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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
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: r.j.ross{at}sheffield.ac.uk.
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Abstract |
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Introduction |
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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 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, 12, 13), and it has been generally assumed that the prolonged plasma half-life 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.
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Materials and Methods |
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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 µg/ml streptomycin, 2 mg/ml fungizone, 2 mM L-glutamine, and 400 µg/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 µg/ml of each) mix in PBS for 2 h before plating cells. 293GHR cells were plated at 3 x 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 µg/ml) that recognizes an epitope independent of the GHR binding domain, a secondary biotinylated goat antimouse antibody (40 µg/ml), or the Alexa488-labeled goat antimouse antibody. Fixed cells were incubated with streptavidin-Alexa488 (4 µg/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.
Fluorescence microscopy imaging of slides
Fluorescence was visualized by confocal microscopy using a x60 or x40 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.
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Results |
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)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).
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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).
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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.
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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 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).
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Discussion |
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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 intensity 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 pegylated 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.
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Footnotes |
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Abbreviations: GHR, GH receptor; PEG, polyethylene glycol.
Received October 10, 2003.
Accepted June 9, 2004.
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References |
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-helix of bovine growth hormone dramatically affect its intracellular distribution in vitro and growth enhancement in transgenic mice. J Biol Chem 266:2252–2258This article has been cited by other articles:
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  J. O. L. Jorgensen, U. Feldt-Rasmussen, J. Frystyk, J.-W. Chen, L. O. Kristensen, C. Hagen, and H. Orskov Cotreatment of Acromegaly with a Somatostatin Analog and a Growth Hormone Receptor Antagonist J. Clin. Endocrinol. Metab., October 1, 2005; 90(10): 5627 - 5631. [Abstract] [Full Text] [PDF]
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