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Cloning of cDNA and genomic DNA encoding fibroblast growth factor receptor-4 of xenopus laevis
Gene, 152 (1995) 215-219 © 1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$9.50
215
GENE 08483
Cloning of cDNA and genomic DNA encoding fibroblast growth factor receptor-4 of Xenopus laevis (Alternative splicing; FGF; development; genomic organization; immunoglobulin-like domain; receptor tyrosine kinase; signal transduction)
Chiori Shiozaki a, Kosuke Tashiro a, Misaki Asano-Miyoshi a, Kaoru Saigo b, Yasufumi Emori b and Koichiro Shiokawa a aLaboratory of Molecular Embryology, Zoological Institute, Department of Biology, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan; and bDepartment of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan Received by A. Nakazawa: 29 June 1994; Revised/Accepted:22 August/26 August 1994; Received at publishers: 6 October 1994
SUMMARY
We have isolated and characterized the cDNA and genomic DNA encoding fibroblast growth factor receptor-4 of Xenopus laevis (XFGFR-4). The gene encompassing the total coding sequence spans about 10 kb, consists of 17 exons, and has an organization very similar to those of mammalian genes encoding FGFR-1 and -2, except that the XFGFR-4 gene does not contain an alternative exon for the third immunoglobulin-like domain nor an internal poly(A)-addition site. Thus, XFGFR-4 appears not to generate multiple forms of mRNA, as are identified for the mammalian FGFR-1, -2 and -3 genes. The amino-acid sequence of XFGFR-4 shows high homology to other vertebrate FGFR-4 species, but the similarity was significantly lower than in the cases of FGFR-1 and -2. Northern blot analysis showed the XFGFR-4 mRNA to occur throughout X. laevis early embryogenesis in a profile different from those of X. laevis FGFR-1 and -2.
INTRODUCTION
In the induction and development of Xenopus laevis ( X l ) mesodermal structures, basic fibroblast growth factor (bFGF) is considered to be one of the key extracellular factors (Slack et al., 1987; Amaya et al., 1993; Cornell and Kimmeiman, 1994). Signals from bFGF are transferred into the intracellular tyrosine kinase cascade via its specific receptors, termed FGFRs (Burgess and Maciag, Correspondence to: Dr. Y. Emori, Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan. Tel.(81-3) 3812-2111, ext, 4406; Fax (81-3) 5684-2394. Abbreviations: aa, amino acid(s);bp, base pair(s); bFGF, basic FGF; FGF, fibroblast growth factor; FGFR, FGF receptor; FGFR, gene (DNA, RNA) encoding FGFR; kb, kilobase(s) or 1000 bp; nt, nucleotide(s); RT-PCR, reverse transcription-polymerase chain reaction; TM, transmembrane (domain); X., Xenopus; XFGFR-4, X. laevis FGFR-4; XFGFR-4, gene (DNA, RNA) encoding XFGFR-4; X1, X. laevis.
SSD1 0378-1119(95)00694-6
1989; Jaye et al., 1992). In mammals, four distict types of FGFR genes are known, and all of the encoded proteins have been shown to bind bFGF with diverse affinities (Jaye et al., 1992; Dell and Williams, 1992; Ron et al., 1993; Zimmer et al., 1993). Consequently, to understand the precise cellular processes mediated by bFGF in X! embryogenesis, each of the four FGFRs need to be analyzed. Until now, however, only two distinct FGFR cDNAs, considered to be the counterparts to mammalian FGFR-1 and -2, have been identified in Xl (Friesel and Dawid, 1991; Friesel and Brown, 1992). Several lines of study on these two Xl FGFRs (XFGFRs) have shown that XFGFR-I and -2 are important in the signalling pathways of Xl mesoderm induction, as well as in later developmental processes (Amaya et al., 1991; Friesel and Dawid, 1991; Friesel and Brown, 1992). However, unidentified receptors, tentatively termed XFGFR-3 and -4, may also be involved in the above processes as in the case of mammalian species.
216 Especially, presumptive XFGFR-4 may have unique features with regard to both structure and function in the X l system, because mammalian FGFR-4 has been shown to exhibit unique ligand-binding and signalling activities (Partanen et al., 1991; Vainikka et al., 1992: Ron et al., 1993; Wang et al., 1994~. As a step toward the total understanding of FGFmediated phenomena during X l development, we undertook the isolation and characterization of yet unidentified XFGFRs. We here describe the molecular cloning and characterization of the cDNA and genomic DNA for a XFGFR-4, which shows unique features in terms of both structure and expression.
E X P E R I M E N T A L A N D DISCUSSION
(a) Cloning of XI FGFR-4 We adopted RT-PCR to obtain cDNA clones encoding unidentified XFGFRs using two oligodeoxyribonucleotide primers corresponding to kinase subdomains I and VIII (Emori et al., 1992). Two distinct types of RT-PCR clones were obtained, and one of them was identified as previously reported X F G F R - 2 (Friesel and Brown, 1992) from its nt and deduced aa sequences. The other clone has a novel sequence and encodes a polypeptide that is highly homologous to urodele FGFR-4 (Shi et al., 1992) and Medaka FGFR-4 (Emori et al., 1992) including the kinase insert (data not shown), and is thus considered to be a clone encoding a X l homologue of FGFR-4. Next, we screened genomic DNA and cDNA libraries using the above RT-PCR clone for the putative XFGFR-4 as a probe. A genomic DNA clone, termed ~.gXFR4-1, containing a 13-kb insert and a cDNA clone, termed LcXFR4-1, containing a 3.2-kb insert, were obtained. Comparison of the restriction maps of these two clones, assignment of regions hybridizing with the RT-PCR clone, and mutual hybridization analysis, revealed the direction of transcription and provided a rough assignment of the exons. The results also suggested that both clones might contain the total coding sequence. We then determined the nt sequences of the relevant regions of ~gXFR4-1. Alignment of the cDNA sequence
with the genomic nt sequence showed the positions of intron breakpoints. From this, the overall organization and nt sequence covering the whole coding region of X F G F R - 4 were determined as described below in section b (Fig. 1). The start codon was postulated to be at nt 1 3 I Fig. 1B), because the deduced aa sequence following the ATG codon had a sequence characteristic of a signal sequence (yon Heijne, 1983) and was homologous to known FGFR-4 sequences as described below in section e.
(b) Genomic organization of XFGFR-4 The X F G F R - 4 gene spans about 10 kb and consists of at least 17 exons (Fig. 1). Most of the introns are smaller than 500 bp, and only the first and fifth introns are longer than 1 kb (Fig. 1A). The exon-intron boundary sequences match the GT-AG rule (Breathnach and Chambon, 1981). The positions of intron insertion points of the XFGFR-4 gene and the human FGFR-2 gene (Johnson et al., 1991) are identical at the nt level, indicating that these vertebrate FGFR genes are derived from a common ancestral gene and that the organization has been maintained during the evolutionary process. However, the genes encoding mammalian FGFR-1, -2 and -3 have been shown to contain an alternative exon encoding the C-terminal half of the third immunoglobulin-like (Ig-3) domain and a poly(A) addition site in an intron splitting the Ig-3 domain (Johnson et al., 1991; Dell and Williams, 1992; Jaye et al., 1992; Werner et al., 1992; Zimmer et al., 1993; Chellaiah et al., 1994); these are not detected in XFGFR-4. The genomic organization of X l FGFR-4 coincided with the organization of the human FGFR-4 gene (Vainikka et al., 1992). In conclusion, there appears to be no alternative splicing in the X F G F R - 4 gene, unlike that in other FGFR genes.
(c) Amino-acid sequence of XFGFR-4 We then compared the deduced aa sequence of XFGFR-4 with those of human and urodele FGFR-4 (Partanen et al., 1991; Shi et al., 1992). As shown in Fig. 2, a high degree of homology was observed in the intracellu-
Fig. 1. Genomic structure of XFGFR-4 and the nt sequence of its eDNA. (A) A restriction m a p of the genomic D N A fragment encoding X F G F R - 4 (LgXFR4-1 insert) is shown for several restriction enzymes. Positions and lengths of exons deduced by comparison with the cDNA sequence are shown by numbered boxes linked by V-shaped introns. Positions of the start and stop codons are shown. Other distinct domains (Ig-1 to Ig-3 and the tyrosine kinase domainl are noted below the exons. Two TM sequences (signal sequence, SG; TM domain, T M ) and an acidic domain (AD) are shown by filled boxes in each exon and indicated below the exons. (B) The nt sequence of XFGFR-4 cDNA and the deduced aa sequence of the protein are numbered from the first letter of the start codon and the start Met, respectively. The signal sequence is underlined and the transmembrane domain is boxed. Positions of introns are shown by arrows. The 5' and 3' ends of the RT-PCR clone are indicated by open and closed triangles, respectively. The nt sequence data has been submitted to E M B L / G e n B a n k / D D B J data libraries under the accession No. D31761. Methods: An Xl genomic library and a tailbud cDNA library constructed by standard procedures (Sambrook et al., 1989) were each screened with the RT-PCR clone. The complete nt sequence of the cDNA clone and relevant regions of the genomic clone were determined and compared (Sambrook et al., 1989).
217
A
1 kb
EcoRI
~mHI I
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I AT(I ~1
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-i 120 40
~C~G~GG~CCTGAGGT~G~CATCTACTTCTAGACCCCGG~TGC~TGCGA~TGTTTTGTGACACC~CC~GC~CAGCATC~C~GTACCGCG~CA~A~GTTTA N W K E P E V E E H L L L D P G N A L R L F C D T N Q S N S I N W Y R E Q
240 80
D
R
L
TTGCCAGGAGGG~GATT~GCA~GTGGGGA~TGTGCTAG~GTCTCAGATGTGACATA~GAC~AGGACTCTACATATG~GTGGTCAGAGG~CAGGCAAAA~TTAG~T~ L P G G K I R M V G T V L E V S D V T Y E D S G L Y I C V V R G T G K I L R E F
360 120
T~CATATCCGTTGTTGACTCGTT~CATCTGGAGATG~G~GA~ATGAGGATGGCCGTAGGGAGGACAC~cCG~TGATATT~TGAGGAGCCAGTTTATTTTTTCC~GCA~CATAC S I S V V D S L A S G D E E D D E D G R R E D T T A D I N E E P V Y F F Q A P y
480 160
TGGACACAG~C~ACCGCATGGAC~GAAACTTCATGCTGTGCCAGCT~T~CACAGTC~GTTCCGCTGTCCAGCTGGTGGGAGTCCCCTTC~CACTATTCGAT~CTTAAA/%A~ W T Q P H R M D K K L H A V P A G N T V K F R C P A G G S P L P T I R W L K N G
600 200
A~G~TTTCGAGGAG~cA~AG~TTGG~GGATCCGGCTTCGACACC~CAC~GAGTCTGGT~TGGAGAGTGTGGTTCCATCAGACCGTG~CTACACCTG~GTAG~C R E F R G E H R I G G I R L R H Q H W S L V M E S V V P S D R G N Y T C V V E N
720 240
AGAGTTGGCAG~ACATATA~CTACTTTCTGGATG~T~AGAGGT~TTCTCACCGTCCTATCCTAcAG~TG~CTTCCAGC~AC~CA~A~G~GT~GTAGCGATG~ R V G S L T Y T Y F L D V L E R S S H R P I L Q A G L P A N T T A R V G S D V E
840 280
TTCTACTGC~GTATACAGCGATGCTCAGCCACATATCC~TGGCTT~CACATCG~GTG~TGG~GCCGATTTGGTCCTGATGATTTTCCATATGTACAGGT~CTGCA F Y C K V Y S D A Q P H I Q W L K H I E V N G S R F G P D D F P Y V Q V L K T A
960 320
GATA~CACTTCGGAGGTAGAGG~CT~ACCTAC~TATCACTATGGAGGATGCAGGGG~TACACATGTCTGGCGGGC~TTCTATTGGTCTTTCTCATCAGTC~CTTG~TG D I N T S E V E V L H L R N I T M E D A G E Y T C L A G N S I G L S H Q S A W L AcTG~cTTTc~cG~GATTTCCTTGAGc~GcTGAGcCA~AGAGTc~GATATATGGAc~T~TATAcAcTTcTGGATTccTGGcTGTAGccATG~cATcG~ATAG~G~ T V L S N E D F L E Q A E P A E S R Y M D ~ I I I Y T S G F ~ A V A M A I V I V V I 4 0
1080 360 1200 0
C~TGCC~AT~AGACACC~ACAGC~GCAGACTCTGC~CCACCAGCTGTTCAT~CTGGCC~GTTCCCCCTCATACGGCAGTTC~TT~GAGTCCAGCTCATCTGGG~GTCC ~ - - ~ R M Q T P H S K Q T L Q P P A V H K L A K F P L I R Q F S L E S S S S G K S AGTGCTCCAC~AT~GCAT~C~GTCTCTCCTC~GCTG~CCCCATGCTGCC~GTGTCAT~A~TTGAGTTACCACTGGACGCT~GGAG~CCCTAGAGACAGGCTTG~ S A P L I R I T R L S S S C A P M L P G V M E V E L P L D A K W E F P R D R L
1320 440
V
1440 480
v CTGGG~GCCGCT~GAGA~T~TTTGGCC~GTGGT~GAGCAGA~GATA~G~TTGAG~GAC~GGCCTGAG~CCAGTTACCGTTGCAGTT~GA~C~AAAGAT~T L G K P L G E G C F G Q V V R A E G Y G I E K D R P E K P V T V A V K M L K D
N
1560 520
GGCACTGAC~GGACTTATCAGA~TGATCTCTG~AGTTGATG~TCATTGGAAACACAAAAATAT~TAAACTTGCTC~CGTCAGCACTC~GA~GGCCATTATT~TT G T D K D L S D L I S E M E L M K V I G K H K N I I N L L G V S T Q E G P L F V
1680 560
ATAGTTG~TA~CTTCT~GGGG~TCTGCGTGAGTT~TACGTGCCAGGCGCCCTCCCACACCGG~GATGCCTTTGATATCACC~GGTTCCA~TG~CTTT~TCCTTT~AC I V E Y A S K G N L R E F L R A R R P P T P E D A F D I T K V P D E L L S F K D
1800 600
CTAG~TCTTGTG~TTATCAGGTTG~C~GTGG~ATGGAGTAC~TTG~TCT~GAGGTGCATACACCGGGATCTGGCAGCCAG~TGTTCTTGTGGCAG~GAT~TCA~GA~ L V S C A Y Q V A R G M E Y L E S K R C I H R D L A A R N V L V A E D N V M K I
1920 640
GCAGATTTTGGCTTGGCACGAGG~TTCACGATATTGACTATTAC~GAAAACTAGT~TGGTCGACTGCC~TT~GA~GCTCCAG~GCTTTATTT~CAGAGTTTACACCCAC A D F G L A R G V H D I D Y Y K K T S N G B L P V K W M A P E A L F D R V Y T H
2040 680
C~GTGACATTT~TCATT~GAGTGTTGACATG~AGATCTTTACTCT~GA~G~TCCATATCC~G~TTCCAGTAG~G~CTATTT~CTCTTAC~G~G~CATCG~ Q S D I W S F G V L T W E I F T L G G S P Y P G I P V E E L F K L L R E G H R
2160 720
M
GAC~CCATCC~CTGTACTCATG~TTGTACATGTTGATGAGGGAGTGCTGGCATGCAGTTCCATCTCAGAGACC~CATTT~CAGCTGGTTG~C~CTGGATAGGATCCTCACA D K P S N C T H E L Y M L M R E C W H A V P S Q R P T F K Q L V E Q L D R I L T
2280 760
GCTGTTTCTG~GAGTATCTGGACTTATCTATGCCATTTG~CAGTATTCTCCCTCCTGTG~GATTCTTC~GCACATGCTCT~ATCCGATGAC~TG~TTTGCCCACGA~CAGTG A V S E E Y L D L S M P F E Q Y S P S C E D S S S T C S S S D D S V F A H D P V
2400 800
CCATCCTCTCCCTGTGTCTTT~TTACCAC~TGTTCACACTCACCT~GGACTTGAAAAC~GGAGACGGTTTGCC~TATTGGGAGATCT~AC~TGTAAACAGAGTGTTATTTC P S S P C V F N Y H N V H T H L G T *
2520 818
CTT~CT~TA~CGTATTTATATAAAAAAAAAGCCATATGCGGTATTATG~CTAT~GG~TCACTGTGC~TTTTTCTGCTGTTC~GCAG~TAGAAAAATCCT~CAGTT ~TCGCTGGTACAGCGGATGCATAGCAcTG~GCC~GCCACCCCGGTCCGTGACGAGATGTCGCCTTGCTCCAGATTCC~GGTCTAG
2640 2734
218
XFGFR4 PFR4 HFGFR4
XFFGR4 PFR4 HFGFR4
1:
MSGSVRRSYTAMONFARLLL-GVLLVATLSSCRPALSED-EANWKE--PEVEEHLLLDPpNAIRLFC DTNQSNS INWYREQDRLLPGGKIRMVGTVLEVSDVTYEDSGLYICVVRGT 1 :M G V Q K D . R D I R W N R T T R P L A . . . C. L . A F S A . - . . A R T . P. G R K . . L A . L V S . E . . . F , . . . 1 . . . . . . . . . . . . T T I V . . . T. S T . . Q H . . R . . L T D . . . . I A . . . . . . . . . . L... P.. I: MRLLL/tLLGV..SVP-GPPV..LEASEEV.LEPCLAPSL-EQQ.QE.TVA~.QPV..C.GRAE-RGGH..K GS A.A RV GWRGR. IASFLP..A.R.L.LA..S[
120: .H...N.T ......... 107 :MIV. QNLTLITG...T.,
[
~ ¢ ~ . . . . . S A G , M G . V. P. S T S Y R . . F . S . . Q ...... Y'"T ........ SA.N.T.G .......... G ........................... T . . . . . . [,SH. -. - P S N - R H - S - . P - - Q . . . . . . H . Q , .E . . . . . . . . [ . . . . . . . . . A . N . T . . . . . . . D . Q A . H . . N . . . . . . . . . . . . . . . . . . . . . . . .
/ XFGFR4 PFR4
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239: ..... L.. ,~F.. IS. S.L ....... 220: .T...L...].... IR.N.L .......
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3 5 9 : . . .Y . . . . . . . . 340 :... Y ........
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4 7 9 : . . . . . IE . . . . . . . . . . . . . . . . . . . . 460: ..... .....................
XFGFR4
P ............... P ...............
FYCKVYS
DAQPH
I QWLKH
I E VNGSRFGPDDFPYVQVL
K TAD I NTSEVEVLHLRNITMEDAGE
ML[...Q.F ....................... Y...GV.F .......... V ....... | LL ................. VI...S..AVG .............
S ...... Y.H.VSF S ...... Y...VSA
YTC LAGNSI
.............. ..............
TM D FLE QAEPAESRYHIII
I YTSGFLAVAMAIVIVVL~RMQTPHSKQTLQP
PAVHKLAKFPL
I RQFSLESS
S SGKSSAP
L I R ITRLSSSCAPMLPGVMEVE
L PL DAK
PE...AKE..GP.T..T ........ S..LL..A . . . . . . I' ' .L. P T . T H . E . A T .... SR...M .............. T S , V . V . . . . . . . T . . . . . . L . F D . . . . S. F E . . p T W T . A A P . A . . T [I" • L. A . . S . . L. V L L L L A G . ~. G. A L . G R H P R P . A T . Q . . S R . . . A . . . . . . . G . . . . . . S S . V. G V . . . . . G P A L . A . L V S L D . . . . P L m
Tyrosine kinase ~DRLVLGKPLGE
GC FGQWRAEGYG
I EK DRP EKpVTVAVKMLKDNGT A...N..Q.D.AI... I.IV..K AF. MDPA. .DQAS ..........
DKDLS DL I SEMELMKV
IGKHKNI
K1 I NLLGVSTQEGPLFVIVE
.... E ............ LM ............ A S . . . . A. .V . . . . V . . L. . R . . . . . . . . . .
yASKGNLRE
FLRARRP
PTPE DAFDI TKV
C..D...YM .................... C . . . . . . Y . . . . C. A . . . . . . . . . . . . . .
S. D Y T . . M . . . G. D L S P . G P R S
592:
?DELLSFKDLVSCAYQVARGMEYLESKRCIHRDLA~NVLVAEDNVMKIADFGLARGVHDIDYYKKT~NGRL~VKWMAPEALFDRVYTHQSDIWSFGVLTWEIFTLGGSPYPGIPVEELF
PFR4
599
.E.Q. . .Q ..... S .......
A ...................
TGE ................................................
V ...........................
HFGFR4
580:
;EGP...PV
Q .... RK .............
T .................
V .... I.L ....................
XFGFR4
712
~LLREGHRHDKPSNCTHELYMLMRECWHAVPSQRPTFKQLVEQL
PFR4 HFGFR4
719 700
............................. ......... R.PH.PP,..G ........
............
H ................................
. A ............ A ............
ILTAVSEEYLDLSMPFEQYSPSCEDSSSTCSSSDDSVFAHDPVPSSPCVFNYHNVHTHLGT T..[...AT.A ............... A...TT.......... T.E--.DV.SL.TH.TTTSMV, A..[KV.I .......... RLT.GP .... GG.A ....... - .... S...L.LGSSS.PFG---SGVQ. !
818 . 822 802
Fig. 2. Comparison of the deduced aa sequence of XFGFR-4 with those of urodele FGFR-4 (PFR4: Shi et al., 1992) and human FGFR-4 (H FGFR4; Partanen et al., 1991 ). Residues identical to XFGFR-4 are represented by dots. Signal peptides are underlined. Three immunoglobulin-like domains (Ig-1, lg-2 and Ig-3), an acidic domain (AD) and a TM domain (TM) are boxed and indicated. The intracellular tyrosine kinase domain (tyrosine kinase) is boxed, and the kinase insert (KI) is overlined. A possible autophosphorylation site in the C-terminal region, which may be a binding site for phosphoinositide-specific phospholipase-C7 (Mohammad et al., 1991 ), is indicated by a closed circle above the sequence. Conserved Cys residues are marked by # above the sequence.
lar kinase domain and the extracellular Ig-2 and Ig-3 domains that have been shown to be important for ligand-binding activity and specificity (Johnson et al., 1991; Dell and Williams, 1992; Jaye et al., 1992; Werner et al., 1992; Zimmer et al., 1993; Chellaiah et al., 1994). This suggests that X l and mammalian FGFR-4s have essentially the same characteristics in terms of intracellular signalling and ligand binding. However, significant differences can be observed in the N-terminal 100 aa, including half of the Ig-1 domain, the acidic domain including both borders, borders of the transmembrane domain, and the C-terminal 20 aa, although the significance of these differences is not yet known. The overall aa sequence identity between XFGFR-4 and human FGFR-4 is 68%, which is significantly lower than between Xl and mammalian F G F R - l s and -2s (about 80%; Friesel and Dawid, 1991; Friesel and Brown, 1992). On the other hand, homology among the three XFGFRs was relatively low as follows: XFGFR-1 (XFGFR1A) vs. XFGFR2, 64%; XFGFR-1 vs. XFGFR-4, 62%: XFGFR-2 vs. XFGFR-4, 63%.
(d) Temporal changes of XFGFR-4 expression during X! development We finally examined the temporal profile of the amount o f X F G F R - 4 mRNA by Northern blot analysis. As shown
in Fig. 3, a single mRNA species of about 4 kb is observed throughout the developmental stages. However, the amounts vary signifiantly; XFGFR-4 mRNA is detected at the beginning of the cleavage stage, suggesting that the mRNA is inherited as a maternal mRNA. This maternal inheritance was confirmed by using the oocyte mRNA (data not shown). Subsequently, the mRNA decreases gradually to the late blastula stage, and then increases probably by zygotic expression reaching a maximum at the blastula and the late gastrula stages. The mRNA is also observed during later stages in lesser amounts. This profile is somewhat similar to that of urodele FGFR-4 mRNA, but it is not detected in early cleavage stage (Shi et al., 1992). It is also noteworthy that the expression profile of XFGFR-4 mRNA differs from those of X F G F R - I and -2 (Friesel and Dawid, 1991; Friesel and Brown, 1992), suggesting that each X F G F R gene is under different developmental control and has distinct roles.
(c) Conclusions (1) The nt sequences of the cDNA and genomic DNA of XFGFR-4 were determined. (2) The gene organization of X F G F R - 4 is similar in overall structure to those of mammalian FGFR-1 and -2, although XFGFR-4 lacks an alternative exon encoding
219
1 2 3 4 5 6 7 8 910
(a)
(b) Fig. 3. Northern blot analysis ofXFGFR-4 mRNA in early embryogenesis of X1. Northern blot analysis was carried out using a cDNA insert of ~.cXFR4-1 as a probe (a), and the same blot was subsequently hybridized with histone H4 DNA (h) (Sambrook et al., 1989). Total RNA (10 ~tg) prepared from embryos at each stage was electrophoresed in 2.2 M HCOH-I% agarose. Embryos were used at two-cell stage (lane 1 ), 16-cell stage (lane 2), early blastula stage (lane 3), late blastula stage (lane 4), early gastrula stage (lane 5), late gastrula stage (lane 6), early neurula stage (lane 7), late neurula stage (lane 8), early tailbud stage (lane 9) and late tailbud stage (lane 10).
the Ig3 domain and a poly(A)-addition site as found in the genes encoding FGFR-1 and -2. (3) The deduced aa sequence of XFGFR-4 is homologous to those of mammalian FGFR-4, but the homology (68% identity) is lower than those (about 80% identity) between X l and mammals FGFR-1 and -2. (4) The X F G F R - 4 mRNA occurs throughout X l development and is regulated in a manner different from X F G F R - I and -2 mRNAs.
REFERENCES Amaya, E., Musci, TJ. and Kirschner, M.W.: Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus laevis. Cell 66 (1991) 257-270. Amaya, E., Stein, P., Musci, T. and Kirschner, M.W.: FGF signalling in the early specification of mesoderm in Xenopus laevis. Development 118 (1993) 477-487. Avivi, A., Yayon, A. and Givol, D.: A novel form of FGF receptor-3 using an alternative exon in the immunoglobulin domain III. FEBS Lett. 330(1993) 249-252. Breathnach, R. and Chambon, P.: Organization and expression of eucaryotic split genes coding for proteins. Annu. Rev. Biochem. 50 (1981) 349-383. Burgess, W.H. and Maciag, T.: The heparin-binding (fibroblast) growth factor family of protein. Annu. Rev. Biochem. 58 (1989) 575-606. Chellaiah, A.T., McEwen, D.G., Werner, S., Xu, J. and Ornitz, D.M.: Fibroblast growth factor receptor (FGFR) 3; alternative splicing in immunoglobulin-like domain III creates a receptor highly specific for acidic FGF/FGF-1. J. BioL Chem. 269 (1994) 11620-11627.
Cornell, R.A. and Kimmeiman, D.: Activin-mediated mesoderm induction requires FGF. Development 120 (1994) 453-462. Dell, K.R. and Williams, L.T.: A novel form of fibroblast growth factor receptor 2; alternative splicing of the third immunoglobulin-like domain confers ligand binding specificity. J. Biol. Chem. 267 (1992) 21225-21229. Emori, Y., Yasuoka, A. and Saigo, K.: Identification of four FGF receptor genes in Medaka fish (Oryzias latipes). FEBS Lett. 314 (1992) 176-178. Friesel, R. and Brown, S.A.: Spatially restricted expression of fibroblast growth factor receptor-2 during Xenopus laevis development. Development 116 (1992) 1051-1058. Friesel, R. and Dawid, I.B.: cDNA cloning and developmental expression of fibroblast growth factor receptors from Xenopus laevis. Mol. Cell. Biol. 11 (1991) 2481-2488. Jaye, M., Schlessinger, J. and Dionne, C.A.: Fibroblast growth factor receptor tyrosine kinases; molecular analysis and signal transduction. Biochem. Biophys. Acta 1135 (1992) 185-199. Johnson, D.E., Lu, J., Chen, H., Werner, S. and Williams, L.T.: The human fibroblast growth factor receptor genes; a common structural arrangement underlies the mechanisms for generating receptor forms that differ in their third immunoglobulin domain. Mol. Cell. Biol. 11 (1991) 4267-4634. Mohammadi, M., Honnegger, A.M., Rotin, D., Fischer, R., Bellot, F., Li, W., Dionne, C.A., Jaye, M., Rubinstein, M. and Schlessinger, J.: A tyrosine-phosphorylated C-terminal peptide of the fibroblast growth factor receptor (fig) is a binding site for the SH2 domain of phospholipase C-71. Mol. Cell. Biol. 11 (1991) 5068-5078. Partanen, J., Makada, T.P., Eerola, E., Korhonen, J., Hirvonen, H., Claesson-Welsh, L. and Alitalo, K.: FGFR-4, a novel acidic fibroblast factor receptor with a distinct expression. EMBO J. 10 (1991) 1347 1354. Ron, D., Reich, R., Chedid, M., Lengel, C., Cohen, O.E., Chan, A.M.-L., Neufeld, G., Miki, T. and Tronick, S.R.: Fibroblast growth factor receptor 4 is a high affinity receptor for the acidic and basic fibroblast growth factor but not for keratinocyte growth factor. J. Biol. Chem. 268 (1993) 5388-5394. Sambrook, J., Fritsch, E.F. and Maniatis, T.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Shi, D.-L., Feige, J.-J., Riou, J.-F., DeSimone, D.W. and Boucaut, J.-C.: Differential expression and regulation of two distinct fibroblast growth factor receptors during early development of the urodele amphibian Pleurodeles waltl. Development 116 (1992) 261-273. Slack, J.M.W., Darlington, B.G., Heath, J.K. and Godsave, S.F.: Mesoderm induction in early Xenopus laevis embryos by heparinbinding growth factors. Nature 326 (1987) 197-200. Vainikka, S., Partanen, J., Bellosta, P., Coulier, F., Basilico, C., Jaye, M. and Alitalo, K.: Fibroblast growth factor receptor-4 shows novel features in genomic structure, ligand binding and signal transduction. EMBO J. 11 (1992) 4273-4280. von Heijne, G.: Patterns of amino acids near signal sequence cleavage sites. Eur. J. Biochem. 133 (1983) 17-21. Wang, J.-K., Gao, G. and Goldfarb, M.: Fibroblast growth factor receptors have different signalling and mitogenic potentials. Mol. Cell. Biol. 14 (1994) 181-188. Werner, S., Duan, D.-H.R., de Vries, C., Peters, K.G., Johnson, D.E. and Williams, LT.: Differential splicing in the extracellular region of fibroblast growth factor receptor 1 generates receptor variants with different ligand-binding specificities. Mol. Cell. Biol. 12 (1992) 82-88. Zimmer, Y., Givol, D. and Yayon, A.: Multiple structural elements determine ligand binding of fibroblast growth factor receptors; evidence that both Ig domain 2 and 3 define receptor specificity. J. Biol. Chem. 268 (1993) 7899-7903.
215
GENE 08483
Cloning of cDNA and genomic DNA encoding fibroblast growth factor receptor-4 of Xenopus laevis (Alternative splicing; FGF; development; genomic organization; immunoglobulin-like domain; receptor tyrosine kinase; signal transduction)
Chiori Shiozaki a, Kosuke Tashiro a, Misaki Asano-Miyoshi a, Kaoru Saigo b, Yasufumi Emori b and Koichiro Shiokawa a aLaboratory of Molecular Embryology, Zoological Institute, Department of Biology, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan; and bDepartment of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan Received by A. Nakazawa: 29 June 1994; Revised/Accepted:22 August/26 August 1994; Received at publishers: 6 October 1994
SUMMARY
We have isolated and characterized the cDNA and genomic DNA encoding fibroblast growth factor receptor-4 of Xenopus laevis (XFGFR-4). The gene encompassing the total coding sequence spans about 10 kb, consists of 17 exons, and has an organization very similar to those of mammalian genes encoding FGFR-1 and -2, except that the XFGFR-4 gene does not contain an alternative exon for the third immunoglobulin-like domain nor an internal poly(A)-addition site. Thus, XFGFR-4 appears not to generate multiple forms of mRNA, as are identified for the mammalian FGFR-1, -2 and -3 genes. The amino-acid sequence of XFGFR-4 shows high homology to other vertebrate FGFR-4 species, but the similarity was significantly lower than in the cases of FGFR-1 and -2. Northern blot analysis showed the XFGFR-4 mRNA to occur throughout X. laevis early embryogenesis in a profile different from those of X. laevis FGFR-1 and -2.
INTRODUCTION
In the induction and development of Xenopus laevis ( X l ) mesodermal structures, basic fibroblast growth factor (bFGF) is considered to be one of the key extracellular factors (Slack et al., 1987; Amaya et al., 1993; Cornell and Kimmeiman, 1994). Signals from bFGF are transferred into the intracellular tyrosine kinase cascade via its specific receptors, termed FGFRs (Burgess and Maciag, Correspondence to: Dr. Y. Emori, Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan. Tel.(81-3) 3812-2111, ext, 4406; Fax (81-3) 5684-2394. Abbreviations: aa, amino acid(s);bp, base pair(s); bFGF, basic FGF; FGF, fibroblast growth factor; FGFR, FGF receptor; FGFR, gene (DNA, RNA) encoding FGFR; kb, kilobase(s) or 1000 bp; nt, nucleotide(s); RT-PCR, reverse transcription-polymerase chain reaction; TM, transmembrane (domain); X., Xenopus; XFGFR-4, X. laevis FGFR-4; XFGFR-4, gene (DNA, RNA) encoding XFGFR-4; X1, X. laevis.
SSD1 0378-1119(95)00694-6
1989; Jaye et al., 1992). In mammals, four distict types of FGFR genes are known, and all of the encoded proteins have been shown to bind bFGF with diverse affinities (Jaye et al., 1992; Dell and Williams, 1992; Ron et al., 1993; Zimmer et al., 1993). Consequently, to understand the precise cellular processes mediated by bFGF in X! embryogenesis, each of the four FGFRs need to be analyzed. Until now, however, only two distinct FGFR cDNAs, considered to be the counterparts to mammalian FGFR-1 and -2, have been identified in Xl (Friesel and Dawid, 1991; Friesel and Brown, 1992). Several lines of study on these two Xl FGFRs (XFGFRs) have shown that XFGFR-I and -2 are important in the signalling pathways of Xl mesoderm induction, as well as in later developmental processes (Amaya et al., 1991; Friesel and Dawid, 1991; Friesel and Brown, 1992). However, unidentified receptors, tentatively termed XFGFR-3 and -4, may also be involved in the above processes as in the case of mammalian species.
216 Especially, presumptive XFGFR-4 may have unique features with regard to both structure and function in the X l system, because mammalian FGFR-4 has been shown to exhibit unique ligand-binding and signalling activities (Partanen et al., 1991; Vainikka et al., 1992: Ron et al., 1993; Wang et al., 1994~. As a step toward the total understanding of FGFmediated phenomena during X l development, we undertook the isolation and characterization of yet unidentified XFGFRs. We here describe the molecular cloning and characterization of the cDNA and genomic DNA for a XFGFR-4, which shows unique features in terms of both structure and expression.
E X P E R I M E N T A L A N D DISCUSSION
(a) Cloning of XI FGFR-4 We adopted RT-PCR to obtain cDNA clones encoding unidentified XFGFRs using two oligodeoxyribonucleotide primers corresponding to kinase subdomains I and VIII (Emori et al., 1992). Two distinct types of RT-PCR clones were obtained, and one of them was identified as previously reported X F G F R - 2 (Friesel and Brown, 1992) from its nt and deduced aa sequences. The other clone has a novel sequence and encodes a polypeptide that is highly homologous to urodele FGFR-4 (Shi et al., 1992) and Medaka FGFR-4 (Emori et al., 1992) including the kinase insert (data not shown), and is thus considered to be a clone encoding a X l homologue of FGFR-4. Next, we screened genomic DNA and cDNA libraries using the above RT-PCR clone for the putative XFGFR-4 as a probe. A genomic DNA clone, termed ~.gXFR4-1, containing a 13-kb insert and a cDNA clone, termed LcXFR4-1, containing a 3.2-kb insert, were obtained. Comparison of the restriction maps of these two clones, assignment of regions hybridizing with the RT-PCR clone, and mutual hybridization analysis, revealed the direction of transcription and provided a rough assignment of the exons. The results also suggested that both clones might contain the total coding sequence. We then determined the nt sequences of the relevant regions of ~gXFR4-1. Alignment of the cDNA sequence
with the genomic nt sequence showed the positions of intron breakpoints. From this, the overall organization and nt sequence covering the whole coding region of X F G F R - 4 were determined as described below in section b (Fig. 1). The start codon was postulated to be at nt 1 3 I Fig. 1B), because the deduced aa sequence following the ATG codon had a sequence characteristic of a signal sequence (yon Heijne, 1983) and was homologous to known FGFR-4 sequences as described below in section e.
(b) Genomic organization of XFGFR-4 The X F G F R - 4 gene spans about 10 kb and consists of at least 17 exons (Fig. 1). Most of the introns are smaller than 500 bp, and only the first and fifth introns are longer than 1 kb (Fig. 1A). The exon-intron boundary sequences match the GT-AG rule (Breathnach and Chambon, 1981). The positions of intron insertion points of the XFGFR-4 gene and the human FGFR-2 gene (Johnson et al., 1991) are identical at the nt level, indicating that these vertebrate FGFR genes are derived from a common ancestral gene and that the organization has been maintained during the evolutionary process. However, the genes encoding mammalian FGFR-1, -2 and -3 have been shown to contain an alternative exon encoding the C-terminal half of the third immunoglobulin-like (Ig-3) domain and a poly(A) addition site in an intron splitting the Ig-3 domain (Johnson et al., 1991; Dell and Williams, 1992; Jaye et al., 1992; Werner et al., 1992; Zimmer et al., 1993; Chellaiah et al., 1994); these are not detected in XFGFR-4. The genomic organization of X l FGFR-4 coincided with the organization of the human FGFR-4 gene (Vainikka et al., 1992). In conclusion, there appears to be no alternative splicing in the X F G F R - 4 gene, unlike that in other FGFR genes.
(c) Amino-acid sequence of XFGFR-4 We then compared the deduced aa sequence of XFGFR-4 with those of human and urodele FGFR-4 (Partanen et al., 1991; Shi et al., 1992). As shown in Fig. 2, a high degree of homology was observed in the intracellu-
Fig. 1. Genomic structure of XFGFR-4 and the nt sequence of its eDNA. (A) A restriction m a p of the genomic D N A fragment encoding X F G F R - 4 (LgXFR4-1 insert) is shown for several restriction enzymes. Positions and lengths of exons deduced by comparison with the cDNA sequence are shown by numbered boxes linked by V-shaped introns. Positions of the start and stop codons are shown. Other distinct domains (Ig-1 to Ig-3 and the tyrosine kinase domainl are noted below the exons. Two TM sequences (signal sequence, SG; TM domain, T M ) and an acidic domain (AD) are shown by filled boxes in each exon and indicated below the exons. (B) The nt sequence of XFGFR-4 cDNA and the deduced aa sequence of the protein are numbered from the first letter of the start codon and the start Met, respectively. The signal sequence is underlined and the transmembrane domain is boxed. Positions of introns are shown by arrows. The 5' and 3' ends of the RT-PCR clone are indicated by open and closed triangles, respectively. The nt sequence data has been submitted to E M B L / G e n B a n k / D D B J data libraries under the accession No. D31761. Methods: An Xl genomic library and a tailbud cDNA library constructed by standard procedures (Sambrook et al., 1989) were each screened with the RT-PCR clone. The complete nt sequence of the cDNA clone and relevant regions of the genomic clone were determined and compared (Sambrook et al., 1989).
217
A
1 kb
EcoRI
~mHI I
XbaI
I AT(I ~1
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17
[ T ~ o s i n e kinase
B GTTTGTCCATACGCGCCAGCC ATGTCT~ATCTGT~G~G~GCTATACTGCCA~CAG~CTTTGCCAGGTTGCTGTT~GAGT~TGTTGGTTGCTACTTT~GCTCGT~AGACCAGCGTTATCCG~GA~GCC M S G S V R R S Y T A M Q N F A R L L L G V L L V A T L S S C R P A L S E D E A
-i 120 40
~C~G~GG~CCTGAGGT~G~CATCTACTTCTAGACCCCGG~TGC~TGCGA~TGTTTTGTGACACC~CC~GC~CAGCATC~C~GTACCGCG~CA~A~GTTTA N W K E P E V E E H L L L D P G N A L R L F C D T N Q S N S I N W Y R E Q
240 80
D
R
L
TTGCCAGGAGGG~GATT~GCA~GTGGGGA~TGTGCTAG~GTCTCAGATGTGACATA~GAC~AGGACTCTACATATG~GTGGTCAGAGG~CAGGCAAAA~TTAG~T~ L P G G K I R M V G T V L E V S D V T Y E D S G L Y I C V V R G T G K I L R E F
360 120
T~CATATCCGTTGTTGACTCGTT~CATCTGGAGATG~G~GA~ATGAGGATGGCCGTAGGGAGGACAC~cCG~TGATATT~TGAGGAGCCAGTTTATTTTTTCC~GCA~CATAC S I S V V D S L A S G D E E D D E D G R R E D T T A D I N E E P V Y F F Q A P y
480 160
TGGACACAG~C~ACCGCATGGAC~GAAACTTCATGCTGTGCCAGCT~T~CACAGTC~GTTCCGCTGTCCAGCTGGTGGGAGTCCCCTTC~CACTATTCGAT~CTTAAA/%A~ W T Q P H R M D K K L H A V P A G N T V K F R C P A G G S P L P T I R W L K N G
600 200
A~G~TTTCGAGGAG~cA~AG~TTGG~GGATCCGGCTTCGACACC~CAC~GAGTCTGGT~TGGAGAGTGTGGTTCCATCAGACCGTG~CTACACCTG~GTAG~C R E F R G E H R I G G I R L R H Q H W S L V M E S V V P S D R G N Y T C V V E N
720 240
AGAGTTGGCAG~ACATATA~CTACTTTCTGGATG~T~AGAGGT~TTCTCACCGTCCTATCCTAcAG~TG~CTTCCAGC~AC~CA~A~G~GT~GTAGCGATG~ R V G S L T Y T Y F L D V L E R S S H R P I L Q A G L P A N T T A R V G S D V E
840 280
TTCTACTGC~GTATACAGCGATGCTCAGCCACATATCC~TGGCTT~CACATCG~GTG~TGG~GCCGATTTGGTCCTGATGATTTTCCATATGTACAGGT~CTGCA F Y C K V Y S D A Q P H I Q W L K H I E V N G S R F G P D D F P Y V Q V L K T A
960 320
GATA~CACTTCGGAGGTAGAGG~CT~ACCTAC~TATCACTATGGAGGATGCAGGGG~TACACATGTCTGGCGGGC~TTCTATTGGTCTTTCTCATCAGTC~CTTG~TG D I N T S E V E V L H L R N I T M E D A G E Y T C L A G N S I G L S H Q S A W L AcTG~cTTTc~cG~GATTTCCTTGAGc~GcTGAGcCA~AGAGTc~GATATATGGAc~T~TATAcAcTTcTGGATTccTGGcTGTAGccATG~cATcG~ATAG~G~ T V L S N E D F L E Q A E P A E S R Y M D ~ I I I Y T S G F ~ A V A M A I V I V V I 4 0
1080 360 1200 0
C~TGCC~AT~AGACACC~ACAGC~GCAGACTCTGC~CCACCAGCTGTTCAT~CTGGCC~GTTCCCCCTCATACGGCAGTTC~TT~GAGTCCAGCTCATCTGGG~GTCC ~ - - ~ R M Q T P H S K Q T L Q P P A V H K L A K F P L I R Q F S L E S S S S G K S AGTGCTCCAC~AT~GCAT~C~GTCTCTCCTC~GCTG~CCCCATGCTGCC~GTGTCAT~A~TTGAGTTACCACTGGACGCT~GGAG~CCCTAGAGACAGGCTTG~ S A P L I R I T R L S S S C A P M L P G V M E V E L P L D A K W E F P R D R L
1320 440
V
1440 480
v CTGGG~GCCGCT~GAGA~T~TTTGGCC~GTGGT~GAGCAGA~GATA~G~TTGAG~GAC~GGCCTGAG~CCAGTTACCGTTGCAGTT~GA~C~AAAGAT~T L G K P L G E G C F G Q V V R A E G Y G I E K D R P E K P V T V A V K M L K D
N
1560 520
GGCACTGAC~GGACTTATCAGA~TGATCTCTG~AGTTGATG~TCATTGGAAACACAAAAATAT~TAAACTTGCTC~CGTCAGCACTC~GA~GGCCATTATT~TT G T D K D L S D L I S E M E L M K V I G K H K N I I N L L G V S T Q E G P L F V
1680 560
ATAGTTG~TA~CTTCT~GGGG~TCTGCGTGAGTT~TACGTGCCAGGCGCCCTCCCACACCGG~GATGCCTTTGATATCACC~GGTTCCA~TG~CTTT~TCCTTT~AC I V E Y A S K G N L R E F L R A R R P P T P E D A F D I T K V P D E L L S F K D
1800 600
CTAG~TCTTGTG~TTATCAGGTTG~C~GTGG~ATGGAGTAC~TTG~TCT~GAGGTGCATACACCGGGATCTGGCAGCCAG~TGTTCTTGTGGCAG~GAT~TCA~GA~ L V S C A Y Q V A R G M E Y L E S K R C I H R D L A A R N V L V A E D N V M K I
1920 640
GCAGATTTTGGCTTGGCACGAGG~TTCACGATATTGACTATTAC~GAAAACTAGT~TGGTCGACTGCC~TT~GA~GCTCCAG~GCTTTATTT~CAGAGTTTACACCCAC A D F G L A R G V H D I D Y Y K K T S N G B L P V K W M A P E A L F D R V Y T H
2040 680
C~GTGACATTT~TCATT~GAGTGTTGACATG~AGATCTTTACTCT~GA~G~TCCATATCC~G~TTCCAGTAG~G~CTATTT~CTCTTAC~G~G~CATCG~ Q S D I W S F G V L T W E I F T L G G S P Y P G I P V E E L F K L L R E G H R
2160 720
M
GAC~CCATCC~CTGTACTCATG~TTGTACATGTTGATGAGGGAGTGCTGGCATGCAGTTCCATCTCAGAGACC~CATTT~CAGCTGGTTG~C~CTGGATAGGATCCTCACA D K P S N C T H E L Y M L M R E C W H A V P S Q R P T F K Q L V E Q L D R I L T
2280 760
GCTGTTTCTG~GAGTATCTGGACTTATCTATGCCATTTG~CAGTATTCTCCCTCCTGTG~GATTCTTC~GCACATGCTCT~ATCCGATGAC~TG~TTTGCCCACGA~CAGTG A V S E E Y L D L S M P F E Q Y S P S C E D S S S T C S S S D D S V F A H D P V
2400 800
CCATCCTCTCCCTGTGTCTTT~TTACCAC~TGTTCACACTCACCT~GGACTTGAAAAC~GGAGACGGTTTGCC~TATTGGGAGATCT~AC~TGTAAACAGAGTGTTATTTC P S S P C V F N Y H N V H T H L G T *
2520 818
CTT~CT~TA~CGTATTTATATAAAAAAAAAGCCATATGCGGTATTATG~CTAT~GG~TCACTGTGC~TTTTTCTGCTGTTC~GCAG~TAGAAAAATCCT~CAGTT ~TCGCTGGTACAGCGGATGCATAGCAcTG~GCC~GCCACCCCGGTCCGTGACGAGATGTCGCCTTGCTCCAGATTCC~GGTCTAG
2640 2734
218
XFGFR4 PFR4 HFGFR4
XFFGR4 PFR4 HFGFR4
1:
MSGSVRRSYTAMONFARLLL-GVLLVATLSSCRPALSED-EANWKE--PEVEEHLLLDPpNAIRLFC DTNQSNS INWYREQDRLLPGGKIRMVGTVLEVSDVTYEDSGLYICVVRGT 1 :M G V Q K D . R D I R W N R T T R P L A . . . C. L . A F S A . - . . A R T . P. G R K . . L A . L V S . E . . . F , . . . 1 . . . . . . . . . . . . T T I V . . . T. S T . . Q H . . R . . L T D . . . . I A . . . . . . . . . . L... P.. I: MRLLL/tLLGV..SVP-GPPV..LEASEEV.LEPCLAPSL-EQQ.QE.TVA~.QPV..C.GRAE-RGGH..K GS A.A RV GWRGR. IASFLP..A.R.L.LA..S[
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P ............... P ...............
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DAQPH
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S ...... Y.H.VSF S ...... Y...VSA
YTC LAGNSI
.............. ..............
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I RQFSLESS
S SGKSSAP
L I R ITRLSSSCAPMLPGVMEVE
L PL DAK
PE...AKE..GP.T..T ........ S..LL..A . . . . . . I' ' .L. P T . T H . E . A T .... SR...M .............. T S , V . V . . . . . . . T . . . . . . L . F D . . . . S. F E . . p T W T . A A P . A . . T [I" • L. A . . S . . L. V L L L L A G . ~. G. A L . G R H P R P . A T . Q . . S R . . . A . . . . . . . G . . . . . . S S . V. G V . . . . . G P A L . A . L V S L D . . . . P L m
Tyrosine kinase ~DRLVLGKPLGE
GC FGQWRAEGYG
I EK DRP EKpVTVAVKMLKDNGT A...N..Q.D.AI... I.IV..K AF. MDPA. .DQAS ..........
DKDLS DL I SEMELMKV
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K1 I NLLGVSTQEGPLFVIVE
.... E ............ LM ............ A S . . . . A. .V . . . . V . . L. . R . . . . . . . . . .
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592:
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599
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V ...........................
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580:
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712
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PFR4 HFGFR4
719 700
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............
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ILTAVSEEYLDLSMPFEQYSPSCEDSSSTCSSSDDSVFAHDPVPSSPCVFNYHNVHTHLGT T..[...AT.A ............... A...TT.......... T.E--.DV.SL.TH.TTTSMV, A..[KV.I .......... RLT.GP .... GG.A ....... - .... S...L.LGSSS.PFG---SGVQ. !
818 . 822 802
Fig. 2. Comparison of the deduced aa sequence of XFGFR-4 with those of urodele FGFR-4 (PFR4: Shi et al., 1992) and human FGFR-4 (H FGFR4; Partanen et al., 1991 ). Residues identical to XFGFR-4 are represented by dots. Signal peptides are underlined. Three immunoglobulin-like domains (Ig-1, lg-2 and Ig-3), an acidic domain (AD) and a TM domain (TM) are boxed and indicated. The intracellular tyrosine kinase domain (tyrosine kinase) is boxed, and the kinase insert (KI) is overlined. A possible autophosphorylation site in the C-terminal region, which may be a binding site for phosphoinositide-specific phospholipase-C7 (Mohammad et al., 1991 ), is indicated by a closed circle above the sequence. Conserved Cys residues are marked by # above the sequence.
lar kinase domain and the extracellular Ig-2 and Ig-3 domains that have been shown to be important for ligand-binding activity and specificity (Johnson et al., 1991; Dell and Williams, 1992; Jaye et al., 1992; Werner et al., 1992; Zimmer et al., 1993; Chellaiah et al., 1994). This suggests that X l and mammalian FGFR-4s have essentially the same characteristics in terms of intracellular signalling and ligand binding. However, significant differences can be observed in the N-terminal 100 aa, including half of the Ig-1 domain, the acidic domain including both borders, borders of the transmembrane domain, and the C-terminal 20 aa, although the significance of these differences is not yet known. The overall aa sequence identity between XFGFR-4 and human FGFR-4 is 68%, which is significantly lower than between Xl and mammalian F G F R - l s and -2s (about 80%; Friesel and Dawid, 1991; Friesel and Brown, 1992). On the other hand, homology among the three XFGFRs was relatively low as follows: XFGFR-1 (XFGFR1A) vs. XFGFR2, 64%; XFGFR-1 vs. XFGFR-4, 62%: XFGFR-2 vs. XFGFR-4, 63%.
(d) Temporal changes of XFGFR-4 expression during X! development We finally examined the temporal profile of the amount o f X F G F R - 4 mRNA by Northern blot analysis. As shown
in Fig. 3, a single mRNA species of about 4 kb is observed throughout the developmental stages. However, the amounts vary signifiantly; XFGFR-4 mRNA is detected at the beginning of the cleavage stage, suggesting that the mRNA is inherited as a maternal mRNA. This maternal inheritance was confirmed by using the oocyte mRNA (data not shown). Subsequently, the mRNA decreases gradually to the late blastula stage, and then increases probably by zygotic expression reaching a maximum at the blastula and the late gastrula stages. The mRNA is also observed during later stages in lesser amounts. This profile is somewhat similar to that of urodele FGFR-4 mRNA, but it is not detected in early cleavage stage (Shi et al., 1992). It is also noteworthy that the expression profile of XFGFR-4 mRNA differs from those of X F G F R - I and -2 (Friesel and Dawid, 1991; Friesel and Brown, 1992), suggesting that each X F G F R gene is under different developmental control and has distinct roles.
(c) Conclusions (1) The nt sequences of the cDNA and genomic DNA of XFGFR-4 were determined. (2) The gene organization of X F G F R - 4 is similar in overall structure to those of mammalian FGFR-1 and -2, although XFGFR-4 lacks an alternative exon encoding
219
1 2 3 4 5 6 7 8 910
(a)
(b) Fig. 3. Northern blot analysis ofXFGFR-4 mRNA in early embryogenesis of X1. Northern blot analysis was carried out using a cDNA insert of ~.cXFR4-1 as a probe (a), and the same blot was subsequently hybridized with histone H4 DNA (h) (Sambrook et al., 1989). Total RNA (10 ~tg) prepared from embryos at each stage was electrophoresed in 2.2 M HCOH-I% agarose. Embryos were used at two-cell stage (lane 1 ), 16-cell stage (lane 2), early blastula stage (lane 3), late blastula stage (lane 4), early gastrula stage (lane 5), late gastrula stage (lane 6), early neurula stage (lane 7), late neurula stage (lane 8), early tailbud stage (lane 9) and late tailbud stage (lane 10).
the Ig3 domain and a poly(A)-addition site as found in the genes encoding FGFR-1 and -2. (3) The deduced aa sequence of XFGFR-4 is homologous to those of mammalian FGFR-4, but the homology (68% identity) is lower than those (about 80% identity) between X l and mammals FGFR-1 and -2. (4) The X F G F R - 4 mRNA occurs throughout X l development and is regulated in a manner different from X F G F R - I and -2 mRNAs.
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