Evidence for two distinct classes of high affinity growth hormone binding proteins in pregnant rat serum

Growth Hormone & IGF Research 2000, 10, 275–289 doi:10.1054/ghir.2000.0169, available online at http://www.idealibrary.com on

Evidence for two distinct classes of high affinity growth hormone binding proteins in pregnant rat serum S. I. Ymer1, J. L. Stevenson1 and A. C. Herington2 1 Centre for Hormone Research, Royal Children’s Hospital, Melbourne, Australia and 2Centre for Molecular Biotechnology, Queensland University of Technology, Brisbane, Australia

Summary These studies have established the presence of two major classes of high affinity growth hormone binding proteins in pregnant rat serum, designated GHBPa and GHBPb, with apparent native Mr of 257 K and 98 K respectively. GHBPa, which has not been identified previously, exhibits a binding affinity (2–5 nM–1) that is up to 20-fold higher than GHBPb (0.2–0.8 nM–1) and is the least abundant form, being ~15–20% of total serum GH-binding capacity. Western immunoblot analysis revealed that each GHBP is composed of several immunoreactive proteins which were reactive with carboxy-terminal (RB1615) and/or N-terminal (MAb263) domain antibodies, suggesting the presence of GHBPs with and without the hydrophilic tail. Of importance is that GHBPa exhibited significantly higher Mr (78–182 K, +DTT) than that predicted by GHBP cloning, suggesting that they may be covalently bound to other non-GH-binding proteins or may be distinct entities. GHBPb, on the other hand, was composed of smaller Mr (43/48 K, +DTT) “hydrophilic” tail-containing proteins, some of which were disulphide linked to a larger complex of ~110 K. These novel findings challenge the current view of the mechanism for generation of the rat serum GHBP and raise the intriguing possibility that the two classes of GHBP may play distinct and important roles in GH physiology. © 2000 Harcourt Publishers Ltd Key words: growth hormone binding protein, rat serum GHBP, novel isoforms

INTRODUCTION The growth hormone receptor (GHR) is a member of the superfamily which includes receptors for prolactin, cytokines and haematopoietic growth factors1 and, like most of these, exists both as a membrane bound form and a soluble form, the GHBP. The GHR has been cloned and sequenced from several species but the GHBP has only been purified and partially sequenced from rabbit serum. The GHBP data revealed that its amino-terminal Received 28 February 2000 Revised 24 July 2000 Accepted 18 August 2000 Correspondence to: Professor Adrian C. Herington, Centre for Molecular Biotechnology, School of Life Sciences, Queensland University of Technology, GPO Box 2434, Brisbane, Queensland, Australia 4001. Tel: 617 3864 2554; Fax: 617 3864 1534; E-mail: [email protected]

1096–6374/00/050275+15 $35.00/0

amino acid sequence was identical to that of the extracellular domain of the membrane GHR2. The complete sequence of the rabbit GHBP, however, including the carboxyl terminus, remains unknown. On the other hand, the full sequences of the rat and mouse GHBP have been deduced from analysis of a truncated, alternatively spliced GHR cDNA, found to date only in these rodent species3,4. They predict a protein containing an identical extracellular domain to the rat/mouse membrane GHR and a “hydrophilic” tail at its carboxyl terminus which has replaced the transmembrane and cytoplasmic domains. Studies using antibodies raised against this unique tail demonstrated the presence of a soluble GHBP in rat serum5 and there is some recent evidence to suggest that it may also be associated with target cell membranes6. There is increasing evidence that the rabbit and human GHBPs are produced by proteolytic cleavage © 2000 Harcourt Publishers Ltd

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from membrane-bound GHR7,8. Until recently it was thought that this mechanism did not operate in rats and mice. Recent studies, however, identified a human GHR mRNA transcript that coded for a protein lacking over 90% of the cytoplasmic domain and had an increased capacity to generate a soluble GHBP9,10. A similar transcript was also identified by PCR in the rat and mouse, suggesting that GHBPs in these species may also be produced from the membrane bound receptor by a comparable mechanism9. Such GHBPs would lack the “hydrophilic” tail produced by alternative mRNA splicing. In the rat, therefore, both proteolytic cleavage of the membrane bound GHR (full-length or truncated) and translation from an alternative GHR mRNA transcript may operate to produce GHBPs that differ in their carboxyl terminal sequence, thus producing heterogeneity in GHBP structure. Mr heterogeneity is a common feature of GHBPs in rabbit, human, rat, mouse and guinea pig sera11–17. Growth hormone (GH) binding activity in rat serum and other mammalian sera is associated with proteins which exhibit significantly higher native Mr than that predicted from cloning studies. This suggests that they may undergo post-translational modification, yet nothing is known about their biosynthesis11–14,16. Serum GHBPs in several species have been classified into Types 0–4 on the basis of their GH binding properties using human GH (hGH), or bovine prolactin (bPRL) as ligands18. This is intriguing given the presumed sequence identity in their core protein and raises a number of questions regarding their post-translational modification and possible association with other proteins. Human GH has been widely used as a ligand to measure GH binding activity in various mammalian sera. We recently demonstrated, however, that hGH did not bind specifically to guinea pig serum GHBP, whereas abundant GH binding activity could be demonstrated using 125I-oGH16. We also observed differential binding specificity towards hGH and oGH by rat and mouse, but not rabbit, serum GHBPs16. Very recent studies by Leung et al17 have highlighted the presence of a high molecular weight (Mr), but very low affinity, 125I-hGH binding protein in rat serum, which appeared to be immunologically distinct from the classical low Mr GHBP. In contrast, studies on the native Mr of the GH binding activity in rat serum have demonstrated the presence of a single species using 125I-hGH13, whereas at least two specifically labelled complexes have been identified using labelled bovine GH (bGH)14. Recently, we have shown that 125 I-oGH binding to rat serum also revealed the presence of multiple specifically bound complexes and, furthermore, that 125I-hGH did not detect all of the GH binding activity present19. These observations prompted us to

examine in more detail the nature and binding characteristics of rat serum GHBPs using 125I-oGH, a purely somatotrophic ligand, and to determine their immunological relationship to the cloned rat GHBP, using specific antibodies which can distinguish GHBPs with and without a “hydrophilic” tail.

MATERIALS AND METHODS Hormones hGH (NIDDK hGH I-1) and oGH (I-1-4) used for iodination and oGH (NIH-GH-S-15), bGH (NIH-GH-B-18), and oPRL (NIH-P-S-20), used for unlabelled preparations were gifts of the National Hormone and Pituitary Program (NIADDKD, NIH, Bethseda, MD, USA). Recombinant and pituitary hGH were obtained from the Commonwealth Serum Laboratories, Melbourne, Australia. Recombinant rat GH (rGH) was obtained from Bresatec, Adelaide, Australia. Antibodies MAb 263, which was raised against rat liver membrane GHR20 and recognizes the extracellular GH binding domain of the GHR, was a gift of Dr M J Waters. Affinity purified polyclonal antibody RB1615 was raised against a synthetic peptide corresponding to the 17 “hydrophilic” amino acids predicted from the rat GHBP mRNA and was a gift of Dr P Frick6. Control MAb C (against chicken immunoglobulin, IgG1 phenotype) was from Silenius, Melbourne, Australia. Control polyclonal antibody, anti-limonin, was from Sigma, St Louis, MO, USA and non-immune rabbit serum was obtained from Vector, Burlingame, USA.

Iodination oGH and hGH were iodinated as previously described using the Iodogen method21. Specific activities of 18–30 and 30–40 µCi/µg were achieved for oGH and hGH respectively.

Animals Serum and liver tissue were collected from pregnant (19 days) Sprague Dawley rats housed at the Animal Laboratory of the Royal Children’s Hospital, Melbourne, Australia. The study was approved by the Animal Ethics Committee of the Royal Children’s Hospital, Melbourne, Australia. Serum and liver were stored at –20°C prior to analysis.

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Receptor preparation Microsomal membranes (100 000 × g) were prepared as described previously16 with the addition of benzamidine (10 mM) during preparation and resuspension of pellet. Protein estimations for membranes and serum were carried out by the methods of Lowry22 and Bradford23, respectively. Binding studies Binding studies were performed overnight (16–24 h) at 21–23°C using 25 mM HEPES buffer pH 7.5 containing 10 mM MgCl2, 0.02% (wt/vol) sodium azide and 0.1% (wt/vol) BSA (assay buffer) in a final volume of 250 µL. Pregnant rat serum (0.2–50 µL), or liver membrane (100–300 µg protein) was incubated with 125I-oGH or 125 I-hGH (20–30 000 cpm; 12–20 fmol) in the presence and absence of unlabelled GH (0.1 µM). For membrane preparations, bound and free hormones were separated by centrifugation16. For serum, bound and free hormone were separated by gel filtration on AcA54 mini-columns (0.6 × 22 cm) as described previously11. Detection of GH binding activity by gel filtration chromatography Serum was incubated with 125I-GH as described above and the entire incubation mixture was chromatographed on an Ultrogel AcA34 column (1 × 96 cm) as previously described16. The Ultrogel column was calibrated with the following protein molecular weight (Mr) markers: ferritin (horse spleen, Mr, 440 K); β-amylase (sweet potato; Mr, 200 K); alcohol dehydrogenase (yeast; Mr, 150 K); hexokinase (yeast, Mr, 99 K); lactoperoxidase (bovine milk, Mr, 85 K); albumin (bovine serum, Mr, 68 K) or albumin (chicken egg, Mr 45 K); trypsin inhibitor (soya bean, Mr, 20 K). Blue dextran (Mr, 2,000 K) and 125I-Nal were used to determined the void volume (Vo) and total volume (Vt) of the column, respectively. Alternatively, serum alone (1.5 mL) was chromatographed on the same AcA34 column and the GH binding profile was determined by taking 10–100 µL aliquots from each 1 mL column fraction, incubating with 125I-GH, as described above, and measuring specific binding by separation of bound and free GH on AcA54 mini-columns11. The Mr of each fraction was determined from the elution volume. Immuno-precipitation of preformed 125I-oGH serum complex The binding assay incubation volume was scaled up to allow multiple aliquots of each mixture to be removed for the subsequent treatments. Serum fractions (equivalent

to 5–10 µL of serum fraction in a total of 250 µL incubation volume) obtained by chromatography on Ultrogel AcA34 as described above, were incubated with 125 I-oGH in the presence and absence of unlabelled oGH (0.1 µM) in assay buffer for 6 h at 21–23°C. Aliquots were taken from each sample and added to tubes containing buffer, control MAb C, MAb 263, polyclonal antibody control or RB1615 and incubated for a further 18 h at 21–23°C. The GHBP-MAb or GHBP-polyclonal antibody complexes were immunoprecipitated as described previously by the addition of ice-cold polyethylene glycol 6 000 and γ-globulin24 or protein A crosslinked to agarose beads6, respectively. The radioactivity was measured in each pellet using an LKB gamma counter. For all samples containing buffer or RB1615, bound and free hormone were also separated by gel filtration on AcA54 minicolumns11. “Pore limit” native PAGE Whole rat serum (10 µL) or serum fractions (100 µL) obtained by gel chromatography on Ultrogel AcA34, as described above, were incubated with 125I-oGH in the absence or presence of unlabelled (0.1 µM) oGH or hGH as described above in Binding Studies. An aliquot (10–15 µL) of each mixture was then electrophoresed to their restricted pore size25 on Gradipore (North Ryde, Australia) pre-cast micro gels (concave gradient, 5–40% w/vol acrylamide; linear Mr range of 1000 K–5 K) in TEB buffer [1 × TEB = 1% (w/v) Tris, 0.5% (w/v) Boric Acid and 0.05% (w/v) EDTA] at pH 8.3 for 1 h at 150 volts. Gels were stained in Gradipure stain (Gradipore) for 1–2 h, rinsed in H2O, treated with gel drying solution (Novex, San Diego, USA) and dried, followed by exposure to XAR film (Eastman Kodak, NY, USA) and Lightning Plus enhancing screens (Dupont, DE, USA) at –75°C. Gels were calibrated with Gradipore and Pharmacia (Castle Hill, Australia) native protein Mr markers (800 K–20 K) and (669 K–67 K), respectively. Western immunoblot analysis of serum GHBP Native PAGE: Serum fractions corresponding to GHBPa (Mr ~ 257 K) and GHBPb (Mr ~ 98 K) were obtained by gel chromatography on Ultrogel AcA34 as described above. Each GHBP isoform (10 µL) was electrophoresed on Gradipore precast micro-gels as described above in “Pore limit” native PAGE, transferred to PVDF membrane (Amersham, Castle Hill, Australia), immuno-blotted and assessed by enhanced chemiluminescence detection (ECL) using Amersham’s Western ECL kit as previously described16. The following primary antibodies were used: control MAb C and Rat GHR MAb 263 (final

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concentration, 1–7 µg/mL), or rat GHBP polyclonal antibody, RB1615 or non-immune rabbit serum (1/1000). Anti-mouse or anti-rabbit horseradish peroxidase (HRP) labelled secondary antibodies (1/2000 dilution) were used to detect primary monoclonal or polyclonal antibodies, respectively. Blots were stained with Ponceau S prior to immunodetection in order to determine the Mr of the standard proteins. After blotting, gels were stained with Gradipure (Gradipore) to ensure that proteins were adequately transferred. SDS PAGE: Serum fractions (10 µL) corresponding to GHBPa (Mr ~ 257 K) and GHBPb (Mr ~ 98 K) were boiled for 5 min in sample buffer containing Tris-HCl/2% SDS with or without 100 mM dithiothreitol (DTT). Electrophoresis was performed on Gradipore precast, mini-gels (concave gradient, 4–20% (wt/vol) acrylamide), in Tris/glycine/SDS buffer as described by the manufacturers. Samples were blotted and immunodetected as described above for native PAGE except for the addition of methanol (20% v/v) to the transfer buffer. Both Sigma broad Mr standards (205 K–29 K) and Life Technologies (Melbourne, Australia) Benchmark standards (220 K– 10 K) were used to calibrate the gels and estimate the Mr of immunospecific bands. However, it is important to note that both products generally gave similar Mr estimation but the Mr differed significantly below 50 K, with the Benchmark standards giving lower values. The Mr reported in these studies has been estimated using Benchmark standards. Biorad (Hornsby, Australia) prestained (203 K–7 K calibrated) markers were used to monitor protein transfer and at times were used as an extra check for Mr estimation. RESULTS

Fig. 1 Radioactive elution profile of pregnant rat serum complexed to 125I-GH chromatographed on Ultrogel AcA34 (1 × 96 cm): (a) Serum (10 µL) was incubated with 125I-oGH (~20–30 000 cpm) in the absence (total binding) or in the presence of excess (0.1 µM) unlabelled oGH or hGH for 24 h at 21°C. The entire incubation mixtures were gel chromatographed and the radioactive elution profiles were determined by counting the radioactivity of each 1ml fraction. (b) Serum (50 µL) was incubated with 125I-hGH as described above and separated on a similar sized Ultrogel AcA34 column. The Mr of the 125I-GH-GHBP complexes have been estimated from protein standards (440 K–20 K) used to calibrate the column as described.

The presence and native Mr of GHBPs in rat serum was determined by gel filtration chromatography of either a) serum preincubated with 125I-GH or b) serum alone followed by measurement of specific 125I-oGH binding in each elution fraction. a) A representative radioactive elution profile of the chromatography of preformed 125I-oGH serum-complex is shown in Fig. 1A. We consistently observed specifically labelled complexes eluting with apparent Mr of 257 K, 123 K and 54 K (a shoulder) which were displaced by excess unlabelled oGH but only poorly by hGH. A labelled complex eluting at the void volume of the column was not displaced by either oGH or hGH but, invariably, total 125I-oGH binding was increased in the presence of excess unlabelled oGH. In contrast, gel chromatography of rat serum pre-incubated with 125I-hGH resulted in the formation of a 191 K complex, which was specifically displaced by excess oGH and hGH, and a non-displaceable complex at the void volume (Fig. 1B).

Thus, three specifically displaced complexes were identified with labelled oGH but only one was identified with labelled hGH. b) In order to examine the molecular size of native (i.e. uncomplexed, free) GHBP in rat serum, serum alone was first chromatographed on the same AcA34 column as used above and the specific binding of 125I-GH in each fraction was subsequently measured using Ultrogel AcA54 mini-columns (0.6 × 20 cm) to separate bound and free hormone11. The specific oGH-binding profile is shown in Fig. 2a. GH binding activity covering a wide Mr range (~ 400 K–50 K) was identified. The broad distribution of GH-binding species may reflect a mixture of GHBPs which are structurally distinct, aggregated and/or bound to carrier proteins. Two major peaks of GH binding activity, however, eluted at an apparent Mr of 220K and 100K. If one adds the Mr of GH (~ 22 K) to these peak GHBP Mr then they are similar in size to the 125 I-GH/GHBP complexes of 257 K and 123 K shown in

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Whole serum A

B

C

Serum fraction (Mr 176K) D

E

F 10–3 × Mr

10–3 × Mr

195

195 102 xs oGH xs hGH Fig. 2 Specific GH binding profiles of pregnant rat serum partially purified on Ultrogel AcA 34 (1 × 96 cm). Serum (1.5 mL) alone was chromatographed on the same AcA 34 column and the specific binding profiles of 125I-oGH (a) or 125I-hGH (b) were determined as follows: 10 µL or 100 µL aliquots from each 1 mL fraction were incubated with 125I-oGH or 125I-hGH, respectively, as described above. Each data point is derived from AcA54 mini-column separation of bound and free 125I-GH (0.6 × 22 cm) as described previously11 and is the mean of duplicates.

Fig 1a. Nonetheless, we were interested to know if the 257 K complex was a dimer of the ~123 K complex. Thus, a serum fraction corresponding to the Mr 100 K peak (Fig 2) was incubated with labelled GH as described above and the whole incubation mixture was chromatographed on the same AcA34 column from which the fraction had been semi-purified. The data (not shown) indicated that it eluted at a Mr of ~145 K and not at 257 K. Various other fractions were similarly analysed and little change in their elution Mr (other than the addition of 22 K for the bound GH) was observed. These data clearly suggest that, within the tolerance limits (10–15%) of Mr determination by gel filtration techniques, the binding stoichiometry of oGH to these binding species was 1:1. In contrast, we did find that serum fractions corresponding to the smallest GHBPs (Mr 40–70 K) increased significantly (~ two-fold) in Mr following binding to GH. This may suggest that dimerisation (binding stoichiometry of 1 GH:2 GHBP) or a conformational change had occurred (data not shown). These data also indicate that the high Mr form was already present in serum and existed naturally as a large complex, either as a naturally occurring aggregate of the smaller Mr GHBP or in covalent or noncovalent association with other proteins. We also determined binding of 125I-hGH to the same partially purified serum fractions as used for 125I-oGH binding in Fig 2a. In order to maximize detection of hGH binding activity, we used 100 µL of serum fraction rather than 10 µL which was used for 125I-oGH. A similarly wide Mr distribution of specific binding was observed. Binding was significantly lower, however, with hGH than with oGH to the same fractions (Fig. 2b). Maximum specific GH binding activity was associated with proteins exhibiting Mr ~180 K, while a smaller peak of binding was

– –

+ –

– +

Whole serum A 10–3

B

C

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Serum fraction (Mr 54K) D

E

F 10–3 × Mr

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186 98 xs oGH xs hGH

89 – –

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Fig. 3 Autoradiograph of electrophoretic patterns of preformed 125 I-oGH-serum complexes run on native micro-gels. Pregnant rat serum or serum fractions were incubated with 125I-oGH in the presence and absence of unlabelled oGH or hGH (0.1 µM). The incubation mixtures (10 µL) were subjected to “pore limit” native PAGE (5–40%; concave gradient) in 1 × TEB pH 8.3. Whole serum is shown in upper and lower panels (lanes A–C) and serum fractions corresponding to Mr ~ 176 K and ~ 54 K, obtained by gel chromatography on Ultrogel AcA34 as shown in Fig. 2A, are shown in upper and lower panels (lanes D–F), respectively. Total binding and non-specific binding lanes are marked – and + excess unlabelled oGH or hGH, respectively. The Mr values for the specifically labelled 125I-oGH-binding protein complexes are shown by the arrows. Autoradiography was carried out for 5 days at –70°C using Kodak Xomat AR film. Abbreviations: xs, excess unlabelled.

associated with GHBPs of ~100 K. Given the potential errors in measurement of Mr using gel chromatography, both ligands gave similar native Mr for peak GH binding activity. However, 125I-oGH binding was poorly displaced by hGH suggesting the presence of variant GHBPs which may be ligand specific. We also observed that very high Mr fractions (eluting in the void volume) constituted over 80% of total 125I-hGH bound, but binding was not displaced by excess hGH (data not shown). Given that Mr estimation by gel filtration chromatography may give anomalous results, together with the possibility of dissociation of the preformed 125I-oGHcomplexes during chromatography, we used an alternative technique – “pore limit” native gel electrophoresis – to estimate the native Mr of GHBPs in whole serum and in serum fractions corresponding to the higher (176 K)

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and lower (54 K) Mr peaks. A representative autoradiograph of the electrophoretic pattern of preformed 125IoGH serum complexes is shown in Fig. 3. Binding of 125 I-oGH to whole serum resulted in the formation of two specifically labelled complexes of Mr 195 K/186 K and 102 K/98 K (lane A, upper and lower panels) which were displaced by excess oGH (lane B, upper and lower panels) but only poorly by hGH (lane C, upper and lower panels). These values are somewhat lower than the native Mr of the 125I-oGH-serum complexes determined by gel filtration chromatography, as shown in Fig. 1a. Binding of 125 I-oGH to individual serum fractions corresponding to Mr ~176 K and ~54 K (obtained by chromatography as shown in Fig. 2a) resulted in the formation of 195 K and 89 K complexes, respectively, as shown in Fig. 3 (lanes D-F, upper and lower panels). Both complexes demonstrate the same binding specificity for oGH and hGH shown by whole rat serum. A binding stoichiometry of 1:1 of oGH to the 176 K GHBP or the 54 K GHBP is suggested by the data. Measurements of 125I-oGH specific binding to whole rat serum determined by chromatography on the large AcA34 column were significantly less (up to 50%) than those obtained using AcA54 mini columns, suggesting that some dissociation of bound GHBP had occurred during chromatography. Similar results were obtained for GHBPs exhibiting Mr ~ 100 K. We hypothesized that some GHBPs may dissociate more readily due to a lower affinity for 125I-oGH. Thus, rat serum was size fractionated on an Ultrogel AcA34 column as shown in Fig. 2a and full Scatchard analysis of each elution fraction was used to determine the binding affinity and capacity of GHBPs for 125I-oGH binding. The Scatchard data generally revealed a single class of GH binding sites but curvilinear plots were also observed for serum fractions eluting with a Mr of 160 K–180 K (data not shown). A two-site model, however, was not accommodated by the LIGAND program 26. As clearly illustrated in Fig. 4, binding affinity (0.2–5.4 nM–1) and capacity (300– 40 000 fmoles/mL serum fraction) varied significantly and inversely across the Mr profile. The two major peaks of GH binding activity corresponded to proteins of Mr 257 K and 98 K, similar to those observed for the two major GHBPs identified by gel chromatography of serum preincubated with 125I-oGH or serum alone (as shown in Fig. 1A and 2A respectively). The data of Fig. 4 demonstrate the presence of two major classes of GHBPs, designated GHBPa (Mr 257K) and GHBPb (98K), which, in addition to their distinct Mr, can be further distinguished by their higher (2–5 nM–1) and lower binding affinities (0.2–0.8 nM–1), respectively. The designation of “high” and “low” is only relative since the affinity of both classes of GHBPs is still high. GHBPa represents ~15–20% of total GHBPs in pregnant rat serum

Fig. 4 Representative profile of the binding affinity and capacity of GHBPs in individual pregnant rat serum fractions as estimated by Scatchard analysis. Dose response curves of 125I-oGH binding for each serum fraction were determined to ensure that the percentage specific binding was on the linear part of the curve. The data were obtained using the Ligand-PC program of Munson and Rodbard25 based on duplicate dose response curves, using 5 µL of pregnant rat serum fractions (from rat serum purified as shown in Fig. 2a), increasing concentrations of unlabelled oGH and a fixed concentration of 125I-oGH. Bound and free hormone were separated by gel chromatography on Ultrogel AcA54 mini columns. Left and right y axes correspond to binding affinity and capacity, respectively. Abbreviation B/F, Bound to Free ratio.

(total = 168,468 ± 22,845 fmol/mL serum, mean ± SEM, n = 3), with GHBPb representing the remaining and, therefore, major form of circulating GHBP. Similar profiles of Mr (± 10%), and relative binding affinity and capacity were observed for two other samples of pregnant rat serum fractionated on the same column, although the relative binding affinities (0.5–2 nM–1 and 5–10 nM–1) were somewhat higher for both classes of GHBPs. The delineation of high and low affinity GHBPs, as determined by Scatchard analysis, provides a possible explanation for the significant dissociation of the lower affinity GHBPs of Mr < 120 K on the large AcA34 column. Since rat serum contains two major classes of GHBPs, the binding characteristics were examined in serum fractions corresponding to these isoforms and compared to those in whole rat serum. Within these two classes, designated GHBPa and GHBPb, serum fractions corresponding to peak GH binding activity (Fig. 4) which eluted at Mr ~257 K and ~98 K, respectively, were characterized. Specific binding was dependent on concentration and incubation time and was readily detectable with as little as 0.2 µL of pregnant rat serum (14%), 1ul of GHBPa (8.34%) and 1 µL of GHBPb (6.86%) in a total incubation volume of 250 µL (Fig. 5A, left panels). The association/dissociation characteristics for serum and GHBP isoforms are shown in Fig. 5B (right panels). Equilibrium was achieved by 20 h at 21–23°C for whole serum and the reaction was reversible with a t 1-2 of 2 h following addition of a large excess of unlabelled oGH. Association

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Fig. 5 125I-oGH binding characteristics of whole serum and partially purified serum. The effect of increasing serum concentration (A) and the time course of association and dissociation (B) for the specific binding of 125I-oGH to pregnant rat serum or the GHBP isoforms are shown in left and right panels respectively. In (B) binding was performed at 21°C for 16 h and dissociation was initiated in one set of tubes by addition of an excess (2 µg) of unlabelled oGH in a 10-µL volume to avoid dilution effects. Each data point is derived from AcA54 minicolumn separation of bound and free 125I-oGH and is the mean of duplicates.

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Fig. 6 The hormonal specificity of binding of 125I-oGH to pregnant rat serum or liver membranes. All unlabelled hormones were added at a final concentration of 0.1 µM. Binding is expressed as a percentage of total binding (T = 100%) in the absence of unlabelled hormone. For 125 I-oGH binding: T = 14% for pregnant rat serum (0.2 µL) and 18% for liver membrane (150 µg protein).

Fig. 7 Specific 125I-oGH binding profiles of serum fractions determined by gel chromatography or immunoprecipitation. Pregnant rat serum was size fractionated by gel chromatography as shown in Fig. 2A and each fraction was incubated with 125I-oGH in the presence and absence of excess unlabelled oGH. The oGH specific binding profile for each fraction was determined as follows: bound and free hormone were separated by gel chromatography (control) on Ultrogel AcA54 mini-columns11 or by immunoprecipitation with MAb26323 or polyclonal antibody RB16156. The percentage specific binding shown is for 10 µL of serum fraction and each data point is the mean of duplicates.

rate at 21°C was faster for GHBPb (equilibrium reached in 6 h) than for GHBPa which required approximately 24 h to reach equilibrium. The binding reaction for GHBPb was reversible with a t-12 of 1 h whereas the dissociation reaction for GHBPa was slow with only 34% of bound complex dissociated by 6 h. The dissociation profile for

whole serum was not linear and is likely to be due to the presence of the two classes of GHBPs. The hormonal specificity of 125I-oGH binding to rat serum and liver membranes is illustrated in Fig. 6. While oGH and bGH were equally effective in displacing 125 I-oGH bound to rat serum GHBP, hGH was less

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effective than rGH and no competition was observed for 20 K hGH and oPRL. Similar results were obtained with higher concentrations of rat serum (5–10 µL) but, noticeably, hGH and rGH became extremely poor competitors (data not shown). Binding to rat liver membranes was displaced similarly by all the GHs, but hGH was the least effective. In contrast to serum, 20 K hGH displaced ~50% of total binding to membranes and some cross-reactivity was observed with oPRL. Thus, in contrast to the purely somatogenic nature of the serum GHBPs, pregnant liver GHR exhibited some lactogenic specificity. However, 125I-oGH binding to the membrane GHR in adult male and female liver exhibited purely somatogenic specificity with hGH being the least potent (data not shown). Previous studies27 also found that hGH was less effective than oGH or bGH in displacing 125 I-bGH binding to rat liver membranes. Immunoprecipitation was used as an alternative method to determine if the GH binding profile obtained by gel filtration could be replicated and whether the detected GHBPs were immunologically related to the cloned GHBP. Rat serum was fractionated on an Ultrogel AcA34 column as shown in Fig. 2A and each fraction was incubated with 125I-oGH (± unlabelled oGH). Bound and free hormone was separated either by chromatography on AcA54 mini-columns (control), or by immunoprecipitation of the 125I-oGH-GHBP complex with antibodies which recognise the N-terminal (MAb 263) or carboxyterminal (RB1615) sequence of the GHBP. As shown in Fig. 7, the binding profile obtained by immunoprecipitation across the Mr range is generally similar to gel filtration and both antibodies recognised proteins within the high and low Mr forms of the GHBPs. The level of specific binding, however, was significantly lower than that obtained using the “control” AcA54 mini-column method. This observation is a little surprising and may be related to methodology or, alternatively, GHBPs may be present which are not recognised by either antibody. It is clear from the data that both antibodies recognise the native forms of the GHBPs when complexed to 125I-oGH, but the percentage specific binding immunoprecipitated by each antibody varied within each fraction. Furthermore, the percentage of the control specific binding (as measured by AcA54 mini-columns) which was immunoprecipitated for each fraction, was not constant (30–75%) and was considerably higher for lower Mr serum fractions precipitated with antibody RB1615. Since Mab263 recognizes the extracellular GH-binding domain of the GHR it will identify GHBPs with and without a hydrophilic tail, whereas RB1615 recognises only the hydrophilic tail. Both antibodies immunoprecipitated GHBPs complexed to labelled GH in each elution fraction. However, this observation does not necessarily imply that the serum fractions contain only GHBPs with

Fig. 8 Effect of rat GHBP antibody on 125I-oGH specific binding to serum fractions. Serum fractions (10 µL) were incubated with 125 I-oGH in the absence and presence of excess unlabelled oGH for 6 h. Aliquots were transferred to duplicate tubes containing rat GHBP antibody RB1615, control polyclonal antibody or buffer and all tubes were incubated for a further 18 h. After a total of 24 h incubation the whole incubation mixtures were gel chromatographed on Ultrogel AcA 54 mini-columns to separate bound and free hormone. For each fraction, “control” is the specific binding of 125I-oGH measured in the absence of antibody. Shown is the percentage enhancement of 125I-oGH specific binding of control in the presence of RB1615.

a hydrophilic tail. GHBPs which lack the hydrophilic tail could also be present in the same elution fractions. This has been examined specifically by Western blot analysis in Figs. 10–12 below. Given that immunoprecipitation did not detect all of the GH binding activity determined by chromatography on AcA54 mini-columns, we examined whether gel chromatography on the large AcA34 column of preformed 125I-oGH-serum complexes, which had been incubated in the presence of MAb263 or RB1615, would allow recognition of all the bound complexes by these antibodies. We consistently observed that oGH specific binding was enhanced (20–30%) in the presence of RB1615 but inhibited (20–30%) in the presence of MAb 263. Similar results were obtained using AcA54 minicolumns (data not shown). Inhibition of 125I-oGH binding by MAb 263 is characteristically observed for the proposed Type 2 rat and rabbit GHR20. The effect of RB1615 on oGH specific binding to individual serum fractions across the Mr range (~400 K–50 K) was determined and the data are shown in Fig. 8. We consistently observed a significant increase (10–107%) of the percentage specific binding of 125I-oGH in the presence of RB1615 and this was dependent on the Mr of the GHBP, enhancement being observed for fractions up to ~190 K but not for those of 190–400 K. The % specific binding of the <190 K fractions was also enhanced using MAb 4.3, another antibody which

284 S. I. Ymer et al.

GHBPa

10–3 × Mr

A

B

C

GHBPb

D

E

F

G

H

10–3 × Mr

447 240

Fig. 9 Scatchard plots of 125I-oGH binding to GHBPa and GHBPb in the presence and absence of rat GHBP antibody RB1615. The data were obtained using the Ligand-PC program of Munson and Rodbard25 based on duplicate dose response curves, using 5 µL of GHBPa (left panel) and 4 µL of GHBPb (right panel), increasing concentrations of unlabelled oGH with or without a fixed concentration of RB1615 (1/1000 final concentration) and 125I-oGH (12–20 fmol). Bound and free hormone were separated by gel chromatography on Ultrogel AcA54 mini columns. Abbreviation B/F, Bound to Free ratio.

recognizes the “hydrophilic” tail (data not shown). Subsequent Scatchard analysis of various Mr serum fractions belonging to the two major classes of GHBPs (± RB1615), revealed that the RB1615-induced increase in binding was manifested by a doubling of the affinity constant. For GHBPs of Mr ~60 K binding capacity decreased by about 19% whereas other GHBPs exhibited up to a 50% decrease. Scatchard analysis on the higher Mr serum fractions (~400 K–190 K) validated the lack of enhancement of specific binding in the presence of RB1615, since binding affinity and capacity were not significantly altered. A representative example of Scatchard plots corresponding to the two major classes of GHBPs ± RB1615 is shown in Fig. 9. Thus, although the two major classes of GHBPs were recognized by RB1615, the enhancement of 125I-oGH binding by RB1615 was observed only for the GHBPb isoform (Fig. 9b). This suggests some difference in the structural composition between the GHBPa and GHBPb isoforms. Further immunological analysis was used to examine in more detail the native and denatured structural characteristics of the two classes of GHBPs. The chemiluminescent signal of a representative Western immunoblot analysis of GHBPa and GHBPb subjected to “pore limit” native PAGE is shown in Fig. 10. Several proteins (Mr 447 K, 240 K and 110 K), which were strongly immunoreactive with MAb 263 (lane B) but weakly or not at all with RB1615 (lane C), were associated with GHBPa. On longer exposures significant cross-reactivity was observed with RB1615 for the 110 K protein. Interestingly, a protein of 102 K (lane C), which appears clearly distinct from the 110 K protein, was detected only with RB1615 and this finding has been consistently observed. In contrast, GHBPb (right panel) is associated with proteins of Mr 110 K, 48 K and 44 K which were strongly immunoreac-

110

110

102

48

85

44

Fig. 10 Western immunoblot analysis of GHBP isoforms using ECL detection. Samples (10 µL) containing GHBPa (lanes A–D) or GHBPb (lanes E–H) were subjected to “pore limit” native PAGE on micro-gels (concave gradient: 5–40%) in 1× TEB buffer pH 8.3. Proteins were electroeluted onto PVDF membrane, incubated with control MAb C (lanes A and E), rat liver membrane GHR MAb 263 (lanes B and F), polyclonal rat GHBP RB1615 (lanes C and G), or normal rabbit serum (lanes D and H) followed by immunodetection with an antimouse (lanes A, B, E and F) or anti-rabbit (lanes C, D, G and H) HRP labelled secondary antibody using the Amersham ECL detection system. The chemiluminescent signal shown was evaluated after 2 min exposure of the blot using Amersham Hyperfilm-ECL. Mr values for the specific immunodetected GHBPs are shown by the arrows.

tive with MAb 263 (lane F), and were also recognised by RB1615 (lane G). These data suggest that GHBPa is associated with proteins (447 K, 240 K) which may lack the “hydrophilic” tail, whereas all of the immunoreactive proteins associated with GHBPb contain the “hydrophilic” tail. Interestingly, the lack of cross-reactivity of MAb 263 with a GHBPa protein of Mr 102 K (lane C) containing the “hydrophilic” tail raises the possibility of a structurally distinct GH binding domain from the cloned GHBP. A lack of immunoreactivity needs to be interpreted with caution, however, as accessibility of the antibody epitope site may be blocked when the protein is in a native conformational state. In order to delineate the composition of the immunoreactive proteins associated with GHBP isoforms, GHBPa and GHBPb were subjected to denaturing SDS PAGE in the presence and absence of DTT, followed by immunodetection with the same antibodies as described above in Fig. 10. The specificity of the antibodies was always examined in relation to the control antibodies. A chemiluminescent signal of the Western immunoblot analysis of GHBPa and GHBPb is shown in Fig. 11 and Fig. 12, respectively. GHBPa was associated with several proteins which were strongly immunoreactive with MAb263 and exhibited high Mr (229 K–81 K) in the absence (lane C) and even in the presence (182 K–

Novel rat serum growth hormone binding proteins 285

MAbC

10–3 × Mr

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B

MAb263

C

RB1615

D

229

10–3 × Mr

182 123 105 78 68

135 112 81

E

F

MAbC

NRS

G

H

98 78

47 41 35

27

26

–

+

–

+

A

B

C

–

+

–

+

Fig. 11 Western immunoblot analysis of GHBPa isoforms using ECL. Samples (10 µL) containing GHBPa were subjected to SDS PAGE (4–20% concave gradient) in the absence (–) and presence (+) of 100 mM DTT. Proteins were electroeluted onto PVDF membrane, incubated with control MAb C (lane A, B), rat liver membrane GHR MAb 263 (lane C, D), rat GHBP polyclonal antibody RB1615 (lane E, F), or normal rabbit serum (lane G, H) followed by immunodetection with an anti-mouse (lanes A–D) or anti-rabbit (lanes E–H) HRP labelled secondary antibody using the Amersham ECL detection system. The chemiluminescent signals shown were evaluated after 2 min exposure of the blots using Amersham Hyperfilm-ECL. The blots are derived from the same gel and have been divided to allow clear identification of the specific immunodetected proteins. The Mr values for –DTT and +DTT lanes are shown by the arrows on the left and right side of the blot respectively.

78 K) of DTT (Fig 11 lane D). Smaller MAb 263 weakly immunoreactive proteins of Mr (68 K–27 K) were primarily present under reducing conditions (lane D). RB1615 was strongly immunoreactive with GHBPa associated proteins (98 K, 78 K –DTT) and (47 K, 41 K +DTT), lane E and F respectively. Several proteins (110 K–76 K) which were weakly immunoreactive with RB1615 were identified only under reducing conditions (lane F). The identification of immunoreactive proteins only under reducing conditions suggests that they are normally disulphide linked to other proteins as part of a larger complex. The origin of these particular proteins is not clear. Comparisons of the Mr of the immunoreactive proteins detected by each antibody suggest that several GHBPaassociated proteins [e.g. 112–229 K (–DTT), 123 & 182 K (+DTT)] lack the “hydrophilic” tail. Interestingly, the presence of a protein of Mr 47 K (+DTT lane F) which was recognised by RB1615 but not by MAb 263 raises the possibility that it may contain a different N-terminal sequence from the cloned GHBP. This protein may be the denatured form of the similarly immunoreactive protein of Mr 102 K identified above by “pore limit” native PAGE (Fig. 10 lane C). A similarly sized protein of Mr 98 K (Fig 11. lane E, –DTT), which disappeared under reducing conditions (Fig 11. lane F), was detectable with RB1615. However, it is unclear if it was also detectable with MAb

RB1615

D

E

10–3 × Mr

159 138

110

46

48 43

26

26

DTT

F

NRS

G

H

10–3 × Mr

224 110 105 76

42 35

DTT

10–3 × Mr

MAb263

–

+

–

+

10–3 × Mr

138 110

46 43

48 43

15

17

–

+

–

+

Fig. 12 Western immunoblot analysis of GHBPb. Samples (10 µL) containing GHBPb were subjected to SDS PAGE (4–20% concave gradient) in the absence (–) and presence (+) of 100 mM DTT. Proteins were blotted and immunodetected as for Fig. 11 excepting the chemiluminescent signals shown were evaluated after 5 min exposure of the blots using Amersham Hyperfilm-ECL. The blots are derived from the same gel and have been divided to allow clear identification of the specific immunodetected proteins. The Mr values for –DTT and +DTT lanes are shown by the arrows on the left and right side of the blot respectively.

263 as several MAb 263 immunoreactive proteins of Mr between 81 K and 112 K (lane C –DTT) could have obscured its presence. Examination of GHBPb (Fig. 12) revealed that MAb 263 was strongly immunoreactive with a protein of 110 K (lane C –DTT) which disappeared in the presence of DTT to be replaced with less immunoreactive proteins of 48 K and 43 K (lane D). Rat serum albumin, which is present in GHBPb, often results in non-specific interaction with the antibodies and migrates as a broad band between 40 K and 60 K thus obscuring the immunoreactive proteins in this region (data not shown). A short exposure time of the chemiluminescent signal (as is shown) identifies the immunoreactive bands in this region more precisely. Additional proteins of 224 K and 46 K (– DTT lane C) and 159 K and 138 K (+DTT lane D) were weakly immunoreactive with MAb 263. RB1615 was strongly immunoreactive with proteins 110 K, 46 K and 43 K (–DTT, lane E) and 48 K and 43 K (+DTT lane F). These findings suggest that the protein of Mr 110 K (–DTT lanes C and E), which was recognized by both MAb 263 and RB1615, is a disulphide linked complex of the GHBPs of Mr 43 and/or 48 K (+DTT lane F), either as a dimer or in association with another similarly-sized, non-GH binding protein. The protein of Mr 48 K (+DTT lanes D and F) is likely to be the same protein as the 46 K (–DTT lanes C and E) with the slight increase in Mr attributed to intra-disulphide bonds. The presence of an RB1615 immunoreactive

286 S. I. Ymer et al.

protein of 43 K in the absence of DTT (lane E) was not consistently observed and may be due to cross contamination from the +DTT sample. The small sized proteins of Mr 15–17 K (–/+DTT lanes E and F) were immunoreactive only with RB1615 and may be proteolysed fragments of GHBP. A strong band at ~56 K–60 K (–DTT lane G) was detected only in the presence of non-immune rabbit serum and its appearance was inconsistent. DISCUSSION These studies have identified and characterized GHBPs in pregnant rat serum and established the presence of GHBP isoforms which can be clearly distinguished on the basis of binding characteristics (affinity, specificity, kinetics) and structural characteristics (immunoreactivity, native and denatured molecular size). Gel chromatographic techniques have demonstrated specific oGH binding activity across a broad Mr spectrum, varying in native Mr from ~400 K to ~50 K. This binding profile, however, reflects the presence of two major classes of GHBPs, with apparent native Mr of ~257 K and ~98 K. These two binding proteins, designated GHBPa and GHBPb, respectively, were clearly distinguished on the basis of detailed Scatchard analyses across the column fractions. GHBPa, which has not been identified previously, represents ~15–20% of total serum GH-binding capacity but exhibits a binding affinity (2.5–5.4 nM–1) that is up to 20-fold higher than GHBPb (0.2–0.8 nM–1). The presence of two classes of purely somatogenic binding sites which also differed significantly in their relative affinities (Ka – 0.5 nM–1 and 12–21 nM–1) has been previously demonstrated in rat liver27. The presence of these two isoforms raises a major note of caution for interpretation of simple GH binding studies in rat serum/liver in the absence of affinity data. Although these studies have been carried out with heterologous ligands, it is clear that both isoforms show cross-reactivity with native rat GH in competition studies. Since the affinity of the GHBPs for endogenous rat GH is unknown, the concentration of the GHBP isoforms may only be indicative of unoccupied rather than total GHBPs present in rat serum. Based on GH binding studies and epitope mapping with a variety of GHR antibodies, Barnard et al20 also suggested the presence of two classes of oGH binding sites in rat liver membranes. Given that GHBPs are structurally related to the membrane GHR it is conceivable that the GHR also exhibits similar heterogeneity. Binding of 125I-hGH to chromatographic fractions of pregnant rat serum identified two major peaks of GH binding with apparent native Mr of 180 K and 100 K (Fig. 2b). These compare in size to the two major isoforms

(220 K and 100 K) identified using 125I-oGH (Fig. 2a) but are somewhat smaller than the low affinity (~105 M–1) high Mr (260 K) hGH-binding protein reported by Leung et al17. Interestingly, in the present study, 125I-hGH binding to these isoforms was displaced equally well by unlabelled hGH or oGH whereas 125I-oGH binding was poorly displaced by unlabelled hGH, suggesting that the proteins recognised by each ligand, although of similar Mr, may not be identical. These observations suggest the presence of distinct oGH- and hGH-specific binding proteins. Measurement of hGH specific binding was significantly lower than that using oGH and it is unclear if this is a reflection of the lower affinity for hGH and/or the presence of distinct ligand-specific GHBPs. The effect of the rat GHBP “hydrophilic” tail specific antibody, RB1615, on binding of oGH to GHBP isoforms has revealed enhancement of oGH specific binding (up to 107%), primarily for the GHBPb isoforms. This phenomenon was particularly pronounced for the smaller GHBPs of Mr ~50 K. Scatchard analysis revealed that the increase in binding was due to a doubling of the affinity constant and an accompanying smaller decrease (19–50%) in binding capacity. One explanation may be GHBP dimerization (via the divalency of the antibody) with a consequent increase in binding affinity. However, an alternative and intriguing possibility is that the “hydrophilic” tail may influence GH binding, a phenomenon that would have potential physiological sequelae arising from the endogenous mechanism(s) used for GHBP generation – alternative mRNA splicing or proteolytic cleavage. In previous studies, examination of GH binding determinants has relied on the use of recombinant GHBP which is the expressed extracellular domain of the membrane GHR28,29. Recombinant GHBP is likely to differ at the C terminus from wild type GHBP since the true carboxyl terminus sequence of the native cleaved GHBP remains unknown for all mammalian species. The possible effect of specific sequences in the GHBP carboxyl terminus on GH binding has not been previously addressed. The presence or absence of the “hydrophilic” tail, as suggested here, or the presence of a different carboxyl terminal sequence, may have significant consequences for GH binding. In the rat, the GHBPs are present in a soluble form in the circulation and there is some evidence to suggest that the GHBP containing a “hydrophilic” tail may also be cell – associated6,30. Thus, it is also possible that free or cell-associated GHBP may exhibit different characteristics in regulation of GH binding. Although it has been established that hGH possesses two binding sites which are recognised equally well by recombinant GHBP29 nothing is known about the interaction of native GHBPs with these two GH binding sites. Given that the endogenous rat serum GHBPs exhibit significant differences in

Novel rat serum growth hormone binding proteins 287

Mr, affinity, abundance and binding kinetics it is conceivable that they may interact differentially with the two GH binding sites. This may impact on the formation of homo-dimeric complexes in the circulation and/or hetero-dimeric complexes with the GHR at the cell surface. These aspects may be critically important for modulation of GH binding and the subsequent cellular response to GH. The suggestion that significant structural differences might exist between the two GHBP isoforms is supported by evidence from Western immunoblot analysis of the GHBPa and b isoforms, subjected to denaturing and nondenaturing PAGE. The GHBPa and GHBPb isoforms are each a complex of several proteins containing disulphide bond-dependent and -independent GHBP immunoreactive proteins. A significant finding was that some proteins were identified with RB1615 and/or MAb263, thus suggesting the presence of GHBPs with and without the “hydrophilic” tail. Although several immunoreactive proteins associated with each isoform exhibit similar Mr they are not identical and may differ due to glycosylation. Several studies have demonstrated that rat serum GHBPs are differentially glycosylated5,31,32. The high sensitivity of chemiluminescent detection, together with the use of both N-terminal (Mab263) and C-terminal (RB1615) GHBP antibodies, has allowed the identification of several immunoreactive proteins. Given the welldescribed specificity of Mab263 for GH receptor-related proteins20 and the absence of any non-specific banding when control antisera were used, it is likely that each of these immunoreactive proteins is indeed GHBP-related, however, we have not formally demonstrated that all of these retain oGH-binding activity and, therefore, they may not be functional. Confirmation of the identity of each of these proteins will require purification and sequence analysis. GHBPa is associated with several MAb 263 immunoreactive proteins, some of which have not been previously identified and which exhibit notably higher Mr (78 K–182 K, +DTT) than that predicted from cloning studies. An important finding is that some of these proteins appear to lack the “hydrophilic” tail. These observations suggest novel alternative mRNA splicing arrangements, extensive post-translational modification and/or the presence of other GHR/GHBP genes. These higher Mr forms may also arise through covalent complexing of a GHBP with other non-GH-binding proteins. The identification of a protein which contains the “hydrophilic” tail but was not immunoreactive with MAb 263 using two different methods [native PAGE (Mr 102 K) and SDS PAGE (Mr 47 K + DTT)] was surprising but is consistent with the proposal that the N-terminal amino acid sequence of this protein may be structurally distinct from the cloned GHBP and may result from alternative

splicing of exons. This protein, of Mr 47 K (+DTT), which is disulphide linked as part of a larger complex (102 K), as well as a protein of 135 K (–DTT) lacking the “hydrophilic” tail, were consistently observed to be associated with the GHBPa but not the GHBPb isoform. Most of the proteins that were associated with the GHBPb isoform were immunoreactive with both RB1615 and MAb 263, as would be predicted by the currently accepted mechanism for generation of the low Mr rat serum GHBP. The proposed structural differences in the N-terminal domain of at least one GHBPa protein and the presence or absence of the hydrophilic tail in other GHBPa proteins are differences which may provide an explanation for the lack of enhancement of oGH specific binding to the GHBPa isoform by the hydrophilic tail Ab, RB1615, compared to its distinct enhancing effect on the GHBPb isoform. The GHBPb isoform, is composed of proteins of Mr 43 K –48 K (–/+DTT) containing the “hydrophilic” tail, of which a significant proportion exists as a disulphide linked complex of Mr 110 K. Previous studies by Sadeghi et al5 and very recent studies by Frick et al32 did not make this observation because they did not subject rat serum to denaturing SDS PAGE in the absence of DTT. The immunoreactive proteins of Mr 43 K and 48 K associated with the GHBPb isoform were similar in size to the Mr 44 K/54 K and 44 K/52 K proteins identified in rat serum by immunoblotting with a rat GHBP “hydrophilic” tail antibody, MAb 4.3, and a rat GHBP polyclonal antibody, respectively5,32. Similar sized GHBPs of Mr 42 K and 58 K were identified using covalent crosslinking techniques31. In rat adipocyte extracts, RB1615 immunoprecipitable proteins of Mr 50 K and 150 K (+DTT) have been identified in high speed supernatants and proteins of Mr 38 K and 42 K (+DTT) have been identified in membranes, but only the latter two proteins were shown to elute from an Affigel-hGH affinity column6. However, this observation alone does not preclude lack of GH binding by the 50 K and 150 K isoform as we have shown the presence of oGH binding proteins which are poorly recognized by hGH. The presence of GHBP distinct isoforms has implications for the measurement of serum GHBP levels. This is due to the presence of GHBPs with significantly different binding affinities, together with the presence of isoforms with and without the “hydrophilic” tail, some of which may elude detection because of the choice of antibody employed for an immuno-functional assay. Although Scatchard analysis is able to distinguish GHBPs of different affinities it requires that rat serum is first fractionated which is time consuming, not readily applicable for routine use and, unlike immuno-functional assays, is only a measure of unoccupied GHBP levels. When using Scatchard analysis or ligand mediated immuno-

288 S. I. Ymer et al.

functional analysis the choice of ligand is a major consideration, particularly given the differences in binding between oGH and hGH for rat serum GHBP. This is also true for guinea pig serum GHBP16. Obviously, other methodologies need to be devised to allow physiological interpretation of the levels of the GHBP isoforms which may be significantly altered during development, and in normal or pathophysiological states. These studies provide evidence for the presence of two major classes of rat serum GHBPs which are structurally distinct and exhibit significantly different binding kinetics. Thus, rat serum GH binding activity cannot be assumed to represent a single protein. The presence of multiple GHBP isoforms suggest that they may be posttranscriptional variants, be differentially post-translationally modified or raise the possibility that they are the product of other GHR/GHBP genes. Our data do not support the concept that the native serum GHBP is simply the extracellular domain of the membrane GHR. Immunological differences with carboxyl-and/or Nterminal sequence directed antibodies suggest that at least two mechanisms operate to generate the serum GHBP isoforms. This observation cannot be reconciled with a single cloned GHBP mRNA of 1.2 kb encoding a GHBP with a “hydrophilic” tail. A significant finding is that the “hydrophilic” tail may be involved in modulating GH binding, and, thus, its presence or absence may have significant implications for binding and subsequent cellular responses to GH. These novel findings raise new questions about the nature and origin of the GHBP isoforms and suggest that they may impact on the formation of complexes with GH in the circulation and/or with GH/GH receptor at the cell surface and, therefore, may have distinct and important functional consequences for the diverse actions of GH. ACKNOWLEDGEMENTS These studies were supported by research grants from the National Health and Medical Research Council (ACH and SIY), and the Royal Children’s Hospital Research Foundation (SIY). The generous infrastructure support of the Centre for Hormone Research is also gratefully acknowledged.

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21. Salacinski PRP, Mclean C, Sykes JEC, Clement-Jones VV, Lowry PJ. Iodination of proteins, glycoproteins and peptides using solid-phase oxidizing agent 1, 3, 4, 6-tetrachloro-3a, 6adiphenylglycoluril (Iodogen). Anal Biochem 1981; 117: 136–146. 22. Lowry OH, Rosebrough NJ, Farr AC, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265–275. 23. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 1976; 72: 248–254. 24. Ho KK, Vaiontis E, Waters MJ, Rajkovic IA. Regulation of growth hormone binding protein in man: comparison of gel chromatography and immunoprecipitation methods. J Clin Endocrinol Metab 1993; 76: 302–308. 25. Wrigley CW, Margolis J. Rapid (ten-minute) pore-gradient electrophoresis of proteins and peptides in Micrograd gels. Appl Theor Electrophor 1992; 3: 13–16. 26. Munson PJ, Rodbard D. LIGAND: a versatile computerised approach for characterization of ligand binding systems. Anal Biochem 1980; 107: 220–239.

27. Baxter RC, Bryson JM, Turtle JR. Somatogenic receptors of rat liver: Regulation by insulin. Endocrinology 1980; 107: 1176–1181. 28. Bass SH, Mulkerrin MG, Wells JA. A systematic mutational analysis of hormone-binding determinants in the human growth hormone receptor. Proc Natl Acad Sci USA 1991; 88: 4498–4502. 29. de Vos AM, Ultsch M, Kossiakoff AA. Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Science 1992; 255: 306–312. 30. Frick GP, Tai LR, Goodman HM. Subcellular distribution of the long and short isoforms of the growth hormone (GH) receptor in rat adipocytes: Both isoforms participate in specific binding of GH. Endocrinology 1994; 134: 307–314. 31. Haldosen LA, Gustafsson JA. Detection of glycosylated growth hormone-binding protein in rat serum. Mol Cell Endocrinol 1990; 68: 187–194. 32. Frick P, Tai LR, Baumbach WR, Goodman MH. Tissue distribution, turnover, and glycosylation of the long and short growth hormone receptor isoforms in rat tissues. Endocrinology 1998; 139: 2824–2830.