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The Journal of Clinical Endocrinology & Metabolism 89(9):4532– 4537
Copyright © 2004 by The Endocrine Society
doi: 10.1210/jc.2003-031781
Pegvisomant, a Growth Hormone-Specific Antagonist,
Undergoes Cellular Internalization
M. MAAMRA, J. J. KOPCHICK, C. J. STRASBURGER,
AND
R. J. M. ROSS
Division of Clinical Sciences (M.M., R.J.M.R.), Edison Biotechnology Institute (J.J.K.), and Department of Biomedical
Sciences, Ohio University, Athens, Ohio 45701; and Division of Clinical Endocrinology, Department of Medicine, Charitè
Campus Mitte (C.J.S.), Berlin, Germany
GH binding to a receptor (GHR) dimer triggers signaling and
internalization of the receptor/ligand complex. Pegvisomant
is a specific GH antagonist developed for the treatment of
acromegaly, and the basic molecule is GH with an amino acid
substitution that blocks the conformational change necessary
to generate functional GHR dimerization required for signal
transduction. Pegvisomant has additional polyethylene glycol moieties to prolong its half-life in the circulation and improve clinical efficacy through reduced renal clearance.
Pegvisomant has a long plasma half-life, and its mode of clear-
P
EGVISOMANT IS A specific GH antagonist used in the
treatment of acromegaly (1, 2). GH initiates intracellular signaling by binding two molecules of its receptor
(GHR) at the cell surface through two distinct binding sites.
The affinity of GH binding site 1 for GHR is high, whereas
the affinity of site 2 is lower. An amino acid substitution at
residue G120 in binding site 2 of GH has been shown to
inhibit GH-induced signal transduction (3–5). The genetically engineered GH receptor antagonist, B2036, is mutated
at GH binding site 2 with G120K. B2036 has eight additional
mutations at site 1 that increase the affinity of B2036 for
soluble GHR. The pegylated counterpart of B2036, pegvisomant [polyethylene glycol (PEG)], is generated by the conjugation of B2036 with four or five moieties of PEG 5000
(pegylation).
The GHR is devoid of intrinsic kinase activity and acts
through recruitment of the tyrosine kinase, Janus kinase 2, at
the conserved cytokine receptor motif, box 1, leading to the
activation of the Janus kinase-signal transducer and activator
of transcription and mitogen-activated protein kinase signaling pathways. GH binding occurs sequentially, with the
first receptor binding to GH through site 1, and the second
GHR binding to GH through binding site 2. Recent evidence
suggests that the GHR may exist as a preformed dimer (6, 7),
and that GH triggers a conformational change required for
signaling similar to that demonstrated for the erythropoietin
receptor (8, 9). However, the evidence of a GHR preformed
dimer has not been demonstrated with the same rigor as that
for the erythropoietin receptor. The antagonist B2036 binds
Abbreviations: GHR, GH receptor; PEG, polyethylene glycol.
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ance has not been established. We have studied pegvisomant
internalization and demonstrate that despite its size and prolonged plasma half-life, it is internalized by cells expressing
the GHR. As pegvisomant does not activate intracellular signal transduction systems, our results support the concept that
the conformational changes required for GHR signaling are
not essential for the intracellular trafficking of the ligand and
establish one potential contributing mechanism for pegvisomant clearance. (J Clin Endocrinol Metab 89: 4532– 4537, 2004)
to a receptor dimer, but does not trigger the essential change
in conformation required for signaling (7, 10).
Little is known about the biology of pegvisomant, because
pegylation makes it difficult to generate specific antibodies
and inhibits radiolabeling of the molecule. Renal excretion is
considered the major route for GH clearance (11–13), and it
has been generally assumed that the prolonged plasma halflife of pegvisomant is due to reduced renal clearance. In this
study we developed an immunofluorescence method to examine the internalization of GH, B2036, and pegvisomant.
We present evidence that despite being pegylated, pegvisomant is internalized in cells expressing the GHR.
Materials and Methods
Reagents
Recombinant human GH (genotropin) was obtained from Pharmacia
Biotech (Uppsala, Sweden). B2036 and pegvisomant were supplied by
Sensus Corp. (Austin, TX). The anti-GH 10A7 antibody has cross-reactivity with pegvisomant, although the affinity is 25–50% less than that
for GH (13, 14). The avidin-biotin blocking kit and Vectashield mounting
solution were purchased from Vector Laboratories, Inc. (Peterborough,
UK). The biotin-xx Fab goat antimouse antibody, Alexa488-streptavidin,
Alexa488-goat antimouse antibody, and Alexa594-transferrin were purchased from Molecular Probes (Leiden, The Netherlands).
Cell culture
The HEK293 cell line stably expressing the full-length human GHR
(293GHR) has been previously described (15). The cells were maintained
in DMEM-Nut F-12, supplemented with 10% fetal calf serum, 100 IU
penicillin, 100 ␮g/ml streptomycin, 2 mg/ml fungizone, 2 mm lglutamine, and 400 ␮g/ml geneticin, buffered with HEPES, and routinely grown at 37 C in a 95% humidified atmosphere of 5% CO2.
Immunocytochemistry
Four-well chambered glass slides (Nunc Labtech, Paisley, UK)
were coated with a polylysine/fibronectin (20 ␮g/ml of each) mix in
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Maamra et al. • Internalization of Pegvisomant
J Clin Endocrinol Metab, September 2004, 89(9):4532– 4537 4533
FIG. 1. Internalization of GH, B2036, and pegvisomant. To study the internalization of GH, B2036, and
pegvisomant, cells were incubated with ligand (200,
200, and 5000 ng/ml, respectively) for 30 min and fixed
in paraformaldehyde. Immunofluorescence detection
of ligand was performed on permeabilized and nonpermeabilized cells. All three ligands show both membrane binding (nonpermeabilized) and internalization
(permeabilized), although the percent internalization
of GH was greater than those of B2036 and pegvisomant (P ⬍ 0.001).
PBS for 2 h before plating cells. 293GHR cells were plated at 3 ⫻ 104
cells/well and allowed to settle for 24 – 48 h before treatment. Cells
were exposed to GH, B2036, or pegvisomant in serum-free medium.
After ligand exposure, cells were fixed in a nonpermeabilized state
(to show cell membrane binding) in 2% paraformaldehyde for 20 min
or for an additional 3 min in paraformaldehyde with 0.1% Triton
when permeabilization was required to allow immunostaining within
the cell (to demonstrate internalized ligand). The buffer used for
immunostaining was PBS supplemented with 0.1% BSA. For intracellular staining, the buffer was supplemented with 0.1% saponin to
reduce nonspecific staining. After fixing, cells were treated for 1 h
with buffer and 5% goat serum to block nonspecific binding sites.
Cells were rinsed briefly and incubated for 15 min with avidin-
blocking solution, followed by rinsing and incubation for 15 min with
biotin-blocking solution to ensure that all endogenous avidin- and
biotin-binding sites were blocked [according to the manufacturer’s
instructions (Vector Laboratories, Inc.)]. Subsequent antibody incubations were performed in buffer supplemented with 1% goat serum.
Ligands were detected with the specific monoclonal anti-GH antibody 10A7 (4 ␮g/ml) that recognizes an epitope independent of the
GHR binding domain, a secondary biotinylated goat antimouse antibody (40 ␮g/ml), or the Alexa488-labeled goat antimouse antibody.
Fixed cells were incubated with streptavidin-Alexa488 (4 ␮g/ml) in
buffer supplemented with 1% goat serum. Five- to 10-min washes
were performed between incubations using PBS (nonpermeabilized)
or PBS supplemented with 0.1% saponin.
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J Clin Endocrinol Metab, September 2004, 89(9):4532– 4537
Maamra et al. • Internalization of Pegvisomant
Fluorescence microscopy imaging of slides
Fluorescence was visualized by confocal microscopy using a ⫻60 or
⫻40 oil lens with a CLSM2010 confocal fluorescent microscope (Molecular Dynamics, Sunnyvale, CA) equipped with dual fluorescence and
transmission detection. Images were file transfer protocoled from the
INDY (Silicon Graphics, Mountain View, CA) on the CSLM 2010, converted to MAC 8-bit Tagged Image File Format files (Apple Computer,
Cupertino, CA), and analyzed with NIH Image, and the data were
exported to EXCEL (Microsoft, Redmond, WA) for further analysis.
Image analysis and statistics
Image analysis was performed with the NIH Image program (http://
rsb.info.nih.gov/nih.image). For the percent internalization studies, the
fluorescence of 10 cells in each permeabilized and nonpermeabilized
well was analyzed, and the results were expressed as a percentage of the
total fluorescence. Analysis of the internalization rates between GH and
pegvisomant was performed using ANOVA.
Results
Internalization of GH, B2036, and pegvisomant (Fig. 1)
To study internalization 293GHR cells were exposed for 30
min to GH, B2036, or pegvisomant at concentrations similar
to those found in the treatment of acromegaly (200, 200, and
5000 ng/ml, respectively). After ligand exposure, cells were
fixed either in a nonpermeabilized state (to show cell surface
binding) or after permeabilization to allow immunostaining
within the cell (to demonstrate internalized GH or antagonist). Cells exposed to PBS alone showed no specific fluorescence. GH showed less labeling at the cell surface, and the
majority of GH was internalized. In cells exposed to B2036,
there was clear plasma membrane binding and internalization. Similar results were seen in cells exposed to pegvisomant (5000 ng/ml). Analysis of at least 10 cells in each condition revealed that the percentage of total intracellular
fluorescence was 91.4.0 ⫾ 2.1% for GH, 55.6 ⫾ 3.5% for B2036,
and 58.3 ⫾ 6.0% for pegvisomant (P ⬍ 0.001 for B2036 and
pegvisomant vs. GH). The mean size of intracellular particles
was not significantly different among GH-, B2036-, and
pegvisomant-treated cells (47.0 ⫾ 3.0, 42.0 ⫾ 3.2, and 35.0 ⫾
4.7 pixels, respectively).
Dose-dependent intracellular accumulation of pegvisomant
(Fig. 2)
The above experiment was performed with a single dose
of pegvisomant (5000 ng/ml). In this experiment, we investigated whether intracellular accumulation of pegvisomant
was influenced by dose. To determine whether pegvisomant
would accumulate inside the cell, 293GHR cells were exposed for 30 and 120 min to increasing concentrations of
pegvisomant. After exposure, cells were fixed and permeabilized to allow immunostaining of internalized antagonist.
Pegvisomant intracellular localization was detected both after 30 and 120 min of treatment (Fig. 2A). Analysis of at least
10 cells at each dose and time point showed that increasing
pegvisomant concentrations led to a dose-dependent increase in intracellular mean cell fluorescence (Fig. 2B shows
data from 120 min). No uptake of Pegvisomant was observed
in parental 293 cells not expressing the GHR (data not
shown).
FIG. 2. Dose-dependent intracellular staining with pegvisomant. A,
To study the intracellular uptake of pegvisomant, 293GHR cells were
incubated with increasing concentrations of pegvisomant for 30 min
and 2 h. Cells were then fixed in a permeabilized state, and immunostaining was performed. B, 293GHR cells were incubated with
increasing concentrations of pegvisomant for 2 h. Ten cells were
analyzed for fluorescence intensity (mean ⫾ SEM). There was a dosedependant increase in intracellular staining (P ⬍ 0.001).
Maamra et al. • Internalization of Pegvisomant
Intracellular colocalization of GH and B2036 with
transferrin (Fig. 3)
To assess whether mutations in B2036 alter intracellular
trafficking in 293GHR, cells were treated with GH or B2036
in the presence of Alexa594-labeled transferrin. Immunostaining was performed immediately after incubation with
ligand, and fluorescence was analyzed by superimposing
transferrin fluorescence (red) on that of ligand (green; Fig. 3
shows results at 30 min). After 10- and 30-min incubations
with ligand, similar colocalization with transferrin was observed for both GH and B2036, suggesting that intracellular
compartment trafficking does not differ between GH and the
antagonist.
Analysis of trafficking after ligand pulse exposure (Fig. 4)
In the above experiment, fixation of cells was performed
while cells were still being exposed to ligand, that is cells
were exposed to ligand throughout the experiment. Thus, at
the time of fixation (10 and 30 min), some ligand would still
be being internalized while the early internalized ligand
would be entering the recycling pathway. To follow the
passage of ligand within the cell, we exposed cells to a pulse
of ligand, followed by a washing step and then incubation for
varying periods before fixation. 293GHR cells were exposed
to a 15-min pulse of GH, pegvisomant, or B2036 in the presence of Alexa594 transferrin. After the ligand pulse, cells
were rinsed and incubated in starvation medium for 10, 15,
30, 60, 120, and 180 min before fixing and staining. Intracellular fluorescent vesicles were detectable in all GH-, B2036-,
and pegvisomant-treated cells. Intracellular localization was
similar for all three ligands, with a vesicular pattern and
accumulation in the central part of the cells observed 10 min
J Clin Endocrinol Metab, September 2004, 89(9):4532– 4537 4535
postpulse. All three ligands showed colocalization with
transferrin. Figure 4 shows results at 60 min. There was no
difference between the ligands at the other time points (10,
15, 30, and 120 min).
Discussion
Pegylation increases the plasma half-life of hormones by
reducing renal clearance and intravascular proteolysis (16).
Pegvisomant has a prolonged plasma half-life, and high levels are achieved after administration to humans (14). Our
results demonstrate that pegvisomant is internalized into
cells expressing the GHR, thus providing one mechanism for
pegvisomant clearance. The dynamics of pegvisomant internalization differed from those of GH; however, intracellular
trafficking was similar.
The kidney is responsible for clearance of up to 60 –70% of
circulating GH, and until recently the GHR was also thought
to be a determinant of GH clearance (12, 17). Studies with
pegvisomant, however, demonstrate that GHR blockade
does not alter the metabolic clearance rate of GH (13). Thus,
for GH, GHR-mediated cellular internalization does not appear to contribute to the metabolic clearance rate. In contrast,
pegvisomant has a greatly reduced renal clearance rate; thus,
minor contributors to clearance may become more relevant.
We suggest that GHR-mediated cellular internalization may
be one possible mechanism of pegvisomant clearance from
the circulation.
The percentages of pegvisomant and B2036 internalized
were less than that of GH; more than 90% of GH was within
the cell after a 30-min incubation. The intensity of binding on
the cell surface for B2036 and pegvisomant was greater than
that for GH. One needs to be careful in comparing the in-
FIG. 3. Transferrin colocalization with GH and B2036. To study the trafficking after internalization of GH and B2036, cells were incubated
with ligand (500 ng/ml) for 30 min in the presence of Alexa594-transferrin before fixing and immunostaining. Ligand fluorescence is shown
in green, and transferrin in red. In the overlay (ligand⫹transferrin), colocalization is shown in yellow.
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J Clin Endocrinol Metab, September 2004, 89(9):4532– 4537
Maamra et al. • Internalization of Pegvisomant
FIG. 4. Analysis of trafficking after ligand pulse exposure. 293GHR cells were incubated for 15 min with GH, B2036, and pegvisomant. Cells
were then carefully rinsed, and the medium was replaced with ligand-free medium and incubated for 60 min before fixing and staining. Ligand
fluorescence is shown in green, and transferrin in red. In the overlay (ligand⫹transferrin), colocalization is shown in yellow.
tensity of fluorescence between ligands, because the affinity
of the detection antibody differed between the ligands. However, in the calculation of percent internalization, we are
comparing the same ligand at the cell surface with that internalized under identical experimental conditions. Therefore, we can be confident that there is a difference among GH,
B2036, and pegvisomant. It is perhaps not surprising that
B2036 and pegvisomant do have different cell surface binding, because conformation of the antagonist-receptor complex must be different from that of the GH-receptor complex.
We know that B2036 binds to a receptor dimer, but does not
induce the conformational change required for signal transduction (7), that GH accelerates GHR internalization (18),
that internalization is not required for signaling (19), and we
now recognize that signaling is not required for internalization. Our results confirm previous observations that although receptor dimerization is required for GHR internalization, the trafficking process is disassociated from receptor
signaling (7, 10). The cytoplasmic domain of the receptor is
required for GHR internalization, because an amino acid
substitution in the rat receptor at box 1 prevents receptor
internalization (19). Also, the truncated human GHR lacking
the cytoplasmic domain of the receptor fails to internalize
(15). The observation that B2036 and pegvisomant show
greater fluorescence at the cell surface may relate to a different conformation of the ligand-GHR complex delaying
triggering of internalization and increasing residence of antagonist at the cell surface.
We accept that all of our data relate to studies in cells
overexpressing the GHR. We have attempted studies in primary hepatocytes (data not shown); however, the sensitivity
of immunofluorescence was not sufficient to obtain meaningful results. It is possible that overexpression of receptor
could lead to a change in intracellular trafficking; however,
our observations do demonstrate that pegvisomant is internalized by a GHR-mediated process and that intracellular
trafficking is similar to that seen for GH.
GH, B2036, and pegvisomant accumulated within the cell
during prolonged exposure, and colocalization with transferrin suggests that the majority of ligand enters the recycling
pathway associated with the receptor (20), although in previous studies with iodinated GH, only 25% of GH was recycled out of the cell intact (21). It is possible that during the
trafficking of ligand there is degradation, with loss of pegy-
Maamra et al. • Internalization of Pegvisomant
lated moieties; however, our results demonstrate that despite
its increased size and reduced binding affinity compared
with GH, pegvisomant is internalized into GHR-expressing
cells. To our knowledge, this is the first demonstration of
internalization of a pegylated hormone and provides one
mechanism for pegylated hormone clearance.
Acknowledgments
Received October 10, 2003. Accepted June 9, 2004.
Address all correspondence and requests for reprints to: Dr. Richard
J. M. Ross, Sheffield University, Clinical Sciences, Northern General Hospital, Sheffield, United Kingdom S5 7AU. E-mail: [email protected].
This work was supported by an Endocrine Care Prize from Pharmacia
(to M.M.).
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JCEM is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the
endocrine community.