International Journal of Biological Macromolecules 79 (2015) 864–870
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Development and characterization of a novel GHR antibody antagonist, GF185 Fengjuan Sun a , Yaping Liu a , Hui Sun b , Baofang Tian c,∗ a b c
Department of Endocrinology, The First People’s Hospital, Jining 272000, Shandong, PR China Central Laboratory, The First People’s Hospital, Jining 272000, Shandong, PR China Traumatology Department, The First People’s Hospital, Jining 272000, Shandong, PR China
a r t i c l e
i n f o
Article history: Received 14 December 2014 Received in revised form 20 May 2015 Accepted 20 May 2015 Available online 4 June 2015 Keywords: Growth hormone Growth hormone receptor Anti-GHR antagonist
a b s t r a c t Here, we describe the development of a panel of monoclonal antibodies targeting the growth hormone receptor (GHR). Of these monoclonal antibodies (Mabs), GF185 was selected for further characterization due to its activities. Competitive receptor-binding assays and Western blotting analyses were used to demonstrate that GF185’s epitopes are localized within subdomain 1 of the growth hormone receptor extracellular domain (GHR-ECD). Subsequently, we evaluated GF185’s antagonistic activities in vivo and in vitro and showed that GF185 was able to neutralize growth hormone (GH) signalling and inhibit GHinduced Ba/F3-GHR proliferation. Our findings suggest that GF185 may serve as an attractive tool for GHR-related research and has a potential future application for the treatment of GH-dependent disease. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Growth hormone (GH) is the major regulator of postnatal somatic growth and metabolism. GH activities are mediated via the GHR, which is widely expressed by GH target cells [1]. Binding of GH to a preformed dimer of GHR at the plasma membrane induces a specific conformational change within the GHR and leads to activation of the janus-activated kinase 2 (JAK2) and the Srchomology 2 (SH2) domains of the GHR monomers. Downstream of GHR lie signalling proteins, such as signal transducer and activator of transcription (STAT) 5, 3 and 1, which are activated by tyrosine phosphorylation and lead to the translocation of homoor heterodimers into the nucleus, where they bind specific target sequences and modulate gene transcription [2]. GHR belongs to the cytokine receptor superfamily, which includes prolactin receptor, erythropoietin receptor, granulocyte colony-stimulating factor receptor, and others [3,4]. Structurally, full-length GHR is a 620-residue, single membrane-spanning protein, which consists of an extracellular domain (246 amino acids), a single-pass transmembrane domain (24 amino acids), and an intracellular domain (350 amino acids). The GHR extracellular domain (ECD, residues 1–246) consists of two subdomains (residues 1–123, referred to as subdomain 1; residues128–238, referred to as subdomain 2) that are linked by a four-residue hinge region. Each
∗ Corresponding author. Tel.: +86 0537 2106208; fax: +86 0537 2106208. E-mail address: [email protected] (B. Tian). http://dx.doi.org/10.1016/j.ijbiomac.2015.05.039 0141-8130/© 2015 Elsevier B.V. All rights reserved.
subdomain is composed of seven strands that are arranged into two antiparallel  sheets [5,6]. Structural and mutagenesis studies indicate that GH binds to two identical GHR-ECDs via two distinct sites on the GH molecule and that GH contacts GHR-ECD mainly via residues in subdomain 1 and in the hinge, although tryptophan 169 in subdomain 2 also contributes to binding [5,6]. In addition, the crystal structure of the GH-(GHR)2 complex suggests that an interaction mediated by subdomain 2 leads to the formation of a 500-A˚ 2 contact interface, which is thought to stabilize the GH (GHR)2 complex [6]. Furthermore, mutagenesis of conserved amino acids within this domain demonstrates that subdomain 2 plays a critical role in GH-induced signalling [7]. It is necessary to develop and prepare GH antagonists in situations of excess GH (e.g., acromegaly), and these may have applications in malignancies and other special research needs [8]. A series of studies on GHR antagonists have been reported over the past few decades. One approach has used a GH analogue. One of the first reports of a GH antagonist was observed in transgenic mice expressing the gene encoding bovine GH with a mutation in the third helix of the region of binding site 2 (bGH-M8), which led to a reduction in circulating IGF-I and a dwarf-like phenotype [9,10]. Subsequently, studies from Chen at al. [11] and Fuh et al. [12] indicated that a single point mutation in GH at position 120 (glycine) in the third helix with any amino acid other than alanine leads to a change in GH from an agonist (growth enhancer) to an antagonist (growth suppressor), such as G120R, G120K, B2036 and pegvisomant [8]; anti-GHR antibodies have also been developed as potential GH antagonists. Mab5, a monoclonal anti-GHR antibody,
F. Sun et al. / International Journal of Biological Macromolecules 79 (2015) 864–870
was initially reported as a GHR antibody antagonist, which recognized GHR on an epitope in the receptor dimerization region of subdomain 2 of the GHR-ECD [13,12,14]. Similarly, another monoclonal anti-GHR antibody (GHRext-Mab) was also shown to bind subdomain 2 of the GHR-ECD [15–17]. Each of these anti-GHR antagonists (Mab5 and GHRext-Mab) were successful antagonist towards GHR but not GH due to their epitope localization in subdomain 2 of GHR-ECD, whereas GH’s epitopes have been shown to be mainly localized in subdomain 1 of GHR-ECD. In the present study, we screened and characterized a novel neutralizing monoclonal anti-GHR antibody (designated GF185). We found that GF185 effectively antagonized GHR signalling in vitro and in vivo. In addition, we showed that GF185 shares common GHR-binding epitopes with GH and inhibited GH binding to GHR. Collectively, the current findings suggest that GF185 represents a novel GHR/GH antagonist that serves as an attractive tool for GHR-related research and may have potential application for the treatment of GH-dependent disease in the future. 2. Materials and methods 2.1. Antibodies, reagents and cell line Anti-phospho-JAK2 (Tyr1007/1008), anti-JAK2, anti-phosphoSTAT5 (Tyr 694), anti-STAT5, anti-phospho-STAT3 (Tyr705), anti-STAT3, anti-phospho-STAT1 (Tyr 701), anti-STAT1, antiphospho-ERK1/2 (Thr 202/Tyr 204) and anti-ERK1/2 antibodies were purchased from Cell Signaling Technology (USA). The antiphosphotyrosine antibody (clone 4G10) was obtained from Upstate Biotechnology. HRP-conjugated goat anti-rabbit and anti-mouse antibodies and recombinant hGH were purchased from Sigma (USA). [3 H] thymidine was purchased from Amersham Biosciences. The ImmunoPure Fab Preparation kit, Cell Lysis Buffer, IP kit, enhanced chemiluminescence (ECL) and BCA kit were purchased from Pierce (USA). The extracellular domain of rat GHR (GHBP) was obtained from Sino Biological Inc (Jinang, China). GHRECD1–123 and GHR-ECD124–246 were provided by Top-Peptide Co., Ltd. (China). The fluorescein isothiocyanate (FITC) conjugation kit was obtained from Sigma–Aldrich. The FITC Conjugation Kit was used to label recombinant GH according to the manufacturer’s directions. Conjugates with F/P (FITC/protein) ratios of 1 exhibited the greatest sensitivity and reproducibility and were chosen for further use in this study. Unless otherwise stated, all reagents were from Sigma–Aldrich (USA). CHO and Ba/F3 cells were stably transfected with a rat GH receptor cDNA that corresponded to amino acids 1–638 and was provided by Xin Liu (HuaChen Medical and Biological Co., Ltd.). CHO and Ba/F3 cells stably expressing full-length rGHR were cultured as previously described [18]. In addition, rat hepatocytes that endogenously express GHR were isolated and cultured as described previously [20]. 2.2. Production of anti-GHR monoclonal antibody Six 8-week-old female BALB/c mice were immunized intraperitoneally with 0.2 mg of GHBP in Freund’s complete adjuvant and received similar booster injections of GHBP in incomplete Freund’s adjuvant at 14 days intervals. The animals were immunized four times. Four mice were chosen for hybridoma preparation. Three days following the final injection, splenocytes were harvested from four GHBP-immunized BALB/c mice and were fused with sp2/0 myeloma cells at a 4:1 ratio using 50% (w/v) polyethylene glycol (PEG) 1500. Post-fusion, the cells were distributed in 96-well cell culture plates at 1 × 105 cells/well in HAT selection medium. The cultured supernatants of hybridomas selected
865
on hypoxanthine–aminopterin–thymidine (HAT) medium were screened for the presence of antibodies against GHBP using an ELISA. Antibody-secreting hybridomas were subcloned four times by limiting dilution. 2.3. Immunoprecipitation and Western immunoblotting To determine whether GF185 specifically binds to GHR, immunoprecipitation experiments were performed according to the method of Lan et al. [18]. Briefly, CHO-GHR cells were collected and lysed with cell lysis buffer according to the manufacturer’s instructions. The immunoprecipitation was conducted by treating cell lysates with GF185 (10 g), B-10 (10 g) or control (10 g) antibodies. The immunoglobulin-bound proteins were obtained using an IP Kit according to the manufacturer’s instructions. Immunoprecipitated proteins were subjected to SDS-PAGE and transferred to PVDF membranes, which were then blocked using 5% non-fat milk and incubated with GF185, B-10 or a control antibody overnight at 4 ◦ C. The membranes were then washed three times with TBST and incubated with HRP-conjugated goat anti-mouse IgG antibody in blocking buffer for 1 h at 37 ◦ C. Following the incubation, the membranes were washed three times and the signals were detected using the ECL detection system. 2.4. Preliminary screen of anti-GHR antibodies by FACS analysis Flow cytometric analysis was used to screen for potential antiGHR antibodies that bound to the GHR that was exogenously expressed in CHO cells (CHO-GHR). Before experimental incubations, the media of the CHO-GHR cells were replaced with the fresh serum-free containing 1% BSA and incubated for 12 h at 37 ◦ C. CHOGHR cells were then detached from the tissue culture flasks, washed twice with sterile PBS, and resuspended in either negative control media or an anti-GHBP hybridoma supernatant for 1 h at 4 ◦ C in separate 1.5-ml microcentrifuge tubes (2 × 104 cells/tube). The cells were then washed and incubated with an FITC-conjugated secondary antibody (1:200 dilution) for 60 min in the dark at 4 ◦ C. Following the incubation, the cells were washed twice and resuspended in 0.5 ml of FACS buffer (1% BSA and 0.01% sodium azide in PBS, pH 7.4). The samples were analyzed using a FACS Calibur flow cytometry device (Becton Dickenson). The data were analyzed using Cell Quest software. 2.5. Competitive receptor-binding assay To determine whether GF185 inhibited GH binding to GHBP, a competitive ELISA was performed. First, 96-well microtiter plates coated with 1 g/ml GHBP in a volume of 100 l were incubated at 4 ◦ C for 12 h and then washed and blocked with 2% BSA for 1 h at 37 ◦ C. The wells were then washed three times with PBS containing 1% BSA and 0.05% Tween 20, and a constant amount of GH was added to each well with increasing concentrations of either GF185-F(ab )2 (as the competitor) or F(ab )2 of control antibody (negative control) for 0.5 h. After incubation for 1 h at 37 ◦ C, all of the wells were washed three times with PBST, and then anti-hGH was added. After incubation for 1 h at 37 ◦ C, a secondary antibody (Fc-specific) (1:2000 dilution) was added and incubated for another 1 h at 37 ◦ C. After three final washes, the TMB substrate was applied to develop the colorimetric reaction, which was then terminated by the addition of 3 M H2 SO4 to each well (50 l/well). The optical density at 450 nm was measured using a Microplate Reader (Multiskan FC, Thermo Fisher Scientific). Binding of GH to GHBP without any competitor was defined as 100% binding. To further determine whether GF185 competed with GH for specific binding to full-length GHR, a competitive receptor-binding assay was performed and analyzed by flow cytometry. Prior to
866
F. Sun et al. / International Journal of Biological Macromolecules 79 (2015) 864–870
experiments, CHO-GHR cells were incubated in serum-free F12 media containing 1% BSA for 12 h. The cells were then detached, collected by centrifugation, washed three times with PBS, resuspended in binding buffer (Williams E medium containing 1 mg/ml BSA), pipetted into FACS tubes (1 × 106 cells/tube) and incubated with FITC-GH together with increasing concentrations of unlabelled GH, GF185, or a control antibody for 1 h in the dark at 4 ◦ C. After the incubation, the cells were washed three times and resuspended in 0.5 ml FACS Buffer and analyzed using a FACS Calibur Flow Cytometer (Becton Dickenson). The data were analyzed using Cell Quest software. 2.6. Evaluation of GF185’s antagonistic effects in vitro The CHO-GHR or rat hepatocytes were seeded onto 6-well plates and cultured as described as above. Prior to the experiments, CHO-GHR cells or rat hepatocytes were serum-starved for 10 h. A constant amount of GH (50 ng) was incubated together with increasing concentrations of GF185 and then added to the plates for 20 min (CHO-GHR) or 30 min (rat hepatocytes) at 37 ◦ C. Unless noted, all of the stimulations were carried out at 37 ◦ C. At the end of the treatment, the medium was discarded and the cells were washed three times with ice-cold PBS. The cells were solubilized in lysis buffer on ice for 30 min, and cell debris was removed by centrifugation under refrigeration at 12,000 × g for 30 min. The resulting supernatants were harvested and subjected to ultrafiltration. Samples were lysed directly in SDS-PAGE sample buffer and boiled for 5 min, and the protein concentration of the lysate was determined with the BCA protein assay according to the manufacturer’s instruction. The samples (50 g of protein per lane) were subjected to SDS-PAGE (10% gel) and then transferred to a PVDF membrane. The membranes were washed and blocked with 5% non-fat milk for 1 h at 37 ◦ C. After three washes with TBST, the membranes were incubated with phospho-STAT5, phospho-STAT3, phospho-STAT1 or phospho-ERK1/2 antibodies according to the manufacturer’s protocols. After three washes with TBST, the membranes were incubated with an HRP-conjugated goat anti-rabbit IgG antibody (secondary antibody) for 1 h at 37 ◦ C. After three final washes, the signals were detected by ECL detection system. After ECL detection, the membranes were stripped of primary and secondary antibodies according to the manufacturer’s instructions, and then blocked and reprobed for JAK2, STAT1/3/5 or ERK1/2. Experiments were carried out in triplicate. 2.7. Evaluation of GF185’s antagonistic effects in vitro The [3 H] thymidine incorporation assay was performed according to the method of Lan et al. [18]. Briefly, Ba/F3-GHR cells were grown in RPMI1640 medium containing 10% foetal calf serum (FCS) for 16 h. The cells were then suspended, counted and adjusted to 1 × 106 cells/ml. After washing two times with serum-free RPMI 1640, 50 l of the cell suspension was seeded into the wells of 96-well tissue culture plates. A constant amount of GH was incubated together with increasing concentrations of GF185 or a control antibody and then added to selected wells in a final volume of 0.2 ml for16 h at 37 ◦ C. [3 H] thymidine was then added to each well (1 Ci/well), and after 6 h at 37 ◦ C, [3 H] thymidine incorporation was determined by liquid scintillation counting. 2.8. Preliminary evaluation of GF185’s antagonistic effects in vivo The above experiments were carried out in vitro. As described in this section, the antagonistic activity of GF185 was also determined in vivo according to the method of Lan et al. [21]. It has been reported that the liver from intact, but not hypophysectomized, rats can be used as a model to investigate GH signalling [21–23]. Male
Sprague Dawley rats (∼200 g, 6 wk old) were housed at three individuals per cage in the conventional animal room with free access to standard rodent chow and water. The rats were acclimated for 3 days prior to the start of the experiments. The rats were anesthetized with ether. The abdominal cavity was opened, the portal vein was exposed, and GH (0.5 mg), GF185 (10 mg), a control antibody (10 mg), the mixture of GH (0.5 mg) plus GF185 (10 mg) or the mixture of GH (0.5 mg) plus a control antibody (10 mg) was injected in a volume of 1.5 ml of physiological saline. After 20 min, the livers were quickly removed from rats killed by decapitation and the liver samples were homogenized in ice-cold lysis buffer and incubated on a shaker platform for 30 min at 4 ◦ C. The samples were then centrifuged at 12,000 × g, and the supernatant was collected. The samples were then lysed and quantitated. The samples (50 g of protein per lane) were subjected to SDS-PAGE (10% gel) and then transferred to a PVDF membrane, followed by Western blot analysis as described above. 2.9. Statistical analysis The data are presented as the mean values ± standard error (S.E.). 3. Results 3.1. Generation and characterization of GF185 Immunized mice were shown to produce high-titre sera against GHBP, as determined by ELISA. Spleen cells from the immunized mice were fused with the murine myeloma cell line SP2/0. ELISAs of each well’s culture supernatant revealed 55 positive antibodysecreting clones (data not shown). Subsequently, flow cytometric analysis was performed to screen for anti-GHR monoclonal antibodies that were capable of binding GHR on CHO-GHR cells and inhibiting GH binding to GHR expressed in CHO-GHR cells. Eight of the 55 clones were shown to exhibit a strong positive signal. Of these clones, the Mab named GF185 was selected for further characterization based on its biological properties described below (Fig. 1A). GF185 was classified as an IgG1 isotype using a commercial mouse Mab isotyping kit. To further confirm the specificity of GF185 binding to GHR, immunoprecipitation experiment using GF185 highlighted a 130 kD protein that cross-reacted with B-10, a commercial antiGHR monoclonal antibody that was prepared by Santa Cruz Biotechnology (Santa Cruz, California, USA), which specifically recognizes Rghr and is suitable for IP and WB experiments. Taken together, these data demonstrate that GF185 specifically recognizes the GHR (Fig. 1B). 3.2. Characterization of GF185 binding epitopes on GHR To characterize the epitopes on GHR that mediate GF185 binding, competitive ELISA and competitive receptor-binding assays were performed. As shown in Fig. 2A, GF185 was shown to inhibit GH binding to GHBP in a dose-dependent manner. Because GHBP is composed of the extracellular domain (ECD) of GHR, a competitive receptor-binding assay was carried out at the cellular level (CHO-GHR) to assess full-length GHR binding. GF185 was shown to compete with GH for binding to GHR, while a control antibody had no effect (Fig. 2B), suggesting that GF185 binds at residues that over-lap with the GH-binding site within subdomain 1 of GHR-ECD. To directly confirm that GF185’s epitopes are localized within subdomain 1 of GHR ECD, Western-blot analyses were used to show that GF185 reacted with GHR1–123 but not with GHR124–246.
F. Sun et al. / International Journal of Biological Macromolecules 79 (2015) 864–870
867
Fig. 1. Screening and identification of anti-GHBP monoclonal antibodies that can bind to subdomain 1 of GHR-ECD. (A) Flow cytometric screening of potential anti-GHBP monoclonal antibodies that can bind to CHO-GHR. CHO-GHR were cultured and pretreated as described in Section 2. The cells were then incubated with GF185supernatant or control media for 1 h at 4 ◦ C. After the incubation and washing, the cells were incubated with FITC-conjugated secondary antibody for 1 h in the dark at 4 ◦ C. After three washes, the cells were resuspended in 0.5 ml of FACS buffer, and the samples were analyzed using a FACS Calibur flow cytometry device (Becton Dickenson). The data were analyzed using Cell Quest software. (B). GF185 was determined to interact with full-length GHR expressed on CHO-GHR cells by immunoprecipitation experiments. CHO-GHR were cultured and pretreated as described in Section 2. Immunoprecipitation experiments were performed by treating cell lysates with GF185 (10 g), B-10 (10 g) or control antibody (10 g). The immunoglobulin-bound proteins were obtained using an IP kit according to the manufacturer’s instructions. Immunoprecipitated proteins were subject to SDS-PAGE and transferred to PVDF membranes. Proteins transferred to PVDF membranes were detected with GF185, B-10 or control antibodies. Signals were detected using the ECL detection system.
Fig. 2. Characterization of the GF185-binding epitopes on GHR-ECD. (A) GF185 competes with GH for binding to GHBP. First, 96-well microtiter plates were coated with GHBP and blocked with 2% BSA. Before the experiments, a constant amount of GH was incubated together with increasing concentrations of GF185-F(ab )2 (as the competitor) or control antibody (as the negative control) for 0.5 h, and the mixture was then added to the plates. After incubation for 1 h, anti-hGH was added to the plates, and incubated for 1 h at 37 ◦ C. Secondary antibody was then added and incubated for another 1 h at 37 ◦ C. After washing, the TMB substrate was applied to develop the colour in a colourimetric reaction. (B) GF185 inhibits GH binding to GHR expressed on CHO-GHR. Cells were incubated with FITC-GH (15 nM) or increasing concentrations of unlabelled GH, GF185, or control antibody for 1 h in the dark at 4 ◦ C. After the incubation, the cells were washed three times and resuspended in 0.5 ml of FACS Buffer and analyzed using a FACS Calibur Flow Cytometer. The data were analyzed using Cell Quest software. (C) GF185 reacts with subdomain 1 of GHR-ECD but not with subdomain 2. The structure GHR-ECD is diagrammed, and subdomain 1, hinge region and subdomain 2 are indicated. Recombinant proteins (GHR-ECD 1–123 and 124–246) were subjected to SDS-PAGE and transferred to PVDF membranes. The proteins transferred to the PVDF membranes were detected with GF185. Signals were detected using the ECL detection system. After detection, the membranes were stripped and re-probed with polyclonal anti-GHBP antibody to verify equal protein loading in each lane (In the pre-experiment, it was demonstrated that the polyclonal anti-GHBP could recognize GHR-ECD1–123 and GHR-ECD124–246 ). The results are representative of at least three separate experiments.
3.3. Inhibition of GHR signalling by GF185 in CHO-GHR cells and rat hepatocytes To determine whether GF185 possesses agonistic activity, CHOGHR cells were treated with GH, Mab263 (an anti-rGHR monoclonal antibody that can activate GHR-mediated signalling [21]), GF185 or
a control antibody. As determined by Western-blot analysis, GH and Mab263 activatedJAK2 (Fig. 3), in contrast, GF185 and the control antibody were unable to induce tyrosine phosphorylation of JAK2, indicating that GF185 does not possess agonistic activity. We then tested whether GF185 inhibited GH signalling in CHO-GHR cells. CHO-GHR cells were treated as described as
868
F. Sun et al. / International Journal of Biological Macromolecules 79 (2015) 864–870
Fig. 3. GF185 does not display agonistic activity. CHO-GHR cells were cultured and pretreated as described in Section 2. The cells were then stimulated with the indicated concentrations of GF185control antibody or GH for 20 min at 37 ◦ C. The cell extracts were harvested, and samples (50 g of protein per lane) were subjected to SDS-PAGE (10% gel), transferred to a PVDF membrane and immunoblotted with the indicated antibodies. Detection was performed using the ECL detection system. After ECL detection, the membranes were stripped of primary and secondary antibodies according to the manufacturer’s instructions. The stripped membranes were blocked and reprobed for JAK2 to verify equal protein loading in each lane. The figures represent at least three independent experiments.
in the materials and methods, and Western-blot analyses was used to show that the antagonistic effect of GF185 was initially observed at 0.05 g/ml and that the phosphorylation levels of STAT5/3/1 and ERK1/2 gradually declined with increasing concentrations of GF185. Maximal antagonist effects of GF185 were reached at a concentration of approximately 5 g/ml when GH signalling was completely inhibited. These results demonstrate that GF185 efficiently neutralize styrosine phosphorylation of GHinduced intracellular signalling molecules in a dose-dependent manner. The above studies were conducted in a CHO cell line that was stably transfected with full-length GHR. To confirm these results, rat hepatocytes that endogenously express abundant GHRs were used as a model to further assess the antagonistic effect of GF185. Rat hepatocytes were treated as described in Section 2 and Western-blot analysis was used to show that antagonistic effects of GF185 were easily observed at a concentration of 0.05 g/ml. Increasing concentrations of GF185 were shown to exhibit stronger antagonistic effects on GH signalling, with a complete blockage of GH-mediated intracellular signalling observed at 5 g/ml of GF185. In contrast, a control antibody showed no effect. These data indicate that GF185 efficiently inhibited tyrosine phosphorylation of GH intracellular signalling in a dose-dependent manner in rat hepatocytes (somatic cells). 3.4. Inhibition of GH-induced proliferation of BaF3-GHR638 cells by GF185 To further characterize GF185’s effects on GH action, we analyzed GF185’s impacts on GH’s ability to promote cell proliferation. Ba/F3-GHR cells were used and treated as described as in Section 2. As indicated in Fig. 6, GF185, but not a control antibody, was shown to dramatically inhibit GH-induced cell proliferation of Ba/F3-GHR in a dose-dependent manner. These results indicate that the anti-GHR monoclonal antibody (GF185) specifically inhibits GH-dependent Ba/F3-GHR proliferation. 3.5. Inhibition of GHR signalling by GF185 in vivo In the experiments mentioned above, we evaluated GF185’s antagonistic activity in vitro. In this section, to determine the impact of GF185 on GH signalling in vivo, rats were injected with GH and intracellular signalling pathways were assessed by Western blot analyses. Injection with GH alone was sufficient to promote a strong phosphorylation of JAK2, and STAT1/3/5. However, rats injected with a mixture of GH and GF185 (but not the mixture of GH and control antibody) experienced a complete block in the phosphorylation of intracellular signalling protein molecules (such as JAK2 and STAT1/3/5). In addition, GF185 (alone) or control antibody (alone) showed no effect. These results indicate that GF185 inhibits GH signalling in vivo.
4. Discussion GH is an asymmetric molecule whose downstream function is mediated via the GHR. After GH binding, downstream signalling events are triggered by specific conformational changes within the pre-dimerized GHR. Structurally, the full-length GHR consists of an extracellular domain (ECD), a single-pass transmembrane domain (TMD), and an intracellular domain (ICD). The GHR extracellular domain (ECD, residues 1–246) consists of two subdomains (residues 1–123, referred to as subdomain 1; residues 128–238, referred to as subdomain 2) [5,6]. Structural and mutagenesis studies indicate that GH’s epitopes are mainly distributed in subdomain 1 of GHR ECD [5,6]. The use of anti-GHR antibodies as GHR antagonists has been extensively reported over the past few decades. To the best of our knowledge, Mab5 represents the first commercially available GHR monoclonal antibody antagonist whose epitopes are localized in subdomain2 of GHR ECD [13,12,14]. Recently, another well-characterized GHR antibody antagonist (anti-GHRext-Mab) was reported [15–17]. A common feature of Mab5 and anti-GHR ext-Mab is their lack of effect on GH binding to GHR, which is due to epitope distribution in subdomain 2 of GHR-ECD. It has been suggested that the action mechanism of Mab5 and anti-GHRextMab is mediated by the inhibition of “active” GHR conformational change(s) following GH binding. In the present study, we have characterized a novel GHR antibody antagonist (GF185), which inhibited GH binding to GHR (Fig. 2) and neutralized GHR signalling (Figs. 4 and 5). To the best of our knowledge, this is the first report of an anti-GHR antibody that mediated GHR antagonist activity via epitopes on subdomain1 of GHR-ECD (Fig. 7). In a previous study, Lan et al. [18] reported that the anti-GHR antibody CG-172 showed agonistic activity. Similar results have been reported by the Waters’ group, who showed that only one of the 14 anti-GHR Mabs, which were directed to the GH binding site and competed with GH for binding GHR, was able to activate the full-length GHR [19]. In the current work, we have screened and characterized a panel of anti-GHR antibodies, among which we found that GF185 was suitable to serve as a GHR antagonist. Importantly, we show that two additional anti-GHR antibodies (GF153 and GF176) that were directed to the GH binding site on GHR also showed antagonistic activity (data not shown), which suggests that the preparation of GHR antagonists may be more easily produced than agonists. Similar observations were reported by Wu et al. [24], who showed that of the 21 Mabs developed against GHR-ECD, only one Mab (termed as GHRA1) exhibited agonistic property; however, the Mabs harbouring the highest degree of epitopes that overlapped with the GH binding site on GHR did not show agonistic activity. Taken together, these results suggest that antibodies designed with epitopes on the GH binding site may represent the natural antagonist for GH/GHR. Why do anti-GHR antibodies (such as GF185) containing epitopes on the GH binding site of GHR show antagonistic but not agonistic properties? It is has been accepted that special
F. Sun et al. / International Journal of Biological Macromolecules 79 (2015) 864–870
Fig. 4. Inhibition of GH-induced signalling by GF185 in CHO-GHR cells. CHO-GHR cells were cultured and pretreated as described in Section 2. A constant amount of GH (50 ng) was incubated together with increasing concentrations of GF185 (0.05–5 g), and the mixtures were then added to CHO-GHR cells and incubated for 20 min. Cell extracts were harvested, and the samples (50 g of protein per lane) were subjected to SDS-PAGE (10% gel), transferred to a PVDF membrane and immunoblotted with the indicated antibodies. Detection was performed using the ECL detection system. After ECL detection, the membranes were stripped of primary and secondary antibodies according to the manufacturer’s instructions. The stripped membranes were blocked and reprobed for JAK2, STAT1/3/5 or ERK1/2 to verify equal protein loading in each lane. The figures represent at least three independent experiments.
Fig. 5. Inhibition of GH-induced signalling by GF185 in rat hepatocytes. Rat hepatocytes were cultured and pre-treated as described in Section 2. A constant amount of GH (50 g) was incubated together with increasing concentrations of GF185 (0.05–5 g), and the mixtures were then added to rat hepatocytes and incubated for 20 min. The cells were solubilized in lysis buffer on ice for 30 min. Cell extracts were then harvested, and the samples (50 g of protein per lane) were subjected to SDS-PAGE (10% gel), transferred to a PVDF membrane and immunoblotted with the indicated antibodies. Detection was performed using the ECL detection system. After ECL detection, the membranes were stripped of primary and secondary antibodies according to the manufacturer’s instructions. The stripped membranes were blocked and reprobed for JAK2, STAT1/3/5 or ERK1/2 to verify equal protein loading in each lane. The figures represent at least three independent experiments.
conformation change(s) are required for GHR activation, although the nature of this conformational change is still not fully understood. The Waters’ group has recently proposed a new model suggesting that the GH receptor exists predominantly as a dimer held together by its transmembrane helices and that GH binding to
869
Fig. 6. Inhibition of GH-induced proliferation of Ba/F3-GHR by GF185. Ba/F3-GHR cells were cultured and pre-treated as described in Section 2. A constant amount of GH was pre-incubated together with increasing concentrations of GF185 or control antibody for 30 min, and then they were added to selected wells in a final volume of 0.2 ml for 16 h at 37 ◦ C. [3 H] thymidine was then added to each well (1 Ci/well), and after 6 h at 37 ◦ C, [3 H] thymidine incorporation was then determined by liquid scintillation counting. The results are representative of at least three separate experiments.
Fig. 7. Inhibition of GH-induced signalling by GF185 in vivo. SD rats were pretreated as described in Section 2. The rats were then injected with GH alone (0.5 mg), GF185 alone (10 mg), control antibody alone (10 mg), the mixture of GH (10 mg) plus GF185 (10 mg), or the mixture of GH (0.5 mg) plus control antibody (10 mg). Twenty minutes after the injection, the livers were quickly removed from rats killed by decapitation, and the liver samples were homogenized in ice-cold lysis buffer and incubated on a shaker platform for 30 min at 4 ◦ C. The samples were then centrifuged at 12,000 × g, and the supernatant was collected. The samples were lysed directly in SDS-PAGE sample buffer, boiled for 5 min and quantitated with the BCA Protein Assay according to the manufacturer’s instructions. The samples (50 g of protein per lane) were subjected to SDS-PAGE (10% gel) and then transferred to a PVDF membrane. The membranes were probed with the indicated antibodies. Signals were detected using the ECL detection system. The results are representative of at least three separate experiments.
GHR induces a rotation of subunits that subsequently converts the transmembrane helices into a left-hand crossover state and causes a repositioning of the ICDs to activate JAK2 [25,26]. Based on the above activation mechanism of GHR, we speculated that the degree of subunit rotation induced by anti-GHR antibodies (such as GF185) with the epitopes on the GH binding site of GHR is not suitable for GHR activation and therefore is unable to induce an “active” conformation change(s) of GHR. Of course, this represents only one possible explanation, and further research is required to explore and elucidate answers to this question. Thus, we have developed and characterized a novel GHR antibody antagonist (GF185) that not only acts as a full competitor for GH binding but also inhibits GHR signalling. These findings have
870
F. Sun et al. / International Journal of Biological Macromolecules 79 (2015) 864–870
future implications for the use of GF185 as a potential therapeutic option in GH-dependent diseases. Acknowledgement We are grateful to Li Qiang for excellent technical assistance. References [1] N. Møller, O.L.J. Jens, Effects of growth hormone on glucose, lipid, and protein metabolism in human subjects, Endocr. Rev. 30 (2009) 152–177. [2] A.J. Brooks, M.J. Waters, The growth hormone receptor: mechanism of activation and clinical implications, Nat. Rev. Endocrinol. 6 (2010) 515–525. [3] S. Moutoussamy, P.A. Kelly, J. Finidori, Growth hormone receptor and cytokine receptor family signalling, Eur. J. Biochem. 255 (1998) 1–11. [4] X. Wang, P. Lupardus, S.L. Laporte, K.C. Garcia, Structural biology of shared cytokine receptors, Annu. Rev. Immunol. 27 (2009) 29–60. [5] B.C. Cunningham, M. Ultsch, A.M. De Vos, M.G. Mulkerrin, K.R. Clauser, J.A. Wells, Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule, Science 254 (1991) 821–825. [6] A.M. de Vos, M. Ultsch, A.A. Kossiakoff, Human growth hormone and extracellular domain of its receptor: crystal structure of the complex, Science 255 (1992) 306–312. [7] J. Gent, M. van den Eijnden, P. van Kerkhof, G.J. Strous, Dimerization and signal transduction of the growth hormone receptor, Mol. Endocrinol. 17 (2003) 967–975. [8] J.J. Kopchick, C. Parkinson, E.C. Stevens, P.J. Trainer, Growth hormone receptor antagonists: discovery, development, and use in patients with acromegaly, Endocr. Rev. 23 (2002) 623–646. [9] W.Y. Chen, D.C. Wight, T.E. Wagner, J.J. Kopchick, Expression of a mutated bovine growth hormone gene suppresses growth of transgenic mice, Proc. Natl. Acad. Sci. U. S. A. 87 (1990) 5061–5065. [10] W.Y. Chen, M.E. White, T.E. Wagner, J.J. Kopchick, Functional antagonism between endogenous mouse growth hormone (GH) and a GH analog results in dwarf transgenic mice, Endocrinology 129 (1991) 1402–1408. [11] W.Y. Chen, D.C. Wight, B.V. Mehta, T.E. Wagner, J.J. Kopchick, Glycine 119 of bovine growth hormone is critical for growth-promoting activity, Mol. Endocrinol. 5 (1991) 1845–1852. [12] G. Fuh, B.C. Cunningham, R. Fukunaga, S. Nagata, D.V. Goeddel, J.A. Wells, Rational design of potent antagonists to the human growth hormone receptor, Science 256 (1992) 1677–1680. [13] R. Barnard, P.G. Bundesen, D.B. Rylatt, M.J. Waters, Monoclonal antibodies to the rabbit liver growth hormone receptor: production and characterization, Endocrinology 115 (1984) 1805–1813.
[14] R.J. Ross, K.C. Leung, M. Maamra, W. Bennett, N. Doyle, M.J. Waters, K.K. Ho, Binding and functional studies with the growth hormone receptor antagonist, B2036-PEG (pegvisomant), reveal effects of pegylation and evidence that it binds to a receptor dimer, J. Clin. Endocrinol. Metab. 86 (2001) 1716–1723. [15] Y. Zhang, J. Jiang, J.J. Kopchick, S.J. Frank, Disulfide linkage of growth hormone (GH) receptors (GHR) reflects GH-induced GHR dimerization. Association of JAK2 with the GHR is enhanced by receptor dimerization, J. Biol. Chem. 274 (1999) 33072–33084. [16] J. Jiang, X. Wang, K. He, X. Li, C. Chen, P.P. Sayeski, M.J. Waters, S.J. Frank, A conformationally-sensitive GHR (growth hormone (GH) receptor) antibody: impact on GH signaling and GHR proteolysis, Mol. Endocrinol. 18 (2004) 2981–2996. [17] J. Jiang, Y. Wan, X. Wang, J. Xu, J.M. Harris, P.E. Lobie, S.J. Frank, Inhibitory GH receptor extracellular domain monoclonal antibodies: three-dimensional epitope mapping, Endocrinology 152 (2011) 4777–4788. [18] H. Lan, W. Li, Z. Fu, Y. Yang, T. Wu, Y. Liu, X. Zheng, Differential intracellular signalling properties of the growth hormone receptor induced by the activation of an anti-GHR antibody, Mol. Cell. Endocrinol. 390 (2014) 54–64. [19] S.W. Rowlinson, S.N. Behncken, J.E. Rowland, R.W. Clarkson, C.J. Strasburger, Z. Wu, Activation of chimeric and full-length growth hormone receptors by growth hormone receptor monoclonal antibodies. A specific conformational change may be required for full-length receptor signalling, J. Biol. Chem. 273 (1998) 5307–5314. [20] W. Li, H. Lan, H. Liu, Z. Fu, Y. Yang, W.W. Han, X. Zheng, The activation and differential signalling of the growth hormone receptor induced by pGH or anti-idiotypic monoclonal antibodies in primary rat hepatocytes, Mol. Cell. Endocrinol. 376 (2013) 51–59. [21] H. Lan, W. Li, H. Jiang, Y. Yang, X. Zheng, Intracellular signaling transduction pathways triggered by a well-known anti-GHR monoclonal antibody, Mab263, in vitro and in vivo, Int. J. Mol. Sci. 15 (2014) 20538–20554. [22] V. Beauloye, B. Willems, V. de Coninck, S.J. Frank, M. Edery, J.P. Thissen, Impairment of liver GH receptor signaling by fasting, Endocrinology 143 (2002) 792–800. [23] F. Schaefer, Y. Chen, T. Tsao, P. Nouri, R. Rabkin, Impaired JAK-STAT signal transduction contributes to growth hormone resistance in chronic uremia, J. Clin. Invest. 108 (2001) 467–475. [24] Z. Wu, G. Lima, C.J. Strasburger, Explore the mechanism of growth hormone signaling with growth hormone receptor monoclonal antibodies, Growth Horm. IGF Res. 20 (2010) S32–S33. [25] R.J. Brown, J.J. Adams, R.A. Pelekanos, Y. Wan, W.J. McKinstry, K. Palethorpe, R.M. Seeber, T.A. Monks, K.A. Eidne, M.W. Parker, M.J. Waters, Model for growth hormone receptor activation based on subunit rotation within a receptor dimer, Nat. Struct. Mol. Biol. 12 (2005) 814–821. [26] A.J. Brooks, W. Dai, M.L. O’Mara, D. Abankwa, Y. Chhabra, R.A. Pelekanos, M.J. Waters, Mechanism of activation of protein kinase JAK2 by the growth hormone receptor, Science 344 (2014) 1249783.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Development and characterization of a novel GHR antibody antagonist, GF185 Fengjuan Sun a , Yaping Liu a , Hui Sun b , Baofang Tian c,∗ a b c
Department of Endocrinology, The First People’s Hospital, Jining 272000, Shandong, PR China Central Laboratory, The First People’s Hospital, Jining 272000, Shandong, PR China Traumatology Department, The First People’s Hospital, Jining 272000, Shandong, PR China
a r t i c l e
i n f o
Article history: Received 14 December 2014 Received in revised form 20 May 2015 Accepted 20 May 2015 Available online 4 June 2015 Keywords: Growth hormone Growth hormone receptor Anti-GHR antagonist
a b s t r a c t Here, we describe the development of a panel of monoclonal antibodies targeting the growth hormone receptor (GHR). Of these monoclonal antibodies (Mabs), GF185 was selected for further characterization due to its activities. Competitive receptor-binding assays and Western blotting analyses were used to demonstrate that GF185’s epitopes are localized within subdomain 1 of the growth hormone receptor extracellular domain (GHR-ECD). Subsequently, we evaluated GF185’s antagonistic activities in vivo and in vitro and showed that GF185 was able to neutralize growth hormone (GH) signalling and inhibit GHinduced Ba/F3-GHR proliferation. Our findings suggest that GF185 may serve as an attractive tool for GHR-related research and has a potential future application for the treatment of GH-dependent disease. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Growth hormone (GH) is the major regulator of postnatal somatic growth and metabolism. GH activities are mediated via the GHR, which is widely expressed by GH target cells [1]. Binding of GH to a preformed dimer of GHR at the plasma membrane induces a specific conformational change within the GHR and leads to activation of the janus-activated kinase 2 (JAK2) and the Srchomology 2 (SH2) domains of the GHR monomers. Downstream of GHR lie signalling proteins, such as signal transducer and activator of transcription (STAT) 5, 3 and 1, which are activated by tyrosine phosphorylation and lead to the translocation of homoor heterodimers into the nucleus, where they bind specific target sequences and modulate gene transcription [2]. GHR belongs to the cytokine receptor superfamily, which includes prolactin receptor, erythropoietin receptor, granulocyte colony-stimulating factor receptor, and others [3,4]. Structurally, full-length GHR is a 620-residue, single membrane-spanning protein, which consists of an extracellular domain (246 amino acids), a single-pass transmembrane domain (24 amino acids), and an intracellular domain (350 amino acids). The GHR extracellular domain (ECD, residues 1–246) consists of two subdomains (residues 1–123, referred to as subdomain 1; residues128–238, referred to as subdomain 2) that are linked by a four-residue hinge region. Each
∗ Corresponding author. Tel.: +86 0537 2106208; fax: +86 0537 2106208. E-mail address: [email protected] (B. Tian). http://dx.doi.org/10.1016/j.ijbiomac.2015.05.039 0141-8130/© 2015 Elsevier B.V. All rights reserved.
subdomain is composed of seven strands that are arranged into two antiparallel  sheets [5,6]. Structural and mutagenesis studies indicate that GH binds to two identical GHR-ECDs via two distinct sites on the GH molecule and that GH contacts GHR-ECD mainly via residues in subdomain 1 and in the hinge, although tryptophan 169 in subdomain 2 also contributes to binding [5,6]. In addition, the crystal structure of the GH-(GHR)2 complex suggests that an interaction mediated by subdomain 2 leads to the formation of a 500-A˚ 2 contact interface, which is thought to stabilize the GH (GHR)2 complex [6]. Furthermore, mutagenesis of conserved amino acids within this domain demonstrates that subdomain 2 plays a critical role in GH-induced signalling [7]. It is necessary to develop and prepare GH antagonists in situations of excess GH (e.g., acromegaly), and these may have applications in malignancies and other special research needs [8]. A series of studies on GHR antagonists have been reported over the past few decades. One approach has used a GH analogue. One of the first reports of a GH antagonist was observed in transgenic mice expressing the gene encoding bovine GH with a mutation in the third helix of the region of binding site 2 (bGH-M8), which led to a reduction in circulating IGF-I and a dwarf-like phenotype [9,10]. Subsequently, studies from Chen at al. [11] and Fuh et al. [12] indicated that a single point mutation in GH at position 120 (glycine) in the third helix with any amino acid other than alanine leads to a change in GH from an agonist (growth enhancer) to an antagonist (growth suppressor), such as G120R, G120K, B2036 and pegvisomant [8]; anti-GHR antibodies have also been developed as potential GH antagonists. Mab5, a monoclonal anti-GHR antibody,
F. Sun et al. / International Journal of Biological Macromolecules 79 (2015) 864–870
was initially reported as a GHR antibody antagonist, which recognized GHR on an epitope in the receptor dimerization region of subdomain 2 of the GHR-ECD [13,12,14]. Similarly, another monoclonal anti-GHR antibody (GHRext-Mab) was also shown to bind subdomain 2 of the GHR-ECD [15–17]. Each of these anti-GHR antagonists (Mab5 and GHRext-Mab) were successful antagonist towards GHR but not GH due to their epitope localization in subdomain 2 of GHR-ECD, whereas GH’s epitopes have been shown to be mainly localized in subdomain 1 of GHR-ECD. In the present study, we screened and characterized a novel neutralizing monoclonal anti-GHR antibody (designated GF185). We found that GF185 effectively antagonized GHR signalling in vitro and in vivo. In addition, we showed that GF185 shares common GHR-binding epitopes with GH and inhibited GH binding to GHR. Collectively, the current findings suggest that GF185 represents a novel GHR/GH antagonist that serves as an attractive tool for GHR-related research and may have potential application for the treatment of GH-dependent disease in the future. 2. Materials and methods 2.1. Antibodies, reagents and cell line Anti-phospho-JAK2 (Tyr1007/1008), anti-JAK2, anti-phosphoSTAT5 (Tyr 694), anti-STAT5, anti-phospho-STAT3 (Tyr705), anti-STAT3, anti-phospho-STAT1 (Tyr 701), anti-STAT1, antiphospho-ERK1/2 (Thr 202/Tyr 204) and anti-ERK1/2 antibodies were purchased from Cell Signaling Technology (USA). The antiphosphotyrosine antibody (clone 4G10) was obtained from Upstate Biotechnology. HRP-conjugated goat anti-rabbit and anti-mouse antibodies and recombinant hGH were purchased from Sigma (USA). [3 H] thymidine was purchased from Amersham Biosciences. The ImmunoPure Fab Preparation kit, Cell Lysis Buffer, IP kit, enhanced chemiluminescence (ECL) and BCA kit were purchased from Pierce (USA). The extracellular domain of rat GHR (GHBP) was obtained from Sino Biological Inc (Jinang, China). GHRECD1–123 and GHR-ECD124–246 were provided by Top-Peptide Co., Ltd. (China). The fluorescein isothiocyanate (FITC) conjugation kit was obtained from Sigma–Aldrich. The FITC Conjugation Kit was used to label recombinant GH according to the manufacturer’s directions. Conjugates with F/P (FITC/protein) ratios of 1 exhibited the greatest sensitivity and reproducibility and were chosen for further use in this study. Unless otherwise stated, all reagents were from Sigma–Aldrich (USA). CHO and Ba/F3 cells were stably transfected with a rat GH receptor cDNA that corresponded to amino acids 1–638 and was provided by Xin Liu (HuaChen Medical and Biological Co., Ltd.). CHO and Ba/F3 cells stably expressing full-length rGHR were cultured as previously described [18]. In addition, rat hepatocytes that endogenously express GHR were isolated and cultured as described previously [20]. 2.2. Production of anti-GHR monoclonal antibody Six 8-week-old female BALB/c mice were immunized intraperitoneally with 0.2 mg of GHBP in Freund’s complete adjuvant and received similar booster injections of GHBP in incomplete Freund’s adjuvant at 14 days intervals. The animals were immunized four times. Four mice were chosen for hybridoma preparation. Three days following the final injection, splenocytes were harvested from four GHBP-immunized BALB/c mice and were fused with sp2/0 myeloma cells at a 4:1 ratio using 50% (w/v) polyethylene glycol (PEG) 1500. Post-fusion, the cells were distributed in 96-well cell culture plates at 1 × 105 cells/well in HAT selection medium. The cultured supernatants of hybridomas selected
865
on hypoxanthine–aminopterin–thymidine (HAT) medium were screened for the presence of antibodies against GHBP using an ELISA. Antibody-secreting hybridomas were subcloned four times by limiting dilution. 2.3. Immunoprecipitation and Western immunoblotting To determine whether GF185 specifically binds to GHR, immunoprecipitation experiments were performed according to the method of Lan et al. [18]. Briefly, CHO-GHR cells were collected and lysed with cell lysis buffer according to the manufacturer’s instructions. The immunoprecipitation was conducted by treating cell lysates with GF185 (10 g), B-10 (10 g) or control (10 g) antibodies. The immunoglobulin-bound proteins were obtained using an IP Kit according to the manufacturer’s instructions. Immunoprecipitated proteins were subjected to SDS-PAGE and transferred to PVDF membranes, which were then blocked using 5% non-fat milk and incubated with GF185, B-10 or a control antibody overnight at 4 ◦ C. The membranes were then washed three times with TBST and incubated with HRP-conjugated goat anti-mouse IgG antibody in blocking buffer for 1 h at 37 ◦ C. Following the incubation, the membranes were washed three times and the signals were detected using the ECL detection system. 2.4. Preliminary screen of anti-GHR antibodies by FACS analysis Flow cytometric analysis was used to screen for potential antiGHR antibodies that bound to the GHR that was exogenously expressed in CHO cells (CHO-GHR). Before experimental incubations, the media of the CHO-GHR cells were replaced with the fresh serum-free containing 1% BSA and incubated for 12 h at 37 ◦ C. CHOGHR cells were then detached from the tissue culture flasks, washed twice with sterile PBS, and resuspended in either negative control media or an anti-GHBP hybridoma supernatant for 1 h at 4 ◦ C in separate 1.5-ml microcentrifuge tubes (2 × 104 cells/tube). The cells were then washed and incubated with an FITC-conjugated secondary antibody (1:200 dilution) for 60 min in the dark at 4 ◦ C. Following the incubation, the cells were washed twice and resuspended in 0.5 ml of FACS buffer (1% BSA and 0.01% sodium azide in PBS, pH 7.4). The samples were analyzed using a FACS Calibur flow cytometry device (Becton Dickenson). The data were analyzed using Cell Quest software. 2.5. Competitive receptor-binding assay To determine whether GF185 inhibited GH binding to GHBP, a competitive ELISA was performed. First, 96-well microtiter plates coated with 1 g/ml GHBP in a volume of 100 l were incubated at 4 ◦ C for 12 h and then washed and blocked with 2% BSA for 1 h at 37 ◦ C. The wells were then washed three times with PBS containing 1% BSA and 0.05% Tween 20, and a constant amount of GH was added to each well with increasing concentrations of either GF185-F(ab )2 (as the competitor) or F(ab )2 of control antibody (negative control) for 0.5 h. After incubation for 1 h at 37 ◦ C, all of the wells were washed three times with PBST, and then anti-hGH was added. After incubation for 1 h at 37 ◦ C, a secondary antibody (Fc-specific) (1:2000 dilution) was added and incubated for another 1 h at 37 ◦ C. After three final washes, the TMB substrate was applied to develop the colorimetric reaction, which was then terminated by the addition of 3 M H2 SO4 to each well (50 l/well). The optical density at 450 nm was measured using a Microplate Reader (Multiskan FC, Thermo Fisher Scientific). Binding of GH to GHBP without any competitor was defined as 100% binding. To further determine whether GF185 competed with GH for specific binding to full-length GHR, a competitive receptor-binding assay was performed and analyzed by flow cytometry. Prior to
866
F. Sun et al. / International Journal of Biological Macromolecules 79 (2015) 864–870
experiments, CHO-GHR cells were incubated in serum-free F12 media containing 1% BSA for 12 h. The cells were then detached, collected by centrifugation, washed three times with PBS, resuspended in binding buffer (Williams E medium containing 1 mg/ml BSA), pipetted into FACS tubes (1 × 106 cells/tube) and incubated with FITC-GH together with increasing concentrations of unlabelled GH, GF185, or a control antibody for 1 h in the dark at 4 ◦ C. After the incubation, the cells were washed three times and resuspended in 0.5 ml FACS Buffer and analyzed using a FACS Calibur Flow Cytometer (Becton Dickenson). The data were analyzed using Cell Quest software. 2.6. Evaluation of GF185’s antagonistic effects in vitro The CHO-GHR or rat hepatocytes were seeded onto 6-well plates and cultured as described as above. Prior to the experiments, CHO-GHR cells or rat hepatocytes were serum-starved for 10 h. A constant amount of GH (50 ng) was incubated together with increasing concentrations of GF185 and then added to the plates for 20 min (CHO-GHR) or 30 min (rat hepatocytes) at 37 ◦ C. Unless noted, all of the stimulations were carried out at 37 ◦ C. At the end of the treatment, the medium was discarded and the cells were washed three times with ice-cold PBS. The cells were solubilized in lysis buffer on ice for 30 min, and cell debris was removed by centrifugation under refrigeration at 12,000 × g for 30 min. The resulting supernatants were harvested and subjected to ultrafiltration. Samples were lysed directly in SDS-PAGE sample buffer and boiled for 5 min, and the protein concentration of the lysate was determined with the BCA protein assay according to the manufacturer’s instruction. The samples (50 g of protein per lane) were subjected to SDS-PAGE (10% gel) and then transferred to a PVDF membrane. The membranes were washed and blocked with 5% non-fat milk for 1 h at 37 ◦ C. After three washes with TBST, the membranes were incubated with phospho-STAT5, phospho-STAT3, phospho-STAT1 or phospho-ERK1/2 antibodies according to the manufacturer’s protocols. After three washes with TBST, the membranes were incubated with an HRP-conjugated goat anti-rabbit IgG antibody (secondary antibody) for 1 h at 37 ◦ C. After three final washes, the signals were detected by ECL detection system. After ECL detection, the membranes were stripped of primary and secondary antibodies according to the manufacturer’s instructions, and then blocked and reprobed for JAK2, STAT1/3/5 or ERK1/2. Experiments were carried out in triplicate. 2.7. Evaluation of GF185’s antagonistic effects in vitro The [3 H] thymidine incorporation assay was performed according to the method of Lan et al. [18]. Briefly, Ba/F3-GHR cells were grown in RPMI1640 medium containing 10% foetal calf serum (FCS) for 16 h. The cells were then suspended, counted and adjusted to 1 × 106 cells/ml. After washing two times with serum-free RPMI 1640, 50 l of the cell suspension was seeded into the wells of 96-well tissue culture plates. A constant amount of GH was incubated together with increasing concentrations of GF185 or a control antibody and then added to selected wells in a final volume of 0.2 ml for16 h at 37 ◦ C. [3 H] thymidine was then added to each well (1 Ci/well), and after 6 h at 37 ◦ C, [3 H] thymidine incorporation was determined by liquid scintillation counting. 2.8. Preliminary evaluation of GF185’s antagonistic effects in vivo The above experiments were carried out in vitro. As described in this section, the antagonistic activity of GF185 was also determined in vivo according to the method of Lan et al. [21]. It has been reported that the liver from intact, but not hypophysectomized, rats can be used as a model to investigate GH signalling [21–23]. Male
Sprague Dawley rats (∼200 g, 6 wk old) were housed at three individuals per cage in the conventional animal room with free access to standard rodent chow and water. The rats were acclimated for 3 days prior to the start of the experiments. The rats were anesthetized with ether. The abdominal cavity was opened, the portal vein was exposed, and GH (0.5 mg), GF185 (10 mg), a control antibody (10 mg), the mixture of GH (0.5 mg) plus GF185 (10 mg) or the mixture of GH (0.5 mg) plus a control antibody (10 mg) was injected in a volume of 1.5 ml of physiological saline. After 20 min, the livers were quickly removed from rats killed by decapitation and the liver samples were homogenized in ice-cold lysis buffer and incubated on a shaker platform for 30 min at 4 ◦ C. The samples were then centrifuged at 12,000 × g, and the supernatant was collected. The samples were then lysed and quantitated. The samples (50 g of protein per lane) were subjected to SDS-PAGE (10% gel) and then transferred to a PVDF membrane, followed by Western blot analysis as described above. 2.9. Statistical analysis The data are presented as the mean values ± standard error (S.E.). 3. Results 3.1. Generation and characterization of GF185 Immunized mice were shown to produce high-titre sera against GHBP, as determined by ELISA. Spleen cells from the immunized mice were fused with the murine myeloma cell line SP2/0. ELISAs of each well’s culture supernatant revealed 55 positive antibodysecreting clones (data not shown). Subsequently, flow cytometric analysis was performed to screen for anti-GHR monoclonal antibodies that were capable of binding GHR on CHO-GHR cells and inhibiting GH binding to GHR expressed in CHO-GHR cells. Eight of the 55 clones were shown to exhibit a strong positive signal. Of these clones, the Mab named GF185 was selected for further characterization based on its biological properties described below (Fig. 1A). GF185 was classified as an IgG1 isotype using a commercial mouse Mab isotyping kit. To further confirm the specificity of GF185 binding to GHR, immunoprecipitation experiment using GF185 highlighted a 130 kD protein that cross-reacted with B-10, a commercial antiGHR monoclonal antibody that was prepared by Santa Cruz Biotechnology (Santa Cruz, California, USA), which specifically recognizes Rghr and is suitable for IP and WB experiments. Taken together, these data demonstrate that GF185 specifically recognizes the GHR (Fig. 1B). 3.2. Characterization of GF185 binding epitopes on GHR To characterize the epitopes on GHR that mediate GF185 binding, competitive ELISA and competitive receptor-binding assays were performed. As shown in Fig. 2A, GF185 was shown to inhibit GH binding to GHBP in a dose-dependent manner. Because GHBP is composed of the extracellular domain (ECD) of GHR, a competitive receptor-binding assay was carried out at the cellular level (CHO-GHR) to assess full-length GHR binding. GF185 was shown to compete with GH for binding to GHR, while a control antibody had no effect (Fig. 2B), suggesting that GF185 binds at residues that over-lap with the GH-binding site within subdomain 1 of GHR-ECD. To directly confirm that GF185’s epitopes are localized within subdomain 1 of GHR ECD, Western-blot analyses were used to show that GF185 reacted with GHR1–123 but not with GHR124–246.
F. Sun et al. / International Journal of Biological Macromolecules 79 (2015) 864–870
867
Fig. 1. Screening and identification of anti-GHBP monoclonal antibodies that can bind to subdomain 1 of GHR-ECD. (A) Flow cytometric screening of potential anti-GHBP monoclonal antibodies that can bind to CHO-GHR. CHO-GHR were cultured and pretreated as described in Section 2. The cells were then incubated with GF185supernatant or control media for 1 h at 4 ◦ C. After the incubation and washing, the cells were incubated with FITC-conjugated secondary antibody for 1 h in the dark at 4 ◦ C. After three washes, the cells were resuspended in 0.5 ml of FACS buffer, and the samples were analyzed using a FACS Calibur flow cytometry device (Becton Dickenson). The data were analyzed using Cell Quest software. (B). GF185 was determined to interact with full-length GHR expressed on CHO-GHR cells by immunoprecipitation experiments. CHO-GHR were cultured and pretreated as described in Section 2. Immunoprecipitation experiments were performed by treating cell lysates with GF185 (10 g), B-10 (10 g) or control antibody (10 g). The immunoglobulin-bound proteins were obtained using an IP kit according to the manufacturer’s instructions. Immunoprecipitated proteins were subject to SDS-PAGE and transferred to PVDF membranes. Proteins transferred to PVDF membranes were detected with GF185, B-10 or control antibodies. Signals were detected using the ECL detection system.
Fig. 2. Characterization of the GF185-binding epitopes on GHR-ECD. (A) GF185 competes with GH for binding to GHBP. First, 96-well microtiter plates were coated with GHBP and blocked with 2% BSA. Before the experiments, a constant amount of GH was incubated together with increasing concentrations of GF185-F(ab )2 (as the competitor) or control antibody (as the negative control) for 0.5 h, and the mixture was then added to the plates. After incubation for 1 h, anti-hGH was added to the plates, and incubated for 1 h at 37 ◦ C. Secondary antibody was then added and incubated for another 1 h at 37 ◦ C. After washing, the TMB substrate was applied to develop the colour in a colourimetric reaction. (B) GF185 inhibits GH binding to GHR expressed on CHO-GHR. Cells were incubated with FITC-GH (15 nM) or increasing concentrations of unlabelled GH, GF185, or control antibody for 1 h in the dark at 4 ◦ C. After the incubation, the cells were washed three times and resuspended in 0.5 ml of FACS Buffer and analyzed using a FACS Calibur Flow Cytometer. The data were analyzed using Cell Quest software. (C) GF185 reacts with subdomain 1 of GHR-ECD but not with subdomain 2. The structure GHR-ECD is diagrammed, and subdomain 1, hinge region and subdomain 2 are indicated. Recombinant proteins (GHR-ECD 1–123 and 124–246) were subjected to SDS-PAGE and transferred to PVDF membranes. The proteins transferred to the PVDF membranes were detected with GF185. Signals were detected using the ECL detection system. After detection, the membranes were stripped and re-probed with polyclonal anti-GHBP antibody to verify equal protein loading in each lane (In the pre-experiment, it was demonstrated that the polyclonal anti-GHBP could recognize GHR-ECD1–123 and GHR-ECD124–246 ). The results are representative of at least three separate experiments.
3.3. Inhibition of GHR signalling by GF185 in CHO-GHR cells and rat hepatocytes To determine whether GF185 possesses agonistic activity, CHOGHR cells were treated with GH, Mab263 (an anti-rGHR monoclonal antibody that can activate GHR-mediated signalling [21]), GF185 or
a control antibody. As determined by Western-blot analysis, GH and Mab263 activatedJAK2 (Fig. 3), in contrast, GF185 and the control antibody were unable to induce tyrosine phosphorylation of JAK2, indicating that GF185 does not possess agonistic activity. We then tested whether GF185 inhibited GH signalling in CHO-GHR cells. CHO-GHR cells were treated as described as
868
F. Sun et al. / International Journal of Biological Macromolecules 79 (2015) 864–870
Fig. 3. GF185 does not display agonistic activity. CHO-GHR cells were cultured and pretreated as described in Section 2. The cells were then stimulated with the indicated concentrations of GF185control antibody or GH for 20 min at 37 ◦ C. The cell extracts were harvested, and samples (50 g of protein per lane) were subjected to SDS-PAGE (10% gel), transferred to a PVDF membrane and immunoblotted with the indicated antibodies. Detection was performed using the ECL detection system. After ECL detection, the membranes were stripped of primary and secondary antibodies according to the manufacturer’s instructions. The stripped membranes were blocked and reprobed for JAK2 to verify equal protein loading in each lane. The figures represent at least three independent experiments.
in the materials and methods, and Western-blot analyses was used to show that the antagonistic effect of GF185 was initially observed at 0.05 g/ml and that the phosphorylation levels of STAT5/3/1 and ERK1/2 gradually declined with increasing concentrations of GF185. Maximal antagonist effects of GF185 were reached at a concentration of approximately 5 g/ml when GH signalling was completely inhibited. These results demonstrate that GF185 efficiently neutralize styrosine phosphorylation of GHinduced intracellular signalling molecules in a dose-dependent manner. The above studies were conducted in a CHO cell line that was stably transfected with full-length GHR. To confirm these results, rat hepatocytes that endogenously express abundant GHRs were used as a model to further assess the antagonistic effect of GF185. Rat hepatocytes were treated as described in Section 2 and Western-blot analysis was used to show that antagonistic effects of GF185 were easily observed at a concentration of 0.05 g/ml. Increasing concentrations of GF185 were shown to exhibit stronger antagonistic effects on GH signalling, with a complete blockage of GH-mediated intracellular signalling observed at 5 g/ml of GF185. In contrast, a control antibody showed no effect. These data indicate that GF185 efficiently inhibited tyrosine phosphorylation of GH intracellular signalling in a dose-dependent manner in rat hepatocytes (somatic cells). 3.4. Inhibition of GH-induced proliferation of BaF3-GHR638 cells by GF185 To further characterize GF185’s effects on GH action, we analyzed GF185’s impacts on GH’s ability to promote cell proliferation. Ba/F3-GHR cells were used and treated as described as in Section 2. As indicated in Fig. 6, GF185, but not a control antibody, was shown to dramatically inhibit GH-induced cell proliferation of Ba/F3-GHR in a dose-dependent manner. These results indicate that the anti-GHR monoclonal antibody (GF185) specifically inhibits GH-dependent Ba/F3-GHR proliferation. 3.5. Inhibition of GHR signalling by GF185 in vivo In the experiments mentioned above, we evaluated GF185’s antagonistic activity in vitro. In this section, to determine the impact of GF185 on GH signalling in vivo, rats were injected with GH and intracellular signalling pathways were assessed by Western blot analyses. Injection with GH alone was sufficient to promote a strong phosphorylation of JAK2, and STAT1/3/5. However, rats injected with a mixture of GH and GF185 (but not the mixture of GH and control antibody) experienced a complete block in the phosphorylation of intracellular signalling protein molecules (such as JAK2 and STAT1/3/5). In addition, GF185 (alone) or control antibody (alone) showed no effect. These results indicate that GF185 inhibits GH signalling in vivo.
4. Discussion GH is an asymmetric molecule whose downstream function is mediated via the GHR. After GH binding, downstream signalling events are triggered by specific conformational changes within the pre-dimerized GHR. Structurally, the full-length GHR consists of an extracellular domain (ECD), a single-pass transmembrane domain (TMD), and an intracellular domain (ICD). The GHR extracellular domain (ECD, residues 1–246) consists of two subdomains (residues 1–123, referred to as subdomain 1; residues 128–238, referred to as subdomain 2) [5,6]. Structural and mutagenesis studies indicate that GH’s epitopes are mainly distributed in subdomain 1 of GHR ECD [5,6]. The use of anti-GHR antibodies as GHR antagonists has been extensively reported over the past few decades. To the best of our knowledge, Mab5 represents the first commercially available GHR monoclonal antibody antagonist whose epitopes are localized in subdomain2 of GHR ECD [13,12,14]. Recently, another well-characterized GHR antibody antagonist (anti-GHRext-Mab) was reported [15–17]. A common feature of Mab5 and anti-GHR ext-Mab is their lack of effect on GH binding to GHR, which is due to epitope distribution in subdomain 2 of GHR-ECD. It has been suggested that the action mechanism of Mab5 and anti-GHRextMab is mediated by the inhibition of “active” GHR conformational change(s) following GH binding. In the present study, we have characterized a novel GHR antibody antagonist (GF185), which inhibited GH binding to GHR (Fig. 2) and neutralized GHR signalling (Figs. 4 and 5). To the best of our knowledge, this is the first report of an anti-GHR antibody that mediated GHR antagonist activity via epitopes on subdomain1 of GHR-ECD (Fig. 7). In a previous study, Lan et al. [18] reported that the anti-GHR antibody CG-172 showed agonistic activity. Similar results have been reported by the Waters’ group, who showed that only one of the 14 anti-GHR Mabs, which were directed to the GH binding site and competed with GH for binding GHR, was able to activate the full-length GHR [19]. In the current work, we have screened and characterized a panel of anti-GHR antibodies, among which we found that GF185 was suitable to serve as a GHR antagonist. Importantly, we show that two additional anti-GHR antibodies (GF153 and GF176) that were directed to the GH binding site on GHR also showed antagonistic activity (data not shown), which suggests that the preparation of GHR antagonists may be more easily produced than agonists. Similar observations were reported by Wu et al. [24], who showed that of the 21 Mabs developed against GHR-ECD, only one Mab (termed as GHRA1) exhibited agonistic property; however, the Mabs harbouring the highest degree of epitopes that overlapped with the GH binding site on GHR did not show agonistic activity. Taken together, these results suggest that antibodies designed with epitopes on the GH binding site may represent the natural antagonist for GH/GHR. Why do anti-GHR antibodies (such as GF185) containing epitopes on the GH binding site of GHR show antagonistic but not agonistic properties? It is has been accepted that special
F. Sun et al. / International Journal of Biological Macromolecules 79 (2015) 864–870
Fig. 4. Inhibition of GH-induced signalling by GF185 in CHO-GHR cells. CHO-GHR cells were cultured and pretreated as described in Section 2. A constant amount of GH (50 ng) was incubated together with increasing concentrations of GF185 (0.05–5 g), and the mixtures were then added to CHO-GHR cells and incubated for 20 min. Cell extracts were harvested, and the samples (50 g of protein per lane) were subjected to SDS-PAGE (10% gel), transferred to a PVDF membrane and immunoblotted with the indicated antibodies. Detection was performed using the ECL detection system. After ECL detection, the membranes were stripped of primary and secondary antibodies according to the manufacturer’s instructions. The stripped membranes were blocked and reprobed for JAK2, STAT1/3/5 or ERK1/2 to verify equal protein loading in each lane. The figures represent at least three independent experiments.
Fig. 5. Inhibition of GH-induced signalling by GF185 in rat hepatocytes. Rat hepatocytes were cultured and pre-treated as described in Section 2. A constant amount of GH (50 g) was incubated together with increasing concentrations of GF185 (0.05–5 g), and the mixtures were then added to rat hepatocytes and incubated for 20 min. The cells were solubilized in lysis buffer on ice for 30 min. Cell extracts were then harvested, and the samples (50 g of protein per lane) were subjected to SDS-PAGE (10% gel), transferred to a PVDF membrane and immunoblotted with the indicated antibodies. Detection was performed using the ECL detection system. After ECL detection, the membranes were stripped of primary and secondary antibodies according to the manufacturer’s instructions. The stripped membranes were blocked and reprobed for JAK2, STAT1/3/5 or ERK1/2 to verify equal protein loading in each lane. The figures represent at least three independent experiments.
conformation change(s) are required for GHR activation, although the nature of this conformational change is still not fully understood. The Waters’ group has recently proposed a new model suggesting that the GH receptor exists predominantly as a dimer held together by its transmembrane helices and that GH binding to
869
Fig. 6. Inhibition of GH-induced proliferation of Ba/F3-GHR by GF185. Ba/F3-GHR cells were cultured and pre-treated as described in Section 2. A constant amount of GH was pre-incubated together with increasing concentrations of GF185 or control antibody for 30 min, and then they were added to selected wells in a final volume of 0.2 ml for 16 h at 37 ◦ C. [3 H] thymidine was then added to each well (1 Ci/well), and after 6 h at 37 ◦ C, [3 H] thymidine incorporation was then determined by liquid scintillation counting. The results are representative of at least three separate experiments.
Fig. 7. Inhibition of GH-induced signalling by GF185 in vivo. SD rats were pretreated as described in Section 2. The rats were then injected with GH alone (0.5 mg), GF185 alone (10 mg), control antibody alone (10 mg), the mixture of GH (10 mg) plus GF185 (10 mg), or the mixture of GH (0.5 mg) plus control antibody (10 mg). Twenty minutes after the injection, the livers were quickly removed from rats killed by decapitation, and the liver samples were homogenized in ice-cold lysis buffer and incubated on a shaker platform for 30 min at 4 ◦ C. The samples were then centrifuged at 12,000 × g, and the supernatant was collected. The samples were lysed directly in SDS-PAGE sample buffer, boiled for 5 min and quantitated with the BCA Protein Assay according to the manufacturer’s instructions. The samples (50 g of protein per lane) were subjected to SDS-PAGE (10% gel) and then transferred to a PVDF membrane. The membranes were probed with the indicated antibodies. Signals were detected using the ECL detection system. The results are representative of at least three separate experiments.
GHR induces a rotation of subunits that subsequently converts the transmembrane helices into a left-hand crossover state and causes a repositioning of the ICDs to activate JAK2 [25,26]. Based on the above activation mechanism of GHR, we speculated that the degree of subunit rotation induced by anti-GHR antibodies (such as GF185) with the epitopes on the GH binding site of GHR is not suitable for GHR activation and therefore is unable to induce an “active” conformation change(s) of GHR. Of course, this represents only one possible explanation, and further research is required to explore and elucidate answers to this question. Thus, we have developed and characterized a novel GHR antibody antagonist (GF185) that not only acts as a full competitor for GH binding but also inhibits GHR signalling. These findings have
870
F. Sun et al. / International Journal of Biological Macromolecules 79 (2015) 864–870
future implications for the use of GF185 as a potential therapeutic option in GH-dependent diseases. Acknowledgement We are grateful to Li Qiang for excellent technical assistance. References [1] N. Møller, O.L.J. Jens, Effects of growth hormone on glucose, lipid, and protein metabolism in human subjects, Endocr. Rev. 30 (2009) 152–177. [2] A.J. Brooks, M.J. Waters, The growth hormone receptor: mechanism of activation and clinical implications, Nat. Rev. Endocrinol. 6 (2010) 515–525. [3] S. Moutoussamy, P.A. Kelly, J. Finidori, Growth hormone receptor and cytokine receptor family signalling, Eur. J. Biochem. 255 (1998) 1–11. [4] X. Wang, P. Lupardus, S.L. Laporte, K.C. Garcia, Structural biology of shared cytokine receptors, Annu. Rev. Immunol. 27 (2009) 29–60. [5] B.C. Cunningham, M. Ultsch, A.M. De Vos, M.G. Mulkerrin, K.R. Clauser, J.A. Wells, Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule, Science 254 (1991) 821–825. [6] A.M. de Vos, M. Ultsch, A.A. Kossiakoff, Human growth hormone and extracellular domain of its receptor: crystal structure of the complex, Science 255 (1992) 306–312. [7] J. Gent, M. van den Eijnden, P. van Kerkhof, G.J. Strous, Dimerization and signal transduction of the growth hormone receptor, Mol. Endocrinol. 17 (2003) 967–975. [8] J.J. Kopchick, C. Parkinson, E.C. Stevens, P.J. Trainer, Growth hormone receptor antagonists: discovery, development, and use in patients with acromegaly, Endocr. Rev. 23 (2002) 623–646. [9] W.Y. Chen, D.C. Wight, T.E. Wagner, J.J. Kopchick, Expression of a mutated bovine growth hormone gene suppresses growth of transgenic mice, Proc. Natl. Acad. Sci. U. S. A. 87 (1990) 5061–5065. [10] W.Y. Chen, M.E. White, T.E. Wagner, J.J. Kopchick, Functional antagonism between endogenous mouse growth hormone (GH) and a GH analog results in dwarf transgenic mice, Endocrinology 129 (1991) 1402–1408. [11] W.Y. Chen, D.C. Wight, B.V. Mehta, T.E. Wagner, J.J. Kopchick, Glycine 119 of bovine growth hormone is critical for growth-promoting activity, Mol. Endocrinol. 5 (1991) 1845–1852. [12] G. Fuh, B.C. Cunningham, R. Fukunaga, S. Nagata, D.V. Goeddel, J.A. Wells, Rational design of potent antagonists to the human growth hormone receptor, Science 256 (1992) 1677–1680. [13] R. Barnard, P.G. Bundesen, D.B. Rylatt, M.J. Waters, Monoclonal antibodies to the rabbit liver growth hormone receptor: production and characterization, Endocrinology 115 (1984) 1805–1813.
[14] R.J. Ross, K.C. Leung, M. Maamra, W. Bennett, N. Doyle, M.J. Waters, K.K. Ho, Binding and functional studies with the growth hormone receptor antagonist, B2036-PEG (pegvisomant), reveal effects of pegylation and evidence that it binds to a receptor dimer, J. Clin. Endocrinol. Metab. 86 (2001) 1716–1723. [15] Y. Zhang, J. Jiang, J.J. Kopchick, S.J. Frank, Disulfide linkage of growth hormone (GH) receptors (GHR) reflects GH-induced GHR dimerization. Association of JAK2 with the GHR is enhanced by receptor dimerization, J. Biol. Chem. 274 (1999) 33072–33084. [16] J. Jiang, X. Wang, K. He, X. Li, C. Chen, P.P. Sayeski, M.J. Waters, S.J. Frank, A conformationally-sensitive GHR (growth hormone (GH) receptor) antibody: impact on GH signaling and GHR proteolysis, Mol. Endocrinol. 18 (2004) 2981–2996. [17] J. Jiang, Y. Wan, X. Wang, J. Xu, J.M. Harris, P.E. Lobie, S.J. Frank, Inhibitory GH receptor extracellular domain monoclonal antibodies: three-dimensional epitope mapping, Endocrinology 152 (2011) 4777–4788. [18] H. Lan, W. Li, Z. Fu, Y. Yang, T. Wu, Y. Liu, X. Zheng, Differential intracellular signalling properties of the growth hormone receptor induced by the activation of an anti-GHR antibody, Mol. Cell. Endocrinol. 390 (2014) 54–64. [19] S.W. Rowlinson, S.N. Behncken, J.E. Rowland, R.W. Clarkson, C.J. Strasburger, Z. Wu, Activation of chimeric and full-length growth hormone receptors by growth hormone receptor monoclonal antibodies. A specific conformational change may be required for full-length receptor signalling, J. Biol. Chem. 273 (1998) 5307–5314. [20] W. Li, H. Lan, H. Liu, Z. Fu, Y. Yang, W.W. Han, X. Zheng, The activation and differential signalling of the growth hormone receptor induced by pGH or anti-idiotypic monoclonal antibodies in primary rat hepatocytes, Mol. Cell. Endocrinol. 376 (2013) 51–59. [21] H. Lan, W. Li, H. Jiang, Y. Yang, X. Zheng, Intracellular signaling transduction pathways triggered by a well-known anti-GHR monoclonal antibody, Mab263, in vitro and in vivo, Int. J. Mol. Sci. 15 (2014) 20538–20554. [22] V. Beauloye, B. Willems, V. de Coninck, S.J. Frank, M. Edery, J.P. Thissen, Impairment of liver GH receptor signaling by fasting, Endocrinology 143 (2002) 792–800. [23] F. Schaefer, Y. Chen, T. Tsao, P. Nouri, R. Rabkin, Impaired JAK-STAT signal transduction contributes to growth hormone resistance in chronic uremia, J. Clin. Invest. 108 (2001) 467–475. [24] Z. Wu, G. Lima, C.J. Strasburger, Explore the mechanism of growth hormone signaling with growth hormone receptor monoclonal antibodies, Growth Horm. IGF Res. 20 (2010) S32–S33. [25] R.J. Brown, J.J. Adams, R.A. Pelekanos, Y. Wan, W.J. McKinstry, K. Palethorpe, R.M. Seeber, T.A. Monks, K.A. Eidne, M.W. Parker, M.J. Waters, Model for growth hormone receptor activation based on subunit rotation within a receptor dimer, Nat. Struct. Mol. Biol. 12 (2005) 814–821. [26] A.J. Brooks, W. Dai, M.L. O’Mara, D. Abankwa, Y. Chhabra, R.A. Pelekanos, M.J. Waters, Mechanism of activation of protein kinase JAK2 by the growth hormone receptor, Science 344 (2014) 1249783.
