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Endocrinology Vol. 138, No. 9 3577-3586
Copyright © 1997 by The Endocrine Society

ARTICLES

Guinea Pig Serum Contains a Specific High Affinity Growth Hormone-Binding Protein with Novel Ligand Specificity1

Susie I. Ymer, Janet L. Stevenson and Adrian C. Herington

Center for Hormone Research and the Department of Clinical Biochemistry, Royal Children’s Hospital, Melbourne; and Center for Molecular Biotechnology, Queensland University of Technology, Brisbane, Australia

Address all correspondence and requests for reprints to: Prof. Adrian C. Herington, Department of Biochemistry and Molecular Biology, Queensland University of Technology, PO Box 2434, Brisbane, Queensland 4001, Australia. E-mail: a.herington{at}qut.edu.au


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous workers have suggested that guinea pig serum does not contain a GH-binding protein (GHBP) or that it is defective. The current studies, however, have identified and characterized the presence of GH-binding activity in guinea pig serum using gel chromatography to separate bound and free hormone. The detection of GH-binding activity is critically reliant on the type of radioligand used to measure binding. Clear identification of GH-binding activity was demonstrated with [125I]ovine GH (oGH), but specific binding could not be measured with [125I]human GH. The novel specificity was also shared by guinea pig liver membrane GH receptor (GHR) and cytosol GHBP, suggesting structural similarity in the GH-binding domain between the GHR and soluble GHBPs. The binding of oGH was dependent on serum concentration (5 µl serum produced 16.03 ± 0.5% specific binding; mean ± SEM; n = 11) and incubation time (equilibrium was reached by ~6 h at 21 C) and was completely reversible (t1/2, ~2 h). Scatchard analysis revealed linear plots with an affinity constant (Ka) of 0.59 ± 0.09 x 109 M-1 and a capacity of 23,181 ± 4,474 fmol/ml serum. Similar association constants were obtained for liver membrane GHR (0.79 ± 0.22 x 109 M-1) and cytosol GHBPs (0.99 ± 0.15 x 109 M-1), but the capacity, when expressed as femtomoles per g tissue, was significantly increased (4-fold) in cytosol (4,303 ± 505) over that in membranes (1,071 ± 257). There was no sex difference in Ka or level of GHBP in guinea pig serum. Surprisingly, the level of GH-binding activity was very low to undetectable in pregnant guinea pig serum.

Characterization of the native structure of guinea pig GHBPs has indicated the presence of several proteins that are structurally distinct. Although the distribution of GH-binding activity covered a large Mr range (~70–350 kDa) the major form of the circulating GHBP identified by gel chromatography had an apparent native Mr of 150–170 kDa. Partially purified GHBP (approximate Mr, 170 kDa) was covalently cross-linked to [125I]oGH and subjected to nonreducing SDS-PAGE. Specific GHBP complexes of 158 and 85 kDa were detected, suggesting that the partially purified GHBP complex may be composed of a smaller GHBP associated noncovalently with a non-GH-binding protein. "Pore limit" native PAGE (cathodic and anodic) revealed the presence of specific GHBPs of 363, 158, 74, and 55 kDa, which cross-hybridized with the rat liver membrane GHR monoclonal antibody mAb 263 but not with the rat serum GHBP-specific mAb 4.3. Interestingly, although GH binding was undetectable in pregnant guinea pig serum, Western immunoblot analysis with mAb 263 demonstrated the presence of a major immunoreactive GHBP band of 105 kDa in addition to 158- and 55-kDa GHBPs. The data indicate that the GHBPs are immunologically related to the rat membrane GHR, but provide no evidence to support the presence of a hydrophilic tail sequence homologous to that in the rat GHBP.

These studies have identified in guinea pig serum GHBPs that exhibit novel ligand specificity, structural heterogeneity, and an immunological relationship to the rat liver membrane GHR. The identification of serum GHBP and the novel ligand specificity, which is also expressed by the liver membrane GHR, argue against the view that the guinea pig has a defective GHBP.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SOLUBLE FORMS of the cell membrane GH receptor (GHR) have been identified in the circulation of many mammalian species (1) and in phylogenetically distinct species such as birds (2). In rats and mice, the soluble GH-binding protein (GHBP) is generated from translation of an alternatively spliced, truncated form of the GHR messenger RNA (mRNA) (3, 4), in which a short sequence encoding a 17- to 25-amino acid hydrophilic tail replaces the entire transmembrane and cytoplasmic domains of the GHR. Absence of the transmembrane domain appears to allow secretion of the GHBP (3). Based on specific immunoprecipitation studies, GHBP derived from the alternatively spliced GHBP mRNA has been shown to circulate in the rat (5), although studies by Frick and Goodman (6) have also reported membrane association of the GHBP in rat adipocytes.

In other species little is known about the mechanism for the generation of serum GHBP. Although no equivalent alternative splicing mechanism has been demonstrated in other species, truncated GHR mRNAs have been identified in rabbit, ovine, and bovine tissues (7, 8, 9). It is not known whether any of these transcripts can be translated into GHBPs. In humans and rabbits, a number of studies have suggested that proteolytic cleavage of the membrane-bound GHR results in generation of the serum GHBP (10, 11, 12). Recent studies have identified an alternatively spliced human (h) GHR transcript that encodes a hGHR with a truncated cytoplasmic domain (13). The data raise the possibility that this truncated transcript may generate soluble GHBP also via a proteolytic mechanism. Whether cleavage occurs before or after membrane insertion of the GHR remains unknown, although Amit et al. (14) recently proposed an intracellular site of cleavage.

The biological role of the GHBP is not yet established, although it would appear to have an endocrine role in regulating GH transport and metabolism in the circulation (15) as well as having an autocrine/paracrine/intracrine role in modulating local cellular effects of GH (16, 17, 18, 19). The GHBP has been used widely as a marker of GHR status and potential GH responsiveness, although this relationship is not proven. The human syndrome of GH insensitivity (Laron-type dwarfism) is one example of the utility of this approach (20, 21). The guinea pig, which appears to show abnormalities in GH responsiveness, may be a useful animal model for examining further the structural and physiological interrelationship between the GHR and GHBP.

The guinea pig has classically been included with the rat and mouse as belonging to the order Rodentia. However, it has been suggested recently that the guinea pig may have a separate origin within mammalian evolution in a new mammalian order (Caviomorpha) distinct from Rodentia (22). The guinea pig has several endocrine anomalies, for example, within the gastro-entero-pancreatic axis and the pituitary-adrenal axis (23). The GH response axis may also have changed, as growth in the guinea pig appears to be largely independent of GH. Little or no effect of hypophysectomy is seen on the growth rate (24, 25), and there is no growth response to exogenous administration of bovine GH in normal or hypophysectomized guinea pigs (24, 26). The studies have been few, however, and given the purity of the hormones used at that time, the results need to be verified. Nevertheless, these observations raised questions of whether there were defects in or a deficiency of GH or its receptor.

More recent studies have shown that guinea pigs have a normal pulsatile secretory pattern of GH and that both somatostatin and GH-releasing factor play a regulatory role (27). The secretion pattern is very like that in the rat, with large pulses of GH every 3–4 h in male rats and more, smaller pulses in females. Only limited structural data are available, but guinea pig GH shows a comparable overall amino acid composition and a close homology of the N-terminal 20 amino acids with the GH of other species (27). There have been suggestions that guinea pig GH may have evolved as a primarily metabolic hormone rather than a growth-promoting hormone (27, 28), although guinea pig pituitary extracts caused growth in hypophysectomized rats (26), suggesting that guinea pig GH retained somatotropic activity.

GHRs have recently been identified in guinea pig liver (29, 30). Ligand binding studies using [125I]ovine (o) GH demonstrated the presence of high affinity, GH-specific binding sites in guinea pig liver membranes. GHR mRNA expression has also been demonstrated, both by reverse transcription-PCR of an extracellular GHR domain fragment and by Northern blotting (29, 30). There has been, however, no structural characterization of this GHR.

Guinea pig serum GHBP has not been clearly identified. Using [125I]hGH as ligand, GHBP was undetectable in guinea pig serum (31, 32), although soluble GHR immunoreactivity has been reported using a monoclonal antibody raised against rat GHR (mAb 263) (32). These observations led to the suggestion that there may be a defect in the GH-binding domain of GHBP, implying that the GHR and GHBP are structurally distinct. However, this conclusion is inconsistent with current knowledge of the sequence similarity of the extracellular GH-binding domain of the membrane GHR and GHBP in other species (3, 4, 33).

Thus, the aims of this study were to reexamine the presence of serum GHBP in the guinea pig and to determine its binding and structural characteristics, with a view to subsequently examining the relationship between the GHBP and GHR in this animal model of GH insensitivity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Hormones
hGH (NIDDK hGH I-1) and oGH (I-1–4) used for iodination and oGH (NIH GH S-15), bovine GH (NIH GH B-18), rat GH (NIH rGH B-6), and oPRL (NIH P-S-15), used for unlabeled preparations were gifts from the National Hormone and Pituitary Program (NIDDK, NIH, Bethesda, MD). Recombinant and pituitary hGH were obtained from the Commonwealth Serum Laboratories (Melbourne, Australia).

Antibodies
mAb 263, raised against rat liver membrane GHR (34) and recognizing the extracellular GH-binding domain in GHR and GHBP, was a gift from Dr. M. J. Waters (University of Queensland, St. Lucia, Australia). mAb 4.3 (5) and affinity-purified polyclonal antibody RB1615 (6) were raised against a synthetic peptide corresponding to the predicted 17-amino acid hydrophilic tail of the rat GHBP mRNA and recognize only the GHBP. mAb 4.3 and RB1615 were gifts from Dr. Baumbach (American Cyanamid, Princeton, NJ) and Dr. P. Frick (University of Massachusetts Medical School, Worcester, MA), respectively. Control mAb (against chicken immunoglobulin, IgG1 phenotype) was obtained from Silenius (Melbourne, Australia).

Protein molecular mass markers for gel filtration were: ferritin, (horse spleen; Mr, 440 kDa), ß-amylase (sweet potato; Mr, 200 kDa), alcohol dehydrogenase (yeast; Mr, 150 kDa), hexokinase (yeast; Mr, 99 kDa), lactoperoxidase (bovine milk; Mr, 85 kDa), BSA (Mr, 68 kDa) or chicken egg albumin (Mr, 45 kDa), trypsin inhibitor (soybean; Mr, 20 kDa). Blue dextran (Mr, 2000 kDa) and [125I]NaI were used to determine the void volume and the total volume of the column, respectively. Ferritin, hexokinase, and trypsin inhibitor were purchased from Calbiochem-Novabiochem Corp. (San Diego, CA), and the remaining Mr markers were obtained from Sigma Chemical Co. (St. Louis, MO). For "pore limit" native PAGE, the high Mr protein calibration kit (67–669 kDa) and MW-100 kit (14–800 kDa) were obtained from Pharmacia Biotech and Gradipore (North Ryde, Australia); respectively. For SDS-PAGE, the Mark12 kit (2.5–200 kDa) was obtained from Novex (San Diego, CA).

Other reagents
All general chemicals were of analytical grade, and the following products were obtained as indicated: Ultrogel AcA34 and AcA54, LKB Produkter (Bromma, Sweden); Sephadex G-50 (Uppsala, Sweden); precast polyacrylamide gels [micro native, concave gradient (5–40%), mini-tricine SDS (10–20%)], Gradipore (North Ryde, Australia); Cronex Lightning Plus Intensifying screens, DuPont (Wilmington, DE); SDS and dimethylsulfoxide, BDH Chemicals (Melbourne, Australia); disuccinimidyl suberate (DSS), Iodogen (Pierce Chemical Co., Rockford, IL); aprotinin, phenylmethylsulfonylfluoride, mercaptoethanol, and Ponceau S, Sigma; Hyperfilm ECL, ECL Western blotting kit and Hybond-N membrane, Amersham International (Aylesbury, UK); Fuji RX film, Fuji Photo Film Co. (Japan).

Animals
Serum and liver tissue were collected for this study from adult and late pregnant (gestation = 63–68 days) Duncan Hartley guinea pigs (~500–800 g) housed at the Central Animal Laboratory of Monash University (Clayton, Australia). The study was approved by the animal ethics committee of the Royal Children’s Hospital (Melbourne, Australia). Serum was stored at -20 C, and liver was stored at -75 C before analysis.

Iodination
oGH and hGH were iodinated as previously described, using the Iodogen method (35). The 125I-labeled protein peak was separated on a Sephadex G-50 column (0.8 x 19 cm) and subsequently purified on Ultrogel AcA54 (0.8 x 24 cm) to yield a peak of monomeric [125I]oGH or [125I]hGH (~21,000 Mr). Specific activities of 5–20 and 30–50 µCi/µg were achieved for oGH and hGH, respectively.

Receptor preparation
Microsomal membranes (100,000 x g) were prepared as described previously (36) in the presence of 1000 kallikrein inactivator units of aprotinin/ml and phenylmethylsulfonylfluoride (1 mM). The 100,000 x g supernatant was centrifuged at 200,000 x g, and the resulting supernatant was filtered through a 0.22-µm Millipore filter (Millipore Corp., Bedford, MA) to provide a cytosol preparation. Blood was allowed to clot at room temperature for about 2 h, and the serum was obtained after centrifugation at approximately 2,500 x g for 10 min at 4 C. Protein estimations for membranes and soluble GHBPs were carried out by the methods of Lowry (37) and Bradford (38), 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. Guinea pig serum (5–50 µl) or liver cytosol (400 µg protein) or membrane (400 µg protein) were incubated with 20,000–30,000 cpm [125I]oGH (28–150 fmol) or [125I]hGH (11–26 fmol) in the presence and absence of unlabeled GH (0.1 µM). For serum and cytosol, bound and free hormone were separated by gel filtration on AcA54 minicolumns (0.6 x 22 cm) at 21–23 C as described previously (35) or on a large Ultrogel AcA34 column as described below. For membrane preparations, bound and free hormone were separated by centrifugation (36). Specific binding was calculated as the difference in binding in the presence (nonspecific binding) and absence (total binding) of an excess (0.1 µM) of unlabeled GH.

Binding affinity and capacity
The binding affinity and capacity of the GHBPs and GHRs were estimated by Scatchard analysis of dose-response curves for the displacement of [125I]oGH by increasing concentrations of unlabeled oGH. Binding data were determined using the Ultrogel AcA54 minigel filtration system described above, and the data were analyzed by the Ligand program of Munson and Rodbard (39).

Gel chromatography of serum preincubated with [125I]oGH or serum alone
Gel filtration studies were performed at room temperature on an Ultrogel AcA34 column (1 x 96 cm) in 50 mM HEPES buffer, pH 7.5, containing 0.02% sodium azide, as described previously for rabbit serum binding proteins (35). Serum was incubated with [125I]oGH as described above in Materials and Methods, and the entire incubation mixture was chromatographed on the Ultrogel AcA34 column. The radioactive elution profile was determined by measuring the radioactivity in each fraction collected (1 ml). In parallel experiments, [125I]oGH was incubated in assay buffer only (blank) under the same conditions to determine the radioactive profile of the [125I]oGH itself. The Ultrogel column was calibrated with Mr protein standards (20–440 kDa).

The Mr of the GHBP was estimated by subtracting the contribution of [125I]oGH (21 kDa), from the apparent Mr of the specifically bound [125I]oGH-GHBP complex, assuming a 1:1 binding stoichiometry. This stoichiometry has been assumed, although the recent stoichiometric model of 1:2 [GH-(GHBP)2] proposed by Cunningham et al. (40) could also have fit the data. The concentration of [125I]oGH used throughout these studies (~0.15–0.8 nM) favors monomer formation (41); therefore, a 1:1 stoichiometry has been consistently assumed in extrapolation of the Mr of the GHBP from a preformed [125I]oGH complex. This is also applicable when determining the Mr of denatured GHBP from cross-linked [125I]oGH-GHBP complexes. However, it is recognized that this may be a simplistic interpretation given the uncertainties in measuring the exact concentration of GHBPs and, therefore, the GH to GHBP ratio.

When serum alone (1.5 ml) was chromatographed on the same AcA34 column, the GH binding profile was determined by taking 150-µl aliquots from each 1-ml column fraction, incubating with [125I]oGH as described above, and then measuring specific binding by separation of bound and free GH on AcA54 minicolumns (35). The Mr of each fraction was determined from the AcA34 elution volume. A comparison of the Mr determined from gel chromatography of serum alone or serum complexed with [125I]oGH allowed verification of a 1:1 binding stoichiometry.

Western immunoblot analysis of serum GHBP
Pore limit native PAGE on Gradipore precast microgels [concave gradient, 5–40% (wt/vol) acrylamide; linear Mr range, 5–1000 kDa] was used for the separation of serum proteins. This was followed by immunoblotting with GHR/GHBP mAbs and assessment by enhanced chemiluminescent detection (ECL) using Amersham’s ECL Western blotting kit as follows; guinea pig serum (5 µl, male or pregnant) or pregnant rat serum (1 µl) was subjected to anodic electrophoresis [1 x TBE, pH 8.3 = 1% (wt/vol) Tris, 0.5% (wt/vol) boric acid, and 0.05% (wt/vol) EDTA] or cathodic electrophoresis [1 x sodium lactate, pH 3.1 = 0.025% (wt/vol) lactic acid, and 0.1% (wt/vol) NaOH] at 150 V for 60 min (twice the pore limit). Preelectrophoresis (5 min at 200 V) was applied for cathodic electrophoresis only. Samples were run in triplicate on the same gel. Gels were equilibrated in transfer buffer (25 mM Tris and 192 mM glycine buffer, approximate pH 8.3, without methanol) and electroeluted onto DuPont polyvinylidene difluoride membrane using a Bio-Rad Mini Trans-Blot system (Bio-Rad, Richmond, CA). Blots were cut into three and blocked in TBS-T buffer [0.1 M NaCl, 10 mM Tris-HCl (pH 7.5), and 0.1% (vol/vol) Tween-20] plus 5% (wt/vol) nonfat skim milk (Carnation, Nestle Australia, Ltd., Sydney, NSW, Australia) at 4 C for approximately 16–24 h, followed by incubation with rat GHR mAb 263, rat GHBP mAb 4.3, or control mAb (final concentration, 1–3 µg/ml) for 1 h at about 21 C with gentle shaking. As an additional control, blots were also incubated in the absence of primary antibody to assess nonspecific interactions with the secondary antibody. After four 15-min washes with TBS-T buffer, blots were incubated with an antimouse horseradish peroxidase-labeled secondary antibody (1:2000 dilution) and washed as described for primary antibody. The chemiluminescent signal was evaluated after various time exposures (30 sec to 30 min) of the blots using Amersham Hyperfilm ECL. Gels were calibrated with Gradipore and Pharmacia native protein Mr markers (800–20 and 669–67 kDa, respectively). Blots were stained with Ponceau S before immunodetection to determine the Mr of the standard proteins.

Affinity cross-linking of serum GHBP to [125I]oGH and SDS-PAGE
Serum was partially purified by gel chromatography on Ultrogel AcA34 as described above in the gel filtration protocol. Fractionated serum (150 µl) corresponding to a Mr of approximately 170 kDa was incubated with [125I]oGH in assay buffer without BSA in the absence and presence of unlabeled oGH (0.1 µM) as described above. The cross-linking studies were carried out as reported previously (36) using DSS (0.5 mM). After cross-linking for 15 min at 21–23 C, 50-µl aliquots were removed, and the reaction was stopped by boiling in the presence of Tris-HCl buffer containing 2% SDS without dithiothreitol (DTT). SDS-PAGE was performed on Gradipore gradient minigels [precast, 10–20% (wt/vol) acrylamide and Tris-tricine] as described by the manufacturers. Gels were stained with Gradipore Gradipure, dried, and then subjected to autoradiography at -70 C using Fuji film and DuPont Lightning Plus intensifying screens. The Novex Mark12 protein standards (200–2.5 kDa) were used to calibrate the gels and estimate the Mr of cross-linked complexes.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Figure 1aGo shows a typical radioactive elution profile of an incubation mixture of [125I]oGH and guinea pig serum. Three peaks were observed with peaks 2 and 3 eluting in the same position as the markers for free [125I]oGH and [125]NaI, respectively. Peak 1, eluting at a Mr of approximately 170 kDa, was displaced by excess unlabeled oGH (panel 1), but not by hGH (panel 2), indicating the presence of a specific oGH-binding protein with a native Mr of about 150 kDa assuming a 1:1 binding stoichiometry of GH:GHBP (see explanation in section on gel chromatography of serum in Materials and Methods). GH-binding activity was also measured in liver cytosol, and the size and specificity of the GHBP were the same (data not shown).



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  		Figure 1. Representative gel filtration profile of serum preincubated with [125I]oGH or serum alone on Ultrogel AcA34 (1 x 96 cm). a, Adult male guinea pig serum complexed to [125I]oGH. Serum (10 µl) was incubated with [125I]oGH (~25,000 cpm) in the absence (total binding) or presence of excess (0.1 µM) unlabeled oGH (panel 1), or hGH (panel 2) 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 1-ml fraction. b, Serum (1.5 ml) was chromatographed on the same column, and the GH binding profile was determined by taking 150-µl aliquots from each 1-ml fraction and measuring specific binding by separation of bound and free [125I]oGH on AcA54 minicolumns as described previously (35). The Mr of the [125I]oGH-GHBP complex and GHBPs have been estimated from the protein standards (20–440 kDa) used to calibrate the column as described in Materials and Methods.

 
To determine if the binding stoichiometry was 1:1 (GH:GHBP), or whether the apparent Mr of the GH-GHBP complex was different from the expected Mr due to a GH-induced conformational change, serum was fractionated on the same Ultrogel AcA34 column followed by measurement of GH-binding activity in each fraction (Fig. 1bGo). A peak with maximum specific binding eluting at a Mr of about 170 kDa was observed. However, the peak was quite broad, covering the approximate Mr range 70–350 kDa. The specific binding in this experiment reflects the Mr of unbound GHBP in the original serum and not the sizes of [125I]oGH-GHBP complexes as does Fig. 1aGo. The 170-kDa binding protein corresponds closely in size to the GHBP (150 kDa) estimated from the [125I]oGH-GHBP complex in Fig. 1aGo.

GHBP eluting at a Mr of about 170 kDa was partially purified from serum by gel chromatography on Ultrogel AcA34. After incubation with [125I]oGH and rechromatography on the same column, the bound complexes eluted at a Mr of 170 kDa (Fig. 2aGo). The formation of this peak was inhibited by the inclusion of oGH (panel 1), but not hGH (panel 2), in the incubation buffer. Thus, this GHBP exhibited the same novel specificity as that shown for whole serum. Furthermore, as there was little change in the Mr of the complexed GHBP, it suggests an absence of dimerization. This provides further verification that under the experimental conditions used, [125I]oGH bound to serum GHBPs with a 1:1 binding stoichiometry. The percent specific binding differed little regardless of whether minicolumns or large columns were used, suggesting that dissociation during chromatography is minimal (data not shown).



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  		Figure 2. a, Representative gel filtration profile of partially purified guinea pig serum preincubated with [125I]oGH on Ultrogel AcA34 (1 x 96 cm). Adult male guinea pig serum was partially purified on Ultrogel AcA34 as described in Fig. 1bGo, and a 150-µl aliquot of the serum fraction corresponding to a GHBP with a Mr of 170 kDa was incubated with [125I]oGH (~25,000 cpm) in the absence (total binding) or presence of excess (0.1 µM) unlabeled oGH (panel 1) or hGH (panel 2) for 24 h at 21 C. A duplicate set of tubes containing partially purified serum GHBP (170 kDa) and [125I]oGH was preincubated for 6 h at 21 C before the addition of rat liver membrane GHR mAb 263 (panel 3) or rat GHBP mAb 4.3 (panel 4), and the binding reaction was continued for an additional 18 h at 21 C. The entire incubation mixtures were gel chromatographed, and the radioactive elution profiles were determined by counting the radioactivity of each 1-ml fraction. b, Representative gel filtration profile of pregnant rat serum preincubated with [125I]oGH on Ultrogel AcA34 (1 x 96 cm): Pregnant rat serum (50 µl) was incubated with [125I]oGH in the presence and absence of excess unlabeled oGH as described above for 24 h at 21 C (upper panel). Another tube was incubated for 6 h at 21 C before the addition of mAb 4.3 and further incubated for 18 h at 21 C (lower panel).

 
Preformed [125I]oGH-GHBP complex (Mr, 170 kDa) was further incubated with rat membrane GHR mAb 263 (Fig. 2aGo, panel 3) or rat GHBP mAb 4.3 (Fig. 2aGo, panel 4). The data show that in the presence of mAb 263, the Mr of the bound GHBP increased to about 350 kDa, indicating that the antibody binds to the GHBP. This finding suggests that GHBP shares structural homology with the rat membrane GHR. However, in the presence of mAb 4.3, the Mr of the bound GHBP complex remained unchanged, suggesting that the guinea pig GHBP does not contain a hydrophilic tail epitope homologous with that present in rat GHBP. The use of another rat GHBP hydrophilic tail polyclonal antibody (RB1615), addition of RB1615 or mAb 4.3 to serum before addition of labeled oGH, or increasing the concentration of these antibodies also had no effect (data not shown), indicating that these Abs did not bind to the GHBP. These data provide additional verification of the absence or lack of accessibility of rat GHBP tail epitopes. However, as expected, mAb 4.3 did recognize GHBPs present in rat serum Fig. 2bGo. A typical radioactive elution profile of rat serum preincubated with [125I]oGH in the presence and absence of excess unlabeled oGH is shown in Fig. 2bGo, top panel, whereas incubation of preformed rat serum [125I]oGH complexes with mAb 4.3 is shown in the bottom panel of Fig. 2bGo. It is evident that both [125I]oGH-rat serum complexes (90 and 200 kDa) are recognized by mAb 4.3, as both undergo a major shift (~150 kDa) in their elution positions to higher Mr.

The binding characteristics of guinea pig GHBP have been examined and shown to be classically dependent on the incubation time and serum concentration used. Maximum specific binding (28%) was achieved with 50 µl serum in a total incubation volume of 250 µl (Fig. 3aGo) Binding was readily detected with as little as 5 µl (18.0%). Equilibrium was reached by 6 h at 21–23 C, and the reaction was completely reversible with a t1/2 of about 2 h after the addition of a large excess of unlabeled oGH (Fig. 3bGo). The time of association was longer than that observed for other mammalian species (35, 42, 43). The presence of cations (Ca/Mg, 5–40 mM) did not enhance the binding of [125I]oGH, although high concentrations (40 mM) of either cation led to a decrease (20%) in specific oGH binding (data not shown).



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  		Figure 3. Effect of increasing serum concentration (a) and the time course of association and dissociation (b) on the specific binding of [125I]oGH to guinea pig serum. Each data point is derived from AcA54 minicolumn separation of bound and free [125I]oGH and is the mean of duplicates. In b, binding was performed at 21 C for 16 h, and dissociation was initiated in one set of tubes by the addition of an excess (2 µg) of unlabeled oGH in a 10-µl volume to avoid dilution effects.

 
The unusual species specificity for GH ligands is again illustrated in Fig. 4Go. The left and right panels show [125I]oGH and [125I]hGH binding, respectively, in serum (a) and liver membranes (b). The hormonal specificity demonstrated that although oGH and bGH were effective in displacing [125I]oGH bound to guinea pig serum GHBP and liver GHR, hGH, rGH, and oPRL were ineffective. oGH and hGH were equally effective in displacing [125I]oGH bound to rabbit serum GHBP and membrane GHR. On the other hand, [125I]hGH (right panels), which bound well to rabbit serum GHBP (39.82%) and GHR (42.83%), had very low total binding (2–4%) and no binding specificity at all for guinea pig serum GHBP or membrane GHR.



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  		Figure 4. The hormonal specificity of binding of [125I]oGH and [125I]hGH to guinea pig serum GHBP (a) and membrane GHR (b). Also included as a control for the ability of the labeled ligands to bind to GHR/GHBP are pregnant rabbit serum and liver membranes. All unlabeled hormones were added at a final concentration of 2 µg/ml. Binding is expressed as a percentage of the total binding (T = 100%) in the absence of unlabeled hormone. For [125I]oGH binding: T = 17.37% for guinea pig serum, 27.46% for rabbit serum, 8.16% for guinea pig membrane, and 36.26% for rabbit membrane. For [125I]hGH binding: T = 4.59% for guinea pig serum, 39.82% for rabbit serum, 2.66% for guinea pig membrane, and 42.83% for rabbit membrane.

 
A representative Scatchard plot of [125I]oGH binding to female guinea pig serum is shown in Fig 5Go. Linear and parallel plots for male guinea pig serum were also observed, indicating a single class of sites with similar affinities. Occasionally, curves were observed, but a two-site model could not be accommodated by the Ligand analysis program. Table 1Go shows that there was no sex difference in the association constants or in the level of GHBPs. The affinity and capacity of guinea pig serum GHBP were comparable to those of rat serum (42). However, the capacity was much higher than that in some other species, such as the human (43) and rabbit (35). The binding affinity and capacity were also determined for liver membrane GHRs and cytosol GHBPs, and the data are shown in Table 1Go. Although the association constant was not significantly different between membrane-bound GHR and soluble GHBPs, the capacity of cytosol GHBP when expressed as femtomoles per g tissue was significantly greater (4-fold) than that of GHRs present in liver membranes. The binding affinity and capacity of the guinea pig liver GHR were significantly lower than the surprisingly high values reported by Harvey and Fraser (29) for [125I]oGH binding to the membrane-bound GHR in guinea pig liver (Ka, ~400 nM-1).



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  		Figure 5. Representative Scatchard plot of [125I]oGH specific binding to female guinea pig serum GHBP. The data were obtained using the Ligand-PC program of Munson and Rodbard (39) based on duplicate dose-response curves, using 5 µl guinea pig serum, increasing concentrations of unlabeled oGH, and a fixed concentration of [125I]oGH. B/F, Bound to free ratio. Bound is expressed as moles per liter incubation volume. The binding capacity (x-axis intercept) is equivalent to about 23,000 fmol/ml serum, assuming a 1:1 GH:GHBP binding stoichiometry.

 

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  		Table 1. Scatchard analysis of [125I]oGH binding to guinea pig soluble GHBPs and membrane-bound GHR

 
The broad gel filtration elution profile (Fig. 1bGo) suggests the presence of structurally distinct GHBPs in guinea pig serum. However, the Mr sizes determined by gel filtration of serum alone (Fig. 1bGo) may be artifactual or inaccurate, as they can be influenced by the nature of the protein. Mammalian GHBPs are known to be glycosylated (44, 45), and guinea pig GHBPs may be no exception. As glycoproteins are known to exhibit anomalous behavior on gel filtration (46), verification of the Mr size was sought using pore limit native PAGE. Using microgels (5–40% concave gradient), proteins were electrophoresed under nondenaturing conditions for sufficient time to reach their pore-restricted size, where charge is negated and their separation is based solely on molecular size (47). From the gel chromatography data it would appear that there are multiple guinea pig GHBP isoforms, and as there is no knowledge of the isoelectric points (pI) of these proteins, both anodic and cathodic electrophoreses were performed to ensure representation of all GHBPs present.

Pore limit PAGE was performed at pH 8.3 or pH 3.1 (shown in upper and lower panels of Fig. 6Go, respectively), and GHR/GHBP immunoreactive bands were detected with rat membrane GHR mAb 263 (A), rat GHBP mAb 4.3 (B), or control mAb (C). At pH 8.3, two major bands corresponding to Mr of 55 and 74 kDa were detected in male guinea pig serum with mAb 263, but not mAb 4.3 or control mAb (Fig. 6Go, upper panel). On the other hand, pregnant rat serum contained two proteins corresponding to Mr of 47 and 53 kDa, which were detected with both mAb 263 and mAb 4.3 and were similar in size to those reported by others (5, 45). Interestingly, although we were unable to measure [125I]oGH-binding activity in pregnant guinea pig serum, two major bands corresponding to Mr of 105 and 55 kDa were specifically detected with mAb 263, but not mAb 4.3. There was some concern about the presence of strong immunoreactivity in all gels at Mr of 200 kDa or more (pH 8.3). Therefore, as a control, the detection was also performed in the absence of primary antibody. Even at dilutions of 1:10,000 of secondary antibody, immunoreactivity was present, albeit weaker than with the working concentration of 1:2,000, and changed little when the primary control antibody was used. Therefore, the high Mr background immunoreactivity observed was essentially due to interaction with the secondary antibody.



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  		Figure 6. Western immunoblot analysis of guinea pig serum GHBP using ECL after pore limit PAGE. Guinea pig serum (5 µl) was electrophoresed in 1 x TBE buffer, pH 8.3 (upper panels), or 1 x sodium lactate buffer, pH 3.1 (lower panels), on native microgels (concave gradient, 5–40%). Proteins were electroeluted onto polyvinylidene difluoride membrane and incubated with rat liver membrane GHR mAb 263 (A), rat GHBP mAb 4.3 (B), or control mAb (C), followed by immunodetection with an antimouse horseradish peroxidase-labeled secondary antibody using the Amersham ECL detection system. Rat serum (1 µl) was included as a positive control for the presence of GHBP. The chemiluminescent signal shown was evaluated after 2- or 5-min exposure (upper and lower panels, respectively) of the blots using Amersham Hyperfilm-ECL. preg, Pregnant. Mr markers are indicated by the horizontal lines, and Mr values for the specific immunodetected GHBPs are shown by the arrows.

 
When serum was subjected to cathodic electrophoresis (pH 3.1; Fig. 6Go, lower panel), a different pattern was observed. Two proteins of 363 and 158 kDa were detected in male guinea pig serum with mAb 263, but not mAb 4.3 or control mAb. A protein corresponding to a Mr of 158 kDa, which was weakly immunoreactive with mAb 263, was present in pregnant guinea pig serum. Interestingly, rat serum contained two proteins of 178 and 316 kDa that were strongly immunoreactive with mAb 4.3, but not mAb 263. The specificity of the antibodies was always examined in relation to that of the control antibody.

Data obtained from investigations of the denatured structure of the guinea pig serum GHBPs using Western immunoblot and SDS-PAGE proved unsatisfactory. This was mainly due to strong background, particularly in the high Mr region, which prevented examination of specific immunoreactivity in this region. However, smaller Mr proteins (40–65 kDa) that cross-reacted with mAb 263, but not mAb 4.3, were consistently identified in both male and pregnant guinea pig serum (data not shown). An alternative approach to determine the denatured structure of the GHBP involved the use of the chemical cross-linking agent DSS. GHBP with a Mr of about 170 kDa was partially purified from guinea pig serum using gel chromatography. Covalent cross-linking of [125I]oGH to this preparation, followed by SDS-PAGE (without DTT) revealed the presence of two bands of 158 and 85 kDa that were completely suppressed by the inclusion of excess unlabeled oGH in the original incubation mixture (Fig. 7Go). If one assumes a contribution of 20 kDa for [125I]oGH, the Mr of the binding proteins themselves are 138 and 65 kDa. Although the 85-kDa complex was more strongly cross-linked, it does not necessarily indicate greater abundance. The presence of the 158-kDa complex was not consistently seen, and it may represent a poorly cross-linked complex composed of the 65-kDa GHBP noncovalently associated with GH and another protein(s) that does not bind GH.



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  		Figure 7. Autoradiograph of SDS-polyacrylamide gel [10–20% (wt/vol) acrylamide gradient] electrophoretic pattern of [125I]oGH cross-linked (0.5 mM DSS) to partially purified male guinea pig serum (GHBP; Mr, ~170 kDa) as described in Materials and Methods. Lane A, Total binding; lane B, nonspecific binding (both in the absence of DTT). Autoradiography was carried out for 10 days at -70 C using DuPont Lightning Plus intensifying screens and Fuji RX film. Mr markers are indicated by the horizontal lines; Mr values for the specifically labeled [125I]oGH complexes and free [125I]oGH are shown by the arrows.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These data provide clear evidence for the existence of multiple GHBP isoforms in guinea pig serum that are immunologically related to the rat liver membrane GHR, but can be distinguished from the rat GHBP due to a lack of cross-reactivity with a rat GHBP hydrophilic tail antibody. The native form of the major GHBP isoform, which has an apparent Mr of 150 kDa, exhibits a high affinity for oGH (Ka, 0.59 nM-1) and is abundant (~23 pM) in guinea pig serum.

Of critical importance in detection of GH-binding activity in guinea pig serum is the type of ligand used to measure GH binding. Despite its cross-reactivity with lactogenic receptors, hGH has been extensively used for measuring GH-binding activity because it usually binds GHRs much better than other animal GHs. However, hGH does not bind specifically to guinea pig serum GHBP. In contrast, this GHBP can be clearly demonstrated when oGH is used as the ligand. Lack of detectability of the guinea pig GHBP with labeled hGH was also observed by Amit et al. (31) and Hull et al. (32). Previous studies (29, 32) reported the presence of liver membrane GHR, but not GHBP, in guinea pig using labeled oGH to measure the binding. This latter observation is surprising given GHBP abundance in serum and may be due to technical differences, including insufficient incubation time. Recently, the same investigators demonstrated the presence of immunoreactive, but non-GH-binding, protein in the guinea pig using an immunoassay ([125I]hGHBP and mAb 263) and postulated that the guinea pig serum GHBP is defective (32). However, the current studies have demonstrated that novel GH specificity is exhibited by not only the serum GHBP but also by the liver membrane GHR and cytosol GHBP. This suggests that both guinea pig GHRs and soluble GHBPs share structural homology and does not support the hypothesis suggested by Hull et al. (32).

Although oGH and hGH exhibit a high degree of amino acid homology, they exhibit quite different isoelectric points; hGH is acidic, whereas oGH is alkaline. These charge differences may have some impact on the interaction between guinea pig GHBP and GH. The novel specificity was not exhibited by rabbit GHBP or GHR, but recent studies in this laboratory (in preparation) have demonstrated similar ligand specificity for rat serum GHBPs. Differences between serum GHBPs in their ability to bind hGH or bPRL have been used to categorize GHBPs from various species into four types, which allowed distinction among guinea pig, rat, rabbit, and human (31). With the exception of hGHBP, the ability of these GHBPs to bind endogenous GH is not known. It would be interesting to examine the binding characteristics of guinea pig GHBPs using homologous GH, but the lack of availability of purified guinea pig GH precludes such an investigation at this time. Given the sequence homology in the extracellular domains of known GHBPs and GHRs, the observations are intriguing. This points to possible differences within the structure of the GHBPs, such as the presence of other associated proteins (GH or non-GH binding) and/or the type and extent of glycosylation, all of which have the potential to significantly alter the GH binding interaction and may be responsible for the observed differential species binding specificity. In fact, recent studies have suggested that the differences in binding properties between rat and mouse liver PRL receptors are due to the distinct type of glycosylation present within each receptor (48). We have recently cloned the extracellular domain (exons 2–9) of the guinea pig GHR (30), and the major residues for GH binding are conserved, but there are additional potential glycosylation sites present that could contribute to the novel specificity. However, until the guinea pig GHR receptor is expressed in cells and characterized, one cannot draw any conclusions about the importance of the sequence changes in the extracellular domain of the guinea pig GHR.

The native forms of circulating GHBPs in guinea pig serum have been characterized using two chromatographic approaches. Chromatography of the preformed serum-[125I]oGH complex identified a major GHBP with an apparent Mr of 150 kDa, whereas chromatography of serum followed by measurement of binding in each fraction identified a major peak at a Mr of about 170 kDa and multiple GHBPs eluting between 350 and 70 kDa. The identification of additional GH-binding species with this latter approach may reflect differences in the affinity constant and relative concentration of the GHBPs, suggesting that these GHBPs may go undetected when preformed [125I]oGH serum complexes are chromatographed. Similar Mr variations were observed for rabbit and human GHBPs using the two chromatographic approaches described above (35, 43). The native Mr (170 kDa) of the [125I]oGH-guinea pig GHBP complex is similar to that reported for rat serum [125I]-GH complexes (159, 220, and 110 kDa) by Emtner et al. (49) and Massa et al. (42). These GHBPs together with rabbit and human GHBPs exhibit native Mr significantly larger than those predicted from cloning studies, suggesting that they may associate covalently/noncovalently with other proteins. In fact, the present studies using covalent cross-linking raise the possibility that the major guinea pig GHBP (~170 kDa) is composed of a protein complex containing a GHBP with a Mr of 65 kDa associated noncovalently with another protein(s) that does not bind GH.

The structural heterogeneity observed was not produced artifactually by the gel chromatography procedure, as a second analytical approach, pore limit native PAGE and Western immunoblot analysis, also revealed the presence of several proteins corresponding to Mr of 363, 158, 74, and 55 kDa in male guinea pig serum and 158, 105, and 55 kDa in pregnant guinea pig serum. This latter observation is surprising given that GH-binding activity was not detectable in pregnant guinea pig serum by gel chromatography. The 158-kDa protein corresponds in Mr to the major form of the GHBP detected by gel chromatography (~150 kDa). Furthermore, the lack of detectability of this protein in pregnant guinea pig serum is supported by its weak immunoreactivity on native gels. If this protein represents the major form of the high affinity GHBP in guinea pig circulation, then its apparent reduction during pregnancy is unusual. It may be that the 105-kDa protein is a cleaved form of the 158-kDa protein, and that if these GHBPs are occupied by endogenous GH it may prevent [125I]oGH binding under the in vitro incubation conditions used. Despite their heterogeneity, all of the guinea pig GHBPs are immunologically related to the rat liver membrane GHR, but these studies provide no evidence to support the presence of a hydrophilic tail similar in sequence to that of the rat GHBP. The presence of GHBP isoforms has been observed in rabbit, human, rat, and mouse serum (35, 42, 43, 44, 45, 50). Although some of the different GHBPs identified in human and rat serum are due to glycosylation differences, nothing is known about the composition of the remaining GHBPs.

Our data contradict recently published work by Hull et al. (32) that suggests the presence of a guinea pig GHBP with a rat GHBP hydrophilic tail. However, they used a much higher concentration of purified serum (30 µl) and, under denaturing conditions of SDS-PAGE, a smaller protein (GHBP-like), which may be noncovalently associated with a larger protein (for example, Mr >350 kDa), may be released and readily detected. With our analysis by native gel electrophoresis such a protein may be structurally obscured and, therefore, pass detection. However, this was not the case with rat serum GHBPs (316 and 178 kDa), which were clearly detected with mAb 4.3. The specificity of the immunoreactive bands reported by Hull et al. (32) is unclear, as data using a control mAb were not presented.

These studies have demonstrated that high affinity GH-binding sites are present in guinea pig serum, liver membranes, and cytosol and all exhibit differential GH specificity. Although the serum GHBP isoforms are immunologically related to the rat liver membrane GHR, we cannot provide any evidence for the presence of a rat-like GHBP hydrophilic tail. However, these studies do not preclude the existence of a hydrophilic tail in guinea pig GHBPs distinct from that present in the rat GHBP. Further studies are required to address this issue and to determine the mechanism of generation of the guinea pig serum GHBP. The nature of the structural heterogeneity of guinea pig GHBPs remains to be determined and raises questions about the significance and origin of the isoforms and their role in modulating GH actions.


   Footnotes
 
1 This work was supported by grants from the National Health and Medical Research Council of Australia and the Royal Children’s Hospital Research Foundation. Back

Received December 20, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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