Studies with a Growth Hormone Antagonist and Dual-fluorescent Confocal Microscopy Demonstrate that the Full-length Human Growth Hormone Receptor, but Not the Truncated Isoform, Is Very Rapidly Internalized Independent of Jak2-Stat5 Signaling*

Mabrouka Maamra, Joelle FinidoriDagger , Sigward Von Laue, Sylvie Simon, Sue Justice, Jonathan Webster, Steven Dower§, and Richard Ross

From the Divisions of Clinical Sciences and § Molecular and Genetic Medicine, Sheffield University, Sheffield S5 7AU, United Kingdom and Dagger  INSERM Unite 344, Endocrinologie Moleculaire, Faculte de Medecine Necker, 75730 Paris Cedex 15, France

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have investigated trafficking of two negative regulators of growth hormone receptor (GHR) signaling: a human, truncated receptor, GHR1-279, and a GH antagonist, B2036. Fluorescent-labeled growth hormone (GH) was rapidly internalized by the full-length GHR, with >80% of the hormone internalized within 5 min of exposure to GH. In contrast, <5% of labeled GH was internalized by cells expressing truncated GHR1-279. Using another truncated receptor, GHR1-317 fused to enhanced green fluorescent protein (EGFP), we have exploited fluorescence energy transfer to monitor the trafficking of ligand-receptor complexes. The data confirmed that internalization of this truncated receptor is very inefficient. It was possible to visualize the truncated GHR1-317-EGFP packaged in the endoplasmic reticulum, its rapid movement in membrane bound vesicles to the Golgi apparatus, and subsequent transport to the cell membrane. The GH antagonist, B2036, blocked Jak2-Stat5-mediated GHR signaling but was internalized with a similar time course to native GH. The results: 1) demonstrate the rapid internalization of GH when studied under physiological conditions; 2) confirm the hypothesis that internalization of cytoplasmic domain truncated human GHRs is very inefficient, which explains their dominant negative action; and 3) show that the antagonist action of B2036 is independent of receptor internalization.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Growth hormone (GH)1 has diverse biological actions, including promoting cellular growth and differentiation, that are mediated by binding to a specific, high affinity cell surface receptor (GHR). The GHR is a member of the type I cytokine family of receptors, which possess in common a single transmembrane domain and lack intrinsic kinase activity (1). Binding of a single molecule of GH results in receptor dimerization and signaling through a phosphorylation cascade that includes the Jak-Stat pathway. Activation of the Jak-Stat pathway is dependent on an intact cytoplasmic domain of the receptor forming multisubunit complexes with associated tyrosine kinases. Recently, we (2) and others (3) identified a truncated form of the receptor, GHR1-279, in normal human liver and certain human cell lines. This truncated receptor lacks 97% of the cytoplasmic domain of the receptor and has a dominant negative action on Jak-Stat signaling (2). In human tissues the truncated receptor is expressed at a low level compared with the full-length receptor (2, 4). However, in cell transfection experiments, the truncated receptor always demonstrates a greater level of receptor expression at the cell surface compared with the full-length receptor (2). It has been hypothesized that the truncated receptor lacks essential internalization signals in the cytoplasmic domain and therefore remains at the cell surface available to heterodimerize with the full-length receptor and inhibit signaling (2, 5). In addition, the truncated receptor generates large amounts of GH-binding protein (GHBP) (2, 3). Confirmation that these truncated receptors have a dominant negative action on GHR signaling comes from patients who are heterozygous for a mutation that encodes a very similar truncated receptor, GHR1-277 (6, 7). These patients have high levels of GHBP and GH insensitivity, presumably because of increased levels of truncated receptor at the cell surface, which fails to internalize and heterodimerizes with the full-length receptor to inhibit signaling (6).

The GHR is internalized via clathrin-coated pits (8, 9). This internalization is dependent upon an intact ubiquitination system, and GHR ubiquitination is dependent upon an intact endocytic pathway (9). It has been suggested that a small fraction of GHR is constitutively ubiquitinated and internalized but that GH-induced dimerization results in an increase in ubiquitination and internalization (9). The Phe327 residue within the cytosolic tail of the GHR is involved in both GHR ubiquitination and ligand-induced receptor endocytosis (9, 10). In addition, it has been reported that there is an ubiquitin-independent internalization signal, based on a di-leucine repeat (Leu347,348), which is inactive in the full-length receptor but is activated in a truncated receptor, GHR1-349 (11). It has been suggested that this motif may function in the internalization of an as yet unidentified GHR isoforms (11).

Internalization is not required for Jak-Stat signal transduction, as GHR mutated at Phe327 signals but is not internalized (10). However, there is strong evidence for the importance of receptor dimerization for signal transduction, which is derived from a number of experiments. In the presence of a molar excess of GH, which favors the formation of the monomeric GH-GHR complex, there is inhibition of the GH signal (12), truncated receptors that lack the cytoplasmic signaling domain block signal transduction by heterodimerization (2), and receptor dimerization is associated with a conformational change that is required for receptor signaling (13). The strongest evidence, however, comes from work with a GH antagonist mutated at GH binding site 2 (G120R), which blocks GH-stimulated cell proliferation (14) and does not generate the conformational change associated with receptor dimerization and Jak-Stat signaling (13). However, the GH antagonist G120R does appear to dimerize in a (GH)2-(GHR)2 complex and is internalized (15). Another GH antagonist mutated at binding site 2 (G120K) is currently being successfully investigated for its potential as a treatment for acromegaly, a disease resulting from an excess GH secretion from a pituitary tumor (16, 17). This antagonist, B2036, has 8 additional mutations at GH binding site 1, which increases the affinity of the antagonist for GHBP (18). There are no publications on the effect of this antagonist on internalization and cell signaling.

To date, most studies of GHR trafficking have depended on techniques that require the use of nonphysiological conditions, including acid stripping of cell surface receptor (19) and low temperature for ligand binding (9), and have used either the rat or rabbit GHR (the human GHR has proved difficult to clone and express (20)). These experiments have been of great value in defining the components required for internalization but are limited in their ability to establish the real time dynamics of receptor trafficking. Fluorescent labeling of ligand (9) and generation of fusion proteins with green fluorescent protein have made it possible to study the active transport of cellular proteins (21) and receptors, including nuclear receptors (22) and G protein-coupled receptors (23, 24). We have used fluorescent confocal microscopy and a combination of fluorescent-labeled GH and GHR to study GHR trafficking in real time at the level of the single cell. The results show: 1) the previously unrecognized, extremely rapid internalization of GH and of its antagonist, B2036; 2) that this internalization is independent of signaling; and 3) the failure of the naturally occurring truncated GHR1-279 to internalize.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Plasmids-- The full-length GHR and truncated receptor GHR1-279 were subcloned into pcDNA1/ampicillin (Invitrogen) as described previously (2). The LHRE-EGFP reporter construct was generated by subcloning the Stat5-binding element (LHRE) fused to the minimal TK promoter (25) into EGFP-p1 (CLONTECH). The truncated GHR1-317-EGFP chimera was created by subcloning the N terminus of EGFP to the EcoRI site in the cytoplasmic domain of the human GHR in the expression vector pcDNA1/ampicillin (Invitrogen).

Stable clones were generated in 293 cells (human kidney embryonal cell line) by transfection with the calcium phosphate procedure of the GHR plasmid with pcDNA3/Neo (Invitrogen). Selection was made using G418 (400 µg/ml, Sigma).

Binding Assays-- 24 h after plating, cells were serum-starved for 12 h. Cells were then washed with phosphate-buffered saline containing 1% bovine serum albumin and incubated with 125I-hGH (1 × 105 cpm/well) for 3 h at room temperature in the absence or presence of various concentrations of unlabeled hGH. The cells were then washed in the same buffer and solubilized in 1 ml of 1 N NaOH for counting radiation.

The recombinant human GH used was Genotrophin (Pharmacia and Upjohn, Milton Keynes, UK). The GH antagonist, B2036, is mutated at GH binding site 2 with G120K. In addition, to the G120K mutation there are 8 additional mutations at site 1 that are thought to increase the affinity of the receptor (18). The GH antagonist (B2036) was kindly supplied by William Bennett of Sensus Drug Development Corp.(Austin, Texas).

Texas Red labeling was performed using Texas Red-X succinimidyl ester (Molecular Probes, Leiden, Netherlands). GH or antagonist were dialyzed overnight against borate-buffered saline (pH 8.5). 100 µg of GH or antagonist were incubated with Texas Red at a 1:1 molar ratio in a final volume of 200 µl for 1 h at room temperature and away from light. The reaction mixture was separated on a 1-ml-bed volume Bio-Gel P10 5-kDa polyacrylamide gel.

Transcription assays were performed in 293 cells expressing the GHR using either a reporter gene containing a Stat5-binding element (LHRE) fused to a minimal TK promoter and luciferase or the LHRE-TK fused to EGFP. Luciferase activity was measured as previously reported (2). In studies using the LHRE-TK·EGFP, 293 cells were plated on 4-well coverglass slides, precoated with fibronectin at a density of 3 × 104 cells per well (ml), transfected by the calcium phosphate method with LHRE-TK·EGFP (550 ng/slide), and incubated overnight in complete medium before transfer into serum-free medium containing either 500 ng/ml GH and 100 ng/ml dexamethasone (stimulated) or dexamethasone alone (unstimulated). Fluorescence was detected 24 h later on the stage incubator of the confocal microscope, an analysis of images was performed using the public domain NIH Image (http://rsb.info.nih.gov/nih.image).

Fluorescence Imaging-- For experiments on living cells, fluorescence was detected using a Molecular Dynamics CLSM2010 confocal fluorescent microscope equipped with dual fluorescence and transmission detection. Images were transferred from the Silicon Graphics INDY on the CSLM 2010, converted to Macintosh 8-bit Tagged Image File Format, and analyzed with NIH Image, and the data were exported to EXCEL for further analysis. All experiments were performed at 37 °C using a stage incubator enclosure fitted to the microscope. For the detection of Texas Red, cells were excited at 568 nm, and detection was through a 610 nm long pass filter (red channel). For EGFP, cells were excited at 488 nm and detection was through a 530 nm band pass filter (green channel). In all experiments used for quantitative analysis, the laser power was kept below the level of saturation for the fluor and the photomultiplier voltage set so that the brightest pixels were <250 8-bit units. In the fluorescence energy transfer (FRET) experiments cells were excited with the 488 nm line, and data were collected on the red channel.

Calculation of Internalization-- Using NIH Image the fluorescence within the cell membrane was divided by the total fluorescence of the cell after subtracting the background. This number was multiplied by 100 to give a percentage, and the mean of two measurements for each cell was used in the analysis.

Western Ligand Blotting-- 293 cells were starved overnight in serum-free medium and then stimulated for 15 min at 37 °C with GH or antagonist. Cells were lysed in PBS-TDS (phosphate-buffered saline, 1% Triton X-100, 12 mM sodium deoxycholate, 3.5 mM SDS, and 4.7 nM sodium orthovandate), and 1 mg of protein was precipitated with Jak2 antibody (Upstate Biotechnology, Buckingham, UK) at 1:250 dilution with 20 µl of protein A-agarose (Sigma). Precipitated proteins were separated on a 10% SDS-polyacrylamide gel and after blotting onto polyvinylidene difluoride, phosphorylation was detected with an antiphosphotyrosine antibody (1:2500) (4G10, Upstate Biotechnology, Buckingham, UK) and the ECL system (Amersham Pharmacia Biotech).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To compare internalization of the truncated GHR1-279 with the full-length human GHR, we generated stable clones in a human kidney cell line, 293 cells. Cells were transfected with either receptor and selected under G418. Clones were picked that expressed a high level of GH binding. In our experimental conditions (125I-hGH, 1 × 105 cpm, on 3 × 105 cells), the selected full-length human GHR expressing clone showed 16% specific binding, and the GHR1-279 clone showed 28% specific binding.

Characterization of Stable Clone 293 Cells Expressing the Full-length and Truncated GHR and Affinity of the GH Antagonist-- In cells expressing the full-length receptor, competition studies with 125I-hGH and 125I-B2036 showed a single class of receptors (Fig. 1). The antagonist B2036 has 8 mutations in the GH binding site 1, which would be expected to increase its affinity for the extracellular domain of the GHR (18). In an assay based on GHBP immobilized on an enzyme-linked immunosorbent assay plate, the antagonist had an affinity five times greater than native GH.2 We compared the binding of GH and the antagonist, B2036, on intact cells expressing the full-length GHR. In displacement studies using either labeled GH or antagonist, the apparent affinities calculated by Scatchard analysis for GH (Ka = 1.00 × 109 M-1) and B2036 (Ka = 0.96 × 109 M-1) were comparable (Fig. 1). Thus, in intact cells, the antagonist appears to have a similar affinity to native GH. Functional activity of the full-length receptor was measured in transient transfections with Stat5 reporter constructs, consisting of Stat5 binding sites (LHRE) fused to either EGFP or luciferase. In the luciferase assay test, GH stimulation induced a bell-shaped curve with maximal stimulation (5-fold) occurring between 50 and 500 ng/ml and returning to basal values for GH levels >10 µg/ml. These results confirm that excess of GH favors the formation of GH-GHR monomers and blocks signal transduction (12). In the EGFP test, stimulation with 500 ng/ml GH also resulted in a 5-fold induction of fluorescence compared with unstimulated cells (Fig. 2). This induction of fluorescence was seen across the whole range of cell sizes (Fig. 2C). In contrast, the stable clone expressing the truncated GHR1-279, despite a high level of GH binding, showed no induction of fluorescence when transfected with the LHRE-EGFP reporter and no induction of luciferase activity (data not shown).


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Fig. 1.   Displacement curves of hGH and B2036 for the GHR. 293 cells expressing the full-length GHR were incubated with 125I-hGH (A) or 125I-B2036 (B) with increasing amounts of hGH or B2036. The experimental conditions are described under "Material and Methods."


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Fig. 2.   Stimulation of the LHRE-TK·EGFP reporter gene by hGH. Confocal images (×20) are shown of 293 cells stably expressing the full-length human GHR and transiently transfected with a Stat5 reporter construct with the LHRE-TK fused to EGFP. A, cells incubated in the absence of GH showing occasional cells with background fluorescence. B, cells incubated with GH at 500 ng showing induction of fluorescence. C, graph showing intensity of fluorescence plotted against cell size for cells stimulated with GH or unstimulated. The images analyzed were all under the level of saturation for the detector. However, to provide a clear representation and show the backgrounds, the contrast has been enhanced in both A and B.

Labeling of GH and Antagonist with Texas Red-- For internalization studies, GH and the antagonist B2036 were labeled with Texas Red. We first examined the effect of labeling on GH binding to its receptor and then incubated Texas Red with GH at different molar ratios from 1:1 to 1:10 (GH:Texas Red). A ratio greater than 1:3 resulted in a GH molecule with a reduced affinity for the GHR. Therefore, for all of the studies we elected to label GH and antagonist with a 1:1 molar ratio of protein to Texas Red.

Internalization of Texas Red-labeled GH in 293 Cells Expressing the Full-length and Truncated GHR-- To study internalization, 293 cells were plated on coverglass and incubated overnight in serum-free medium. Analysis was performed by confocal microscopy at 37 °C. 10 nM Texas Red-labeled GH was added in new starvation medium, and the cells were incubated for 5 min at 37 °C before washing and the addition of phenol red-free medium. Confocal images were recorded sequentially over time. Images were analyzed with NIH Image, and internalization was assessed by measuring the fluorescence within the cell membrane expressed as a percentage of the total fluorescence associated with the cell. No binding or internalization was seen in 293 cells not transfected with the GHR, and in all experiments binding and internalization were specifically displaced by the simultaneous addition of 2 µg of unlabeled GH.

For cells expressing the full-length GHR, internalization of GH was rapid with more than 80% of labeled GH internalized at 5 min (Fig. 3A), and membrane binding was difficult to detect at 20 min (Fig. 3B). After 15 min, most GH was internalized (mean ± S.E., 92 ± 1.3%; n = 8 cells). In contrast, cells expressing the truncated GHR1-279, which lacks 97% of the cytoplasmic domain of the receptor, showed little internalization. At 5 min there was well defined surface binding with <5% internalization of Texas Red-labeled GH (Fig. 3C), and at 20 min there was <30% internalization (Fig. 3D). After 15 min, internalization of Texas Red-labeled GH was 6.7 ± 1.8% (mean ± S.E.; n = 14 cells). The difference in internalization for the full-length and truncated receptor over time is shown in Fig. 3E.


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Fig. 3.   Internalization of Texas Red-labeled GH by 293 cells stably expressing the full-length GHR or truncated receptor GHR1-279. Cells expressing full-length GHR (A and B) or the truncated GHR1-279 (C and D) were incubated with 10 nM Texas Red-labeled GH for 5 min. Cells were observed immediately after washing (A and C) or 20 min after initial exposure to GH (B and D). E, percentage of Texas Red-labeled GH internalized in individual cells against time after initial exposure to Texas Red-labeled GH. Analysis was performed using NIH image. The fluorescence inside the cell is expressed as a percentage of the total fluorescence of the cell.

Previous publications (19) had reported inhibition of internalization by cooling cells to 4 °C. We attempted to repeat these studies. However, cells incubated for 10 min at 4 °C on coverglass behaved abnormally, showing delayed internalization and frequently separating from the coverglass.

Studies with GHR-EGFP Fusion Proteins-- To further study the trafficking of the GHR, we generated fusion proteins with EGFP. Transient expression of the EGFP expression vector alone (pEGFP-N2, CLONTECH) in 293 cells resulted in a diffuse distribution of fluorescence throughout the cytoplasm as well as in the nucleus of some cells. In contrast, transient and stable transfection of the truncated GHR1-317 fused to EGFP resulted in a cell surface distribution as shown by radioligand binding and laser scanning confocal microscopy (Fig. 4). In addition, the GHR1-317-EGFP was distributed within the cell consistent with an endoplasmic reticulum (ER) localization as well as in a large juxtanuclear fluorescent area representing the Golgi complex (Fig. 4A). The Golgi localization of the GHR represented a large proportion of the total fluorescent GHR in the cell. Control experiments with classical immunofluorescence on fixed cells were performed to verify that the presence of the GFP moiety did not significantly alter the distribution of the receptor. Labeling was done on 293 cells expressing either the full-length or the 279 truncated receptor permeabilized or not with Triton X-100 (Fig. 5). Labeling was present both at the cell surface and in intracellular compartments with a juxtanuclear accumulation in the Golgi apparatus. Such distribution has been described previously in COS-7 cells (26) and 293 cells (27) transfected with the GHR. In cells expressing the GHR1-317-EGFP, the fluorescence resulting from the antibody was superimposed on that from the GFP, so that even in the absence of Triton X-100, some intracellular labeling appeared because of the GFP. We analyzed the labeling distribution with confocal microscopy. Two slices, corresponding respectively to the top of the cell or the middle of the cell, confirm that the fusion receptor was present both at the cell surface and in intracellular compartments. This distribution is comparable with that seen for the truncated receptor GHR 1-279.


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Fig. 4.   Visualization of the truncated GHR1-317-EGFP chimera expressed in 293 cells. A, fluorescence micrograph (×60) showing accumulation of GHR in the Golgi apparatus (arrow). B, fluorescence micrograph (×60) of cell demonstrating vesicles generated in the endoplasmic reticulum (arrow). C, confocal image (×100) demonstrating fusion of vesicles with the cell surface membrane (arrow).


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Fig. 5.   Immunofluorescence for GHR in fixed cells expressing full-length and truncated receptor. 293 cells expressing full-length GHR (GHR FL, A and D), GHR1-279 (B and E), or GHR1-317-EGFP (C and F) were grown on coverslips. Cells were fixed with 3% paraformaldehyde and permeabilized (D-F) or not (A-C) with 0.5% Triton X-100. Cells were then labeled with 5 mg/ml anti-GHR antibody (monoclonal antibody 263), of which the epitope is in the extracellular domain of the receptor, and anti-mouse fluorescein isothiocyanate. Cells were observed under a Zeiss Axiovert confocal microscope (LSM 510). C and F show two different slices of the same cell focused, respectively, at the top of the cell (C) and in the middle of the cell (F). In this cell, the fluorescence of monoclonal antibody 263 and GFP are superimposed.

Monitoring GH/GHR Trafficking Using GHR1-317-EGFP Fusion Protein-- We have shown that Texas Red-labeled GH can be used to monitor GH internalization by confocal microscopy and that internalization is much more rapid than previously reported in experiments in which cells were first incubated at 4 °C, washed and transferred at 37 °C and in which the measure of the acid-stripping resistant radioactivity was the criterion of internalization. Using the receptor fusion protein and the labeled ligand, we exploited FRET to monitor the trafficking of the ligand, the receptor, and the ligand-receptor complexes simultaneously. Thus GHR-EGFP is detected using the 488 nm line to excite and a 530 nm band pass filter for emission (blue to green), the Texas Red-GH is detected using the 568 nm line to excite and a 610 long pass filter for emission (orange to red), and the GHR-EGFP·Texas Red-GH complexes are detected using the 488 nm line to excite and a 610 nm long pass filter for emission (blue to red). A 488 nm scan images the receptor (green) and ligand-receptor complexes (red), and a 568 nm scan images the ligand, whether receptor-bound or not. Ratio images (red/green) of GHR-EGFP alone, using only 488 nm excitation, were used to set the instrument so that there was no detectable spillover of EGFP fluorescence onto the red channel.

The truncated GHR-EGFP fusion would not be expected to internalize based on previous reports (10) suggesting that F327 is required for this process. Cells were incubated with Texas Red-GH as described above and imaged as described previously (Fig. 6). After 30 min at 37 °C, 24% of the cell-associated Texas Red-GH was internalized, whereas 85% of the truncated receptor was within the cell. The FRET image shows that 21% of the GH-GHR complexes were internalized and that the blue to red and the orange to red images are superimposed, confirming that ligand associated with the cell is receptor-bound and internalization of the truncated receptor-ligand complexes is very inefficient compared with that seen with full-length GHR.


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Fig. 6.   FRET in cells expressing the truncated GHR1-317-EGFP chimera and incubated with Texas Red-labeled GH. Images were made 30 min after a 5-min exposure to Texas Red-labeled GH. A, image of a cell stimulated with 568 nm light (absorbance of Texas Red), recorded on the red channel and showing the distribution of Texas Red-labeled GH. B, the same cell stimulated with 488 nm light (absorbance of EGFP). The recording was made on the green channel and shows the distribution of GHR1-317-EGFP chimera. C, the same cell stimulated with 488 nm light (absorbance of EGFP) and recorded on the red channel, showing FRET, which confirms the colocalization of GH with the receptor mainly at the cell surface.

Visualization of GHR1-317-EGFP Trafficking-- The EGFP fusion provided us with the opportunity for real time analysis of protein trafficking events in individual cells (Fig. 4). Packaging and movement of the receptor in vesicles was easily visualized. Budding of vesicles and there transport to the Golgi was rapid, occurring within seconds, and at this stage a large proportion of the GHR remained within the Golgi. Vesicles budding from the Golgi apparatus appeared to move in a random fashion toward the cell surface membrane and fused with the membrane (Fig. 4). A small proportion of the vesicles were moving from the cell surface membrane into the cell, presumably representing membrane turnover.

Studies on the GH Antagonist-- The GH antagonist, B2036, inhibited GH stimulation of the Stat5 reporter construct in a dose-dependent manner with complete inhibition at a 1:5 molar ratio of GH to antagonist (Fig. 7A). Western blotting confirmed that the antagonist fails to induce Jak2 phosphorylation (Fig. 7B). Internalization of the antagonist was studied in a manner similar to native GH. Texas Red-labeled antagonist was incubated with 293 cells stably expressing the full-length GHR for 5 min and then washed; the cells were then imaged by confocal microscopy. The Texas Red-labeled antagonist showed identical internalization dynamics to the native GH with the majority of antagonist internalized by 5 min after exposure to the antagonist (Fig. 7C).


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Fig. 7.   Antagonist effect of B2036. A, the induction of luciferase was measured in lysates from 293 cells stably expressing the full-length GHR and transfected with the Stat5 reporter construct LHRE-TK· luciferase. The cells were either not stimulated (Control) or were stimulated with GH alone (500 ng/ml) or GH plus GH antagonist B2036 (500 ng/ml each or 500 + 2500 ng/ml, respectively). B, lysates from cells stimulated under the same conditions as described above were immunoprecipitated with anti-Jak2 antibody and analyzed by Western blot with antiphosphotyrosine antibodies (lane 1 shows markers). C, graph from confocal microscopy showing percentages of native GH and GH antagonist internalized with time.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The studies reported here demonstrate that internalization of GH with its receptor is a much more rapid process than previously appreciated. Studies using iodinated GH have generally been performed at 4 °C to prevent internalization and allow equilibrium of bound and unbound GH to occur before warming the cells and studying internalization by acid stripping the surface bound ligand (9, 15, 19). In these studies internalization occurred over a period of 1 h with 30 (9) or 65% (15) internalized at 30 min and 85% at 60 min (15). In our experiments using confocal microscopy and Texas Red-labeled GH, surface binding could be demonstrated by briefly incubating the cells at 4 °C and then scanning. However, when cells were incubated with fluorescent GH at 37 °C and immediately scanned, the majority of labeled GH was internalized by 5 min, and after this time point it became difficult to see the cell surface binding as almost all GH bound to its receptor was internalized. It is likely that the previously reported slower course for internalization, as well as the lack of complete internalization, was due to the chilling of cells, as we were able to delay internalization by this procedure. However, in our experiments the chilling of cells was also associated with other changes. In particular, the cells tended to lift from the coverglass after 10 min at 4 °C, and it is probable that these nonphysiological conditions may cause other changes in cell function.

We studied the internalization of a truncated GHR1-279 that is found in normal human tissues (2, 3). This truncated GHR, lacking the major part of the cytoplasmic domain of the receptor, acts as a dominant negative inhibitor of GHR signaling (2) and generates large amounts of GH-binding protein (2, 3). Using confocal microscopy and Texas Red-labeled GH, there was a great difference between internalization of the full-length and of the truncated receptor. At 5 min almost all labeled GH was internalized by the full-length GHR, whereas for the truncated receptor <5% was internalized. A similar but less dramatic difference in internalization between the full-length and truncated receptor (70 versus 10% at 1 h) has been shown by acid stripping (5). This failure of internalization of the truncated human receptor could be predicted from previous studies with the rat and rabbit receptor. In these studies, Phe327, which is deleted in the truncated receptor, proved to be essential for normal ubiquitination and internalization of the receptor (9, 10). In our experiments we also show a great difference in the kinetics of receptor internalization. After 1 h of exposure, we observed some internalization of the truncated receptor (between 5 and 30%). This degree of receptor internalization is similar to that reported for other truncated receptors (5, 9, 10). This internalization may represent cell surface membrane turnover or an uncharacterized internalization signal such as the di-leucine-mediated internalization reported for another truncated GHR (11). Experiments using acid stripping to monitor the cell surface bound GH after 1 h of exposure also reported similar differences in internalization between the full-length and truncated receptor (70 versus 10%) (5). The demonstration that the truncated receptor fails to internalize confirms the hypothesis that a lack of internalization underlies the mechanism for its dominant negative action (2, 6).

Transfection of 293 cells with the EGFP expression vector alone showed diffuse fluorescence throughout the cell including the nucleus, demonstrating that the EGFP protein alone moves between all compartments of the cell. In contrast, the truncated GHR1-317-EGFP fusion protein showed a specific cellular localization, similar to that seen by immunofluorescence in cells expressing full-length and truncated receptor. The GHR-EGFP fusion was visualized within vesicles generated at the ER and transported to the Golgi. The fluorescence seen within the cell represents packaging of the GHR and translocation to the cell surface, which could be observed in real time and was a rapid process. The truncated receptor emerged from the ER at multiple peripheral sites resulting in the accumulation of chimeric protein in numerous peripheral membrane structures. These units, once established, appeared to rapidly (within seconds) translocate to the Golgi apparatus by a process resembling a random walk. From the Golgi apparatus vesicles budded off to then transfer and fuse with the cell membrane. The dynamics of this vesicular transport were very similar to that shown for ER-to-Golgi transport elucidated with a viral glycoprotein tagged to GFP (21). To date, there has been no report analyzing the cellular distribution and dynamic trafficking of a cytokine receptor linked to GFP. The truncated receptor that we studied allowed us mainly to visualize translocation to the cell surface membrane, as the receptor lacked the essential internalization domain (9). The lack of internalization was confirmed by the studies with FRET. However, a proportion of fluorescence was internalized and was presumed to be the result of nonspecific membrane turnover as was the case for the other truncated receptors. Previous receptor studies have reported visualization of the glucocorticoid receptor (22) and the G protein-coupled cholecystokinin and beta 2-adrenergic receptors (23, 24). The internalization of the beta 2-adrenergic receptor is slower than we have demonstrated with the GHR, with only 30% internalized 20 min after exposure to ligand in HeLa cells (24). However, in 293 cells a beta 2-adrenergic·GFP fusion protein was at least partially internalized within 5 min of exposure to agonist (28). The kinetics of internalization are different for these G protein-coupled receptors, which are recycled, and for the cholecystokinin receptor, for which the recycling time is 20 to 60 min (23).

B2036 is an analogue of human GH in which 8 amino acids in binding site 1 are mutated to increase the binding affinity to the receptor, and 1 amino acid in binding site 2 is mutated to prevent binding to a second receptor. Preliminary reports in normal subjects and acromegalic patients suggest that B2036 can reduce IGF-I levels (16, 17). However, to date there are no reports on the effect of this antagonist at the level of receptor internalization and signaling. The mutations selected for site 1 were based on observations made using the extracellular domain of the receptor to select for high affinity variants of hGH (18). Using an assay based on displacement of hGH from immobilized hGHBP, B2036 had five times the affinity to hGH.2 However, in our assay, based on full-length GHR in intact cells, B2036 had an affinity comparable to hGH. This assay presumably measures the affinity of GH for the GHR dimer. It may be that as the GH antagonist (GHa) is thought to dimerize in the complex GHR-GHa-GHa-GHR (15), this could give an affinity similar to GH, which forms a dimer in the complex GHR-GH-GHR. The GH antagonist G120K (B2036) has site 2 for binding GHR mutated, similar to the previously reported G120R, which blocks GH-stimulated cell proliferation (14). Our functional studies demonstrated that the antagonist G120K completely blocked GHR signaling through Stat5 at a 1:5 molar ratio. This was associated with inhibition of Jak2 phosphorylation. Our studies confirm and extend the previous reports that the GH antagonist G120R inhibits signaling (15) by demonstrating that G120K has a similar action, blocks Jak2 phosphorylation, and also completely abolishes Stat5 signaling. The GH antagonist G120R has previously been shown to internalize, suggesting that the abilities of GH to stimulate tyrosine phosphorylation and internalization are separate functions (15). The time course for the internalization of the antagonist was similar to that reported for native GH with approximately 75% internalized within 40 min. We considered the possibility that the time course for internalization may differ between the antagonist and native hormone, but this time lag may have been missed in previous experiments. Under physiological conditions we found an identical level of internalization for the antagonist and native GH, with the major part of both of them internalized within 5 min of exposure to labeled hormone. These results demonstrate the potent antagonist action of G120K on Jak-Stat signaling, indicating that this antagonist action is unrelated to any change in internalization.

Together our results demonstrate that GHR trafficking can be studied by dual-fluorescent confocal microscopy. Using chimeric receptors fused to EGFP, we have visualized the translocation of the GHR from the ER to the cell surface. The studies of truncated GHR, which acts as an antagonist to GHR signaling, confirm that these receptors are unable to internalize rapidly, which explains their dominant negative action and also accounts for the accumulation to high levels of such mutants, a process that would enhance their dominant negative activity. The GH antagonist G120K behaves in a manner similar to G120R, blocking GHR signaling through Jak2-Stat5. Fluorescent labeling of the antagonist G120K confirms that its antagonist action does not effect internalization, which occurs despite inhibition of receptor signaling.

    ACKNOWLEDGEMENT

We are indebted to Prof. P. Kelly for reviewing the manuscript.

    FOOTNOTES

* This work was supported by The Special Trustees of the Former United Hospitals, Trent Regional Research Schemes, Yorkshire Cancer Research, Pharmacia and Upjohn, and Serono Laboratories. The confocal microscope was supported by grants from the Medical Research Council, the Wellcome Trust, and The Arthritis and Rheumatism Campaign.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Clinical Sciences, Northern General Hospital, Sheffield S5 7AU, UK. Tel.: 44-114-2714007; Fax: 44-114-2560458; E-mail: R.J.Ross{at}Sheffield.ac.uk.

2 William Bennett, personal communication.

    ABBREVIATIONS

The abbreviations used are: GH, growth hormone; GHR, GH receptor; GHBP, GH-binding protein; hGH, human GH; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; LHRE, lactogenesis hormone response element; ER, endoplasmic reticulum; TK, tyrosine kinase; FRET, fluorescence energy transfer; Jak, Janus kinase; Stat, signal transducer and activator of transcription.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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