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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
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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.
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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.
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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.
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Results |
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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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). 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. 1a. The 170-kDa binding protein corresponds
closely in size to the GHBP (150 kDa) estimated from the
[125I]oGH-GHBP complex in Fig. 1a.
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. 2a). 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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, panel 3) or rat GHBP mAb 4.3 (Fig. 2a, 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. 2b. 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. 2b, top panel,
whereas incubation of preformed rat serum [125I]oGH
complexes with mAb 4.3 is shown in the bottom panel of Fig. 2b. 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. 3a) 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. 3b). 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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. 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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. 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 1 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 1. 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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) suggests the
presence of structurally distinct GHBPs in guinea pig serum. However,
the Mr sizes determined by gel filtration of
serum alone (Fig. 1b) 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. 6, 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. 6, 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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, 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. 7). 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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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.
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Received December 20, 1996.
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