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The expression of two isoforms of the human fibroblast growth factor receptor (flg) is directed by alternative splicing
Vol.
174,
January
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
31, 1991
Pages
THE EXPRESSION GROWTH FACTOR
Hiroko
Fujita
IDepartment
OF TWO ISOFORMS OF THE HUMAN FIBROBLAST RECEPTOR (fig ) IS DIRECTED BY ALTERNATIVE SPLICING
l, Mitsuhiro
Ohta”, Toshisuke
Kawasaki 1 and Nobuyuki
of Biological Chemistry, Faculty of Pharmaceutical Kyoto University, Kyoto 606, JAPAN
2Clinica1 Research Center, Utano National Received
December
946-951
25,
Hospital,
Itoh 1* Sciences,
Kyoto 603, JAPAN
1990
Structural analysis of the cDNA for the human fibroblast growth factor receptor ( fZg ) revealed the existence of a larger and a shorter isoform of the receptor. The larger form has three extracellular immunoglobulin-like domains. On the other hand, the shorter form deletes the first ( the most external ) immunoglobulin-like domain region. Two consecutive amino acids ( Arg Met ) between the first and second immunoglobulin-like domains are somtimes deleted from the shorter form. In this paper, we isolated and analyzed the gene for the human fibroblast growth factor receptor. Organization of the gene revealed that the isoforms are produced by two different types of alternative splicing ( the cassette and internal donar types ) from the common gene. In human placenta, the shorter form is expressed as 0 1991Academic Pre**, Inc. the major isoform. The acidic and basic fibroblast growth factors ( aFGF and bFGF ) have multiple biological activities in viva and in t~itro : angiogenesis, mitogenesis and cellular differentiation. FGFs mediate their biological response by binding to and activating specific cell surface receptors. FGF receptors were identified by cross-linking with 1251-FGFs to the receptors ( 1 ), and aFGF and bFGF were shown to bind the same receptors ( 2 ). The cDNA for the FGF receptor was first isolated from a chicken cDNA library. The chicken FGF receptor was shown to be a transmembrane protein that contains three extracellular immunoglobulin-like domains and an intracellular tyrosine kinase domain ( 3 ). The cDNAs for the mouse and human homologs ( ffg ) were also isolated ( 4,5,6,7,14,15 ) and their structures revealed that two isoforms of FGF receptor ( the larger and shorter forms ) are expressed in human and mouse. The structure of the larger form of human FGF receptor is similar to that of chicken FGF receptor. The shorter form deletes the first ( the most external ) immunoglobulin-like domain ( 89 amin acids ). Two consecutive amino acid ” To whom correspondence 0006-291X/91 Copyright All rights
should be addressed.
$1.50
0 1991 by Academic Press. Inc. of reproductior7 in nn~ form reserrvd.
946
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
residues ( Arg Met ) between the first and second immunoglobulin-like domains are sometimes deleted from the shorter form ( 5,6,7 ). Recently another putative FGF receptor of human ( hek ) was also identified ( 17,18 ). The human hek also bind to aFGF and bFGF with high affinities. The overall amino acid identity between hek and fig is 7 1 %, with the region of highest identity ( 88 % ) being the kinase domain. The j’lx and hek are located on different chromosomes ( 17 ). These results indicate that f/g and hek are similar yet distinct gene products. To clarify the mechanism of the expression of the isoforms of f/g, we isolated analyzed the gene for the receptor. We also examined the relative abundance of the isoforms in human placenta.
MATERIALS
AND METHODS
Screening of a human genomic DNA library ----- A human genomic DNA library ( Japanease Cancer Research Resources Bank-Gene, LIO18 ) was plated on Escherichia coli LE392, followed by transfer to nylon filters ( 8 ). After denaturation and immobilization of phage DNA, the filters were prehybridized at room temperature for 5 h - 16 h in hybridization solution ( 50 % deionized formamide, SXSSC ( 1XSSC: 0.15 M sodium chloride, 0.015 M sodium citrate ), 50 mM sodium phosphate, pH 7.0, 0.1 % sodium dodecyl sulfate and 0.5 % nonfat dry milk ), then hybridized at 42 “C for 16 h in hybridization solution containing [-?“P]FGF receptor cDNA ( nucleotides 1 - 541 ) ( 3ng/ml, 5X10” cpm/pg ) ( 6 ) labeled with a random primer ( hexadeoxynucleotide mixture, Takara, Kyoto ) ( 9 ). The filters were washed three times at room temperature for 20 min in washing solution I ( 2XSSC and 0.1 % sodium dodecyl sulfate ), then once at room temperature for 20 min and once at 50 “C for 10 min in washing solution II ( O.lXSSC and 0.1% sodium dodecyl sulfate ). The washed filters were exposed to X-ray films at -70 “C using intensifying screens ( Cronex Lightning Plus; Du Pont ). Determination of the nucleotide sequence oj’ the gene fijr the hamarl FGF receptor ----Phage DNA was prepared from positive clones by the plate lysate method ( 10 ). The phage DNA ( 2 pg in 7 pl distilled water ) was denatured at 99 “C for 3 min. The nucleotide sequence of the gene for the FGF receptor was determined by the dideoxy method ( 11 ) using the heat-denatured phage DNA and the oligonucleotide primer ( 20 - 24 mer ) corresponding the nucleotide sequence of the cDNA for the human FGF receptor ( 5,6 ) Amplijication of the FGF receptor mRiXA sequence in human placenta ----For the first strand synthesis, 2.5 pg human placenta poly (A) RNA was reverse incubated in a reaction mixture ( 20 pl ) with 300 units M-MLV transcriptase ( GIBCO-BRL ) and 0.5 pg random primer. For amplification of the cDNA sequences around the deletion regions, the polymerase chain reaction was carried out for 40 cycles in a reaction mixture ( 50 pl ) with 10 pl of the above cDNA solution, 2.5 units Taq DNA polymerase ( Promega ) and IO pmole each the sense and antisense primers which correspond to the nucleotide sequence of the shorter form of human FGF receptor cDNA ( nucleotides 25 1 274 and 585 - 608 ), respectively ( 6 ). Analysis of the amplified prodact _-___ The reaction product was fractionated on 2.5 % agarose gel ( 1.5 % NuSieve GTG agarose ( FMC Bioproducts ) and 1 .O % agarose S ( Wako Pure Chemicals, Osaka ). The gel 947
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
After staining, the amplified DNA was stained with ethidium bromide. fragments on agarose gel were transfered to a nylon filter and hybridized with [ 32P1FGF receptor cDNA as described above. After hybridization, the filter was also washed as described above. The washed filter was exposed to a X-ray film.
RESULTS
AND DISCUSSION
Isolation of the FGF Receptor Gene -----
Using human FGF receptor The cDNA as a probe ( 6 ), we screened a human genomic DNA library. positive clones were further analyzed using several oligonucleotides corresponding to the areas flanking the deletion regions. Two clones ( gF 9 and gF 12 ) in combination were found to cover the two deletion regions, of which the structure was partially determined by the direct sequencing of gF 9 and gF 12 DNA using the oligonucletide primers ( Fig. I ). Organization of the FGF Receptor Gene ----Comparison of the structures of the FGF receptor gene and the FGF receptor cDNA revealed the exon-intron organization around the deletion regions ( Fig. I ). The first immunoglobulinlike domain region which is deleted from the shorter form is encoded by a separate exon ( Exon B in Fig. 1 and 2 ). The gene structure around the deletion region of two consecutive amino acids has two different splice donor sites ( Exon C in Fig. I and 2 ). The sequences of the exon-intron boundaries are shown in Fig. 2. All the 5’ and 3’ ends of the intron sequences adhere to the Thus, they begin at the 5’ end with the nucleotide GT and “GT/AG rule”. As the structures of the cDNAs for the terminate with the nucleotide AG. larger and shorter forms are identical except for the deletion regions ( 5,6,7,17
Clone
I
Gene
gF12
------ gF9 w
4
Exon
-----rig
I
4
119 VNVS
I
120 DALP
-gt
-----ag
w
4
149 PNRM
ngt
B
A
mRNA
*
I
31 AOPW
LPE3i Igt
1
---C
4 150 PVAP
ag I D
_-----___
larger
I**
shorter
‘7
---------tIX
------------
_______-------------
--______
Fig. 1. Organization of the gene for the human FGF receptor and alternative splicing patterns giving rise to the two isoforms of human FGF receptor mRNA. The open- and close-boxes indicate the exons and the deletion regions of the exons, respectively. Exon B encodes the first immunoglobulin-like region. Exon C encodes the two consecutive amino acids at the 3’ end. The dashed lines indicate the introns. Arrows indicate the direction and extent of The corresponding amino sequence determination by the dideoxy method. acids are also indicatedand numbered according to the sequence of the larger isoform ( 5 ). 948
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
A B C
EXOll
I)
II)
Splice
A
E GAACAAG
B
119 v s GTTTCAG
N R AAACCGTATGC] rn)
Splice
donor
30 Q gtaac
. ... . ....
gttgt
.. . . . .. . ... . ... . ... .
120 D
dqgtatgcgtgag
EXOn 0 6
A
catagm
c
149 M 150 P
gtgag...
147
c
acceptor
31 A . . . . . . . . . . . . . agcagjCCCAG
ggcaglm ..... “..
v D
I
Fig. 2.
Nucleotide sequences around splice junctions. Exon sequences and intron sequences are denoted by capital letters and lowercase letters. The corresponding amino acids are also indicated and numbered according to the sequence of the larger form ( 5 ). m
Agarose gel electrophoresis of the polymerase chain reaction product of human placenta cDNA. A. $X 174 DNA- Hae III digest, B. the polymerase chain reaction product stained with ethidium bromide, C. the
ploymerase chain reaction product hybridized with [32P]FGF receptor cDNA.
), these results indicate that two isoforms of the human FGF receptor are generated by two differnt types of alternative splicing ( cassette and internal donar types ) ( 12 ) from the common gene . Expwsion of the Two Isc@ms in the Placerrtu ----As the sizes of the mRNAs for FGF receptors are approximately 4 kilobases. it is difficult to distinguish the mRNAs for the two isoforms by Northern analysis ( 4 ). We examined the relative abundance of the mRNAs for the two isoforms in human placenta by the polymerase chain reaction. Amplification of the gene around the deletion regions of FGF receptor cDNA in the placenta cDNA generated two fragments of -350 and -630 base pairs ( Fig. 3 ). The sizes of the two DNA fragment corresponded the predicted sizes ( 35X and 631 base pairs ) of the shorter and larger forms of the FGF receptor cDNA, respectively ( 5,6 ). The amplified DNA fragments on agarose gel were transferred to a nylon filter and hybridized with [-i’P]FGF receptor cDNA. Both the amplified DNA fragments hybridized with FGF receptor cDNA ( Fig. 3 ). These results indicate that the two DNA fragments originate from the two isoforms of FGF receptor cDNA and that the shorter form is the major isoform in human placenta. We and Johnson et al. isolated several FGF receptor cDNA clones from the human placenta cDNA libraries and determined the nucleotide sequences. The nucleotide sequences indicated that all the cDNA clones were the shorter form ( 6.7 ). These results are consitent with that of the polymerase chain experiment described here. 949
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Both the larger and shorter forms bind and mediate biological responsiveness to aFGF and bFGF, indicating that the first immunoglobulin-like domain may have a function other than binding of aFGF and bFGF ( 7,14,15,16 ). Both aFGF and bFGF bind the common receptors of 125 and 145 kilodaltons in BHK fibroblasts ( 2 ). The two receptors, which are glycoproteins, have protein cores of 100 and 125 kilodaltons ( 13 ). Judging from the molecular masses of the core proteins ( 100 and 125 kilodaltons ). the core proteins should correspond to the shorter form ( 711 amino acids ) and the larger form ( 802 amino acids ) identified by the cDNAs, respectively. Competition with aFGF and bFGF showed preferential affinities to the receptors of 125 and 145 kilodaltons, respectively ( 2 ). Therefore, the first immunoglobulin-like domain may modulate affinity for the ligands. The two consecutive amino acids between the first and second immunoglobulin-like domains are sometimes deleted from the shorter form in human and muose ( 5,6,7 ). The significance of the two consecutive amino acids has also remained to be elucidated.
ACKNOWLEDGMENTS We Noboru and the genomic
thank Mr. Ken-kichi Takagi for human placenta poly (A) RNA, Dr. Tomioka for the oligonucleotide synthesis, and Dr. Yoshiyuki Sakaki Japanease Cancer Research Resources Bank-Gene for the human DNA library.
REFERENCES 1. Burgess,W.H. and Maciag,T. ( 1989)Ann.Rev.Biochem. 58,575-606. 2. Neufeld,G. and Gospodarowicz,D. ( 1986 ) J.BioZ.Chem. 261, 5631-5637. 3. Lee,P.L., Johnson,D.E., Cousens,L.S., Fried,V.A. and Williams,L.T. (1989 ) Science 245, 57-60. 4. Reid,H.H., Wi1kqA.F. and Bernard,O. ( 1990 ) Proc.NutZ.Acad.Sci.USA 87, 1596- 1600. 5. Isacchi,A., Bergonzoli,L. and Sarmientos,P. ( 1990 ) Nucleic Acids Res. 18, 1906. 6. Itoh,N., Terachi,T., Ohta,M. and Kurokawa Seo, M. ( 1990 ) Biochem. Biophys. Res. Commun . 169, 680-685. 7. Johnson,D.E., Lee,P.L., Lu,J. and Williams,L.T. ( 1990)Mol.CelZ.Biol. 10,4728-4736. 8. Maniatis,T., Fritsch,E.F. and Sambrook,J. ( 1982 ) in Moleuclar Cloning, A Laboratory Munud, pp. 320-321, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 9. Feinberg,A.P. and Vogelstein,B. ( 1983 ) And. Biochem. 132, 6-l 3. 10. Manifioletti,G. and Schneider,C. ( 198X ) Nucleic Acids Res.16, 2873-2884. 950
Vol.
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No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
11. Sanger,F., NicklenS. and Coulson,A.R. ( 1977 ) Proc.N~tl.Acad.Sci. 0’,S2t 74,5463-5467. 12. Breitbart,R.F., Andreadis,A. and Nadal-Ginard,B. ( I987 ) Ann.Re~>. Biochem. 56, 467-495. 13. Feige,J.J. and Baird,A. ( 1988 ) J.Biol.Chem. 263, 14023-14029. 14. Safran,A., Avivi,A., Orr-Urtereger,A., Neufeld,G., Lonai,P., Givol,D. and Yarden,Y. ( 1990 ) Oncogene 5, 635-643. 15. Mansukhani,A., Moscatelli,D.. Talarico,D., Levytska,V and Basilico,C. ( 1990 ) Proc.Nutl.Acud.Sci. USA 87, 4378-4382. 16. Ruta,M., Burgess,W., Givol,D., EpsteinJ., Neiger,N., Kaplow,J., Crumley,G., Dionne,C., Jaye,M. and Schlessinger,J. ( 1989 ) Proc.Natl. AcadSci. USA 86, X722-8726. 17. Dionne,C., Crumley,G., Bellot,F., Kaplow,J.M., Searfoss,G., Ruta,M.. Burgess,W.H., Jaye,M. and Schlessinger,J. ( 1990 ) EMBO J. 9, 2685-2692. 1X. Houssaint,E., Blanquet,P.R., Champion-Arnaud,P., Gesnel,M.C., Torriglia,A., Courtois,Y. and Breathnach,R. ( 19%) ) Proc.Nurl.Acud.Sci. USA 87, 81X0-8184.
9.51
174,
January
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
31, 1991
Pages
THE EXPRESSION GROWTH FACTOR
Hiroko
Fujita
IDepartment
OF TWO ISOFORMS OF THE HUMAN FIBROBLAST RECEPTOR (fig ) IS DIRECTED BY ALTERNATIVE SPLICING
l, Mitsuhiro
Ohta”, Toshisuke
Kawasaki 1 and Nobuyuki
of Biological Chemistry, Faculty of Pharmaceutical Kyoto University, Kyoto 606, JAPAN
2Clinica1 Research Center, Utano National Received
December
946-951
25,
Hospital,
Itoh 1* Sciences,
Kyoto 603, JAPAN
1990
Structural analysis of the cDNA for the human fibroblast growth factor receptor ( fZg ) revealed the existence of a larger and a shorter isoform of the receptor. The larger form has three extracellular immunoglobulin-like domains. On the other hand, the shorter form deletes the first ( the most external ) immunoglobulin-like domain region. Two consecutive amino acids ( Arg Met ) between the first and second immunoglobulin-like domains are somtimes deleted from the shorter form. In this paper, we isolated and analyzed the gene for the human fibroblast growth factor receptor. Organization of the gene revealed that the isoforms are produced by two different types of alternative splicing ( the cassette and internal donar types ) from the common gene. In human placenta, the shorter form is expressed as 0 1991Academic Pre**, Inc. the major isoform. The acidic and basic fibroblast growth factors ( aFGF and bFGF ) have multiple biological activities in viva and in t~itro : angiogenesis, mitogenesis and cellular differentiation. FGFs mediate their biological response by binding to and activating specific cell surface receptors. FGF receptors were identified by cross-linking with 1251-FGFs to the receptors ( 1 ), and aFGF and bFGF were shown to bind the same receptors ( 2 ). The cDNA for the FGF receptor was first isolated from a chicken cDNA library. The chicken FGF receptor was shown to be a transmembrane protein that contains three extracellular immunoglobulin-like domains and an intracellular tyrosine kinase domain ( 3 ). The cDNAs for the mouse and human homologs ( ffg ) were also isolated ( 4,5,6,7,14,15 ) and their structures revealed that two isoforms of FGF receptor ( the larger and shorter forms ) are expressed in human and mouse. The structure of the larger form of human FGF receptor is similar to that of chicken FGF receptor. The shorter form deletes the first ( the most external ) immunoglobulin-like domain ( 89 amin acids ). Two consecutive amino acid ” To whom correspondence 0006-291X/91 Copyright All rights
should be addressed.
$1.50
0 1991 by Academic Press. Inc. of reproductior7 in nn~ form reserrvd.
946
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
residues ( Arg Met ) between the first and second immunoglobulin-like domains are sometimes deleted from the shorter form ( 5,6,7 ). Recently another putative FGF receptor of human ( hek ) was also identified ( 17,18 ). The human hek also bind to aFGF and bFGF with high affinities. The overall amino acid identity between hek and fig is 7 1 %, with the region of highest identity ( 88 % ) being the kinase domain. The j’lx and hek are located on different chromosomes ( 17 ). These results indicate that f/g and hek are similar yet distinct gene products. To clarify the mechanism of the expression of the isoforms of f/g, we isolated analyzed the gene for the receptor. We also examined the relative abundance of the isoforms in human placenta.
MATERIALS
AND METHODS
Screening of a human genomic DNA library ----- A human genomic DNA library ( Japanease Cancer Research Resources Bank-Gene, LIO18 ) was plated on Escherichia coli LE392, followed by transfer to nylon filters ( 8 ). After denaturation and immobilization of phage DNA, the filters were prehybridized at room temperature for 5 h - 16 h in hybridization solution ( 50 % deionized formamide, SXSSC ( 1XSSC: 0.15 M sodium chloride, 0.015 M sodium citrate ), 50 mM sodium phosphate, pH 7.0, 0.1 % sodium dodecyl sulfate and 0.5 % nonfat dry milk ), then hybridized at 42 “C for 16 h in hybridization solution containing [-?“P]FGF receptor cDNA ( nucleotides 1 - 541 ) ( 3ng/ml, 5X10” cpm/pg ) ( 6 ) labeled with a random primer ( hexadeoxynucleotide mixture, Takara, Kyoto ) ( 9 ). The filters were washed three times at room temperature for 20 min in washing solution I ( 2XSSC and 0.1 % sodium dodecyl sulfate ), then once at room temperature for 20 min and once at 50 “C for 10 min in washing solution II ( O.lXSSC and 0.1% sodium dodecyl sulfate ). The washed filters were exposed to X-ray films at -70 “C using intensifying screens ( Cronex Lightning Plus; Du Pont ). Determination of the nucleotide sequence oj’ the gene fijr the hamarl FGF receptor ----Phage DNA was prepared from positive clones by the plate lysate method ( 10 ). The phage DNA ( 2 pg in 7 pl distilled water ) was denatured at 99 “C for 3 min. The nucleotide sequence of the gene for the FGF receptor was determined by the dideoxy method ( 11 ) using the heat-denatured phage DNA and the oligonucleotide primer ( 20 - 24 mer ) corresponding the nucleotide sequence of the cDNA for the human FGF receptor ( 5,6 ) Amplijication of the FGF receptor mRiXA sequence in human placenta ----For the first strand synthesis, 2.5 pg human placenta poly (A) RNA was reverse incubated in a reaction mixture ( 20 pl ) with 300 units M-MLV transcriptase ( GIBCO-BRL ) and 0.5 pg random primer. For amplification of the cDNA sequences around the deletion regions, the polymerase chain reaction was carried out for 40 cycles in a reaction mixture ( 50 pl ) with 10 pl of the above cDNA solution, 2.5 units Taq DNA polymerase ( Promega ) and IO pmole each the sense and antisense primers which correspond to the nucleotide sequence of the shorter form of human FGF receptor cDNA ( nucleotides 25 1 274 and 585 - 608 ), respectively ( 6 ). Analysis of the amplified prodact _-___ The reaction product was fractionated on 2.5 % agarose gel ( 1.5 % NuSieve GTG agarose ( FMC Bioproducts ) and 1 .O % agarose S ( Wako Pure Chemicals, Osaka ). The gel 947
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
After staining, the amplified DNA was stained with ethidium bromide. fragments on agarose gel were transfered to a nylon filter and hybridized with [ 32P1FGF receptor cDNA as described above. After hybridization, the filter was also washed as described above. The washed filter was exposed to a X-ray film.
RESULTS
AND DISCUSSION
Isolation of the FGF Receptor Gene -----
Using human FGF receptor The cDNA as a probe ( 6 ), we screened a human genomic DNA library. positive clones were further analyzed using several oligonucleotides corresponding to the areas flanking the deletion regions. Two clones ( gF 9 and gF 12 ) in combination were found to cover the two deletion regions, of which the structure was partially determined by the direct sequencing of gF 9 and gF 12 DNA using the oligonucletide primers ( Fig. I ). Organization of the FGF Receptor Gene ----Comparison of the structures of the FGF receptor gene and the FGF receptor cDNA revealed the exon-intron organization around the deletion regions ( Fig. I ). The first immunoglobulinlike domain region which is deleted from the shorter form is encoded by a separate exon ( Exon B in Fig. 1 and 2 ). The gene structure around the deletion region of two consecutive amino acids has two different splice donor sites ( Exon C in Fig. I and 2 ). The sequences of the exon-intron boundaries are shown in Fig. 2. All the 5’ and 3’ ends of the intron sequences adhere to the Thus, they begin at the 5’ end with the nucleotide GT and “GT/AG rule”. As the structures of the cDNAs for the terminate with the nucleotide AG. larger and shorter forms are identical except for the deletion regions ( 5,6,7,17
Clone
I
Gene
gF12
------ gF9 w
4
Exon
-----rig
I
4
119 VNVS
I
120 DALP
-gt
-----ag
w
4
149 PNRM
ngt
B
A
mRNA
*
I
31 AOPW
LPE3i Igt
1
---C
4 150 PVAP
ag I D
_-----___
larger
I**
shorter
‘7
---------tIX
------------
_______-------------
--______
Fig. 1. Organization of the gene for the human FGF receptor and alternative splicing patterns giving rise to the two isoforms of human FGF receptor mRNA. The open- and close-boxes indicate the exons and the deletion regions of the exons, respectively. Exon B encodes the first immunoglobulin-like region. Exon C encodes the two consecutive amino acids at the 3’ end. The dashed lines indicate the introns. Arrows indicate the direction and extent of The corresponding amino sequence determination by the dideoxy method. acids are also indicatedand numbered according to the sequence of the larger isoform ( 5 ). 948
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
A B C
EXOll
I)
II)
Splice
A
E GAACAAG
B
119 v s GTTTCAG
N R AAACCGTATGC] rn)
Splice
donor
30 Q gtaac
. ... . ....
gttgt
.. . . . .. . ... . ... . ... .
120 D
dqgtatgcgtgag
EXOn 0 6
A
catagm
c
149 M 150 P
gtgag...
147
c
acceptor
31 A . . . . . . . . . . . . . agcagjCCCAG
ggcaglm ..... “..
v D
I
Fig. 2.
Nucleotide sequences around splice junctions. Exon sequences and intron sequences are denoted by capital letters and lowercase letters. The corresponding amino acids are also indicated and numbered according to the sequence of the larger form ( 5 ). m
Agarose gel electrophoresis of the polymerase chain reaction product of human placenta cDNA. A. $X 174 DNA- Hae III digest, B. the polymerase chain reaction product stained with ethidium bromide, C. the
ploymerase chain reaction product hybridized with [32P]FGF receptor cDNA.
), these results indicate that two isoforms of the human FGF receptor are generated by two differnt types of alternative splicing ( cassette and internal donar types ) ( 12 ) from the common gene . Expwsion of the Two Isc@ms in the Placerrtu ----As the sizes of the mRNAs for FGF receptors are approximately 4 kilobases. it is difficult to distinguish the mRNAs for the two isoforms by Northern analysis ( 4 ). We examined the relative abundance of the mRNAs for the two isoforms in human placenta by the polymerase chain reaction. Amplification of the gene around the deletion regions of FGF receptor cDNA in the placenta cDNA generated two fragments of -350 and -630 base pairs ( Fig. 3 ). The sizes of the two DNA fragment corresponded the predicted sizes ( 35X and 631 base pairs ) of the shorter and larger forms of the FGF receptor cDNA, respectively ( 5,6 ). The amplified DNA fragments on agarose gel were transferred to a nylon filter and hybridized with [-i’P]FGF receptor cDNA. Both the amplified DNA fragments hybridized with FGF receptor cDNA ( Fig. 3 ). These results indicate that the two DNA fragments originate from the two isoforms of FGF receptor cDNA and that the shorter form is the major isoform in human placenta. We and Johnson et al. isolated several FGF receptor cDNA clones from the human placenta cDNA libraries and determined the nucleotide sequences. The nucleotide sequences indicated that all the cDNA clones were the shorter form ( 6.7 ). These results are consitent with that of the polymerase chain experiment described here. 949
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Both the larger and shorter forms bind and mediate biological responsiveness to aFGF and bFGF, indicating that the first immunoglobulin-like domain may have a function other than binding of aFGF and bFGF ( 7,14,15,16 ). Both aFGF and bFGF bind the common receptors of 125 and 145 kilodaltons in BHK fibroblasts ( 2 ). The two receptors, which are glycoproteins, have protein cores of 100 and 125 kilodaltons ( 13 ). Judging from the molecular masses of the core proteins ( 100 and 125 kilodaltons ). the core proteins should correspond to the shorter form ( 711 amino acids ) and the larger form ( 802 amino acids ) identified by the cDNAs, respectively. Competition with aFGF and bFGF showed preferential affinities to the receptors of 125 and 145 kilodaltons, respectively ( 2 ). Therefore, the first immunoglobulin-like domain may modulate affinity for the ligands. The two consecutive amino acids between the first and second immunoglobulin-like domains are sometimes deleted from the shorter form in human and muose ( 5,6,7 ). The significance of the two consecutive amino acids has also remained to be elucidated.
ACKNOWLEDGMENTS We Noboru and the genomic
thank Mr. Ken-kichi Takagi for human placenta poly (A) RNA, Dr. Tomioka for the oligonucleotide synthesis, and Dr. Yoshiyuki Sakaki Japanease Cancer Research Resources Bank-Gene for the human DNA library.
REFERENCES 1. Burgess,W.H. and Maciag,T. ( 1989)Ann.Rev.Biochem. 58,575-606. 2. Neufeld,G. and Gospodarowicz,D. ( 1986 ) J.BioZ.Chem. 261, 5631-5637. 3. Lee,P.L., Johnson,D.E., Cousens,L.S., Fried,V.A. and Williams,L.T. (1989 ) Science 245, 57-60. 4. Reid,H.H., Wi1kqA.F. and Bernard,O. ( 1990 ) Proc.NutZ.Acad.Sci.USA 87, 1596- 1600. 5. Isacchi,A., Bergonzoli,L. and Sarmientos,P. ( 1990 ) Nucleic Acids Res. 18, 1906. 6. Itoh,N., Terachi,T., Ohta,M. and Kurokawa Seo, M. ( 1990 ) Biochem. Biophys. Res. Commun . 169, 680-685. 7. Johnson,D.E., Lee,P.L., Lu,J. and Williams,L.T. ( 1990)Mol.CelZ.Biol. 10,4728-4736. 8. Maniatis,T., Fritsch,E.F. and Sambrook,J. ( 1982 ) in Moleuclar Cloning, A Laboratory Munud, pp. 320-321, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 9. Feinberg,A.P. and Vogelstein,B. ( 1983 ) And. Biochem. 132, 6-l 3. 10. Manifioletti,G. and Schneider,C. ( 198X ) Nucleic Acids Res.16, 2873-2884. 950
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
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