Vol. 180, No. 2, 1991 October 31, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 489-495
IDENTIFICATION OF A NOVEL GROWTH HORMONE BINDING PROTEIN mRNA IN RAT LIVER
Tak S. Tiong and Adrian C. Herington Department of Clinical Biochemistry, Royal Children's Hospital and Prince Henry's Institute of Medical Research, Monash Medical Centre, Melbourne 3052. Australia
Received September 6, 1991
The presence of highly specific serum binding proteins for growth hormone (GH) has been well characterized in many species. In the rat the major growth hormone-binding protein (GH-BP) is a truncated, variant form of the target tissue GH receptor and is derived by an alternative mRNA splicing event. The GHBP mRNA is coexpressed in all tissues expressing the full length GH receptor. In the present study, we have made an oligonucleotide probe to the unique hydrophilic tail of the rat GHBP mRNA (l.2kb) and identified a novel GHBPlike mRNA of 2.6 kb transcript in addition to the 1.2 kb transcript. This unique 2.6 kb transcript was expressed/detected only in rat liver. There was no significant difference in abundance between the sexes or during pregnancy, implying that this transcript may be regulated independently of the 1.2 kb mRNA. The 2.6 kb transcript was clearly identifiable in the fetus, as was the 1.2 kb transcript, but showed virtually no change in abundance with age, in sharp contrast to the 1.2 kb mRNA, which has a distinct developmental pattern, being low in the fetus and peaking early postnatally. RNAse H treatment suggested that this 2.6 kb transcript is polyadenylated. A corresponding 2.6 kb mRNA has been detected using a longer eDNA or cRNA probe for the GH-binding domain of the rat GHR/GHBP. These data collectively suggest that the 2.6 kb mRNA transcript is a bonafide but tissue-specific GHBP mRNA and that the 1.2 and 2.6 kb mRNAs are likely to differ primarily with respect to the length of the 3' untranslated region of the sequence. © 1991 Academicpress, Inc.
The GH receptor (GHR) was first cloned and sequenced from rabbit liver in 1987 (1). Cross-hybridization studies subsequently led to the cloning of equivalent GHR cDNAs in human (1), mouse (2), rat (3, 4), bovine (5) and ovine livers (6). All eDNAs had a high degree of nucleotide homology, spanned 3.9 - 4.7 kb in length and encoded a protein of -624 amino acids containing a single, transmembrane-spanning hydrophobic domain. In addition to a plasma membrane receptor it has been demonstrated that quite specific soluble GH binding proteins are present in serum (7-12). Recent evidence indicates that the rabbit and human growth hormone-binding proteins (GH-BP) are related to, and possibly derived from, the hepatic GHR by proteolytic cleavage (1), whereas in rats and mice, the bindingprotein is generated from a variant truncated mRNA resulting from alternative splicing of the GHR mRNA at the exon 7-8 boundary of the hepatic GHR genomic sequence (2, 4, 13). In the rat and mouse GHBP mRNA exons 8-10 of the equivalent human GHR mRNA were substituted by a short alternative exon which encoded a highly hydrophilic protein sequence of just 17 (rat) (13) or 25 amino acids (mouse) (2). This GHBP mRNA was 1.2-1.4 kb in
489
0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in an}, form reserved.
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
size. The GHBP mRNA was found to coexpress with the GHR mRNA in a wide variety of rat tissues, including liver, the most abundant source of GHBP mRNA (14, 15). Recently, our group also demonstrated that the 1.2 kb GHBP mRNA is developmentally regulated and, unlike the full length GH receptor (4.5 kb), is clearly identifiable in fetal liver (16).
In a continuation of our studies on the nature, regulation and possible role(s) of the GHBP, we have made an oligonucleotide probe to the unique "hydrophilic tail" domain of the rat 1.2 kb GHBP eDNA in order to examine specific regulation of this species at the level of gene expression. As a result, in routine Northern blots of rat liver mRNA we have identified a novel GHBP-like mRNA of 2.6 kb in addition to the expected 1.2 kb GHBP mRNA. We now report this finding together with additional studies in the rat to further characterize this novel mRNA transcript and its regulation. MATERIALS AND METHODS Probe A 30-mer oligonucleotide probe was synthesized complementary to nucleotides 805-834 of the unique "hydrophilic tail" domain of the published rat 1.2 kb GHBP cDNA sequence (4). This oligonucleotide had no recognized homology with the published full length (4.5 kb) GHR mRNA. A n i m a l s / C o l l e c t i o n of Tissues
Sprague-Dawley rats were obtained from the Central Animal House, Monash University, Melbourne, Australia. All animals were housed under standard environmental conditions with constant temperature (22 + 2°C) and a controlled 12-hour light-dark illumination cycle. The animals had free access to standard pelleted rat chow and tap water. A.
Tissue distribution :
Tissues (liver, kidney, adrenal, intestine, adipose, muscle, testis, thymus, heart) were obtained from adult male rats (35 days old) whilst ovaries were obtained from adult female rats (35 days old). B.
Physiological Studies :
Livers were obtained from age-matched (80 day old) male, female and pregnant rats (Day 19 gestation). C.
Ontogenic studies :
Livers were obtained from male rats (10-80 days) and from a pool of 10 fetal pups taken on Day 19 of gestation from 80 day old pregnant rats. For RNA processing, tissues were immediately removed, rapidly frozen in liquid nitrogen and stored at -80°C until processing. Preparation of R N A / N o r t h e r n Blot Hybridization
Total RNA was isolated as described previously (17) using the standard Guanidine thiocyanate/Cesium chloride method (18). Northern blot hybridization was performed as previously described (17, 19) except that the nylon membranes were prehybridized at 50°C for 4-5 h in hybridization buffer consisting of 5 X SSC (1 X SSC is 0.15 M sodium chloride and 0.015 M sodium citrate, pH 7.4), 50 mM sodium phosphate pH 7.0, 5 X Denhardt's solution (1 X Denhardt's solution is 0.02% each of bovine serum albumin, Ficoll 400, pyrrolidone), 1% sodium dodecyl sulphate (SDS), 0.1% sodium pyrophosphate and 3mg/ml herring sperm DNA. The probe was labeled by a tailing reaction using terminal transferase 490
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
enzyme (Boehringer Mannheim, Mannheim, F D R ) and 1 X l0 b c p m / m l were added to the buffer. Hybridization then continued at 50°C for 16-20 h. The membranes were washed initially with 2 X SSC/0.1% SDS at room temperature (20 min.) followed by washing at increasing stringency up to 0.1 X SSC/0.1% SDS at 37°C. Relative m R N A levels were determined by scanning densitometry (Isco gel scanner 1312 with a Hewlett Packard integrator), m R N A levels in arbitrary absorbance units, were divided by the integrated densitometry value obtained for a control 18S ribosmal R N A oligonucleotide probe (20). Deadenylation of G H B P mRNAs Deadenylation was performed according to the method previously described (21). Briefly, 20#g of total R N A were incubated for 15 rain. at 20°C in 0.05 M KC1 with 2#g of oligo [(dT) 12-18] to form duplexes with all poly A+ m R N A tails. RNase H (4 #1) was added and the mixture was subsequently incubated for 20 min. at 37°C. Twenty volumes of buffer containing 0.3 M NaCI, 0.01 M Tris-C1 at pH7.4, 0.5% NaDodS04, and 200 #g of proteinase K per ml was added to stop the reaction. The mixture was incubated on ice for 10 min. and at 20°C for 20 min. to allow the digestion of RNase H by proteinase K. The R N A solution was then immediately submitted to batch extraction on oligo (dT)-cellulose at 2°C. After spinning down the oligo (dT) solid phase the supernatant was collected and submitted to phenol extraction. The deadenylated R N A was recovered by ethanol precipitation. Statistics The results are expressed as mean +_ SEM for 3 individual rats. The means were compared by analysis of variance and Student's t test. Differences were considered significant when P
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 489-495
IDENTIFICATION OF A NOVEL GROWTH HORMONE BINDING PROTEIN mRNA IN RAT LIVER
Tak S. Tiong and Adrian C. Herington Department of Clinical Biochemistry, Royal Children's Hospital and Prince Henry's Institute of Medical Research, Monash Medical Centre, Melbourne 3052. Australia
Received September 6, 1991
The presence of highly specific serum binding proteins for growth hormone (GH) has been well characterized in many species. In the rat the major growth hormone-binding protein (GH-BP) is a truncated, variant form of the target tissue GH receptor and is derived by an alternative mRNA splicing event. The GHBP mRNA is coexpressed in all tissues expressing the full length GH receptor. In the present study, we have made an oligonucleotide probe to the unique hydrophilic tail of the rat GHBP mRNA (l.2kb) and identified a novel GHBPlike mRNA of 2.6 kb transcript in addition to the 1.2 kb transcript. This unique 2.6 kb transcript was expressed/detected only in rat liver. There was no significant difference in abundance between the sexes or during pregnancy, implying that this transcript may be regulated independently of the 1.2 kb mRNA. The 2.6 kb transcript was clearly identifiable in the fetus, as was the 1.2 kb transcript, but showed virtually no change in abundance with age, in sharp contrast to the 1.2 kb mRNA, which has a distinct developmental pattern, being low in the fetus and peaking early postnatally. RNAse H treatment suggested that this 2.6 kb transcript is polyadenylated. A corresponding 2.6 kb mRNA has been detected using a longer eDNA or cRNA probe for the GH-binding domain of the rat GHR/GHBP. These data collectively suggest that the 2.6 kb mRNA transcript is a bonafide but tissue-specific GHBP mRNA and that the 1.2 and 2.6 kb mRNAs are likely to differ primarily with respect to the length of the 3' untranslated region of the sequence. © 1991 Academicpress, Inc.
The GH receptor (GHR) was first cloned and sequenced from rabbit liver in 1987 (1). Cross-hybridization studies subsequently led to the cloning of equivalent GHR cDNAs in human (1), mouse (2), rat (3, 4), bovine (5) and ovine livers (6). All eDNAs had a high degree of nucleotide homology, spanned 3.9 - 4.7 kb in length and encoded a protein of -624 amino acids containing a single, transmembrane-spanning hydrophobic domain. In addition to a plasma membrane receptor it has been demonstrated that quite specific soluble GH binding proteins are present in serum (7-12). Recent evidence indicates that the rabbit and human growth hormone-binding proteins (GH-BP) are related to, and possibly derived from, the hepatic GHR by proteolytic cleavage (1), whereas in rats and mice, the bindingprotein is generated from a variant truncated mRNA resulting from alternative splicing of the GHR mRNA at the exon 7-8 boundary of the hepatic GHR genomic sequence (2, 4, 13). In the rat and mouse GHBP mRNA exons 8-10 of the equivalent human GHR mRNA were substituted by a short alternative exon which encoded a highly hydrophilic protein sequence of just 17 (rat) (13) or 25 amino acids (mouse) (2). This GHBP mRNA was 1.2-1.4 kb in
489
0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in an}, form reserved.
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
size. The GHBP mRNA was found to coexpress with the GHR mRNA in a wide variety of rat tissues, including liver, the most abundant source of GHBP mRNA (14, 15). Recently, our group also demonstrated that the 1.2 kb GHBP mRNA is developmentally regulated and, unlike the full length GH receptor (4.5 kb), is clearly identifiable in fetal liver (16).
In a continuation of our studies on the nature, regulation and possible role(s) of the GHBP, we have made an oligonucleotide probe to the unique "hydrophilic tail" domain of the rat 1.2 kb GHBP eDNA in order to examine specific regulation of this species at the level of gene expression. As a result, in routine Northern blots of rat liver mRNA we have identified a novel GHBP-like mRNA of 2.6 kb in addition to the expected 1.2 kb GHBP mRNA. We now report this finding together with additional studies in the rat to further characterize this novel mRNA transcript and its regulation. MATERIALS AND METHODS Probe A 30-mer oligonucleotide probe was synthesized complementary to nucleotides 805-834 of the unique "hydrophilic tail" domain of the published rat 1.2 kb GHBP cDNA sequence (4). This oligonucleotide had no recognized homology with the published full length (4.5 kb) GHR mRNA. A n i m a l s / C o l l e c t i o n of Tissues
Sprague-Dawley rats were obtained from the Central Animal House, Monash University, Melbourne, Australia. All animals were housed under standard environmental conditions with constant temperature (22 + 2°C) and a controlled 12-hour light-dark illumination cycle. The animals had free access to standard pelleted rat chow and tap water. A.
Tissue distribution :
Tissues (liver, kidney, adrenal, intestine, adipose, muscle, testis, thymus, heart) were obtained from adult male rats (35 days old) whilst ovaries were obtained from adult female rats (35 days old). B.
Physiological Studies :
Livers were obtained from age-matched (80 day old) male, female and pregnant rats (Day 19 gestation). C.
Ontogenic studies :
Livers were obtained from male rats (10-80 days) and from a pool of 10 fetal pups taken on Day 19 of gestation from 80 day old pregnant rats. For RNA processing, tissues were immediately removed, rapidly frozen in liquid nitrogen and stored at -80°C until processing. Preparation of R N A / N o r t h e r n Blot Hybridization
Total RNA was isolated as described previously (17) using the standard Guanidine thiocyanate/Cesium chloride method (18). Northern blot hybridization was performed as previously described (17, 19) except that the nylon membranes were prehybridized at 50°C for 4-5 h in hybridization buffer consisting of 5 X SSC (1 X SSC is 0.15 M sodium chloride and 0.015 M sodium citrate, pH 7.4), 50 mM sodium phosphate pH 7.0, 5 X Denhardt's solution (1 X Denhardt's solution is 0.02% each of bovine serum albumin, Ficoll 400, pyrrolidone), 1% sodium dodecyl sulphate (SDS), 0.1% sodium pyrophosphate and 3mg/ml herring sperm DNA. The probe was labeled by a tailing reaction using terminal transferase 490
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
enzyme (Boehringer Mannheim, Mannheim, F D R ) and 1 X l0 b c p m / m l were added to the buffer. Hybridization then continued at 50°C for 16-20 h. The membranes were washed initially with 2 X SSC/0.1% SDS at room temperature (20 min.) followed by washing at increasing stringency up to 0.1 X SSC/0.1% SDS at 37°C. Relative m R N A levels were determined by scanning densitometry (Isco gel scanner 1312 with a Hewlett Packard integrator), m R N A levels in arbitrary absorbance units, were divided by the integrated densitometry value obtained for a control 18S ribosmal R N A oligonucleotide probe (20). Deadenylation of G H B P mRNAs Deadenylation was performed according to the method previously described (21). Briefly, 20#g of total R N A were incubated for 15 rain. at 20°C in 0.05 M KC1 with 2#g of oligo [(dT) 12-18] to form duplexes with all poly A+ m R N A tails. RNase H (4 #1) was added and the mixture was subsequently incubated for 20 min. at 37°C. Twenty volumes of buffer containing 0.3 M NaCI, 0.01 M Tris-C1 at pH7.4, 0.5% NaDodS04, and 200 #g of proteinase K per ml was added to stop the reaction. The mixture was incubated on ice for 10 min. and at 20°C for 20 min. to allow the digestion of RNase H by proteinase K. The R N A solution was then immediately submitted to batch extraction on oligo (dT)-cellulose at 2°C. After spinning down the oligo (dT) solid phase the supernatant was collected and submitted to phenol extraction. The deadenylated R N A was recovered by ethanol precipitation. Statistics The results are expressed as mean +_ SEM for 3 individual rats. The means were compared by analysis of variance and Student's t test. Differences were considered significant when P
