cloning and characterization of the growth hormone-dependent insulin-like growth factor binding...

Vol. 166, No. 2, 1990 January 30, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 892-897

CLONING AND CHARACTERIZATION OFTHE GROWTH HORMONE-DEPENDENT INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN

(IGFBP-3) IN THE RAT

Anthony L. Albiston and Adrian C. Herington

Prince Henry’s Institute of Medical Research, Prhrce Henry’s Hospital Campus, Monash Medical Centm, Melbourne, Australia 3004

Received December 12, 1989

We report for the first time the complete ammo acid sequence for the growth hormone dependent insulin-like growth factor bmding protein (IGFBP3) in the rat. A human IGFBP-3 clone was generated using the polymemse chain reaction (PCR) and used to screen a rat liver cDNA library. cDNA clones of the rat IGFBP-3 were isolated and the full ammo acid sequence deduced. The sequence begins with a putative, 26 amino acid signal peptide followed by a 265 ammo acid binding protein. The amino acid sequence is over 80% homologous with the equivalent human IGFBP-3 form and shows complete conservation of 18 cysteine residues that are clustered at the amino and carboxy ends of the protein. IGFBP3 is the binding subunit of the major circulating IGFBP in the rat, and hence the availability of precise structural data and cDNA probes provides an important opportunity for a detailed study of the control of IGFBP-3 synthesis at the level of gene expression. o 1990 Academic

Press, mc.

The insulin-like growth factors -1 and -II (IGF I, -11) are circulating mitogens that stimulate

growth both in vivo and in vitro (1.2). In serum the IGFs circulate essentially completely

bound to a family of evolutionary te!ated, specific binding proteins (3,4). These binding

proteins prolong the half life of the IGFs and have been shown to regulate IGF action on cells

in culture ($6). In rat or human sera two major forms of binding protein am detected by

radioligand binding studies, a M,lSO,OOO (150 K) form and a smaller M, 35 K form (4.7).

The M, 150 K binding protein is acid-dissociable into an IGF binding subunit, termed

IGFBP-3, of M,-50 K and an acid labile non-binding subunit of M, 80,000-90,000 (8). The

biosynthesis of IGFBPS is regulated by growth hormone (GH) whereas the smaller form,

termed IGFBP-I. is not GH-dependent (3,4). The human forms of IGFBP-1 and -3 have

recently been cloned (9-l 1). A third class of binding protein, IGFBP-2, was originally

isolated from a fetal rat liver cell line (BRL9A) (12). The cDNA for the rat IGFBP-2 has

recently been isolated (13, 14). Although IGFBP-3 which is synthesized primarily by the

liver (7,15) is the predominant IGF binding protein in adult rat serum, it has not been fully

characterized.

0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved. 892

Vol. 166, No. 2, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

In this paper we report the isolation of a cDNA encoding the complete sequence of the rat

IGFBP-3. ‘Ihe deduced amino acid sequence of the mature protein is cysteine rich, 265

residues in length and over 80% homologous with the human equivalent (11). Genomic

Southern analysis suggests that this is a single copy gene and northern analysis shows a single

mRNA species in the liver, approximately 2.4 kb in size.

MATERIALS AND METHODS

Polvruerase chain (PCR)

Based upon the published cDNA sequence of the human IGFBP-3 (11) two oligonucleotide primers were constructed, primer A: 531-550 nt and primer B: the complement of 882-863 nt. cDNA was prepared by reverse transcription of 5~ of human liver poly A+ RNA using 20 pmol of primer B and 110 units of AMV reverse transcriptase (Pharmacia, Uppsala, Sweden) under the conditions recommended by the supplier. A cDNA aliquot corresponding to 0.5% of RNA was used for PCR amplification in 67 mM Tris pH 8.8 at 25-C 17 mM (NH4)2SO4,3mM MgCl2, 10 mM B-mercaptoethanol, 2 mg/ml gelatine, 0.5 M of each primer, 500 M dNTP and 2 U of Taq DNA polymerase (Perkin Elmer Cetus, Norwalk, U.S.A.). Thirty cycles (one cycle: 1 min at 92’C, 2 min at 55’C. 2 min at 72’C) were carried out in a 50@ volume using a thermal cycler (Bartlett, Melbourne. Australia). The 35 1 bp product was subcloned into a Gem 42 plasmid vector and its identity confirmed by dideoxy sequencing.

Isolation of a cDNA clone

A rat liver cDNA library in a&t 10 vector (kindly donated by Professor G. Schreiber, University of Melbourne) was screened for clones of IGFBP-3. The 351 bp PCR product was 32P-labelled by random priming (16), hybridization of duplicate filters was performed at 42-C in 50% fonnamide and washes were in 2 X SSC at 42-C. Among 5 X lo6 plaques screened 7 were found to be positive. The largest clone, 2.0 kb in size, was subcloned into the Gem 42 plasmid vector for further manipulation.

Relevant restriction fragments were subcloned into either Gem 42 or Ml3 vectors. Dideoxy chain termination was performed using SP6 and T7 promoter primers and Ml3 universal primers respectively (17). In regions that lacked convenient restriction sites specifically designed oligonucleotides were synthesized on a DNA synthesizer (Applied Biosystems, Foster City, U.S.A.).

Genomic Southern Analvsis

High molecular weight rat liver DNA was digested with restriction endonucleases according to the supplier’s recommended reaction conditions. The fragments were separated on a 0.7% agarose gel and transferred to a nylon membrane as described by Reed and Mann (18). The restriction fragment probe used was 930 bp in length, containing the entire coding region of the gene, and was 32 P-labelled by random priming. Hybridization was at 42-C in 50% formamide and the filter was given a final wash in 0.1 X SSC, 1% SDS at 5o’C.

Pol A(+) mRNA was purified from adult rat liver as previously described (19). Tenllg of ~01~ A f’) mRNA was glyoxylated and then separated by electmphoresis on a 1.2% agarose gel. The RNA was transferred to a nylon membrane and hybridized with the probe described for genomic Southern analysis. The filter was given a fmal wash in 0.1 X SSC. 0.1% SDS at 55°C.

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RESULTS AND DISCUSSION

A 351 bp polymerase chain reaction product of the human IGPBP-3 was used to screen a

rat liver cDNA library. Approximately 5 x lo6 clones were screened revealing seven

independent positive clones. The largest clone, of 2.0 kb, contained the entire coding region

except for the start codon, ATG. A second overlapping clone, 700 bp in length, contained the

ATG codon plus 36 bp of 5’ untranslated sequence. The total sequence obtained from these

clones was 2058 bp (Pig. 1). Northern blot analysis of liver mRNA showed a single mRNA

species approximately 2.4 kb in size indicating mat we do not have a full length mRNA

sequence (Pig. 2B). The 3’ untranslated sequence obtained does not contain a polyadenylation

signal suggesting that the 3’ region is incomplete. An unusual feature of the 3’ untranslated

sequence is a 36 bp repetitive TC sequence beginning at position 1012. This repeating

sequence is also found in the 3’ untranslated sequence of the rat pmlactin receptor mRNA

(20), however the significance of this sequence is not known. Genomic Southern analysis

after hybridization to the coding region of the rat IGPBP-3 cDNA showed only 2 distinct

bands per lane for each of 4 restriction enzymes suggesting that this is a single copy gene

(Pig. 2 A).

The sequence has an open reading frame of 29 1 amino acids beginning with a methionine

residue. The first 26 residues, containing a hydrophobic core, form a potential secretion

signal peptide (21). The mature peptide is 265 residues in length beginning at Gly 27. The

deduced amino acid sequence of rat IGPBP-3, residues 27-68 (Fig. 1). matches exactly the

NH2-terminal amino acid sequence reported by Zapf et al (22).

The protein has 4 potential N-linked glycosylation sites (NXT or NXS), and 2 short serine-

threonine rich domains which represent potential O-lied glycosylation sites (23). It is

known that rat IGPBP-3 is N-glycosylatcd (22) and this accounts for the discrepancy between

the predicted translated molecular weight of 28.8 K and the electmphoretically determined

molecular weight of -50 K (7).

The rat IGFBP-3 is 83% homologous with the equivalent human BP (11). An insertion of

two amino acids at residue 30 and one deletion at position 103 of the mature protein are

necessary to align the human sequence with the rat. The sequences have complete

conservation of the eighteen cysteine residues present, which am clustered at the N- and C-

termlni of the protein. The cysteine residues am highly conserved across all of the distinct


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-binding protein classes (human and rat) sequenced to date (9-l 1,13,14,24) suggesting

that they may be involved in the binding of IGF. Cysteine-rich domains have been postulated

to constitute the ligand binding domains of several hormone/gmwth factor receptors,

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0.8 -

Fig. Southern and Northern blot analysis of IGF BP3. A) Sprague-Dawley rat genomic DNA (10~) was digested with the following enzymes:

Ecoe (lane l), BamHl (lane 2), Hind111 (lane 3) and PstI (lane 4). The DNA was electrbphorese probed with a h

through a 0.7% agarose gel, transferred to a nylon membrane and P-labelled restriction fragment comprised of the coding sequence of

the rat IGFEP-3. Autoradiography was for 48 hours. B) Ten micrograms of rat liver poly A(+) RNA was glyoxylated and electrophoresed

through a 1.2% agarose gel. The RNA was transferred to a nylon membrane and hybridized with the coding sequence of the rat IGFEP-3. RNA markers (ERL, Gaithersburg, U.S.A.) were run in an adjacent lane and stained with ethidium bromide to assess the size of the IGFEP3 mRNA detected. In this experiment autoradiography was for 12 hours.

including the type I IGF receptor (25), although there is no amino acid homology between

IGFBP3 and the binding domains of the IGF receptors (11).

In addition to absolute conservation of the cysteine residues, all IGFBPs show other

limited sequence homologies at the N- and C- tennini. Rat IGFBP-3 is no exception. A

reported N-terminal sequence for a mouse protein which inhibits serum-free fibroblast growth

(26) is also 83% homologous with rat IGFBP-3 indicating a broad species conservation of this

particular domain. The N-terminal sequence of rat IGFBP-3, determined by protein isolation

and sequencing (22) and conlirrned by the present cDNA-derived sequence, is Gly-Ala-Gly-

Ala-Val-Gly-Ala-Gly-Pro-Val-Val-Arg. Of particular interest is the fact that this sequence is

58% homologous (with padding) with one present at the end of the proposed signal peptide

for the BRL3A rat liver BP, IGFBP-2 (13,14), adjacent to the N-terminal amino acid (Glu)

of the isolated IGFBP-2 protein (12). This may represent an evolutionary shift in the signal

peptide cleavage sites for these proteins (27). a shift which must have occurred tier

divergence of the IGFBP gene family itself. The significance of such a shift for the biological

role and/or regulation of these IGFBPs is not known.

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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

These studies were supported in part by the National Health and Medical Research Council of Australia. We thank Mrs. Joni Law and MS Sue Panckrldge for help with the preparation of the manuscript.

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cloning and characterization of the growth hormone-dependent insulin-like growth factor binding...

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