediated approach we have characterized cDNAs from human and mouse origin representing a novel type of receptor protein tyro-

. sine

kinase (RTK). The deduced amino acid sequence

(855 amino acids) of the Iongest open reading frame has a unique extracellular region encompassing a factor Vlll-like domain, not previously described for RTKs.

The most closely related RTKs are members of the neurotrophin receptors (TRK), which showed 47-49%

homology with the kinase domain of the new RTK.

Therefore, the new gene has been called

TKT (

Tyrosine- Kinase related to 1RK). TKT orthologs from man and mouse were 98% similar. In both species a major tran- script of 10 kb was found to be expressed at high Ievels in heart and lung. Low Ievels of this mRNA-species were detected in human brain, placenta,

Ii

ver, skeletal muscle, kidney and in mouse brain and testis. Analysing human/

mouse somatic cell hybrids we demonstrated that

TKT

segregates with human chromosome

l.

Receptor tyrosine kinases (RTKs) play a key rote in the communication of cells with their microenviron- ment. These molecules are involved in the regulation of cell growth, differentiation and metabolism. In several cases the biochemical mechanism by which RTKs transduce signals across the membrane has been shown to

be

Iigand induced receptor oligomerization and sub- sequent intracellular autophosphorylation. This auto- phosphorylation Ieads to phosphorylation of cytosolic targets as weil as association with other molecules, which are involved in pleiotropic effects of signal trans- duction. RTKs have a tripartite structure with extra- cellular, transmembrane and cytoplasmic regions. The intracellular portion of RTKs harbours the protein tyrosine kinase (PTK) - domain of the molecule. There appear to be at least six subclasses of RTKs: EGF- receptor (Ullrich

et al., 1984); insulin-receptor (Ebina et al.,

1985; Ullrich

et al.,

1985), PDGF-receptor

(Yarden et al.,

1986; Claesson-Welsh

et a/., 1989),

FGF-receptor (Lee

et a/.,

1989; Holtrieb

er a/.,

1991 ), EPH/ELK (Hirai

et a/.,

1987; Böhme

et al.,

1993) and TRK

(Martin-Zanca et a/.,

1989).

RTK-genes were characterized by applying the poly- merase chain reaction (PCR) in combination with

Correspondence: K. Strebhardt

Received 15 June 1993; acccptcd in revised form 4 August 1993

degenerate oligonucleotide primers based upon con- served motifs of the kinase domain of PTKs (Wilks,

.

1989; Ho1trich

et al.,

1991 ). In a more direct approach we identified a new member of the EPH/elk-family of RTKs: we utilized oligonucleotide primers specifically designed to a highly conserved N-terminal motif (CKETFNL) of EPH/elk-RTKs and a motif of the kinase region (SDVWS) in RNA-PCRs. 5' and 3' elon- gation of the primary PCR-product allowed to isolate a new gene

HEK2

as a new member of this family (Böhme

et al.,

1993).

PCR-mediated isolation of a novel RTK-gene

To identify additional members of the EPH/elk-family we utilized a different combination of primers designed according to the above mentioned motifs for PCR with cDNA templates from human embryonie RNA. This amplification gave rise to a fragment of 800 bp which differed from the anticipated PCR-product of 2 kb derived from members of the EPH/elk-family. Nested primers for the PTK-specific motifs HRDLA and SDVWS were used to verify the identity of this PCR- product and gave rise to the expected 200 bp-product.

The original 800 bp-product, designated Kl, was sequenced and subsequently used as a probe to screen

·

cDNA libraries from human heart and thymus (2 x 10

⁶

recombinant clones each). Several overlapping clones spanning 2.3 kb were isolated. Anchored and ligation- mediated PCR was performed to extend the sequence in 3' and 5' direction (Böhme

et a/.,

1993).

TKT represents a new subdass of RTKs

Figure 1 shows the composite nucleotide sequence of 3.1 kb of the K1 cDNA. An open reading frame begins with an ATG codon at nucleotide 354 and ends at an in-frame stop codon at position 2919. Several features of the sequence indicate that the ATG codori at posi- tion 354 is used for the initiation of translation: it is surrounded by a sequence that is in agreement with Kozak's rule (Kozak, 1984) and the following DNA sequence predicts a hydrophobic signal peptide.

Fur~

thermore there are termination codons upstream of the ATG codon in all three reading frames.

The deduced polypeptide contains a second hydro-

phobic stretch of amino acids (residues 400-421),

which represents a transmembrane domain followed by

a basic stop transfer motif. This suggests that the

3434

.

T. KARN et al.

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:r

^{D, H S M N -V} ~M..,._fu~

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Y L ~ V Y D G A V G Y S M T E G L G Q L T D G V S G L D

GATI'ICACCCAGACCCATGAATACCACG'I'GIDXCCOOCTATGACTA~CACCAArro:x:.TACATfGAG

D F T Q T H E Y H V W P G Y D Y V G W R ~ A T N G Y I E

ATCATGTI'IGAATI'TGACCCX:ATCA~ACCATGAACEI'CCN::T.'OC.AACAACATGITrGCTAAAGG'IGTG.~ATCTTTAAG

I M F E F D R I R lli_K_TI T M K V H C N N M F A K G V K I F K

~ACJ:.GT:GerACTTCCGCI'C'IGAPIX:CAG'IGAG'IQX;AACC:TAATGCCATTTCCTTCCCccrroTCC'T(J;;A'IGACGTCAACCCCAaf

E V Q C Y F R S E A S E W E P N A I S F P L V L D D V N P S

GCTCOO'ITfG'ICAC<X:fGCC'IC'ICCACCACCGAA1GGCCAGIGCCA~AATACCA~ATACC'IGGA'IGA'IGTICAGT

A R F V T V P L H H R M A S A I K C Q Y H F A D T W M M F S

GAGATCACCTTCCAATCAGA'IGC'IGCAA'IGTAC~CCTGCCCACC'I'CTCCTA'IGGCACCCACAACCTATGATCCAA'IG

E I T F Q S D A A M Y ~ E A L P T S P M A P T T Y D P M

CTT.AAAGTTGATGAO.GCAACAcrc:ooATCC'IGA~ATCATCTTTATCCTCC'T(J:;CCATCATIG'ICATCATCCTC

L K V D D S N T R I L I G C L V A I I F I L L A I I y I I L

'f(J;;!GXJ\G'I'I'C'I'OJCAAAA'IOCI'G3A~liCGATGC'T(J:;ATGA'IGAAATGACAGICAGCC'ITCCTGCCMGIGAT

_N R Q F W Q K M L E K A S R R M L D D E M T V S L S L P S D

TCTNX:A"''m'TCAACAAT~TCACCT/>GfGAA~AACI'CGACTTACGATCGCATCTTTCCcx:TiaXX:CIGAC

S S M F N N N R S S S P S E Q G S N S T Y D R I F P L R P D

TACCNrJ.Nl/::.cATCCNrJ.CTGATACGAAAN::fCCC.A'I"'TYXTCC~~AlGXJ!GfC

Y Q E P S R L I R K L P E F A P G E E E S G C S G V V K P V

~CCCA~'IGCAGNXX:IGACATAG"fGAJ'..CCTC'A/lro~CACATl'C'I'CJ>GI'G

Q P S G P E G V P H Y A E A D I V N L Q G V T G G N T Y S V

CCTGCCGTCACCA'IOOA~GA~CC~AACTTTCAAAGAGAAGC'IGXA

p A V T M D L L s G K D V A V E E F p R K L L T F K E K L

B

GAAa::J.AC~ATCTC'IGTGAAG'I'GGPGJGAATa::J.AAAAATICAAJ\GACAAAGATITrGCCCTAGA'IGTC;.crGCCAAC

E

m

^Q F

m

E V H L C E V E G M E K F K D K D F A L D V S A N

~'IGCTCCGAGCAGATGCCAACAAGJI.ATGCCAa::J.AATGATlTI'CITA.AtJGAGATAAAGATCA'IGTCT

Q P V L V A V

g

M L R A D A N K N A R N D F L K E I K I M S oxx:rcA.AtJGACC:CAAACATCATCCATCTA'ITATC'I'GIGTGTATCAC'IGATGACCCTCTCIGTA'IGATCAC'IGAATACA'IGGAGAA'IGGA

R L K D P N I I H L L S V C I T D D P L C M I T E Y M E N G GATCTCAATCAGI'I'I'C'ITICCCGCCACGAGCCCCCTAATK'TICCTCCAGCGA'IGTACGCAC'IGTC}IG'ITACACCAATC'IGAAGTTTA'IG

D L N Q F L S R H E P P N S S S S D V R T V S Y T N L K F M

GCTACCCAAAT'IGCCTC'I'GXATGAAGTACCTTICCTCTCTI'AA'l'l'l'IGl'l'CACCGAGATC'l'Q:;CCACACGAAAC'IGTTT~AAG

A T Q I A S G M K Y L S S L N F V H R D L A T R N C L V G K

AACTN:.ACAATCAAGAT~'IGAGC~CC'IGTA~ACTATI'Acox;ATCCAOOGCC'GG:XAG'IGCTCCCT

N Y T I K I A~ M S R N L Y S G D Y Y R I Q G R A V L P

ATCCCI::'IOOATGTCTIOOGAGAm!ATC'ITGCTGOOCAAGTICN:.TACAGCAAG'IGATGTG'IGGXC'I'l'TGGQ}'ITA~CT

I R W M S W E S I L L G K F T T A S D V W A F G V T L W E T

TICACC'l'l'l'IGTCAAGAACAGCCCTATICCCAGC'IGTCAGA'IGAACAm!TATfGNJMT~CGAGACCAAcn:;ACX;

F T F C Q E Q P Y S Q L S D E Q V I E N T G E F F R D Q G R

CAGN:.TI'ACCTCCC."TCAACCAGCCATI'IGTCC'IGACTC'IG'IGTATAAGC'IGA~AGAAG.l\GATN:.GAAGAACCGTCCC

Q T Y L P Q P A I C P D S V Y K L M L S C W R R D T K N R P

TCATTCCAI'CAAA~CAAOOCGACGAG'IGATGCTGTCJ\GTIX'CA'IGTICCTAC"Ga:TCAG:m:crcc

S F Q E I H L L L L Q Q G D E *

CTACAJ>GN:.CTACCAC:T.'CN:.CCA'IGCCTATGCCN:.TCCATC'T(J:;ACATTTAA'IGAAAC'IGAGN:;J'l.O,~::XXTIGT:ITG:c:Trn;cc~:T

Tl'K'C'IOOTCACCCCCAC'ICCCTN:.cccrGACTCATATATN:.T

173 83 263 353 443 30 533 60 623 90 713 120 803 150 893 180 983 210 1073 240 1163 270 1253 300 1343 330 1433 360 1523 390 1613 420 1703 450 1793 480 1883 510 1973 540 2063 570 2153 600 2243 630 2333 660 2423 690 2513 720 2603 750 2693 780 2783 810 2873 840 2963 855 3053 3096

Figure 1 Nuclcotide and deduced amino acid sequence of TKT Thc deduced amino acid sequence in one Ietter code is givcn below the nucleotide sequence. The putative signal peptide and the tran~membranc domain arc undcrlined. Potential sites for N-glycosylation in thc cxtraccllular rcgion are boxed. Tbe invariant motifs of thc kinase domain (Hanh et a/., 1988) are given in invcrtcd lcttcrs: the Consensus sequence GxGxxG of nucleotidc hinding proteins and PTKs, the conserved Iysine residue involvcd in the phosphotransfer reaction and thc invariant residues (DFG) implicated in A TP-binding. Thc factor VIII-Iike sequence in the extracellular part is shaded

putative Kl protein is an integral membrane protein (von Heijne,

1 986;

Singer, 1990). The extracellular region contains 399 amino acids with five potential N-linked glycosylation sites. The cytoplasmic portion consists of 434 amino acids and encompasses a juxta- membrane domain of ·139 amino acids and a kinase domain that contains all characteristic features of

PTKs (Figure 1) (Hanks et al., 1988). A putative auto- phosphorylation site is found at position 740. A kinase insert as weil as a C-terminal tail arc missing. In other RTKs these regions were shown to contain phospho- tyrosine residues which interact with SH2-domains. Thc 3' untranslated sequence encompasses 178 nucleotides.

A potential polyadenylation signal is missing.

TKT, A RECEPTOR TYROSINE KINASE WITH A FACTOR VJII-LIKE DOMAIN 3435

Comparison of the Kl amino acid

sequence

with known sequences

revealed

that K1 is a

member of the

RTK family, but does not belong to one of the known subclasses. The most closely related

RTKs

are mem- bers of the neurotraphin receptors (TRK, Martin- Zanca

et a/.,

1989), which showed 47-49% homology with Kl

in the

kinase domain (Figure 2).

Thus we named

the K

1

gene

TKT

(pronounced ticket):

Tyro- sine-Kinase

related to 7RK. Comparing

the

kinase domains of

TKT

with

those

of the trk-family and

various

types of

insulin

receptors instead of a con-

secutive

alignment TKT exhibits three insertions which are

between

two and

11 amino

ac. ids in

length

(Figure

2).

TKT

contains a

factor VIII-likc domain

The extracellular regions of RTKs contain certain features which distinguish individual farnilies of RTKs:

To date, cysteine-rich regions, immunoglobulin-likc domains and repeats of the EGF-like type and the

m-trkB r-trkB h-TRK TKT INS.R IGF-lR IRR

m-trkB r-trkB h-TRK TKT INS.R IGF-lR IRR

*

** * ** *

*

*

* *** *

flbronectin-type

have been found to be components of the extracellular portion of RTKs

(Hirai et al., 1987;

Yarden

& Ullrich, 1988;

Lindberg

&

Hunter,

1990;

Ziegler et al., 1993).

These motifs could not be detected in

the deduced amino acid sequence of TKT. Interest- ingly, a computer-aided homology search revealed similarities with domains of other proteins. Figure. 3 shows a domain (amino acids 30-

185)

which begins

eight residues after the presumptivc cleavage site of the

signal peptide and

is

homologaus

to

both C-units at the carboxyterminus of

factor

VIII, a component

of

blood coagulation (Gitschier et

al., 1984; Vehar et al.,

1984). These two C-miits within factor VIII have 37%

homology

with

each other. Homology of TKT to the

Cl-unit

and the C2-unit of factor VIII within a stretch of

156

amino

acids

was determined to be 35% and 30% respectively. Homologies to other proteins, which contain factor Vlll-like sequences were also observed:

(a) The 156 amino acid region of TKT

is

27 and 33%

homologaus to the Cl- and C2-unit, respectively, of a surface protein of mouse mammary epithelial cells (MFG-E8: milk fat globule membrane protein, Stubbs

** * * * *

*

* *

** ** *

******

***

* ** **** * * **** * ** ***

** *** **

* ** ***

m-trkB r-trkB h-TRK TKT

INS.R IGF-lR IRR

m-trkB r-trkB h-TRK

TKT

INS.R IGF-lR IRR

** **

*

^{* ** ... ** *}

Figure 2 Comparison of the TKT kinase domain with several others RTKs. Amino acid sequences of the kinase domains (Hanks et a/., 1988) of human TKT, human TRK (h·TRK; Martin-Zanca et al., 1989), mouse trkB (m-trkB; Klein et al., 1989), rat trkB (r-trkB; Middlemas 1!1 al., 1991), the insulin receptor (1NS.R; Ebina er. a/., 1985; UHrich et al., 1985), the insulin like growth factor-1 receptor (IGF-1 R; Ullrich et al., 1986) and the insulin receptor-relatcd rcceptor (IRR; Shier & Watt, 1989) were aligned using the Tree program of the HUSARsoftware package (DKFZ, Heidclbcrg) based on the progressive alignmcnt method ofFeng and Doolittle (1987). Jf all members of at least two of thc three subgroups (i.e. TRK·, insulin receptor· and TKT-subgroup) share identical residues, amino acids are given in invcrted letters

3436 T. KARN er al.

aa * * ** **

TKT 30 107

mfge8 148 225

mfgeS 308 386

A5:.Ag 275 347

A5-Ac;f 431 505

FVIII 2040 2109

FVIII 2193 2268

FV 1907 1982

FV 2066 2142

* **

TKT 108 GGHGIEFAPMYKIN DHSMNVCM1 185

mfge8 226 RAGRAEYLKTFKV 303

mfge8 387 OFGHIQYVESYKV 463

AS~Ag 424

A5-Ag 506 HKENKVFMRKFKI 584

FVIII 2110 QKFSSLYISQFII 2188

FVIII 2269 2345

FV 1983 2061

FV 2143 SLSSEMYVKSYTI 2221

Figure 3 Homology of factor VIII-Iike domains. The amino acid sequence of the factor VIII-like domain of human TKT (aa 30-185) was aligned with homologous regions of other proteins as described in Figure 2. Amino acids are inverted if identical in at least six sequences and macked with asterisks if identical in all nine sequences. The abbreviations are: mfge8 - mouse milk fat globule EGF factor 8 protein (Stubbs et al., 1990) aa 148-303 and aa 308-463; A5-Ag - A5-antigen (Takagi et a/., 1991) aa 275-424 and aa 431 584; FVlll factor VIII (Gitschier et al., 1984; Vehar et al., 1984), aa 2040-2188 (Cl) and aa 2193-2345 (C2); FV- factor V (Jenny ez al., 1987). aa 1907-2061 (Cl) and aa 2066-2221 (C2)

et al., 1990); (b) In addition homologies of 33% and 32% have been found to the factor VIII-Iike motif of a neuronal cell surface protein of Xenopus most likely involved in the neuronal recognition between the optic fibres and the visual center (A5-Antigen, Takagi

et

al., 1991); (c) Factor V, which also participates in blood coagulation, contains factor VIJT-related C-domains as weil (Jenny

et a/.,

1987) with homology to the corres- ponding region of TKT of 29% and 33%

.

The comparison of the kinase domains showed that TKT is clearly distinct from known subclasses of R TKs. Furthermore, the aminoterminal portion · of TKT contains a factor Vlll-like domain, which has not previously been shown to be a component of the Iigand-binding domain of RTKs. Taken together, TKT seems to represent a new subdass of RTKs.

Chromosomal location

Human/mouse somatic cell hybrids (Willecke

et

a/., 1990) were analysed to determine the chromosomal localization of

TKT.

In PCR with primers which amplify human but not mouse · genomic DNA, we demonstrated that

TKT

segregates with the human chromosome 1 and is located in the region I q 12-qter, which is the same as for

TRK

(1q23-lq24, Morris

et

al., 1991).

Comparison of TKT orthologs from human and mouse tissues

TKT

primers that exhibited a product after PCR with mouse DNA were used to generate overlapping frag- ments of the mouse

tkt cDNA.

The combined amino acid sequence of mouse TKT was aligned to its human ortholog (Figure 4). The

.

comparison of the complete human and mouse TKT-amino acid sequences showed a homo1ogy of 98%. We also found the factor VIII- like sequence upstream of the transmembrane domain of the mouse TKT-protein.

T

he locations of the five putative N-linked glycosylation sites are conserved between mouse and human proteins. Although a Iigand for the TKT-protein has not yet been identified, the high degree of homology suggests functional similarity of human and mouse TKT -proteins.

Expression of the TKT gene

in human and mouse tissues

In a Northern blot hybridization experiment we used

poly(A)+ RNA from human adult tissues to determine

the pattern of TKT-expression (Figure 5a). A 229 bp-

TKT-fragment (probe I) representing a portion of the

putative aminoterminal region (nt pos. 667 -895) was

used as probe. In a Southem blot analysis this probe

detected EcoRl-fragments of 7 and 3 kb as weil as

human mouse human mouse human mouse human mouse human mouse human mouse human mouse human mouse human mouse human mouse human mouse human mouse human mouse human mouse human mouse human mouse human mouse human mouse human mouse human mouse human mouse human mouse human mouse human mouse human mouse human mouse human mouse human mouse human mouse

TKT, A RECEPTOR TYROSINE KINASE WITH A FACTOR VIIl-UKE DOMAIN 3437

H I L I P R H L L V L F L L L P I L S S A K A Q V N P A I C

P P L L G

R Y P L G H S G G Q I P 0 E 0 I T A S S Q W S E S T A A K Y B •

G R L 0 S E E G 0 G A W C P E I P V E P 0 0 L K E F L Q I D Q •

L H T L H F I T L V G T Q G R H A G G H G I E F A P M Y K I

R

N Y S R 0 G T R W I S W R N R H G K Q V L D G N S N P Y D I

S V

F L K D L E P P I V A R F V R F I P V T 0 H S M N V C M R V

. D L

E L Y G C V W L D G L V S Y N A P A G Q Q F V L P G G S I I Y L N D S V Y D G A V G Y S M T E G L G Q L T D G V S G L D D F T Q T H E Y H V W P G Y 0 Y V G W R N E S A T N G Y I E

p

I M F E F D R I R N F T T M K V H C N N M F A K G V K I F K E V Q C Y F R S E A S E W E P N A I S F P L V L 0 D V N P S

• T • V Y

A R F V T V P L H H R M A S A I K C Q Y H F A 0 T W M M F S E I T F Q S D A A M Y N N S E A L P T S P M A P T T Y D P M

G •

L K V D D S N T R I L I G C L V A I I F I L L A I I V I I L W R Q F W Q K M L E K A S R R M L 0 0 E M T V S L S L P S 0 E S S M F N N N R S S S P S E Q G S N S T Y 0 R I F P L R P D

• !!

Y Q E P S R L I R K L P E F A P G E E E S G C S G V V K P V A Q P S G P E G V P H Y A E A 0 I V N L Q G V T G G N T Y S V

N S C

P A V T M D L L S G K D V A V E E F P R K L L T F K E K L G

A

E G Q F G E V H L C E V E G M E K F K D K 0 F A L D V S A N Q P V L V A V K M L R A D A N K N A R N D F L K E I K I M S R L K D P N I I H L L

R

D L N Q F L S R H E P A T Q I A S G M K Y L

S V C I T D 0 P L C M I T E Y M E N G

A E

P N S S S S 0 V R T V S Y T N L K F M

L S C A - A

S S L N F V H R 0 L A T R N C L V G K N Y T I K I A 0 F G M S R N L Y S G 0 Y Y R I Q G R A V L P I R W M S W E S I L L G K F T T A S 0 V W A F G V T L W E T F T F C Q E Q P Y S Q L S D E Q V I E N T G E F F R D Q G R Q T Y L P Q P A I C P D S V Y K L M L S C W R R 0 T K N R P

I L E B

S F Q E I H L L L L Q Q G D E - . . . A

Flgure 4 Comparison of the deduced amino acid sequcnces of human and mouse TKT. The nucleotide sequencc of mouse tkt cDNA was determined using RNA-PCR with different primers derived from the human cDNA sequence. The deduced amino acid sequences of human (I -855) and mouse -(1-854) TKT were compared and one gap has been introduced for optimal alignment.

Residues identical to the human sequencc are replaced by dots

Hindlll- and Pstl-fragments of 5 kb each, which indi- cated that the specifity of the probe and the stringency conditions were sufficient for the discrimination be- tween TKT and related genes. The Northern blot was standardized with a glyceraldehyde-3-phosphate de- hydrogenase (GAPDH) probe.

Using probes

I,

II, III and IV in separate hybridiza- tion experiments a major 10 kb-transcript was found at high Ievels in heart and Jung, with lower Ievels in brain,

placenta, liver, skeletal muscle, pancreas and kidney (Figure 5a and c). With the same set of probes a second signal was detected at lower intensity at 4.5 kb in the above mentioned tissues except in brain. Yarious additional weak bands were observed at 8.0, 3.6, 2.4 and 1.7 kb.

In a second Northem blot hybridization experiment we used poly(A)+ RNA from mouse tissues and probe I (Figure Sc) derived from mouse cDNA. As shown in

3438 T. KARN e.t al.

a

2

9.5- 7.5-

4.4-

2.4-

1.35-

3 4

c 5

ATG

ls

6 7 8

FB

b

9.5- 7.5- 4.4-

2.4-

1.35-

TM

2 3

KD

4 5

TGA ~

---~CM»~~~~~s~~~&~~~&~s~·---~CJ~---.

...

~--

II 111 IV

6 7 8

Figure 5 Expression of TKT in human and mouse tissues. (a) Each lane of the Northern blot (Clontech, USA) contained 2Jtg human poly(A)+ RNA. Lanes 1-8: heart, brain. placenta, lung, liver, ske.letal muscle, kidney and pancreas. (b) Eacb lane of the Northcrn blot (Ciontcch, USA) contained 2~tg mousc poly(A)+ RNA. Lanes I -8: heart, brain, spieen. lung, liver, skeletal muscle, kidney a.nd testis. PCR was used to obtain a single stranded specific probe of TKT (probe I, nt 667-895 of the human sequence and the same region of the mouse cDNA, respectively). RadiolabeHing of the antisense strand was performcd using 250 ~tCi

[et.-³²P)dCTP (6000 Ci mmoJ-1). (c) Schematic rcprcscntation of the TKT cDNA. The starl and stop-codons are indicated by arrows, characteristic features of TKT are shown as boxes. The location of various probes (I IV), used in Northern blot experiments are given as vertical lines at the bottom (S: signal peptide: F8: factor Vlll-like domain; TM: Iransmembrane domain; KD: kinase domain)

EGFR INS.R EPH PDGFR FGFR TIE TEK

AXL

ARK

RET TRK

FS

TKT

Figure 6 Structural motifs of receptor tyrosine kinase.s. Yarious Subtypes of RTKs are shown schematically with their intracellular region (KD = kinase domain, KI = kinasc inscrt) as weil as thcir structural motifs in the extracellular regions: cysteinc-rich regions (cys), immunoglobulin-like domains (Ig), EGF-like repeats (EGF) and fibronectin-type-Jil like repeats (FNIII) as weil as the cadhcrin-rclated domain (Cadh.) of RET and tbe factor Vlll-like domain (F8) ofTKT. EGFR (Ullrich er al., 1984), INS.R (Ebina ez al .. 1985; UHrich et al., 1985), EPH (Hirai et al., 1987); PDGFR (Yarden et a/., 1986: Claesson-Welsh et al., 1989), FGFR (Lee et al., 1989), TIE (Pananen et a/., 1982), TEK (Dumont et a/., 1993; Ziegler et al., 1983), AXL (O'Bryan et al., 1991), ARK (Rescigno er al., 1991), RET (Takahashi & Cooper, 1987; lwamoto et al., 1993), TRK (Martin-Zanca et af., 1989)

TKT, A RECEPTOR TYROSINE KINASE WITH A FACTOR VJJJ-LIKE DOMAIN 3439

Figure Sb mouse tki shows high expression of a

10 kb

transcript in heart and

lung.

Low Ievels

of transcripts

were detected in mouse brain and testis. Additional

smaller transcripts were detected at lower frequency of

expression.

These

observations indicate that different

TKT

mRNA species are derived from one gene and may be generated by alternative splicing or by selective use of different polyadenylation sites.

Through molecular cloning and sequencing of a 3096 nt cDNA, we have determined the primary struc-

ture of TKT. The 885 residue polypeptide corresponds

to

a

classical RTK with tripartite

structure.

While all RTKs share a common cytoplasmic kinase domain, the extracellular

Iigand

binding portion of the molecule is

composed

of various structural motifs:

Tg-like, EGF-

like, FNIII-like and cysteine-rich domains (Figure 6).

TKT

enriches this spectrum by a factor VIII-Iike

sequence.

The human factor VIII is a

trace

plasma glycoprotein, which plays a key role in normal blood

coagulation (Gitschier e1 a/., 1984;

Vehar

et al., 1984).

In

addition to the

TKT-receptor the mouse mammary

epithelial cell surface protein

(MFG-E8)

also shows considerable homology to factor VIII. This mouse pro-

tein

is involved in lactogenesis. In this process the apical surface of mammary

epithelial

cells

becomes

highly specialized and participates in the triglyceride

secretion

into milk.

The

triglyceride droplet

is

enclosed

in

the milk fat globule membrane (MFGM), which contains a high percentage of the factor VIII-related protein (Stubbs et al., 1990).

The A5-antigen which has

References

Böhme, B., Holtrich, U., Wolf, G., Luzius, H., Grzeschik, K.-H., Strebhardt, K. & Rübsamen-Waigmann, H. (1993).

Oncogene, in press.

Claesson-Welsh, L., Eriksson, A., Westermark, B. & Heidin, C.-H. (1989). Proc. Natl. Acad. Sei. USA, 86, 4917-4921.

Dumont, D.J., Gradwohl, G.J., Fong, G.-H., Auerbach, R.

& Breitman, M.L. (1993). Oncogene, 8, 1293-1301.

Ebina, Y., Ellis, L., Jarnagin, K., Edery, M., Graf, L., Clauser, E., Ou, J.-H., Masiarz, F., Kan, Y.W., Godfine, l.D., Roth, R.A. & Rutter, W.J. (1985). Cell, 40, 747-758.

Feng, D.-F. & Doolittle, R.F. (1987). J. Mol. Evol., 25, 351-360.

Gitschier, J., Wood, W.I., Goralka, T.M., Wion, K.L., Chen, E.Y., Eaton, D.H., Vehar, G.A., Capon, D . .T. & Lawn, R.M. (1984). Nature, 312, 326-330.

Hanks, S.K., Quinn, A.M. & Hunter, T. (1988). Science, 241, 42-52.

Hirai, H., Maru, Y., Hagiwara, K., Nishida, J. & Takaku, F.

(1987). Science, 238, 1717-1720.

Holtrich, U., Bräuninger, A., Strebhardt, K. & Rübsamen- Waigmann, H. (1991). Proc. Nat!. Acad. Sei. USA, 88,

10411-10415.

lwamoto, T., Taniguchi, M., Asai, N., Ohkusu, K., Naka- shima, I. & Takahashi, M. (1993). Oncogene, 8, 1087-

1091.

Jenny, R.J., Pittman, D.D., Toole, J.J., Kriz, R.W., Aldape, R.A., Hewick, R.M., Kaufman, R.J. & Mann, K.G.

(1987). Proc. Natl. Acad. Sei. USA, 84, 4846-4850.

Klein, R., Parada, L.F., Coulier, F. & Barbacid, M. (1989).

EMBO J., 8, 3701-3709.

Kozak, M. (1984). Nucleic Acids Res., 12, 857-872.

Lee, P.L., Johnson, D.E., Cousens, L.S., Fried, V.A. & Wil- liams, L.T. (1989). Science, 245, 57-60.

homologies to factor VIII as weil,

is

a

neuronal

cell surface protein of Xenopus and seems

to

be

involved

in neuronal recognition

processes

between

optic nerve

fibers and the visual center (Takagi

et al., 1991). Fur-

thermore blood vessels can be highlighted by staining endothelial cells

for

factor VIII, which

is

localized

on

their surface.

Taken

together factor VIII

. and

related molecules are found on or

within the membrane

and seem

to

play a role for cell surface

recognition and

adhesion. The

TKT-receptor

which has a factor VIII- related domain in its extracellular

region may combine

these functions with transmembrane signal transduc- tion.

Note added in proof

TKT-accession numher: X74764.

During the review process of this report a publication by Johnsön et al. (Proc. Nat!. Acad. Sei. USA, 90, 5677-5681) ' appeared describing a RTK with a structure similar to TKT which they named DDR. This protein shares 73'!/o homology with TKT and shows a factor VIII-Iike domain as weil. Thus, the new RTK-subclass contains at least two members.

Acknowlcdgements

Dr K.-H. Grzeschik is gratefully acknowledged for the gifl of chromosomal DNA from humanjmouse somatic cell hybrids.

We thank A. Schimpf for expert technical assistance. The Georg-Speyer-Haus is supported by the Bundesgesundheits- ministerium and the Hessische Ministerium für Wissenschaft und Kunst. This work was supported by a gram from the Deutsche Forschungsgemeinschaft RU 242/11-1. T.K. is sup- ported by lhe Graduiertenförderung of the Johann Wolfgang Goethe - Universität, Frankfurt am. Main.

Lindberg, R.A. & Hunter, T. (1990). Mol. Ce/1. Bio/., 10, 6316-6324.

Martin-Zanca, D., Oskam, R., Mitra, G., Copeland, T. &

Barbacid, M. (1989). Mol. Cell. Bio/., 9, 24-33.

Middlemas, D.S., Lindberg, R.A. & Hunter, T. (1991). Mol.

Cell. Bio/., 11, 143-153.

Morris, C.M., Hao, Q.L., Heisterkamp, N., Fitzgerald, P.H.

& Groffen, J. (1991). Oncogene, 6, 1093-1095.

O'Bryan, J.P., Frye, R.A., Gogswell, P.C., Neubauer, A., Kitch, 8., Prokop, C., Espinosa lll, R., Le Beau, M.M., Earp, H.S. & Liu, E.T. (1991). Mol. Cell. Bio/., 11, 5016-5031.

Partanen, J., Armstrong, E., Mäkelä, T.P., Korhonen, J., Sandberg, M., Renkonen, R., Knuulila, S., Huebner, K. &

Alitalo, K. (1992). Mol. Ce/1. Bio/., 12, 1698-1707.

Rescigno, J., Mansukhani, A. & Basilico, C. (1991). Onco- gene, 6, 1909-1913.

Shier, P. & Watt, V.M. (1989). J. Bio/. Chem., 264, 14605- 14608.

Singer, S.J. (1990). Ann. Rev. Cell. Bio/., 6, 247-296.

Stubbs, J.D., Lekutis, C., Singer, K.L., Bui, A., Yuzuki, D., Srinivasan, U. & Parry, G. (1990). Proc. Natl. Acad. Sei.

USA, 87, 8417-8421.

Takagi, S., Hirata, T., Agata, K., Mochii, M., Eguchi, G. &

Fujisawa, H. (1991). Cell, 7, 295-307.

Takahashi, M. & Cooper, G.M. (1987). Mol. Ce/1. Bio/., 7, 1378-1385.

Ullrich, A., Coussens, L., Hayflick, J.S., Dull, T.J., Gray, A., Tarn, A.W., Lee, J., Yarden, Y., Libermann, T.A., Schless- inger, J., Downward, J., Mayes, E.L.V., Whiltle, N., Waterfield, M.D. & Seeburg, P.H. (1984). Nature, 309, 418-425.

3440 T. KARN et a/.

Ullrich, A., Bell, J.R., Chen, E.Y., Herrera, R., Petruzelli, L.M., Dull, T.J., Gray, A., Coussens, L., Liao, Y.-C., Tsubokawa, M., Mason, A., Seeburg, P.H., Grunfeld, C., Rosen, O.M. & Ramachandran, J. (1985). Nature, 313, 756-761.

Ullrich, A., Gray, A., Tarn, A.W., Yang-Feng, T., Tsubo- kawa, M., Collins, C., Henzel, W., Bon, T.L., Kathuria, S., Chen, E., Jacobs, S., Francke, U., Ramachandran, J. &

Fujita-Yamaguchi, Y. (1986). EMBO J., 5, 2503-2512.

Vehar, G.A., Keyt, B., Eaton, D., Rodriguez, H., O'Brien, D.P., Rotb1at, F., Oppermann, H., Keck, R., Wood, W.l., Harkins, R.N., Tuddenham, E.G.D., Lawn, R.M. &

Capon, D.J. (1984). Nature, 312, 337-342.

von Heijne, M. (1986). Nucleic Acids Res., 14, 4683-4690.

Wilks, A.F. (1989). Proc. Natl. Acad. Sei. USA, 86, 1603-1607.

Willecke, K., Jungb1ut, S., Dah1, .. E., Hennemann, R., Heynkes, R. & Grzeschik, K.-H. (1990). Eur. J. Ce// Bio/., 53, 275-280.

Yarden, Y., Escobedo, J.A., Kuang, W.-J., Yang-Feng, T.L., Daniel, T.O., Tremble, P.M., Chen, E.Y., Ando, M.E., Harkins, R.N., Francke, U., Fried, V.A., Ullrich, A. &

Williams, L.T. (1986). Nature, 323, 226-232.

Yarden, Y. & Ullrich, A. (1988). Ann. Rev. Biochem., 57, 443-478.

Ziegler, S.F., Bird, T.A., Schneringer, J.A., Schooly, K.A. &

Baum, P.R. (1993). Oncogene, 8, 663-670.