BIOCHEMICAL
Vol. 166, No. 2, 1990 January 30, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 892-897
CLONING AND CHARACTERIZATION OFTHE GROWTH HORMONE-DEPENDENTINSULIN-LIKE GROWTH FACTOR BINDING PROTEIN (IGFBP-3) IN THE RAT Anthony L. Albiston andAdrian C. Herington PrinceHenry’sInstituteof Medical Research, PrhrceHenry’sHospitalCampus, MonashMedical Centm,Melbourne,Australia3004 Received
December
12,
1989
We reportfor the first time the completeammoacidsequence for the growthhormone dependentinsulin-likegrowth factor bmdingprotein(IGFBP3) in the rat. A humanIGFBP-3 clonewasgeneratedusingthepolymemsechainreaction(PCR)andusedto screena rat liver cDNA library. cDNA clonesof the rat IGFBP-3 wereisolatedandthefull ammoacid sequence deduced.The sequence beginswith a putative,26 aminoacidsignalpeptide followedby a 265 ammoacidbindingprotein. The aminoacidsequence is over 80% homologous with the equivalenthumanIGFBP-3form andshowscompleteconservationof 18cysteineresiduesthat areclusteredat the aminoandcarboxyendsof theprotein. IGFBP3 is the bindingsubunitof the majorcirculatingIGFBP in the rat, andhencethe availability of precisestructuraldataandcDNA probesprovidesan importantopportunityfor a detailed studyof the control of IGFBP-3 synthesisat the level of geneexpression. o 1990 Academic Press,
mc.
The insulin-likegrowth factors-1and-II (IGF I, -11)arecirculatingmitogensthat stimulate growthboth in vivo andin vitro (1.2). In serumthe IGFs circulateessentiallycompletely boundto a family of evolutionaryte!ated,specificbindingproteins(3,4). Thesebinding proteinsprolongthehalf life of theIGFs andhavebeenshownto regulateIGF actionon cells in culture($6). In rat or humanseratwo majorformsof bindingproteinamdetectedby radioligandbindingstudies,a M,lSO,OOO(150K) form anda smallerM, 35 K form (4.7). The M, 150K bindingproteinis acid-dissociable into anIGF bindingsubunit,termed IGFBP-3,of M,-50 K andanacidlabilenon-bindingsubunitof M, 80,000-90,000 (8). The biosynthesis of IGFBPS is regulatedby growthhormone(GH) whereasthe smallerform, termedIGFBP-I. is not GH-dependent(3,4). The humanformsof IGFBP-1and-3 have recently beencloned(9-l 1). A third classof bindingprotein,IGFBP-2,wasoriginally isolatedfrom a fetal rat liver cell line (BRL9A) (12). The cDNA for the rat IGFBP-2has recentlybeenisolated(13, 14). Although IGFBP-3whichis synthesized primarily by the liver (7,15) is the predominantIGF bindingproteinin adultrat serum,it hasnot beenfully characterized. 0006-291X/90 Copyright All rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
892
Vol.
166, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
In thispaperwereportthe isolationof a cDNA encodingthe completesequence of the rat IGFBP-3. ‘Ihe deducedaminoacidsequence of the matureproteinis cysteinerich, 265 residuesin lengthandover 80%homologous with thehumanequivalent(11). Genomic Southernanalysissuggests that this is a singlecopygeneandnorthernanalysisshowsa single mRNA speciesin the liver, approximately2.4 kb in size.
MATERIALS AND METHODS Polvruerase chain
(PCR)
BaseduponthepublishedcDNA sequence of the humanIGFBP-3(11) two oligonucleotideprimerswereconstructed,primerA: 531-550nt andprimerB: the complementof 882-863nt. cDNA waspreparedby reversetranscriptionof 5~ of human liver poly A+ RNA using20pmol of primerB and110unitsof AMV reversetranscriptase (Pharmacia,Uppsala,Sweden)underthe conditionsrecommended by the supplier.A cDNA aliquotcorresponding to 0.5% of RNA wasusedfor PCRamplificationin 67 mM Tris pH 8.8 at 25-C 17mM (NH4)2SO4,3mMMgCl2, 10mM B-mercaptoethanol, 2 mg/mlgelatine, 0.5 M of eachprimer,500 M dNTP and2 U of Taq DNA polymerase(PerkinElmerCetus, Norwalk, U.S.A.). Thirty cycles(onecycle: 1 min at 92’C, 2 min at 55’C. 2 min at 72’C) werecarriedout in a 50@volumeusinga thermalcycler (Bartlett, Melbourne.Australia). The 351 bp productwassubcloned into aGem42 plasmidvector andits identity confirmed by dideoxy sequencing. Isolationof a cDNA clone A rat liver cDNA library in a&t 10vector (kindly donatedby ProfessorG. Schreiber, University of Melbourne)wasscreenedfor clonesof IGFBP-3. The 351bp PCRproductwas 32P-labelledby randompriming(16), hybridizationof duplicatefilters wasperformedat 42-C in 50%fonnamideandwasheswerein 2 X SSCat 42-C. Among 5 X lo6 plaques screened 7 werefound to bepositive. The largestclone,2.0 kb in size,wassubcloned into the Gem42 plasmidvector for furthermanipulation.
Relevantrestrictionfragmentsweresubclonedinto eitherGem42 or Ml3 vectors. Dideoxy chainterminationwasperformedusingSP6andT7 promoterprimersandMl3 universalprimersrespectively(17). In regionsthat lackedconvenientrestrictionsites specificallydesignedoligonucleotides weresynthesizedon a DNA synthesizer(Applied Biosystems,FosterCity, U.S.A.). GenomicSouthernAnalvsis High molecularweightrat liver DNA wasdigestedwith restrictionendonucleases accordingto the supplier’srecommended reactionconditions.The fragmentswereseparated on a 0.7%agarosegel andtransferredto a nylon membraneasdescribedby ReedandMann (18). The restrictionfragmentprobeusedwas930bp in length,containingthe entirecoding regionof the gene,andwas32P-labelledby randompriming. Hybridizationwasat 42-C in 50% formamideandthe filter wasgiven a final washin 0.1 X SSC, 1%SDSat 5o’C.
Pol A(+) mRNA waspurified from adultrat liver aspreviouslydescribed(19). Tenllgof by electmphoresis on a 1.2%agarose ~01~Af’) mRNA wasglyoxylated andthenseparated gel. The RNA wastransferredto a nylon membrane andhybridizedwith theprobedescribed for genomicSouthernanalysis.The filter wasgiven a fmal washin 0.1 X SSC.0.1% SDSat 55°C. 893
Vol.
166, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
RESULTS AND DISCUSSION A 351bppolymerasechainreactionproductof the humanIGPBP-3wasusedto screena rat liver cDNA library. Approximately 5 x lo6 cloneswerescreened revealingseven independent positiveclones.The largestclone,of 2.0 kb, containedthe entirecodingregion exceptfor the startcodon,ATG. A secondoverlappingclone,700bp in length,containedthe ATG codonplus36 bp of 5’ untranslatedsequence.The total sequence obtainedfrom these cloneswas2058bp (Pig. 1). Northernblot analysisof liver mRNA showeda singlemRNA speciesapproximately2.4 kb in sizeindicatingmatwe donot have a full lengthmRNA sequence (Pig. 2B). The 3’ untranslatedsequence obtaineddoesnot containa polyadenylation signalsuggesting that the 3’ regionis incomplete.An unusualfeatureof the3’ untranslated sequence is a 36 bp repetitiveTC sequence beginningat position1012.This repeating sequence is alsofoundin the 3’ untranslatedsequence of the rat pmlactinreceptormRNA (20), howeverthe significanceof this sequence isnot known. GenomicSouthernanalysis after hybridizationto the codingregionof the rat IGPBP-3cDNA showedonly 2 distinct bandsperlanefor eachof 4 restrictionenzymessuggesting thatthis is a singlecopy gene (Pig. 2A). The sequence hasanopenreadingframeof 291 aminoacidsbeginningwith amethionine residue.The first 26 residues,containinga hydrophobiccore,form a potentialsecretion signalpeptide(21). The maturepeptideis 265residues in lengthbeginningat Gly 27. The deducedaminoacidsequence of rat IGPBP-3,residues 27-68(Fig. 1). matchesexactly the NH2-terminalaminoacidsequence reportedby Zapf et al (22). The proteinhas4 potentialN-linked glycosylationsites(NXT or NXS), and2 shortserinethreoninerich domainswhichrepresentpotentialO-lied glycosylationsites(23). It is knownthat rat IGPBP-3isN-glycosylatcd(22) andthis accountsfor the discrepancybetween the predictedtranslatedmolecularweightof 28.8K andtheelectmphoreticallydetermined molecularweightof -50 K (7). The rat IGFBP-3is 83%homologous with the equivalenthumanBP(11). An insertionof two aminoacidsat residue30 andonedeletionat position103of the matureproteinare necessary to alignthehumansequence with the rat. The sequences have complete conservationof the eighteencysteineresidues present,which amclusteredat the N- andCtermlni of theprotein. The cysteineresiduesamhighly conservedacrossall of the distinct
Vol.
166, No. 2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Fia.Nucleotide sequence and deduced amino acid sequence of the rat IGFBP-3. The putative site of signal peptidase cleavage is indicated by an arrow. Potential glycosylation sites are underlined (N-glycosylatiow; 0-glycosylation - - -). Conserved cystelne residues compared to human IGFBP-3 are indicated(*).
IGF-bindingproteinclasses (humanandrat) sequenced to date(9-l 1,13,14,24) suggesting that they maybe involved in thebindingof IGF. Cysteine-richdomainshavebeenpostulated to constitutetheligandbindingdomainsof severalhormone/gmwth factor receptors, 895
Vol.
166, No. 2, 1990
BIOCHEMICAL
0.8
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
-
Fig. Southern and Northern blot analysis of IGF BP3. rat genomic DNA(10~) wasdigested withthefollowingenzymes: A) Sprague-Dawley
Ecoe (lanel), BamHl(lane2), Hind111 (lane3) andPstI(lane4). TheDNAwas througha0.7%agarose gel,transferred to anylonmembrane and electrbphorese restriction fragment comprised of thecodingsequence of probedwithah P-labelled theratIGFEP-3.Autoradiography wasfor 48hours. B) Tenmicrograms of ratliver poly A(+)RNA was glyoxylatedandelectrophoresed througha 1.2%agarose gel. TheRNAwastransferred to anylonmembrane and hybridizedwiththecodingsequence of theratIGFEP-3.RNAmarkers (ERL, Gaithersburg, U.S.A.)wererunin anadjacent laneandstained withethidium bromide to assess thesizeof theIGFEP3mRNAdetected.In thisexperiment autoradiography wasfor 12hours.
includingthe typeI IGF receptor(25), althoughthereisno aminoacidhomologybetween IGFBP3 andthe bindingdomainsof the IGF receptors(11). In additionto absoluteconservationof the cysteineresidues,all IGFBPsshowother limited sequence homologies at the N- andC- tennini. Rat IGFBP-3is no exception. A reportedN-terminalsequence for a mouseproteinwhich inhibitsserum-freefibroblastgrowth (26) is also83%homologous with rat IGFBP-3 indicatinga broadspecies conservationof this particulardomain.The N-terminalsequence of rat IGFBP-3,determinedby proteinisolation andsequencing (22) andconlirrnedby the presentcDNA-derivedsequence, is Gly-Ala-GlyAla-Val-Gly-Ala-Gly-Pro-Val-Val-Arg. Of particularinterestis the fact that this sequence is 58% homologous (with padding)with onepresentat the endof the proposedsignalpeptide for the BRL3A rat liver BP, IGFBP-2(13,14), adjacentto the N-terminalaminoacid(Glu) of the isolatedIGFBP-2protein(12). This may representanevolutionaryshift in the signal peptidecleavagesitesfor theseproteins(27). a shift whichmusthaveoccurredtier divergenceof the IGFBP genefamily itself. The significanceof sucha shift for the biological role and/orregulationof theseIGFBPsisnot known. 896
Vol.
166,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
In conclusion, we have isolated a cDNA encoding the rat growth hormone dependent insulin-like growth factor binding protein, IGFBP-3, and report for the first time the complete amino acid sequence of this important regulatory rat protein. The deduced amino acid sequence is over 80% homologous to human IGFBP-3. The availability of the rat cDNA will provide an opportunity to examine, in a very convenient model system, the nature and tissue distribution of IGFBP-3 mRNA and its pharmacological/physiological regulation. ACKNOWLEDGMENTS
Thesestudiesweresupportedin part by the NationalHealthandMedical Research Councilof Australia. WethankMrs. JoniLaw andMS SuePanckrldgefor helpwith the preparationof the manuscript. REFJXRENCES
1. Baxter, R.C. (1986) Adv. Clin. Chem.25: 49-115. 2. Froesch,E.R., S&mid, C., Schwander,J, andZapf, J. (1985) Ann. Rev. Physiol. 47: 443468. 3. Ooi, G.T.. andHerington,A.C. (1988) J. Endocrinol. 118:7-18. 4. Baxter, R.C. andMartin, J.L. (1989) Pmg. in GrowthFactorRes. 1: 49-68. 5. Moses,A.C.. Nissley,S.P.,andCohen,K.L. (1976) Nature 263: 137-140. 6. Elgln, R.G., Busby, W.H., Jr. andClemmons, D.R. (1987) Proc.Natl. Acad. Sci. USA 84: 3254-3258. 7. Baxter, R.L. andMartin, J.L. (1987)Biochem.Biophys.Res.Comm. 147:408415. Baxter, R.C. (1988)J. Clin EndocrinolMetab 67: 265-272. ;: Brinkman,A., G&fen, C., Kortleve, D.J., vanKessel,A.G. andDrop, S.L.S. (1988) EmboJ. 7: 2417-2423. lO.Lee,Y.L., Hmtz, R.L., James,P.M., Lee,P.D.K., Shively, J.E. andPowell,D.R. (1988) Mol. Endocrinol 2: 404411. 1l.Wood, W.I., Cachianes, G., Henzel,W.J., Winslow,G.A., Spencer,S.A., Hellmiss,R., Martin, J.L. andBaxter, R.C. (1988)Mol. Endocrinol.2: 1176-l185. 12.Mottola C., MacDonald,R.G., Brackett,J.L., Mole, J.E., Anderson,J.K., andCzech,M.P. (1986)J. Biol. Chem. 261: 11180-11188. 13. Margot. J.B., Binkert. C., Mary, J-L., Landwehr,J., Heinrich,G. andSchwander,J. (1989)Mol. Endocrlnol. 3: 1053-1060. 14. Brown, AL., Chiamtti.L., Orlowski,C.C., Mehhnan,T.. Burgess,W.H.. Ackerman, E.J., Bnmi, C.B., andRechler.M.M. (1989)J. Biol. Chem. 264:5148-5154. 15.Scott.C.D., Martin, J.L. andBaxter, R.C. (1985)Endocrinology 116: 1094-1101. 16.Feinberg,A.P., andVogelstein,B. (1983)Anal. Biochem. 132:6-13. 17ZhangH., Scholl,R., Browse,J. andSomervilleC. (1988)NucleicAcid Res 16: 1220. 18.Reed.K.C. andMann, D.A. (1985)NucleicAcid Res. 13:7207-7221. 19.Aviv, H., andLeder,P. (1972)hoc. Natl. Acad. Sci. USA. 69: 1408-1412. 20.Boutir1,J-M., JolicoeurC., Okamura,H., Gagnon,J., Edery,M.. Shirota,M.. Banville, D., Dusanter-Fourt,I., Djiane,J. andKelly, P.A. (1988)Cell 53: 69-77. 21.Watson,M.E.E. (1984)NucleicAcids Res. 12:5145-5165. 22.Zapf, J., Born, W., Chang,J.-Y., James,P., Froesch,E.R., andFischer,J.A. (1988) Biochem.Biophys.Res.Comm. 156: 1187-1194. 23.Russell,D.W., Schneider,W.J., Yamamoto,T., Luskey,K.L., Brown,M.S. andGoldstein, J.L. (1984)Cell 37: 577-585. 24.Binkei-tC., Landwehr,J., Mary, J.-L., Schwander,J. andHeinrich,G. (1989) EmboJ. 8: 2497-2502. 25.Ullrich, A., Gray, A., Tam, A.W., Yang-Feng,T., Tsubokawa,M., Collins,L., Henzel,W.. Le Bon,T., Kathunia.S., Chen,E., Jacobs,S.,Francke,U., Ramachandnm, J. and Fujita-Yamaguchl,Y. (1986)EmboJ. 10:2503-2512. 26.Blat, C., Bohlen,P., Villaudy, J., Chatelain,G., Golde,A. andHa@, L. (1989)J. Biol. Chem.264: 6021-6024. 27.Weisman,L.S., Krummel,B.M. andWilson,A.C. (1986)J. Biol. Chem. 261: 2309-2313. 897
Vol. 166, No. 2, 1990 January 30, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 892-897
CLONING AND CHARACTERIZATION OFTHE GROWTH HORMONE-DEPENDENTINSULIN-LIKE GROWTH FACTOR BINDING PROTEIN (IGFBP-3) IN THE RAT Anthony L. Albiston andAdrian C. Herington PrinceHenry’sInstituteof Medical Research, PrhrceHenry’sHospitalCampus, MonashMedical Centm,Melbourne,Australia3004 Received
December
12,
1989
We reportfor the first time the completeammoacidsequence for the growthhormone dependentinsulin-likegrowth factor bmdingprotein(IGFBP3) in the rat. A humanIGFBP-3 clonewasgeneratedusingthepolymemsechainreaction(PCR)andusedto screena rat liver cDNA library. cDNA clonesof the rat IGFBP-3 wereisolatedandthefull ammoacid sequence deduced.The sequence beginswith a putative,26 aminoacidsignalpeptide followedby a 265 ammoacidbindingprotein. The aminoacidsequence is over 80% homologous with the equivalenthumanIGFBP-3form andshowscompleteconservationof 18cysteineresiduesthat areclusteredat the aminoandcarboxyendsof theprotein. IGFBP3 is the bindingsubunitof the majorcirculatingIGFBP in the rat, andhencethe availability of precisestructuraldataandcDNA probesprovidesan importantopportunityfor a detailed studyof the control of IGFBP-3 synthesisat the level of geneexpression. o 1990 Academic Press,
mc.
The insulin-likegrowth factors-1and-II (IGF I, -11)arecirculatingmitogensthat stimulate growthboth in vivo andin vitro (1.2). In serumthe IGFs circulateessentiallycompletely boundto a family of evolutionaryte!ated,specificbindingproteins(3,4). Thesebinding proteinsprolongthehalf life of theIGFs andhavebeenshownto regulateIGF actionon cells in culture($6). In rat or humanseratwo majorformsof bindingproteinamdetectedby radioligandbindingstudies,a M,lSO,OOO(150K) form anda smallerM, 35 K form (4.7). The M, 150K bindingproteinis acid-dissociable into anIGF bindingsubunit,termed IGFBP-3,of M,-50 K andanacidlabilenon-bindingsubunitof M, 80,000-90,000 (8). The biosynthesis of IGFBPS is regulatedby growthhormone(GH) whereasthe smallerform, termedIGFBP-I. is not GH-dependent(3,4). The humanformsof IGFBP-1and-3 have recently beencloned(9-l 1). A third classof bindingprotein,IGFBP-2,wasoriginally isolatedfrom a fetal rat liver cell line (BRL9A) (12). The cDNA for the rat IGFBP-2has recentlybeenisolated(13, 14). Although IGFBP-3whichis synthesized primarily by the liver (7,15) is the predominantIGF bindingproteinin adultrat serum,it hasnot beenfully characterized. 0006-291X/90 Copyright All rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
892
Vol.
166, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
In thispaperwereportthe isolationof a cDNA encodingthe completesequence of the rat IGFBP-3. ‘Ihe deducedaminoacidsequence of the matureproteinis cysteinerich, 265 residuesin lengthandover 80%homologous with thehumanequivalent(11). Genomic Southernanalysissuggests that this is a singlecopygeneandnorthernanalysisshowsa single mRNA speciesin the liver, approximately2.4 kb in size.
MATERIALS AND METHODS Polvruerase chain
(PCR)
BaseduponthepublishedcDNA sequence of the humanIGFBP-3(11) two oligonucleotideprimerswereconstructed,primerA: 531-550nt andprimerB: the complementof 882-863nt. cDNA waspreparedby reversetranscriptionof 5~ of human liver poly A+ RNA using20pmol of primerB and110unitsof AMV reversetranscriptase (Pharmacia,Uppsala,Sweden)underthe conditionsrecommended by the supplier.A cDNA aliquotcorresponding to 0.5% of RNA wasusedfor PCRamplificationin 67 mM Tris pH 8.8 at 25-C 17mM (NH4)2SO4,3mMMgCl2, 10mM B-mercaptoethanol, 2 mg/mlgelatine, 0.5 M of eachprimer,500 M dNTP and2 U of Taq DNA polymerase(PerkinElmerCetus, Norwalk, U.S.A.). Thirty cycles(onecycle: 1 min at 92’C, 2 min at 55’C. 2 min at 72’C) werecarriedout in a 50@volumeusinga thermalcycler (Bartlett, Melbourne.Australia). The 351 bp productwassubcloned into aGem42 plasmidvector andits identity confirmed by dideoxy sequencing. Isolationof a cDNA clone A rat liver cDNA library in a&t 10vector (kindly donatedby ProfessorG. Schreiber, University of Melbourne)wasscreenedfor clonesof IGFBP-3. The 351bp PCRproductwas 32P-labelledby randompriming(16), hybridizationof duplicatefilters wasperformedat 42-C in 50%fonnamideandwasheswerein 2 X SSCat 42-C. Among 5 X lo6 plaques screened 7 werefound to bepositive. The largestclone,2.0 kb in size,wassubcloned into the Gem42 plasmidvector for furthermanipulation.
Relevantrestrictionfragmentsweresubclonedinto eitherGem42 or Ml3 vectors. Dideoxy chainterminationwasperformedusingSP6andT7 promoterprimersandMl3 universalprimersrespectively(17). In regionsthat lackedconvenientrestrictionsites specificallydesignedoligonucleotides weresynthesizedon a DNA synthesizer(Applied Biosystems,FosterCity, U.S.A.). GenomicSouthernAnalvsis High molecularweightrat liver DNA wasdigestedwith restrictionendonucleases accordingto the supplier’srecommended reactionconditions.The fragmentswereseparated on a 0.7%agarosegel andtransferredto a nylon membraneasdescribedby ReedandMann (18). The restrictionfragmentprobeusedwas930bp in length,containingthe entirecoding regionof the gene,andwas32P-labelledby randompriming. Hybridizationwasat 42-C in 50% formamideandthe filter wasgiven a final washin 0.1 X SSC, 1%SDSat 5o’C.
Pol A(+) mRNA waspurified from adultrat liver aspreviouslydescribed(19). Tenllgof by electmphoresis on a 1.2%agarose ~01~Af’) mRNA wasglyoxylated andthenseparated gel. The RNA wastransferredto a nylon membrane andhybridizedwith theprobedescribed for genomicSouthernanalysis.The filter wasgiven a fmal washin 0.1 X SSC.0.1% SDSat 55°C. 893
Vol.
166, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
RESULTS AND DISCUSSION A 351bppolymerasechainreactionproductof the humanIGPBP-3wasusedto screena rat liver cDNA library. Approximately 5 x lo6 cloneswerescreened revealingseven independent positiveclones.The largestclone,of 2.0 kb, containedthe entirecodingregion exceptfor the startcodon,ATG. A secondoverlappingclone,700bp in length,containedthe ATG codonplus36 bp of 5’ untranslatedsequence.The total sequence obtainedfrom these cloneswas2058bp (Pig. 1). Northernblot analysisof liver mRNA showeda singlemRNA speciesapproximately2.4 kb in sizeindicatingmatwe donot have a full lengthmRNA sequence (Pig. 2B). The 3’ untranslatedsequence obtaineddoesnot containa polyadenylation signalsuggesting that the 3’ regionis incomplete.An unusualfeatureof the3’ untranslated sequence is a 36 bp repetitiveTC sequence beginningat position1012.This repeating sequence is alsofoundin the 3’ untranslatedsequence of the rat pmlactinreceptormRNA (20), howeverthe significanceof this sequence isnot known. GenomicSouthernanalysis after hybridizationto the codingregionof the rat IGPBP-3cDNA showedonly 2 distinct bandsperlanefor eachof 4 restrictionenzymessuggesting thatthis is a singlecopy gene (Pig. 2A). The sequence hasanopenreadingframeof 291 aminoacidsbeginningwith amethionine residue.The first 26 residues,containinga hydrophobiccore,form a potentialsecretion signalpeptide(21). The maturepeptideis 265residues in lengthbeginningat Gly 27. The deducedaminoacidsequence of rat IGPBP-3,residues 27-68(Fig. 1). matchesexactly the NH2-terminalaminoacidsequence reportedby Zapf et al (22). The proteinhas4 potentialN-linked glycosylationsites(NXT or NXS), and2 shortserinethreoninerich domainswhichrepresentpotentialO-lied glycosylationsites(23). It is knownthat rat IGPBP-3isN-glycosylatcd(22) andthis accountsfor the discrepancybetween the predictedtranslatedmolecularweightof 28.8K andtheelectmphoreticallydetermined molecularweightof -50 K (7). The rat IGFBP-3is 83%homologous with the equivalenthumanBP(11). An insertionof two aminoacidsat residue30 andonedeletionat position103of the matureproteinare necessary to alignthehumansequence with the rat. The sequences have complete conservationof the eighteencysteineresidues present,which amclusteredat the N- andCtermlni of theprotein. The cysteineresiduesamhighly conservedacrossall of the distinct
Vol.
166, No. 2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Fia.Nucleotide sequence and deduced amino acid sequence of the rat IGFBP-3. The putative site of signal peptidase cleavage is indicated by an arrow. Potential glycosylation sites are underlined (N-glycosylatiow; 0-glycosylation - - -). Conserved cystelne residues compared to human IGFBP-3 are indicated(*).
IGF-bindingproteinclasses (humanandrat) sequenced to date(9-l 1,13,14,24) suggesting that they maybe involved in thebindingof IGF. Cysteine-richdomainshavebeenpostulated to constitutetheligandbindingdomainsof severalhormone/gmwth factor receptors, 895
Vol.
166, No. 2, 1990
BIOCHEMICAL
0.8
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
-
Fig. Southern and Northern blot analysis of IGF BP3. rat genomic DNA(10~) wasdigested withthefollowingenzymes: A) Sprague-Dawley
Ecoe (lanel), BamHl(lane2), Hind111 (lane3) andPstI(lane4). TheDNAwas througha0.7%agarose gel,transferred to anylonmembrane and electrbphorese restriction fragment comprised of thecodingsequence of probedwithah P-labelled theratIGFEP-3.Autoradiography wasfor 48hours. B) Tenmicrograms of ratliver poly A(+)RNA was glyoxylatedandelectrophoresed througha 1.2%agarose gel. TheRNAwastransferred to anylonmembrane and hybridizedwiththecodingsequence of theratIGFEP-3.RNAmarkers (ERL, Gaithersburg, U.S.A.)wererunin anadjacent laneandstained withethidium bromide to assess thesizeof theIGFEP3mRNAdetected.In thisexperiment autoradiography wasfor 12hours.
includingthe typeI IGF receptor(25), althoughthereisno aminoacidhomologybetween IGFBP3 andthe bindingdomainsof the IGF receptors(11). In additionto absoluteconservationof the cysteineresidues,all IGFBPsshowother limited sequence homologies at the N- andC- tennini. Rat IGFBP-3is no exception. A reportedN-terminalsequence for a mouseproteinwhich inhibitsserum-freefibroblastgrowth (26) is also83%homologous with rat IGFBP-3 indicatinga broadspecies conservationof this particulardomain.The N-terminalsequence of rat IGFBP-3,determinedby proteinisolation andsequencing (22) andconlirrnedby the presentcDNA-derivedsequence, is Gly-Ala-GlyAla-Val-Gly-Ala-Gly-Pro-Val-Val-Arg. Of particularinterestis the fact that this sequence is 58% homologous (with padding)with onepresentat the endof the proposedsignalpeptide for the BRL3A rat liver BP, IGFBP-2(13,14), adjacentto the N-terminalaminoacid(Glu) of the isolatedIGFBP-2protein(12). This may representanevolutionaryshift in the signal peptidecleavagesitesfor theseproteins(27). a shift whichmusthaveoccurredtier divergenceof the IGFBP genefamily itself. The significanceof sucha shift for the biological role and/orregulationof theseIGFBPsisnot known. 896
Vol.
166,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
In conclusion, we have isolated a cDNA encoding the rat growth hormone dependent insulin-like growth factor binding protein, IGFBP-3, and report for the first time the complete amino acid sequence of this important regulatory rat protein. The deduced amino acid sequence is over 80% homologous to human IGFBP-3. The availability of the rat cDNA will provide an opportunity to examine, in a very convenient model system, the nature and tissue distribution of IGFBP-3 mRNA and its pharmacological/physiological regulation. ACKNOWLEDGMENTS
Thesestudiesweresupportedin part by the NationalHealthandMedical Research Councilof Australia. WethankMrs. JoniLaw andMS SuePanckrldgefor helpwith the preparationof the manuscript. REFJXRENCES
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