0013-7227/91/1293-1628$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 129, No. 3 Printed in U.S.A.
Tissue Distribution, Characterization, and Regulation of Messenger Ribonucleic Acid for Growth Hormone Receptor and Serum Binding Protein in the Rat* TAK S. TIONG AND A. C. HERINGTON Prince Henry's Institute of Medical Research, Prince Henry's Hospital Campus, Monash Medical Centre, Melbourne 3004, Australia
ABSTRACT. The distribution of GH receptor (GHR) and GH-binding protein (GHBP) mRNAs in multiple rat tissues was examined by Northern blotting using a cDNA fragment encoding the common extracellular domain of the GHR and the serum GHBP. Both GHR and GHBP mRNAs [4.5 and 1.2 kilobases (kb), respectively] were present in liver, kidney, adrenal, heart, muscle, ovary, mammary gland, gastrointestinal tract, and adipose tissue, but were barely or not detectable in testis, thymus, or brain. These observations suggest that GH exerts direct effects across a broad spectrum of rat tissues. Nuclease protection analysis also confirmed the presence in extrahepatic tissues of a GHR mRNA with up to 50% of the cytoplasmic domain being identical in sequence to that of the hepatic GHR mRNA. This suggests, but does not prove, that different receptor classes with differing intracellular signalling mechanisms may not exist. It is also clear from our studies that liver was the most abundant
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HE LIVER is a well known target tissue for GH action. The biological effects of GH, mediated by the binding of GH to specific, high affinity receptors present in target tissue(s), have been well studied in adipocytes (1), hepatocytes (2), and IM-9 lymphocytes (3). The hepatic GH receptor (GHR) has been well characterized from several species, including rabbit (46), rat (7), and mouse (8, 9). The primary structure of the GH receptor in rabbit, human, rat, and mouse liver has been deduced from the nucleic acid sequence of cloned cDNA (5, 10-12). The sequences share considerable homology (~70%) across species and, interestingly, also show significant homology with the rat, rabbit, and human PRL receptors, and the family of interleukin receptors (13), but not to other known hormone receptor molecules. The full-length hepatic GHR cDNA [4-4.5 Received February 8,1991. Address requests for reprints to: Dr. A. C. Herington, Department of Clinical Biochemistry, Royal Children's Hospital, Flemington Road, Parkville 3052, Australia. * This work was supported by the William Buckland Foundation (in the form of a postgraduate scholarship to T.S.T.) and a grant from the National Health and Medical Research Council of Australia (to A.C.H.).
source of the truncated (1.2 kb) mRNA and is, therefore, believed to be the primary site of GHBP synthesis. Also, more importantly, at least in the rat, it was only GHBP mRNA that was up-regulated to any extent during pregnancy (female vs. pregnant, P < 0.001). No significant changes were observed in the abundance of the full-length (4.5 kb) GHR mRNA. This pregnancy-related change in GHBP mRNA was also accompanied by a comparable increase in the actual level of measurable serum GHBP (female vs. pregnant, P = 0.005). Little change was seen in hepatic membrane binding. These data suggest that the GHBP and GHR are both widely coexpressed, but that the expression is not always coordinately regulated. This raises the possibility that the GHBP and GHR may have distinct roles in the regulation of GH delivery and action. {Endocrinology 129: 1628-1634, 1991)
kilobases (kb)] consists of an extracellular hormonebinding domain, a single hydrophobic transmembrane domain, and a cytoplasmic domain. Examination of the C-terminal sequence revealed no recognized consensus sequences for the classical receptor second messenger systems. In addition to the large (mol wt, ~ 110,000) membranebound tissue GHR, a truncated soluble GH-binding protein (GHBP) has been reported in serum (14) and cytosolic fractions of rabbit tissues (15). Subsequently, specific serum GHBPs have been identified in several other species, including human (16-18), mouse (19), and rat (20). Recent cloning studies in the rat and mouse (1012) have shown the GHBP in these species to be the product of an alternative splicing event (at the exon 7exon 8 junction) during processing of the full-length GHR mRNA. The resultant mRNA is truncated, being only 1.2-1.4 kb. It has the same extracellular domain (exons 2-7) and lacks a hydrophobic transmembrane domain (exon 8) and the full-length intracellular domain (exons 9 and 10). Earlier studies in the rabbit and human did not identify an equivalent GHBP mRNA, and it was suggested that the serum GHBP was a proteolytically 1628
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mRNA FOR GHR AND GHBP
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cleaved form of the membrane receptor (5). This distinction between rat/mouse and human/rabbit has not yet been resolved. The role of the serum GHBP is not clearly understood. In part to investigate this role studies have begun to examine the regulation of the serum GHBP at both the protein and mRNA level and to compare this with regulation of the full-length membrane-bound GHR. Recent studies by Bick et al. (21) have indicated some changes in rat serum GHBP concentrations in hypophysectomized rats given a continuous sc infusion of hGH, while studies in the mouse indicated a sexual dimorphism (22) and increases in both mRNA and protein levels during pregnancy (23). In the studies reported here, we have used polymerase chain reaction (PCR)-derived fragments of either the common extracellular domain or the GHR-specific cytoplasmic domain of the cloned rat liver GHR cDNA (10) to identify the number, size, and tissue distribution of the mRNAs for the GHR and GHBP. Preliminary studies on the physiological regulation of these particular mRNAs and their correlation with direct [125I]GH binding studies are also reported.
Amersham, Aylesbury, Buckinghamshire, United Kingdom) by capillary blotting. The membranes were baked at 80 C for 2 h and UV cross-linked for 20 min. For riboprobes (cRNA probes), the membranes were prehybridized at 65 C in hybridization solution [50% formamide, 5 x SSPE (1 x SSPE is 0.15 M sodium chloride, 10 mM sodium phosphate, and 1 mM EDTA), 0.15 M Tris-HCl (pH 8.0), 1% sodium dodecyl sulfate (SDS), and 500 mg/ml heparin] before hybridization at 65 C for 16-20 h with about 106 cpm/ml labeled probes. All blots were then washed initially with 2 X SSC-0.1% SDS at room temperature (20 min), followed by washes of increasing stringency up to 0.2 x SSC at 65 C (cytoplasmic probe) or 0.2 x SSC at 50 C (extracellular probe). Membranes were then exposed to Kodak X-AR (Eastman Kodak, Rochester, NY) x-ray film with DuPont Lightning Plus intensifying screens (Wilmington, DE) at -70 C. Before rehybridization with a different probe, blots were stripped by boiling for 3 min in the presence of 0.1% SDS, and the complete removal of previously hybridized probe was determined by autoradiography at -70 C overnight. Relative mRNA levels were determined by densitometry [ISCO gel scanner 1312 (Lincoln, NE) with a Hewlett-Packard integrator (Palo Alto, CA)]. Subsequently, mRNA levels in arbitary absorbance units were divided by the value obtained for a control probe [an 18S rat ribosomal RNA oligonucleotide (30 mer)].
Materials and Methods
cRNA probes: cytoplasmic fragment. Based upon the published cDNA sequence of the rat GHR (10, 11), two oligonucleotide primers were synthesized: primer A, nucleotides (nt) 1401-1430 (which includes a Kpnl restriction site), and primer B, a complementary sequence to nt 1844-1873 (including a Hindlll site). By using cDNA prepared from poly(A)+ mRNA of rat liver, the 472-nt cDNA sequence between the primers A and B was successfully amplified by PCR (25) [40 cycles consisting of 1.5 min at 90 C (denaturation), 2 min at 55 C (annealing) and a 72 C extension (1 min)]. The amplified product gave the expected size on an ethidium bromide-stained nondenaturing gel and was subcloned into a pGEM 4Z plasmid vector (Promega, Madison, WI). Its orientation was confirmed by dideoxy sequencing (26), and a 32P-labeled riboprobe (cRNA) was generated from the T7 promoter as indicated by Promega.
Collection of tissues
All animals were maintained and killed according to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, as published by the National Health and Medical Research Council of Australia. The studies were approved by the Animal Ethics Experimentation Committee of the Monash Medical Centre. To study the tissue distribution, liver, heart, adrenal, kidney, skeletal muscle, thymus, testis, ovary, mammary gland, fat pad, and gastrointestinal tract (GIT) from adult male (35 days), adult female, and pregnant (19th day of gestation) SpragueDawley rats were collected for RNA extraction. Livers from 80day-old male, female, and pregnant (19th day of gestation) Sprague-Dawley rats were also used for both RNA extraction and membrane-binding studies. Trunk blood was collected for binding studies. The tissues for RNA preparation were removed rapidly after decapitation, immediately frozen in liquid nitrogen, and stored at -80 C until processing, whereas for binding studies tissues were collected into 0.3 M sucrose containing 1 mM phenylmethylsulfonylfluoride and 1000 kallikrein inactivator units Trasylol/ml on ice and stored at -20 C until processing. Preparation and analysis of RNA Total RNA was isolated as described in detail previously (24). Northern blot hybridization was also performed as previously described (24). Total RNA was denatured in 1 M glyoxal50% dimethylsulfoxide and electrophoresed on a 1% agarose gel in 10 mM phosphate buffer, pH 7.0. RNA was then transferred to Hybond-N membrane (Amersham International,
Probes
Si-nuclease mapping. A second GHR riboprobe was derived from the cytoplasmic probe by subcloning an approximately 180-basepair (bp) Kpnl-Hincll fragment into the pGEM3Z vector (Promega), and an antisense RNA control probe was prepared by linearizing the pGEM 3Z template with EcoRl. Sinuclease mapping was performed as previously described (27). In brief, 30 ng total RNA from male tissues (liver, heart, kidney, muscle, testis, brain, and adipose tissue) were hybridized with 104-105 cpm uniformly labeled [32P]cRNA probe in a total volume of 30 n\ hybridization buffer [80% formamide, 0.5 M NaCl, 40 mM piperazine-iV,AT-bis-(2-ethanesulfonic acid), sodium salt, and 0.2 mM EDTA, pH 6.4] at 85 C for 5 min, followed by 55 C overnight. Four hundred units of S^nuclease enzyme (Pharmacia/LKB Biotechnology, Uppsala, Sweden) and 470 nl Si-nuclease digestion buffer [0.25 M NaCl, 30 mM Na acetate (pH 4.6) 4 mM ZnSO4, and 2 mg/ml denatured salmon sperm DNA] were added, and the tubes were incubated
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mRNA FOR GHR AND GHBP
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at 37 C for 30 min. After the addition of 100 pi 4 M NH4 acetate/0.1 M EDTA, each sample was extracted with phenolchloroform (1:1) and precipitated with isopropanol. The samples were then electrophoresed on a 6% polyacrylamide sequencing gel in Tris (50 mM)-borate (42 mM)-EDTA (1 mM) buffer. Gels were dried and exposed to Kodak XAR film at room temperature without intensifying screens. cRNA probe: extracellular domain. Based upon the published cDNA sequences of rabbit liver GHR (5) and rat GHR (11), three oligonucleotide primers were used. An antisense primer A (nt 958-986) complementary to rabbit GHR cDNA (5), was used to synthesize the first strand of cDNA using rat liver poly(A)+RNA as template. An approximately 500-bp fragment was successfully derived between an antisense rabbit primer B (nt 919-948 of Ref. 5) and a sense rat primer C (nt 602-629 of ref. 11) by a 40-cycle PCR [1 min (denaturation), 2 min at 60 C (annealing), and 3 min at 72 C (extension), with a final extension of 10 min at 72 C]. The amplified product gave the expected size on an ethidium bromide-stained nondenaturing gel and was subcloned into a pGEM4Z plasmid vector (Promega). Its orientation was confirmed by dideoxy sequencing (26), and a 32P-labeled cRNA probe generated as described above.
4.5kb-
Statistics All results are expressed as the mean ± SEM (n = 3). Means were compared by analysis of variance and Student's t test. Differences were considered significant when P < 0.05.
Results and Discussion Using an extracellular probe encoding the GHR and truncated GHBP mRNAs, the distribution of these mRNAs across different rat tissues was examined. Both the full-length GHR (4.5 kb) and truncated (1.2 kb) mRNAs were detected in liver, kidney, adrenal, heart, muscle, and adipose tissue (Fig. 1A). The 1.2-kb mRNA was most abundant in liver (compared to other tissues), indicating that liver is likely to be the primary source of
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GH binding studies [125I]Bovine GH ([125I]bGH) binding studies measuring membrane GHR were performed using [125I]bGH as ligand, as described previously (24). Separation of bound and free hormone was carried out by centrifugation. For studies measuring serum GHBP 50 pi [125I]bGH (25,000 cpm) were incubated with 10 pi rat serum and 50 pi of various concentrations of unlabeled bGH in a final volume of 250 pi at room temperature for 1 h. The reaction mixture was then loaded onto an Ultrogel ACA 54 column (0.6 X 25 cm; LKB Produtker, Bromma, Sweden), equilibrated, and eluted with Tris-HCl buffer, pH 7.5, containing 0.02% azide, as described by Ymer and Herington (14). The radioactivity of appropriate pooled fractions was then measured in a 7-counter, and specific binding, expressed as a percentage of the total counts per min added/tube, was calculated as the difference between binding in the absence (total binding) and presence of excess unlabeled bGH (nonspecific binding).
Endo • 1991 Vol 129 • No 3
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FIG. 1. A representative Northern blot analysis showing the tissue distribution of GHR/BP in rat tissues from 35-day-old animals. Total RNA (26 /ig/lane) was electrophoresed and transferred as described in the text. The blot was hybridized with a 32P-labeled cRNA probe encoding the common extracellular GHR and GHBP domain. Autoradiography was carried out for 3 days at -80 C with Kodak X-AR film and intensifying screens. The sizes of GHR/BP are shown on the left, as determined by comparison with a commercial RNA ladder (not shown). The blots were subsequently hybridized with an 18S rRNA oligonucleotide probe (bottom panels in both A and B) to assess the equivalence of RNA loaded per lane. Similar data were obtained for individual tissues in several Northern blots. The relative intensities of the 18S bands in A and B differ due to the use of different 18S probes and exposure times in the two experiments.
circulating GHBP. Interestingly, in the liver only, an additional transcript of 2.6 kb estimated size was also detected when the autoradiographs were slightly overexposed (see liver lane in Fig. 1A). This is consistent with the recent report of Carlsson et al. (28). The significance and/or functional role(s) of this lower abundance GHR-related transcript are not known. The two major mRNA species (4.5 and 1.2 kb) were seen in mammary gland and ovary after longer exposure of the autoradiograph, but no hybridization was detected in brain, testis, or thymus. None of the apparent absences was due to inadequate RNA loading, as shown by the 18S ribosomal RNA (rRNA) control hybridizations in
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mRNA FOR GHR AND GHBP
the panels below Fig. 1, A and B. The presence of GHR mRNA in the ovary is consistent with the demonstration of immunoreactive GHR and/or GHBP in ovarian cells (29). A similar demonstration of immunoreactivity in the testis (29) does not match with our Northern blot mRNA data [or the more sensitive nuclease protection assay (see below)] and may reflect accumulation of circulating GHBP rather than cellular GHR/GHBP synthesis. Further studies are required to resolve this issue. Weak hybridization in the brain in the nuclease protection assay (see below) suggests that the GHR/GHBP is present there, although in very low abundance, which would agree with the earlier observation of Mathews et al. (10). The presence of GHR/GHBP in mammary gland is not surprising, despite previous reports (30, 31) that failed to measure any significant GH binding in mammary homogenates by classical binding techniques. Recent molecular evidence clearly demonstrated, by nuclease protection assay, the presence of a full-length GHR mRNA in rabbit mammary gland (32). Furthermore, in situ hybridization was used to identify GHR gene expression in bovine mammary gland (33). The presence of GHR mRNA in the tissues studied was also confirmed using the cytoplasmic probe (data not shown). A detailed study of the distribution of GHR/GHBP mRNA in the GIT was undertaken (Fig. IB). A fulllength (~4.5kb) and a truncated (~1.2kb) mRNA were observed in the antrum and corpus of the stomach, duodenum, jejunum, ileum, caecum, ascending colon, transverse colon, and descending colon (Fig. IB). The ratio of expression of the 1.2- to 4.5-kb mRNAs was relatively constant across the GIT, although significant variation in absolute amounts was seen between regions. In support of our findings, Mathews et al. (10) recently reported that the rat intestine (location not reported) expressed GHR mRNA at high levels. Furthermore, Lobie et al. (34), using immunohistochemistry techniques, have very recently reported the cellular distribution of GHR/GHBP mRNA in the GIT. The study was not able to distinguish between the presence of GHR and/or GHBP. The wide distribution of the expression of GHR mRNAs indicates that GH may act directly on many nonhepatic tissues, a concept in broad agreement with the dual effector hypothesis (35), in which GH and insulin-like growth factor-I can act in concert to promote body growth, with GH promoting the differentiation of precursor cells, and insulin-like growth factors stimulating their subsequent clonal expansion. To examine the relationship (sequence identity or otherwise) between the postulated growth-promoting GHR, e.g. in the liver, and the postulated metabolic GHR, e.g. in adipose tissue, Si-nuclease protection studies were performed using the specific intracellular cRNA probes. The intracellular probes were used because they
1631
cover the transmembrane signalling domain of the receptor and, hence, would probably represent the domains that differ (rather than the hormone-binding domain) between GHRs that had different functions and perhaps different mechanisms of action. As shown in Fig. 2, a protected band (180 bp) corresponding to the GHR mRNA was observed in liver, kidney, muscle, heart, and adipose tissue, but not in testis, although a faint band was seen in brain after longer exposure. These observations agree well with the data reported by Mathews et al. (10). Our data clearly indicate the presence of a common cytoplasmic domain nucleotide sequence in hepatic GHR mRNA as well as in other tissues studied. To further support this view, a longer riboprobe (468 bp), which is entirely within exon 10 and covers about 50% of the cytoplasmic coding region, was also used. However, rather than one protected band as expected, two distinct bands, each 210-220 bp in length, were protected in both liver and adipose tissue (data not shown). The likely explanation for this is the presence of an Si-nucleasesensitive A-T-rich region between bases 220 and 237 of the probe sequence (10). The important issue, however, is that both the liver and adipose tissue mRNA responded
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FIG. 2. Si-nuclease mapping studies. Thirty micrograms of total RNA from liver, heart, muscle, kidney, brain, testis, and adipose tissue of 35-day-old rats were used and hybridized with 32P-labeled riboprobe (210 bp). Samples were digested with 400 U Si-nuclease enzyme for 30 min at 37 C. The protected fragments were then analyzed on 6% acrylamide-urea sequencing gel, with a sequencing ladder as the marker (not shown). The gel was then exposed to X-AR film at room temperature without intensifying screens.
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mRNA FOR GHR AND GHBP
identically, indicating sequence identity across the probe region. These data, therefore, argue against, but do not disprove, the notion that there may be distinct growth and metabolic GHRs, which have differing cytoplasmic sequences and, therefore, differing mechanisms for signal transduction (36). However, the possibility does exist that a second approximately 4.5-kb GHR mRNA may be present in adipose tissue, with a distinct cytoplasmic sequence (and, therefore, is not picked up by nuclease protection assay). Only the complete cloning and sequencing of the adipose GHR mRNA(s) will resolve this question. To assess any potential sex difference in GHR/GHBP expression, Northern blots for total liver RNA isolated from age-matched (80-day-old) male, female, and pregnant rats (19th day of gestation) were hybridized with the 32P-labeled rat PCR-derived common GHR/BP riboprobe (Fig. 3). The results (Fig. 4) indicate that only the truncated GHBP mRNA was up-regulated in liver as a function of sex (female vs. male, P < 0.05) and during pregnancy (female vs. pregnant, P = 0.0009). No significant changes were observed in the full-length GHR mRNA even though there was an upward trend (refer to Fig. 4A). Very similar data were obtained in several other experiments. No significant sex differences in GHR or GHBP mRNA were seen using younger (35-day-old) male and female rats (data not shown). These conclusions were further supported by data obtained with a cytoplasmic domain riboprobe (468 bp), which only de-
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FIG. 3. Northern blot analysis of total RNA (26 pg/lane) extracted from 80-day-old male, female, and pregnant (19th day of gestation) rats. The RNA was electrophoresed on a 1% agarose gel, transferred to nylon membrane, and then hybridized with a 32P-labeled cRNA probe encoding the GHR/GHBP common extracellular domain. Autoradiography was carried out at —80 C overnight with Kodak X-AR film and intensifying screens. The sizes of GHR/GHBP mRNA are shown on the left (n = 3). The blot was subsequently hybridized to the 18S rRNA oligonucleotide probe so as to evaluate the relative amounts of total RNA loaded onto each lane (bottom panel).
tects the full-length (4.5-kb) GHR mRNA (data not shown). This observation is only partly consistent with a previous report (23) that both the 4.5- and 1.2-kb mRNAs were up-regulated during pregnancy in the mouse. The difference may reflect a species difference between rat and mouse. Mathews et al. (10), using a sensitive solution hybridization assay, reported a 2-fold increase in hepatic GHR mRNA expression during pregnancy. However, in contrast to our present approach, because the probe used was for an extracellular GHR domain, this technique does not discriminate the 4.5-kb mRNA for membrane-associated GHR from the 1.2-kb mRNA for the soluble GHBP. To determine if the increase in the GHBP mRNA was accompanied by an increase in the relative concentration of serum GHBP and/or in hepatic GHR, classical 125Ilabeled GH binding studies were also performed using the serum and liver homogenates from the same rats as
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mRNA FOR GHR AND GHBP 10
provide a better understanding of the role of this protein and its interrelationship with the hepatic GH receptor.
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FIG. 5. Binding studies using [ I]bGH as ligand were performed using the liver homogenates (A) or serum (B) obtained from the same rats used for RNA extraction. Data are shown as the mean percentage of radioactivity specifically bound and expressed in arbitrary units as the mean ± SEM (n = 3). For serum (B) male us. female, P = 0.003; *, pregnant us. nonpregnant, P = 0.005.
those used for RNA extraction (Fig. 5). The data suggest that the serum binding protein was elevated during pregnancy (female vs. pregnant, P = 0.005), with no significant change in liver membrane binding. Sexual dimorphism also exists for the GHBP in serum, with adult female rats having significantly more circulating GHBP than male rats (male vs. female, P = 0.003), as reported by Massa et al. (22). Our study also confirms that GHR mRNA is closely related to membrane receptor concentration. At present little is known about the possible physiological role(s) of the serum GHBP. Baumann's group (37) has reported an effect on both the clearance and extravascular distribution of GH, and the GHBP may well be involved in the delivery of GH to its target tissues and/or in regulation of its action at the target membrane receptor (38). The broad tissue distribution of GHBP mRNA expression suggests that it may be synthesized widely and, hence, raises the possibility that it may have a local (autocrine/paracrine) role in modulating GH action or in regulating GH removal from tissues. Further characterization of the GHBP and its regulation should
Acknowledgments The authors thank Janet Stevenson for excellent technical assistance, and Yvonne Ferguson and Sue Panckridge for help in the preparation of the manuscript.
References 1. Herington AC 1981 Identification and characterization of growth hormone receptors on isolated rat adipocytes. J Receptor Res 2:299-316 2. Husman B, Haldosen LA, Andersson G, Gustafsson JA 1988 Characterization of somatogenic receptor in rat liver: hydrodynamic properties and affinity linking. J Biol Chem 263:3963-3970 3. Asakawa K, Hedo JA, McElduff A, Rouiller DG, Waters MJ, Gorden P 1986 The human growth hormone receptor of cultured human lymphocytes. Biochem J 238:379-386 4. Waters MJ, Friesen HG 1979 Purification and partial characterization of a non-primate growth hormone receptor. J Biol Chem 254:6815-6825 5. Leung DW, Spencer SA, Cachianes G, Hammonds GT, Collins C, Henzel WJ, Barnard R, Waters MJ, Wood WI 1987 Growth hormone receptor and serum binding protein: purification, cloning and expression. Nature 330:537-543 6. Spencer SA, Hammonds G, Henzel WJ, Rodriguez H, Waters MJ, Wood WI 1988 Rabbit liver growth hormone receptor and serum binding protein: purification, characterization and sequence. J Biol Chem 263:7862-7867 7. Husman B, Andersson G, Norstedt G, Gustafsson J 1985 Characterization and subcellular distribution of the somatogenic receptor in rat liver. Endocrinology 116:2605-2611 8. Smith WC, Talamantes F 1987 Identification and characterization of a heterogeneous population of growth hormone receptors in mouse hepatic membrane. J Biol Chem 262:2213-2219 9. Smith WC, Colosi P, Talamantes F 1988 Isolation of two molecular weight variants of the mouse growth hormone receptor. Mol Endocrinol 2:108-116 10. Mathews LS, Enberg B, Norstedt G 1989 Regulation of rat growth hormone receptor gene expression. J Biol Chem 264:9905-9910 11. Baumbach WR, Homer DL, Logan JS 1989 The growth hormonebinding protein in rat serum is an alternatively spliced form of the rat growth hormone receptor. Gene 3:1199-1205 12. Smith WC, Kuniyoshi J, Talamantes F 1989 Mouse serum growth hormone binding protein has GH receptor extracellular and substituted transmembrane domains. Mol Endocrinol 3:984-990 13. Bazan JF 1989 A novel family of growth factor receptors. A common binding domain in the growth hormone, prolactin, erythropoeitin and IL-6 receptors and the P75 IL-2 receptor B-chain. Biochem Biophys Res Commun 164:788-795 14. Ymer SI, Herington AC 1985 Evidence for the specific binding of growth hormone to a receptor-like protein in rabbit serum. Mol Cell Endocrinol 41:153-161 15. Ymer SI, Stevenson JL, Herington AC 1984 Identification of a rabbit liver cytosolic binding protein for human growth hormone. Biochem J 221:617-622 16. Baumann G, Stolar MW, Amburn K, Barsano CP, Devries BC 1986 A specific growth hormone-binding protein in human plasma: initial characterisation. J Clin Endocrinol Metab 62:134-141 17. Herington AC, Ymer SI, Stevenson JL 1986 Identification and characterisation of specific binding proteins for growth hormone in normal human sera. J Clin Invest 77:1817-1823 18. Baumann G, Amburn K, Shaw MA 1988 The circulating growth hormone-binding protein complex: a major constituent of plasma GH in man. Endocrinology 122:976-984 19. Peeters S, Friesen HG 1977 A growth hormone binding factor in the serum of pregnant mice. Endocrinology 101:1169-1176
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mRNA FOR GHR AND GHBP
20. Amit T, Barkey RJ, Bick T, Hertz P, Youdim MBH, Hochberg Z 1990 Identification of growth hormone binding protein in rat serum. Mol Cell Endocrinol 70:197-202 21. Bick T, Amit T, Barkey RJ, Hertz P, Youdim MBH, Hochberg Z 1990 The interrelationship of growth hormone (GH), liver membrane GH receptor, serum GH-binding protein activity and insulinlike growth factor I in the male rat. Endocrinology 126:1914-1920 22. Massa G, Mulumba N, Ketelslegers JM, Maes M 1990 Initial characterization and sexual dimorphism of serum growth hormone binding-protein in adult rats. Endocrinology 126:1976-1980 23. Smith WC, Linzer DLH, Talamantes F 1988 Detection of two growth hormone receptor mRNAs and primary translation products in the mouse. Proc Natl Acad Sci USA 85:9576-9579 24. Tiong TS, Herington AC 1989 Identification and tissue distribution of messenger RNA for the growth hormone receptor in the rabbit. Biochem Biophys Res Commun 158:141-148 25. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N 1985 Enzymatic amplification of B-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350-1354 26. Sanger F, Nicklen S, Coulson AR 1977 DNA sequencing with chain terminating inhibitor. Proc Natl Acad Sci USA 74:5463-5467 27. Favaloro J, Triesman R, Kamen R 1980 Transcription maps of polyoma virus specific RNAs: analysis by two dimensional nuclease Si gel mapping methods. Methods Enzymol 65:718-749 28. Carlsson B, Billig H, Rymo L, Isaksson OGP 1990 Expression of the growth hormone binding protein messenger RNA in the liver and extrahepatic tissues in the rat: co-expression with the growth hormone receptor. Mol Cell Endocrinol 73:R1-R6 29. Lobie PE, Breipohl W, Garcia-Aragon J, Waters MJ 1990 Cellular localization of the GH receptor/BP in the male and female repro-
Endo • 1991 Voll29«No3
ductive systems. Endocrinology 126:2214-2220 30. Gertler A, Ashkenazi A, Mador L 1984 Binding sites of human growth hormone and ovine and bovine prolactins in the mammary gland and liver of lactating dairy cows. Mol Cell Endocrinol 345:5157 31. Kazmer GW, Barnes MA, Akers RM, Whittier WD 1986 Lactogenic hormone receptors in mammary preparations from prepartum and 60 and 180 day post-partum Holstein cattle. J Endocrinol 109:175-180 32. Jammes H, Gaye P, Belair L, Djiane J, Identification of poly A+ mRNA of the growth hormone receptor in the rabbit mammary gland. 72nd Annual Meeting of The Endocrine Society, Atlanta GA, p 90 (Abstract) 33. Glimm DR, Baracos VE, Kennelly JJ 1990 Molecular evidence for the presence of growth hormone receptors in the bovine mammary gland. J Endocrinol 126:R5-R8 34. Lobie PE, Breipohl W, Waters MJ 1990 Growth hormone receptor expression in the rat gastrointestinal tract. Endocrinology 126:299306 35. Green H, Morikawa M, Nixon T 1985 A dual effector theory of growth hormone action. Differentiation 29:195-198 36. Roupas P, Herington AC 1989 Cellular mechanisms in the processing of growth hormone and its receptor. Mol Cell Endocrinol 61:1-13 37. Baumann G, Amburn KD, Buchanan TA 1987 The effect of circulating growth hormone binding protein on metabolic clearance, distribution and degradation of human growth hormone. J Clin Endocrinol Metab 64:657-662 38. Lim L, Spencer SA, McKay P, Waters MJ 1990 Regulation of growth hormone (GH) bioactivity by a recombinant human GHbinding protein. Endocrinology 127:1287-1291
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Vol. 129, No. 3 Printed in U.S.A.
Tissue Distribution, Characterization, and Regulation of Messenger Ribonucleic Acid for Growth Hormone Receptor and Serum Binding Protein in the Rat* TAK S. TIONG AND A. C. HERINGTON Prince Henry's Institute of Medical Research, Prince Henry's Hospital Campus, Monash Medical Centre, Melbourne 3004, Australia
ABSTRACT. The distribution of GH receptor (GHR) and GH-binding protein (GHBP) mRNAs in multiple rat tissues was examined by Northern blotting using a cDNA fragment encoding the common extracellular domain of the GHR and the serum GHBP. Both GHR and GHBP mRNAs [4.5 and 1.2 kilobases (kb), respectively] were present in liver, kidney, adrenal, heart, muscle, ovary, mammary gland, gastrointestinal tract, and adipose tissue, but were barely or not detectable in testis, thymus, or brain. These observations suggest that GH exerts direct effects across a broad spectrum of rat tissues. Nuclease protection analysis also confirmed the presence in extrahepatic tissues of a GHR mRNA with up to 50% of the cytoplasmic domain being identical in sequence to that of the hepatic GHR mRNA. This suggests, but does not prove, that different receptor classes with differing intracellular signalling mechanisms may not exist. It is also clear from our studies that liver was the most abundant
T
HE LIVER is a well known target tissue for GH action. The biological effects of GH, mediated by the binding of GH to specific, high affinity receptors present in target tissue(s), have been well studied in adipocytes (1), hepatocytes (2), and IM-9 lymphocytes (3). The hepatic GH receptor (GHR) has been well characterized from several species, including rabbit (46), rat (7), and mouse (8, 9). The primary structure of the GH receptor in rabbit, human, rat, and mouse liver has been deduced from the nucleic acid sequence of cloned cDNA (5, 10-12). The sequences share considerable homology (~70%) across species and, interestingly, also show significant homology with the rat, rabbit, and human PRL receptors, and the family of interleukin receptors (13), but not to other known hormone receptor molecules. The full-length hepatic GHR cDNA [4-4.5 Received February 8,1991. Address requests for reprints to: Dr. A. C. Herington, Department of Clinical Biochemistry, Royal Children's Hospital, Flemington Road, Parkville 3052, Australia. * This work was supported by the William Buckland Foundation (in the form of a postgraduate scholarship to T.S.T.) and a grant from the National Health and Medical Research Council of Australia (to A.C.H.).
source of the truncated (1.2 kb) mRNA and is, therefore, believed to be the primary site of GHBP synthesis. Also, more importantly, at least in the rat, it was only GHBP mRNA that was up-regulated to any extent during pregnancy (female vs. pregnant, P < 0.001). No significant changes were observed in the abundance of the full-length (4.5 kb) GHR mRNA. This pregnancy-related change in GHBP mRNA was also accompanied by a comparable increase in the actual level of measurable serum GHBP (female vs. pregnant, P = 0.005). Little change was seen in hepatic membrane binding. These data suggest that the GHBP and GHR are both widely coexpressed, but that the expression is not always coordinately regulated. This raises the possibility that the GHBP and GHR may have distinct roles in the regulation of GH delivery and action. {Endocrinology 129: 1628-1634, 1991)
kilobases (kb)] consists of an extracellular hormonebinding domain, a single hydrophobic transmembrane domain, and a cytoplasmic domain. Examination of the C-terminal sequence revealed no recognized consensus sequences for the classical receptor second messenger systems. In addition to the large (mol wt, ~ 110,000) membranebound tissue GHR, a truncated soluble GH-binding protein (GHBP) has been reported in serum (14) and cytosolic fractions of rabbit tissues (15). Subsequently, specific serum GHBPs have been identified in several other species, including human (16-18), mouse (19), and rat (20). Recent cloning studies in the rat and mouse (1012) have shown the GHBP in these species to be the product of an alternative splicing event (at the exon 7exon 8 junction) during processing of the full-length GHR mRNA. The resultant mRNA is truncated, being only 1.2-1.4 kb. It has the same extracellular domain (exons 2-7) and lacks a hydrophobic transmembrane domain (exon 8) and the full-length intracellular domain (exons 9 and 10). Earlier studies in the rabbit and human did not identify an equivalent GHBP mRNA, and it was suggested that the serum GHBP was a proteolytically 1628
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mRNA FOR GHR AND GHBP
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cleaved form of the membrane receptor (5). This distinction between rat/mouse and human/rabbit has not yet been resolved. The role of the serum GHBP is not clearly understood. In part to investigate this role studies have begun to examine the regulation of the serum GHBP at both the protein and mRNA level and to compare this with regulation of the full-length membrane-bound GHR. Recent studies by Bick et al. (21) have indicated some changes in rat serum GHBP concentrations in hypophysectomized rats given a continuous sc infusion of hGH, while studies in the mouse indicated a sexual dimorphism (22) and increases in both mRNA and protein levels during pregnancy (23). In the studies reported here, we have used polymerase chain reaction (PCR)-derived fragments of either the common extracellular domain or the GHR-specific cytoplasmic domain of the cloned rat liver GHR cDNA (10) to identify the number, size, and tissue distribution of the mRNAs for the GHR and GHBP. Preliminary studies on the physiological regulation of these particular mRNAs and their correlation with direct [125I]GH binding studies are also reported.
Amersham, Aylesbury, Buckinghamshire, United Kingdom) by capillary blotting. The membranes were baked at 80 C for 2 h and UV cross-linked for 20 min. For riboprobes (cRNA probes), the membranes were prehybridized at 65 C in hybridization solution [50% formamide, 5 x SSPE (1 x SSPE is 0.15 M sodium chloride, 10 mM sodium phosphate, and 1 mM EDTA), 0.15 M Tris-HCl (pH 8.0), 1% sodium dodecyl sulfate (SDS), and 500 mg/ml heparin] before hybridization at 65 C for 16-20 h with about 106 cpm/ml labeled probes. All blots were then washed initially with 2 X SSC-0.1% SDS at room temperature (20 min), followed by washes of increasing stringency up to 0.2 x SSC at 65 C (cytoplasmic probe) or 0.2 x SSC at 50 C (extracellular probe). Membranes were then exposed to Kodak X-AR (Eastman Kodak, Rochester, NY) x-ray film with DuPont Lightning Plus intensifying screens (Wilmington, DE) at -70 C. Before rehybridization with a different probe, blots were stripped by boiling for 3 min in the presence of 0.1% SDS, and the complete removal of previously hybridized probe was determined by autoradiography at -70 C overnight. Relative mRNA levels were determined by densitometry [ISCO gel scanner 1312 (Lincoln, NE) with a Hewlett-Packard integrator (Palo Alto, CA)]. Subsequently, mRNA levels in arbitary absorbance units were divided by the value obtained for a control probe [an 18S rat ribosomal RNA oligonucleotide (30 mer)].
Materials and Methods
cRNA probes: cytoplasmic fragment. Based upon the published cDNA sequence of the rat GHR (10, 11), two oligonucleotide primers were synthesized: primer A, nucleotides (nt) 1401-1430 (which includes a Kpnl restriction site), and primer B, a complementary sequence to nt 1844-1873 (including a Hindlll site). By using cDNA prepared from poly(A)+ mRNA of rat liver, the 472-nt cDNA sequence between the primers A and B was successfully amplified by PCR (25) [40 cycles consisting of 1.5 min at 90 C (denaturation), 2 min at 55 C (annealing) and a 72 C extension (1 min)]. The amplified product gave the expected size on an ethidium bromide-stained nondenaturing gel and was subcloned into a pGEM 4Z plasmid vector (Promega, Madison, WI). Its orientation was confirmed by dideoxy sequencing (26), and a 32P-labeled riboprobe (cRNA) was generated from the T7 promoter as indicated by Promega.
Collection of tissues
All animals were maintained and killed according to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, as published by the National Health and Medical Research Council of Australia. The studies were approved by the Animal Ethics Experimentation Committee of the Monash Medical Centre. To study the tissue distribution, liver, heart, adrenal, kidney, skeletal muscle, thymus, testis, ovary, mammary gland, fat pad, and gastrointestinal tract (GIT) from adult male (35 days), adult female, and pregnant (19th day of gestation) SpragueDawley rats were collected for RNA extraction. Livers from 80day-old male, female, and pregnant (19th day of gestation) Sprague-Dawley rats were also used for both RNA extraction and membrane-binding studies. Trunk blood was collected for binding studies. The tissues for RNA preparation were removed rapidly after decapitation, immediately frozen in liquid nitrogen, and stored at -80 C until processing, whereas for binding studies tissues were collected into 0.3 M sucrose containing 1 mM phenylmethylsulfonylfluoride and 1000 kallikrein inactivator units Trasylol/ml on ice and stored at -20 C until processing. Preparation and analysis of RNA Total RNA was isolated as described in detail previously (24). Northern blot hybridization was also performed as previously described (24). Total RNA was denatured in 1 M glyoxal50% dimethylsulfoxide and electrophoresed on a 1% agarose gel in 10 mM phosphate buffer, pH 7.0. RNA was then transferred to Hybond-N membrane (Amersham International,
Probes
Si-nuclease mapping. A second GHR riboprobe was derived from the cytoplasmic probe by subcloning an approximately 180-basepair (bp) Kpnl-Hincll fragment into the pGEM3Z vector (Promega), and an antisense RNA control probe was prepared by linearizing the pGEM 3Z template with EcoRl. Sinuclease mapping was performed as previously described (27). In brief, 30 ng total RNA from male tissues (liver, heart, kidney, muscle, testis, brain, and adipose tissue) were hybridized with 104-105 cpm uniformly labeled [32P]cRNA probe in a total volume of 30 n\ hybridization buffer [80% formamide, 0.5 M NaCl, 40 mM piperazine-iV,AT-bis-(2-ethanesulfonic acid), sodium salt, and 0.2 mM EDTA, pH 6.4] at 85 C for 5 min, followed by 55 C overnight. Four hundred units of S^nuclease enzyme (Pharmacia/LKB Biotechnology, Uppsala, Sweden) and 470 nl Si-nuclease digestion buffer [0.25 M NaCl, 30 mM Na acetate (pH 4.6) 4 mM ZnSO4, and 2 mg/ml denatured salmon sperm DNA] were added, and the tubes were incubated
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mRNA FOR GHR AND GHBP
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at 37 C for 30 min. After the addition of 100 pi 4 M NH4 acetate/0.1 M EDTA, each sample was extracted with phenolchloroform (1:1) and precipitated with isopropanol. The samples were then electrophoresed on a 6% polyacrylamide sequencing gel in Tris (50 mM)-borate (42 mM)-EDTA (1 mM) buffer. Gels were dried and exposed to Kodak XAR film at room temperature without intensifying screens. cRNA probe: extracellular domain. Based upon the published cDNA sequences of rabbit liver GHR (5) and rat GHR (11), three oligonucleotide primers were used. An antisense primer A (nt 958-986) complementary to rabbit GHR cDNA (5), was used to synthesize the first strand of cDNA using rat liver poly(A)+RNA as template. An approximately 500-bp fragment was successfully derived between an antisense rabbit primer B (nt 919-948 of Ref. 5) and a sense rat primer C (nt 602-629 of ref. 11) by a 40-cycle PCR [1 min (denaturation), 2 min at 60 C (annealing), and 3 min at 72 C (extension), with a final extension of 10 min at 72 C]. The amplified product gave the expected size on an ethidium bromide-stained nondenaturing gel and was subcloned into a pGEM4Z plasmid vector (Promega). Its orientation was confirmed by dideoxy sequencing (26), and a 32P-labeled cRNA probe generated as described above.
4.5kb-
Statistics All results are expressed as the mean ± SEM (n = 3). Means were compared by analysis of variance and Student's t test. Differences were considered significant when P < 0.05.
Results and Discussion Using an extracellular probe encoding the GHR and truncated GHBP mRNAs, the distribution of these mRNAs across different rat tissues was examined. Both the full-length GHR (4.5 kb) and truncated (1.2 kb) mRNAs were detected in liver, kidney, adrenal, heart, muscle, and adipose tissue (Fig. 1A). The 1.2-kb mRNA was most abundant in liver (compared to other tissues), indicating that liver is likely to be the primary source of
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GH binding studies [125I]Bovine GH ([125I]bGH) binding studies measuring membrane GHR were performed using [125I]bGH as ligand, as described previously (24). Separation of bound and free hormone was carried out by centrifugation. For studies measuring serum GHBP 50 pi [125I]bGH (25,000 cpm) were incubated with 10 pi rat serum and 50 pi of various concentrations of unlabeled bGH in a final volume of 250 pi at room temperature for 1 h. The reaction mixture was then loaded onto an Ultrogel ACA 54 column (0.6 X 25 cm; LKB Produtker, Bromma, Sweden), equilibrated, and eluted with Tris-HCl buffer, pH 7.5, containing 0.02% azide, as described by Ymer and Herington (14). The radioactivity of appropriate pooled fractions was then measured in a 7-counter, and specific binding, expressed as a percentage of the total counts per min added/tube, was calculated as the difference between binding in the absence (total binding) and presence of excess unlabeled bGH (nonspecific binding).
Endo • 1991 Vol 129 • No 3
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FIG. 1. A representative Northern blot analysis showing the tissue distribution of GHR/BP in rat tissues from 35-day-old animals. Total RNA (26 /ig/lane) was electrophoresed and transferred as described in the text. The blot was hybridized with a 32P-labeled cRNA probe encoding the common extracellular GHR and GHBP domain. Autoradiography was carried out for 3 days at -80 C with Kodak X-AR film and intensifying screens. The sizes of GHR/BP are shown on the left, as determined by comparison with a commercial RNA ladder (not shown). The blots were subsequently hybridized with an 18S rRNA oligonucleotide probe (bottom panels in both A and B) to assess the equivalence of RNA loaded per lane. Similar data were obtained for individual tissues in several Northern blots. The relative intensities of the 18S bands in A and B differ due to the use of different 18S probes and exposure times in the two experiments.
circulating GHBP. Interestingly, in the liver only, an additional transcript of 2.6 kb estimated size was also detected when the autoradiographs were slightly overexposed (see liver lane in Fig. 1A). This is consistent with the recent report of Carlsson et al. (28). The significance and/or functional role(s) of this lower abundance GHR-related transcript are not known. The two major mRNA species (4.5 and 1.2 kb) were seen in mammary gland and ovary after longer exposure of the autoradiograph, but no hybridization was detected in brain, testis, or thymus. None of the apparent absences was due to inadequate RNA loading, as shown by the 18S ribosomal RNA (rRNA) control hybridizations in
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mRNA FOR GHR AND GHBP
the panels below Fig. 1, A and B. The presence of GHR mRNA in the ovary is consistent with the demonstration of immunoreactive GHR and/or GHBP in ovarian cells (29). A similar demonstration of immunoreactivity in the testis (29) does not match with our Northern blot mRNA data [or the more sensitive nuclease protection assay (see below)] and may reflect accumulation of circulating GHBP rather than cellular GHR/GHBP synthesis. Further studies are required to resolve this issue. Weak hybridization in the brain in the nuclease protection assay (see below) suggests that the GHR/GHBP is present there, although in very low abundance, which would agree with the earlier observation of Mathews et al. (10). The presence of GHR/GHBP in mammary gland is not surprising, despite previous reports (30, 31) that failed to measure any significant GH binding in mammary homogenates by classical binding techniques. Recent molecular evidence clearly demonstrated, by nuclease protection assay, the presence of a full-length GHR mRNA in rabbit mammary gland (32). Furthermore, in situ hybridization was used to identify GHR gene expression in bovine mammary gland (33). The presence of GHR mRNA in the tissues studied was also confirmed using the cytoplasmic probe (data not shown). A detailed study of the distribution of GHR/GHBP mRNA in the GIT was undertaken (Fig. IB). A fulllength (~4.5kb) and a truncated (~1.2kb) mRNA were observed in the antrum and corpus of the stomach, duodenum, jejunum, ileum, caecum, ascending colon, transverse colon, and descending colon (Fig. IB). The ratio of expression of the 1.2- to 4.5-kb mRNAs was relatively constant across the GIT, although significant variation in absolute amounts was seen between regions. In support of our findings, Mathews et al. (10) recently reported that the rat intestine (location not reported) expressed GHR mRNA at high levels. Furthermore, Lobie et al. (34), using immunohistochemistry techniques, have very recently reported the cellular distribution of GHR/GHBP mRNA in the GIT. The study was not able to distinguish between the presence of GHR and/or GHBP. The wide distribution of the expression of GHR mRNAs indicates that GH may act directly on many nonhepatic tissues, a concept in broad agreement with the dual effector hypothesis (35), in which GH and insulin-like growth factor-I can act in concert to promote body growth, with GH promoting the differentiation of precursor cells, and insulin-like growth factors stimulating their subsequent clonal expansion. To examine the relationship (sequence identity or otherwise) between the postulated growth-promoting GHR, e.g. in the liver, and the postulated metabolic GHR, e.g. in adipose tissue, Si-nuclease protection studies were performed using the specific intracellular cRNA probes. The intracellular probes were used because they
1631
cover the transmembrane signalling domain of the receptor and, hence, would probably represent the domains that differ (rather than the hormone-binding domain) between GHRs that had different functions and perhaps different mechanisms of action. As shown in Fig. 2, a protected band (180 bp) corresponding to the GHR mRNA was observed in liver, kidney, muscle, heart, and adipose tissue, but not in testis, although a faint band was seen in brain after longer exposure. These observations agree well with the data reported by Mathews et al. (10). Our data clearly indicate the presence of a common cytoplasmic domain nucleotide sequence in hepatic GHR mRNA as well as in other tissues studied. To further support this view, a longer riboprobe (468 bp), which is entirely within exon 10 and covers about 50% of the cytoplasmic coding region, was also used. However, rather than one protected band as expected, two distinct bands, each 210-220 bp in length, were protected in both liver and adipose tissue (data not shown). The likely explanation for this is the presence of an Si-nucleasesensitive A-T-rich region between bases 220 and 237 of the probe sequence (10). The important issue, however, is that both the liver and adipose tissue mRNA responded
210 bp
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FIG. 2. Si-nuclease mapping studies. Thirty micrograms of total RNA from liver, heart, muscle, kidney, brain, testis, and adipose tissue of 35-day-old rats were used and hybridized with 32P-labeled riboprobe (210 bp). Samples were digested with 400 U Si-nuclease enzyme for 30 min at 37 C. The protected fragments were then analyzed on 6% acrylamide-urea sequencing gel, with a sequencing ladder as the marker (not shown). The gel was then exposed to X-AR film at room temperature without intensifying screens.
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mRNA FOR GHR AND GHBP
identically, indicating sequence identity across the probe region. These data, therefore, argue against, but do not disprove, the notion that there may be distinct growth and metabolic GHRs, which have differing cytoplasmic sequences and, therefore, differing mechanisms for signal transduction (36). However, the possibility does exist that a second approximately 4.5-kb GHR mRNA may be present in adipose tissue, with a distinct cytoplasmic sequence (and, therefore, is not picked up by nuclease protection assay). Only the complete cloning and sequencing of the adipose GHR mRNA(s) will resolve this question. To assess any potential sex difference in GHR/GHBP expression, Northern blots for total liver RNA isolated from age-matched (80-day-old) male, female, and pregnant rats (19th day of gestation) were hybridized with the 32P-labeled rat PCR-derived common GHR/BP riboprobe (Fig. 3). The results (Fig. 4) indicate that only the truncated GHBP mRNA was up-regulated in liver as a function of sex (female vs. male, P < 0.05) and during pregnancy (female vs. pregnant, P = 0.0009). No significant changes were observed in the full-length GHR mRNA even though there was an upward trend (refer to Fig. 4A). Very similar data were obtained in several other experiments. No significant sex differences in GHR or GHBP mRNA were seen using younger (35-day-old) male and female rats (data not shown). These conclusions were further supported by data obtained with a cytoplasmic domain riboprobe (468 bp), which only de-
Endo • 1991 Vol 129 • No 3
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FIG. 3. Northern blot analysis of total RNA (26 pg/lane) extracted from 80-day-old male, female, and pregnant (19th day of gestation) rats. The RNA was electrophoresed on a 1% agarose gel, transferred to nylon membrane, and then hybridized with a 32P-labeled cRNA probe encoding the GHR/GHBP common extracellular domain. Autoradiography was carried out at —80 C overnight with Kodak X-AR film and intensifying screens. The sizes of GHR/GHBP mRNA are shown on the left (n = 3). The blot was subsequently hybridized to the 18S rRNA oligonucleotide probe so as to evaluate the relative amounts of total RNA loaded onto each lane (bottom panel).
tects the full-length (4.5-kb) GHR mRNA (data not shown). This observation is only partly consistent with a previous report (23) that both the 4.5- and 1.2-kb mRNAs were up-regulated during pregnancy in the mouse. The difference may reflect a species difference between rat and mouse. Mathews et al. (10), using a sensitive solution hybridization assay, reported a 2-fold increase in hepatic GHR mRNA expression during pregnancy. However, in contrast to our present approach, because the probe used was for an extracellular GHR domain, this technique does not discriminate the 4.5-kb mRNA for membrane-associated GHR from the 1.2-kb mRNA for the soluble GHBP. To determine if the increase in the GHBP mRNA was accompanied by an increase in the relative concentration of serum GHBP and/or in hepatic GHR, classical 125Ilabeled GH binding studies were also performed using the serum and liver homogenates from the same rats as
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mRNA FOR GHR AND GHBP 10
provide a better understanding of the role of this protein and its interrelationship with the hepatic GH receptor.
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those used for RNA extraction (Fig. 5). The data suggest that the serum binding protein was elevated during pregnancy (female vs. pregnant, P = 0.005), with no significant change in liver membrane binding. Sexual dimorphism also exists for the GHBP in serum, with adult female rats having significantly more circulating GHBP than male rats (male vs. female, P = 0.003), as reported by Massa et al. (22). Our study also confirms that GHR mRNA is closely related to membrane receptor concentration. At present little is known about the possible physiological role(s) of the serum GHBP. Baumann's group (37) has reported an effect on both the clearance and extravascular distribution of GH, and the GHBP may well be involved in the delivery of GH to its target tissues and/or in regulation of its action at the target membrane receptor (38). The broad tissue distribution of GHBP mRNA expression suggests that it may be synthesized widely and, hence, raises the possibility that it may have a local (autocrine/paracrine) role in modulating GH action or in regulating GH removal from tissues. Further characterization of the GHBP and its regulation should
Acknowledgments The authors thank Janet Stevenson for excellent technical assistance, and Yvonne Ferguson and Sue Panckridge for help in the preparation of the manuscript.
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mRNA FOR GHR AND GHBP
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Endo • 1991 Voll29«No3
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