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Original Studies |
Division of Clinical Sciences (R.J.M.R., M.M.), Sheffield University, Sheffield S5 7AU, United Kingdom; Pituitary Research Unit (K.C.L., N.D., K.K.Y.H.), Garvan Institute of Medical Research, Sydney NSW 2010, Australia; Sensus Drug Development Corporation (W.B.), Austin, Texas 78701; and Department of Physiology and Pharmacology (M.J.W.), Centre for Molecular and Cellular Biology, University of Queensland, St. Lucia QLD 4072, Australia
Address all correspondence and requests for reprints to: Professor Richard J. M. Ross, Clinical Sciences, The Northern General Hospital, Sheffield S5 7AU, United Kingdom. E-mail: r.j.ross{at}sheffield.ac.uk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several approaches have shown that a single GH molecule associates with two receptor molecules (5, 6, 7, 8). This occurs through two unique receptor-binding sites on GH and a common binding pocket on the extracellular domain of two receptors. Site 1 on the GH molecule has a higher affinity than site 2, and receptor dimerization is thought to occur sequentially, with one receptor binding to site 1 on GH, followed by recruitment of a second receptor to site 2. It has been proposed that ligand-driven receptor dimerization is the key event leading to signal activation (5), triggering phosphorylation cascades that include the Jak2/Stat5 pathway (9). The importance of receptor dimerization in signal transduction is indicated by a number of experiments. High concentrations of GH, which favor the monomeric GH-GHR complex, inhibit the GH signal (10). Truncated receptors lacking the cytoplasmic domain act as dominant negative inhibitors of signaling by heterodimerization with the full-length receptor (11). Mutations in the interreceptor dimerization domain inhibit signaling without influencing GH binding (12). Finally, the strongest evidence comes from work with a GH molecule mutated at site 2 to prevent receptor dimerization. These GH mutants block GH-stimulated cell proliferation (10, 13, 14, 15, 16), the conformational change associated with receptor dimerization (17), and Jak-Stat signaling (18). These observations form the basis of the molecular drug design of GH antagonists for clinical application (19).
In addition to a site 2 mutation, B2036 possesses eight mutations at site 1, each of which affects affinity for GH binding protein (GHBP) (20) and potentially enhances antagonistic potency. The antagonist is conjugated to 4–5 moieties of PEG 5000 (B2036-PEG) to prolong its circulating half-life and lower immunogenic potential (19, 21). Despite the mutations engineered to enhance its binding activity, B2036-PEG (according to preliminary clinical data) is required at high levels (up to 1000-fold that of endogenous GH) to suppress insulin-like growth factor-I (IGF-I) levels in normal subjects (22), and daily doses of pegvisomant that are 20–40 times the normal daily production of GH are required to control acromegaly (3, 4). Thus, there is a discrepancy between expected and observed in vivo potency of the pegylated antagonist. To date, relatively little is known about the biological actions of B2036 or B2036-PEG at the cellular level.
GHRs are internalized rapidly upon ligand binding (18, 23), with a proportion recycled to the cell surface (24). Because B2036 is rapidly internalized upon binding to GHR (18), it may affect receptor turnover and hence induce a refractory period for GH signaling, another possible mechanism for down-regulating GH responsiveness of cells.
To clarify the actions of B2036 and the impact of pegylation on antagonism at the cellular level, we have compared the binding of GH and the antagonist to GHBP and to GHR and have studied its antagonist action in a transcription assay. We also studied antagonist interactions with GHBP and effects on receptor turnover and down-regulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human (h)GH was produced as previously described (25). Recombinant hGHBP with a molecular mass of 28 kDa (5) was kindly provided by William Wood (Genentech, Inc., South San Francisco, CA). The GH antagonist, G120K, has a single mutation (G120K) in site 2 of the GH molecule, and the antagonist, B2036, is G120K with eight additional mutations at site 1 (H18D, H21N, R167N, K168A, D171S, K172R, E174S, and I179T). B2036-PEG (pegvisomant) is B2036 with the addition of four to five moieties of PEG 5000 (Sensus Drug Development Corporation, Austin, TX). Antibodies Mab263 and Mab5 were supplied by Agen (Acacia Ridge, Queensland, Australia). MAb 263 binds to a cross-species epitope in domain 1 of the GHR, adjacent to the hormone binding site (26) (S. W. Rowlinson and M. J. Waters, unpublished). MAb 5 binds to an epitope in the receptor dimerization region of domain 2 of the GHR, as shown by loss of binding to the D152H dimerization region mutant, which causes GH insensitive dwarfism (27) and the absence of immunoreactivity in immunoblots when domain 2 is deleted from the receptor (S. W. Rowlinson and M. J. Waters, unpublished). Because MAb 5 is able to block receptor dimerization, estimates of soluble receptor concentration, by Scatchard analysis, are increased by 2-fold when receptor is immunoprecipitated with MAb 5, compared with MAb 263 (5).
Cell culture
Stable clones expressing the full-length hGHR were generated in 293 cells, designated 293GHR, as previously described (18).
GHBP binding
Displacement of 125I-labeled GH binding to
GHBP by unlabeled GH and antagonists was studied by an
immunoprecipitation method (28). Briefly, 1 nmol/L
recombinant hGHBP was incubated with 5 x
104 cpm 125I-labeled GH in
300 µL of 25-mmol/L Tris-Cl (pH 7.4) with 0.1% BSA and 10 mmol/L
MgCl2, 20 µg/mL Mab263, and varying amounts of
unlabeled GH or antagonists. After 18 h of incubation at 4 C, the
complex was precipitated by adding 1 mL ice-cold 0.1-mol/L glycine (pH
10) with 28% wt/vol PEG 6000, and 100 µL of PBS with 0.4% bovine
-globulin and 0.2% BSA. Radioactivity of the pellet was
measured.
Receptor binding assays
For cell surface receptor binding, 293GHR cells were serum-starved for 12 h. Cells were then washed with PBS containing 1% BSA and incubated with 125I-labeled GH (105 cpm/well) for 2 h at 23 C in the absence or presence of various concentrations of unlabeled GH or antagonists. The cells were washed in the same buffer and solubilized in 0.5 mol/L NaOH and 0.1% Triton X-100 for counting. For binding studies performed in the presence of Mab5, cells were incubated with antibody (200 µg/mL) for 30 min at 23 C before binding assay. The inhibition constants (Ki) for GH and its antagonists in the binding assays were calculated using the Cheng-Prusoff equation (29), in which Ki = IC50/(1 + [L]/Kd), where IC50 is the concentration for 50% inhibition, [L] is the radioligand concentration, and Kd is the dissociation constant.
For binding to cell membranes, the membranes were prepared from 293GHR cells, and binding assays were performed as previously described (30).
Transcription assays
These were performed in 293GHR cells transiently transfected by the calcium phosphate precipitation method (Gibco BRL, Life Technologies, Inc., Gaithersburg, MD) with a reporter construct containing a Stat5-binding element (lactogenic hormone response element) fused to a minimal TK promoter and luciferase (11), and a ß-galactosidase expression vector as a transfection control. Sixteen hours after transfection, cells were transferred into serum-free medium and treated with GH or antagonists. Luciferase activity is corrected for ß-galactosidase activity and reported as percentage of maximal activity stimulated by GH. The maximal activity stimulated by GH is the fold induction stimulated by GH, i.e. corrected luciferase value in GH-stimulated cells divided by corrected luciferase value in unstimulated cells.
Statistics
For analysis of binding data and functional assays, ANOVA, with post hoc Bonferroni analysis, was used; and the level of significance was accepted as P < 0.05.
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Results |
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and Fig. 1)For binding to GHBP, B2036 had a Ki that was 4.5–5.2-fold less than that of GH (P < 0.05) and G120K. Pegylation of B2036 increased the Ki by 4.6-fold, to a level similar to that of GH. The results for binding to cell surface receptor were different. B2036 had a Ki similar to that of GH and G120K, but pegylation of B2036 increased its Ki by 39-fold, compared with GH (P < 0.0003). G120K-PEG had a Ki further increased by 6.6-fold, compared with B2036-PEG. The binding to cell membrane preparations was similar to that seen for cell surface receptor (monolayer) binding.
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Functional studies with GH antagonist (Fig. 2)
In the Stat5 reporter system, a 6-h exposure to GH at 50 ng/mL induced a 9-fold increase in luciferase activity, compared with unstimulated cells. At equimolar concentrations of GH and antagonist, B2036 resulted in a 50% reduction in luciferase induction by GH, but B2036-PEG had little effect. At a 10:1 ratio of antagonist to GH, B2036 completely blocked signaling, whereas B2036-PEG resulted in only a 42% reduction. A concentration of B2036-PEG approximately 20-fold greater than that of B2036 was required to get similar suppression of GH signaling.
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The effect of GHBP on the inhibitory action of B2036 and B2036-PEG
was examined using the Stat5 functional assay in the presence of GHBP
(2.5 and 10 nmol/L). When 293GHR cells were stimulated with 50 ng/mL GH
alone, addition of GHBP resulted in a dose-dependent reduction in the
luciferase response (P = 0.003). As shown before,
addition of 50 ng/mL B2036 decreased the GH-induced luciferase response
by 50% (Fig. 3a). Coincubation of GH and B2036 with the increasing
concentration of GHBP resulted in a significant increase in the
luciferase response (P < 0.05). The results with
B2036-PEG were different; at a concentration equimolar to that of GH,
B2036-PEG had little antagonistic action, and the inhibitory effect of
GHBP was still present but not significant (P = 0.07).
At a 10:1 concentration ratio of B2036-PEG:GH, the inhibitory effect of
B2036-PEG was evident, whereas the action of GHBP was not. Thus, high
concentrations of GHBP reduce the antagonist action of B2036 but have
no effect on the antagonist action of B2036-PEG.
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)To study the effect of prior exposure to GH or B2036 on GHR binding at the cell surface, 293GHR cells were exposed to unlabeled GH (500 ng/mL) or B2036 (500 ng/mL) for 16 h. Cells were then washed and incubated with 125I-labeled GH at 37 C for 2 h. Prior exposure to GH and B2036 resulted in reduced binding of 125I-labeled GH, which was restored by 2 h to the level without prior exposure to GH or B2036. There was no difference between GH and B2036. Thus, both GH and B2036 induce a temporary down-regulation of membrane receptor.
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)To study the effect of prior exposure to antagonist on subsequent GH signaling, 293GHR cells transfected with the Stat5 reporter were exposed, for 1 or 16 h, to 5000 ng/mL B2036 with or without 500 ng/mL GH. These doses were chosen because previous experiments had shown that a 10:1 ratio of antagonist to GH induced complete inhibition of signaling. The cells were washed before incubation with a 1-h pulse of GH (500 ng/mL), given at 1, 2, and 3 h after the removal of B2036. Coincubation of GH and B2036 completely suppressed signaling. The signal was 16, 50, and 80% of the induction without B2036 pretreatment at 1, 2, and 3 h after removal of B2036, respectively. Thus, B2036 induces a temporary refractory period in signaling, with a time course similar to that seen for membrane receptor down-regulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
Binding to GHBP is increased by the site 1 mutations in B2036, and
pegylation diminishes this binding. However, using GHR as the binding
target, the increased affinity of the site 1 mutations in unpegylated
B2036 is not evident. The pegylated forms of the antagonists show much
lower binding to GHR than their unpegylated counterparts; but when
pegylated, B2036 has a greater affinity than G120K. This can be
explained as follows. There are two lysines in the native binding site
1, which are mutated in B2036 (K168A and K172R). Conjugation of PEG
(pegylation) occurs with primary amines (lysines); thus, in the native
binding site 1 of the pegylated G120K antagonist, it would be likely
that pegylation would interfere with binding. In B2036, however,
pegylation would not occur at site 1, because of the mutated lysines;
and so, the affinity of B2036-PEG would be greater than that of
G120K-PEG. The effect of pegylation on affinity of B2036 was much
greater for membrane GHR than for GHBP. The reduction in affinity of
B2036-PEG at the cell surface was reflected in a 20-fold reduction in
inhibitory activity in the transcription assay. GHBP reduced the
inhibitory activity of B2036, but not that of B2036-PEG, results
compatible with the differential binding of the unpegylated and
pegylated antagonist to GHBP and cell surface receptor. An important finding was the effect of Mab5, a dimerization-blocking antibody (5), on the binding of both GH and B2036 to surface GHRs. Because GHR is expressed as a monomer, it is widely assumed that the receptor exists in a monomeric form in the cell membrane, and that GHR dimerization is ligand driven, with GH binding through site 1 to the first receptor, and then recruiting a second receptor through site 2 (5, 6). Based on this model, site 2-modified antagonists, such as G120K or B2036, would not be expected to bind to a GHR dimer and would be predicted to have an increased number of binding sites, compared with GH. Should B2036 bind to a single molecule of GHR at the cell surface, the number of binding sites for B2036 would be unchanged by the addition of Mab5. Our Scatchard studies revealed, however, that the number of receptor binding sites on the cell surface was similar for GH and B2036; and in the presence of Mab5, the number of binding sites increased for both ligands. This would be expected for GH binding to a GHR dimer that was dissociated by Mab5 [as was shown in (5)] but also implies that B2036 is associated with a receptor dimer. This possibility has previously been proposed for the antagonist G120R (23), where it was suggested that the complex was (G120R)2·(GHR)2, although on size alone it is difficult to differentiate this complex from the trimeric complex (GHR:B2036:GHR) suggested by our results.
B2036 has a mutated site 2, so it would not be expected to recruit a second GHR molecule, and its affinity should be determined by binding to site 1. However, our results of B2036 binding to cell surface receptor in monolayer culture or cell membrane preparations were different from those obtained in studies using GHBP. When studies were performed with GHR in the membrane, the binding for B2036, G120K, and GH were similar, suggesting that, in the trimeric structure, other components of the complex were important for ligand binding apart from site 1. There is evidence of discordance between binding affinity to GHBP and surface receptor for 20-kDa GH, which has a reduced affinity at site 1 (31). Recent studies, using recombinant 20-kDa GH, report its binding affinity to surface receptor to be similar to that of 22-kDa GH (32), although this was not the case for early studies using 20-kDa GH purified from pituitary extract (33). Further support for such a phenomenon is provided by the finding that both site 1 and site 2 mutations, which increase binding to GHBP, do not alter biological activity in a cell proliferation assay (34).
The effect of pegylation on binding of antagonist to cell surface GHR
was much greater than that seen with GHBP. In both monolayer culture
and cell membrane preparations, pegylation of B2036 caused a 30- to
40-fold reduction in binding, compared with the effect of pegylation on
binding to GHBP, where only a 5-fold reduction was seen. The reduced
affinity of B2036 associated with pegylation is likely to be
attributable to steric hindrance, rather than modification of binding
sites as has been reported for pegylated GH (35). Steric
hindrance could also explain the differential binding of GH and
B2036-PEG to GHBP and membrane receptor, where they had similar
affinities for GHBP, but B2036-PEG had a much lower affinity for
membrane receptor than GH. At the membrane, the PEG moieties may
prevent orientation of B2036-PEG for optimal binding to the GHR.
Another plausible explanation for the difference in binding activity of
B2036 and B2036-PEG for GHBP and cell surface receptor could be the
existence of low-affinity preformed GHR dimers, as seen with the
related erythropoetin receptor (36, 37). This model could
explain a number of observations (Fig. 6). B2036 has an increased affinity for
GHBP through its site 1 mutations; but for the GHR dimer, there are
other determinants of affinity in the trimeric structure of
GHR:B2036:GHR (2, 7). In the presence of a GHR dimer,
B2036 may preferentially bind to GHBP through enhanced binding at site
1. This is indirectly revealed in our transcription studies, where GHBP
inhibited the actions of B2036. Pegylation may have a greater effect on
binding of B2036 to a GHR dimer than to GHBP, which essentially binds
as a monomer in dilute solution. The observation that both G120R and
B2036 internalize with similar kinetics to GH (18, 23)
would be compatible with the hypothesis that the antagonists bind to a
GHR dimer, which induces internalization but not signaling. The model
would also explain why 20-kDa GH, which has a reduced affinity at site
1 for GHBP (31), may bind with similar affinity as 22-kDa
GH to membrane receptor (32).
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The interactions of GH and antagonists, in the presence of GHBP, were
complex but were predictable from the binding data. GHBP blocked GH
action in the transcription assay, such that 10 nM GHBP
inhibited the GH-induced luciferase activity by 50%. This is
consistent with previous findings, using other bioassays, that GHBP
attenuates GH action (42, 43). B2036 has a higher affinity
for GHBP than GH (because of site 1 mutations) but similar affinity for
cell surface receptor (advantage of site 1 mutation seems to be lost).
The binding affinity of B2036 for GHBP (Ki, 0.5
nmol/L) was not as great as predicted but was similar to that found in
a solid-phase assay [Ki, 0.4 nmol/L for
recombinant hGHBP and 0.28 nmol/L for serum-derived GHBP (Zida Wu and
Christian Strasburger, personal communication)]. Therefore, at
equimolar concentration of B2036 and GH, GHBP binds preferentially to
B2036; and, as a result, they reverse each other’s inhibitory effect
on GH action. The results with B2036-PEG were different. B2036-PEG has
an affinity for GHBP similar to that of GH but a greatly reduced
affinity for cell surface receptor, compared with GH. At equimolar
concentrations, B2036-PEG had little inhibitory effect on GH action,
compatible with the reduced binding of B2036-PEG to the cell surface
receptor. However, there was a reduction of the inhibitory effect of
GHBP, which would fit with B2036-PEG partially displacing GH from GHBP.
With a 10-fold excess of B2036-PEG, compared with GH, there was a
greater inhibition of GH signaling by B2036-PEG. Also the action of
GHBP was lost, consistent with all GH being displaced from GHBP by
B2036-PEG. In this experiment, we used levels of GHBP greater than that
seen under normal physiological conditions (
1 nmol/L)
(44). However, there is evidence from transgenic mice
overexpressing GH antagonist that GHR expression is increased, as are
levels of GHBP, in the presence of high levels of antagonist
(45).
We investigated whether B2036 could induce a refractory period in signaling as an additional mechanism for antagonism. Prior exposure to GH or antagonist induced a partial receptor down-regulation, which had recovered by 2 h after the removal of GH or B2036. We considered the possibility that the antagonist might block signaling through an action independent of receptor binding, such as the induction of negative regulators of signaling, which could be activated by receptor internalization. To examine this possibility, we measured the effect of preexposure to B2036, on Stat5 activation, by a subsequent pulse of GH. Prior exposure to antagonist reduced Stat5 activation for the next 2 h, and this effect diminished by 3 h. This result is consistent with the observation that prior exposure to antagonist reduces cell surface receptor, which recovers by 2 h. These data suggest that the antagonist action is predominantly related to binding and turnover of receptor.
In conclusion, pegylation reduces affinity of B2036 for the GHR, an effect that is far greater at the cell surface than for GHBP. These results provide an explanation for the high blood concentration of B2036-PEG required to suppress IGF-I levels in normal subjects (22) and in acromegaly (3, 4). However, the reduced bioactivity is compensated for, in vivo, by the positive effect of pegylation on pharmacokinetics. Predicting biological activity using assays based on GHBP has some limitations, because our results suggest that B2036 associates with a GHR dimer on the cell surface.
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Footnotes |
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2 Recipient of a Ph.D. studentship from the Society for
Endocrinology.
Received August 22, 2000.
Revised October 25, 2000.
Revised December 5, 2000.
Accepted December 14, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-helix of bovine
growth hormone dramatically affect its intracellular distribution
in vitro and growth enhancement in transgenic mice. J
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