A 36 residues insertion in the dimerization domain of the growth hormone receptor results in defective trafficking rather than impaired signaling

in Journal of Endocrinology

Growth hormone insensitivity syndrome (GHIS) has been reported in a family homozygous for a point mutation in the GH receptor (GHR) that activates an intronic pseudoexon. The resultant GHR (GHR1–656) includes a 36 amino-acids insertion after residue 207, in the region known to be important for homodimerization of GHR. We have examined the functional consequences of such an insertion in mammalian cells transfected with the wild type (GHRwt) and mutated GHR (GHR1–656). Radio-ligand binding and flow cytometry analysis showed that GHR1–656 is poorly expressed at the cell surface compared with GHRwt. Total membrane binding and Western blot analysis showed no such difference in the level of total cellular GHR expressed for GHR1–656 vs GHRwt. Immunofluorescence showed GHR1–656 to have different cellular distribution to the wild type receptor (GHRwt), with the mutated GHR being mainly perinuclear and less vesicular than GHRwt. Western blot analysis showed GH-induced phosphorylation of Jak2 and Stat5 for both GHR1–656 and GHRwt, although reduced Stat5 activity was detected with GHR1–656, consistent with lower levels of expression of GHR1–656 than GHRwt at the cell surface. In conclusion, we report that GHIS, due to a 36 amino-acids insertion in the extracellular domain of GHR, is likely to be explained by a trafficking defect rather than by a signalling defect of GHR.

Abstract

Growth hormone insensitivity syndrome (GHIS) has been reported in a family homozygous for a point mutation in the GH receptor (GHR) that activates an intronic pseudoexon. The resultant GHR (GHR1–656) includes a 36 amino-acids insertion after residue 207, in the region known to be important for homodimerization of GHR. We have examined the functional consequences of such an insertion in mammalian cells transfected with the wild type (GHRwt) and mutated GHR (GHR1–656). Radio-ligand binding and flow cytometry analysis showed that GHR1–656 is poorly expressed at the cell surface compared with GHRwt. Total membrane binding and Western blot analysis showed no such difference in the level of total cellular GHR expressed for GHR1–656 vs GHRwt. Immunofluorescence showed GHR1–656 to have different cellular distribution to the wild type receptor (GHRwt), with the mutated GHR being mainly perinuclear and less vesicular than GHRwt. Western blot analysis showed GH-induced phosphorylation of Jak2 and Stat5 for both GHR1–656 and GHRwt, although reduced Stat5 activity was detected with GHR1–656, consistent with lower levels of expression of GHR1–656 than GHRwt at the cell surface. In conclusion, we report that GHIS, due to a 36 amino-acids insertion in the extracellular domain of GHR, is likely to be explained by a trafficking defect rather than by a signalling defect of GHR.

Keywords:

Introduction

Laron syndrome or GH insensitivity syndrome (GHIS) is a rare autosomal recessive condition associated with postnatal growth failure leading to extreme short stature, mid-facial hypoplasia, truncal obesity, and hypoglycaemia in infancy. Patients have raised GH levels, associated with deficiency of insulin-like growth factor (IGF-I), and IGF binding protein-3 (IGFBP-3) (Rosenfeld et al. 1994, Woods et al. 1997). These features result from GHR dysfunction and consequent failure of GH action.

The mature wild type human GHR (GHRwt) is 620 amino acids long, possessing 246 extracellular amino acids, a 24 amino acid transmembrane spanning region, and 350 cytoplasmic amino acids (Leung et al. 1987). GH interacts with its receptor in a 1:2 stoichiometry, and this is essential to trigger signalling (Maamra et al. 1999). The GHR extracellular domain constitutes two subdomains, joined by a short hinge region. Subdomain 1 spans from residue 1 to 123 and subdomain 2 from residue 128 to 246. Subdomain 1 is mainly involved in the GH–GHR interaction (Behncken & Waters 1999). The two molecules of GHR also interact, and the dimerization interface of the two receptors is confined to subdomain 2 of the receptors. In this subdomain, the unpaired cysteine 241 is thought to play a role in the conformational change of GHR induced by GH, rather than in the dimerization of the receptor (Zhang et al. 1999).

The GHR lacks intrinsic kinase activity and relies on the regulation and activation of cytosolic Janus kinase 2 (Jak2). Jak2 is constitutively associated with the receptor at the proline rich Box1 site (amino acids 276–287) and it is thought that ligand binding may stabilise the preformed receptor-Jak2 complex (Argetsinger et al. 1993, Colosi et al. 1993). Box2 (amino acids 325–338) of the GHR is thought to be required for full activation of Jak2 by GH (Colosi et al. 1993, Wang & Wood 1995). GH binding results in a conformational change in the receptor dimer, and transphosphorylation of associated Jak2 (Argetsinger et al. 1993, Ross et al. 2001). Following GH stimulation, phosphorylated Jak2 has been reported to activate Stat1, Stat3, Stat5 (Meyer et al. 1994, Campbell et al. 1995, Wood et al. 1995), and the MAP kinase pathway including Erk1/2, p38, and Jnk (Winston & Bertics 1992, Zhu et al. 1998, Zhu & Lobie 2000).

The majority of patients with GHIS have mutations in exons encoding the extracellular domain of the GHR (Rosenfeld et al. 1994, Woods et al. 1997). These mutations usually result in low or absent GH binding protein (GHBP) in serum and failure of GH to bind and signal at GH responsive tissues (Woods et al. 1997, Wojcik et al. 1998). The relatively few mutations described in DNA encoding the transmembrane or intracellular domain of the GHR result in truncated GHRs lacking most of the intracellular domain of the receptor (Ayling et al. 1997, 1999, Woods et al. 1996, Kaji et al. 1997, Iida et al. 1998, Gastier et al. 2000). Rarely, GHIS has been reported to be due to post-receptor defects (Iida et al. 1999, Rosenfeld et al. 2004).

Four patients from one family, with an atypical form of GHIS, have been previously identified having a homozygous point mutation in intron 6 of the GHR. The point mutation lies within the pseudoexon (A_1 to G_1 at the 5′pseudoexon splice site) and may improve the interaction of the pre-mRNA with U1 snRNP resulting in spliceosome action. This results in the activation of a pseudoexon encoding a 36 residue insertion between exons 6 and 7 which lies in the dimerization region of the extracellular domain of the GHR (Metherell et al. 2001). Failure of dimerization was thus thought to be the most likely cause of GHIS. To clarify, the effect of this mutation on GH function, we have expressed the mutant GHR in mammalian cells and investigated its functional properties.

Materials and Methods

Phenotype and genotype

The index cases were four male cousins from a highly consanguineous Pakistani family. The patients presented with marked short stature, and low IGF1 levels (20–29 ng/ml, levels>50 ng/ml), however they had normal levels of GHBP and a normal facial appearance. Genotyping revealed a 108 bp nucleotide insertion between exons 6 and 7, resulting from the inclusion of a pseudoexon, giving a receptor of 656 amino-acids (GHR1–656) (Metherell et al. 2001).

Plasmids

The full-length wild type human GHR (GHRwt) sub-cloned into pcDNA1/amp (Invitrogen) was previously described (Maamra et al. 1999). To amplify the sequence of insertion of GHR1–656, patient cDNA was subjected to PCR using primers within exons 6 and 7 (sense primer 5′- CAT GCA GAT ATC CAA GTG AGA TGG-3′) and reverse primer 5′- GAT CTC ACG CGT ACT TCA TAT TCC TTA TCC ACT TTC AAT GAG -3′). Two fragments were amplified from the patient cDNA. The 302 bp fragment contains the inserted pseudoexon, whereas the lower 194 bp band corresponds to the low levels of GHRwt expressed in the patient. The 302 bp fragment was subcloned in PcDNA1 GHRwt following digestion with EcoRV and MluI. The reporter plasmids, Stat5/Stat3 responsive firefly luciferase reporter vector containing the lactogenic hormone response element LHRE-TK-luc has been described previously (Ross et al. 1997, Maamra et al. 1999).

Cell culture and transfection

Human embryonic kidney (HEK293) cells were routinely grown respectively in DMEM Nut F12 medium supplemented with 10% foetal calf serum (FCS), 100 IU penicillin and 100 μg /ml streptomycin, 2 mg/ml fungi-zone, 2 mM l-glutamine, and buffered with HEPES (all GIBCO). Cells were routinely grown at 37 °C in a 95% humidified atmosphere of 5% CO2. Transfection of HEK293 cells was performed using a calcium phosphate transfection kit (GIBCO) according to the manufacturer’s instructions.

Reporter assays

HEK293 cells were used to investigate the effect on the pseudoexon insertion on signaling. This cellular system was preferentially chosen against Chinese ovary cell line and human hepatoma cell line HuH7 as it responds more strongly to GH-induced signalling (unpublished observations, personal). Cells were incubated in 60% DMEM F12/30% DMEM 4.5 g/l glucose (GIBCO BRL) and 10% FCS for 6 h. Cells were transfected with 5 μg GHR expression vector or empty vector, 1.5 μg LHRE/TK-Luc and 30 ng of a renilla luciferase expression vector per plate. Sixteen h later, the cells were stimulated with GH in the presence or absence of 0.5 μM dexamethasone. The cells were harvested and assayed for firefly (LHRE) and Renilla (internal control) luciferase activities using the Promega dual luciferase kit (Promega). The activities were measured in the same aliquot sample using an Automat plus LB953 luminometer (Berthold, Redbourn, UK). Firefly Luciferase activity reflecting GH-dependent Stat activation was normalised using renilla luciferase activity to adjust for transfection efficiency between samples. Samples were measured in duplicate and experiments were repeated three times. The data are presented as fold induction (mean ± s.e.m.).

GH binding by monolayer cells

HEK293 cells were transfected with 3 μg of GHR expression plasmids and cells incubated in serum free medium 12 h prior to binding. Cells were then washed with PBS containing 1% BSA (Sigma) and incubated with I125-labelled GH (105 c.p.m./well) (Novo Nordisk Bagsvaerd, Denmark) for 3 h at room temperature in the absence (total binding) or presence (non-specific binding) of excess unlabelled hGH (2 μg) (Genotropin, Pharmacia and Upjohn, Milton Keynes, UK). The cells were washed in the same buffer and solubilized in 1 M NaOH for counting on a gamma-counter. Specific binding of the radioligand was calculated as the difference of total and non-specific binding and expressed as percentage of the total count. Samples were measured in duplicate and experiments were repeated three times. The data are presented as percentage specific binding (mean± s.e.m).

Total GHR binding to GH

To estimate the total cellular content of functional GHR (cell surface and intracellular), GH binding activity of surface and intracellular membranes derived from cell sonicates was measured as follows. Cell monolayers were set up in 10-cm dishes. The cells were washed in PBS 0.2% BSA and detached by scraping in 1 ml of ice-cold PBS containing a mixture of protease inhibitors (Roche Diagnostics). After centrifugation at 800 r.p.m., the packed cells were resuspended in 2 ml of ice-cold inhibitor mixture and disrupted by sonication. The cell sonicates were centrifuged at 40 000g for 60 min at 4 °C to yield pellets of total cellular membranes. To measure GH binding, the membrane pellets were resuspended in 6.5 ml of ice-cold PBS 0.2% BSA.

The membrane suspensions were incubated in triplicate with I125-labelled GH (2 × 105 c.p.m./ml) at room temperature for 2 h. Non-specific binding was defined as the binding in the presence of 10 μg/ml unlabelled GH. At the end of the assay, the membrane suspensions were centrifuged, washed twice with ice-cold PBS/0.2% BSA and counted on a gamma-counter. The data are presented as percentage specific binding (mean ± s.e.m). Samples were measured in triplicate and experiments were repeated three times. To confirm equivalence of transfection efficiency, binding experiments were performed under identical conditions with simultaneous transcription assays with renilla luciferase as an internal control. In addition, for total membrane binding experiments. BCA was performed to evaluate the total amount of proteins in the membranes preparation. Membranes were pelleted by centrifugation at 14 000 r.p.m. for 15 mins at 4 °C. The pellet was dissolved in 1 M NaOH/0.2% Triton X-100 and BCA using a the Bio-Rad DC Protein Assay Kit II according to the manufacturer’s instructions (Hemel Hempstead, Hertfordshire, UK). Cell surface binding and total membrane binding experiments were performed on the same transfection samples.

Flow cytometry

HEK293 cells (4 × 105 cells) were transfected with 3 μg of GHR expression plasmid. After preincubation in FCS free medium for 2 h, cells were dislodged from the culture dish using cell dissociation solution (Sigma Genosys). Cells were suspended in 600 μl of PBS 1%BSA (washing buffer). Cells (100 μl) were incubated with 5 μg of an anti-GH receptor antibody (2C8 antibody was a kind gift from Prof. CJ Strasburger, Berlin, Germany) or isotype-matched negative control antibody (R&D Systems, Abingdon, UK) for 30 min on ice. The binding epitope of the 2C8 antibody is on the centre of the hGH binding domain but does not overlap the epitopes of Mab263 (hinge region of GHR) and Mab10B8 (dimerisation domain) (Rowlinson et al. 1998, Z.Wu, unpublished observations). Primary antibody binding was detected by incubation with biotinylated goat anti-mouse IgG polyclonal antibody (1 μg; Calbiochem, Nottingham, UK), followed by incubation with Streptavidin-R.Phycoerythrin (SAV-R-PE) conjugate (10 μl; Serotec, Oxford, UK) for 30 min on ice. Flow cytometry was performed using a FACScan flow cytometer (BD Biosciences, Oxford, UK) and CellQuest data acquisition and analysis software (Beckton Dickinson, San Jose, CA, USA).

Immunofluorescence

Immunofluorescence was performed on fixed cells to determine the cellular distribution of GHR. Labelling was carried out on cells transfected with GHRwt or the mutated receptor GHR1–656. Cells were fixed in 2% paraformaldehyde (PFA) for 15 min and permeabilized or not with Triton X-100 (0.1% in 2% PFA) for 2 min. All washings were performed in PBS with 0.1% saponin. For blocking, cells were incubated in PBS 5% goat serum plus 0.1% saponin for 1 h. Biotin and streptavidin sites were blocked using the avidin–biotin blocking kit (Vector Laboratories UK, Peterborough, England). Cells were rinsed and subsequently incubated with the anti-GHR extracellular domain 2C8 monoclonal antibody (10 μg/ ml) in antibody buffer (PBS 1% goat serum, 0.1% BSA, 0.1% saponin). After washing, cells were incubated with biotinylated Fab fragment of goat anti-mouse antibody and streptavidin Alexa 488 (Molecular probes, Eugene, OR, USA). When immunofluorescence was performed with a polyclonal rabbit antibody against the intracellular domain of GHR (Antibody 12, kindly provided by J Finidori, Paris, France) detection was performed with a secondary antibody directly coupled to Alexa 594. Slides were mounted in Vectashield mounting medium (Vector Laboratories UK). Fluorescence was detected using a LEICA TCS confocal fluorescent microscope (LEICA, Wetzlar, Germany). Cells were excited at 488 nm and detection was through a 530 nm band pass filter for Alexa 488 detection. For Alexa 594 detection, excitation was at 594 nm and detection was through a 610 nm band pass filter.

Jak–Stat Western blotting

Cells (2 × 106 cells per 10 cm dish) transfected with GHR expression plasmids were incubated overnight in serum-free medium and stimulated at 37 °C with GH. Cells were washed in 5 ml of PBS supplemented with 1 mM sodium orthovanadate and lysed in 1 ml of PBS-TDS buffer (PBS, 0.1% Triton X-100, 12 mM sodium deoxycholate, 3.5 mM SDS, 1 mM sodium orthovanadate). Lysates were immunoprecipitated with 10 μl anti-Jak2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-Stat5 antibody clone sc-835 (Santa Cruz) overnight at 4 °C. Lysates were briefly spun and incubated with 40 μl of protein A/G beads suspension (Pierce, Rockford, IL, USA). Western blot analysis was carried out in duplicate with equivalent amounts of protein loaded on gel confirmed by measuring total protein expression. Proteins were separated on a 7.5% SDS–PAGE gel for Jak2 and 10% for Stat5, and blotted onto PVDF membrane. Phosphorylation of Jak2 and Stat5 was detected using an anti-phosphotyrosine antibody (4 G10; 1:650; UBI, Lake Placid, NY, USA). The secondary antibodies were coupled to horseradish peroxidase (HRP; Amersham Pharmacia Biotech.) and the enzyme was detected using an enhanced chemiluminescence (ECL) kit according to the manufacturer’s instructions (Amersham Pharmacia Bitoech). All blots were reprobed for total Jak2 and Stat5.

GHR Western blotting

To estimate the amount of total GHR present and its size in the cells, cells transfected with GHR were incubated overnight in serum free medium and lysed in 250 μl of PBS-TDS lysis buffer. Crude lysates were separated on a 7.5% PAGE and blotted onto PVDF membrane. A rabbit polyclonal antibody against the intracellular domain of GHR (Antibody 12, kindly provided by J Finidori, Paris, France) was used for detection. ECL detection was performed as described for Jak–Stat.

Statistical analysis

The paired t-test was used with Bonferroni correction for multiple comparisons. For analysis of dose–response and time-course studies repeated measures ANOVA was used with Bonferroni correction as appropriate. Data are expressed as the mean ± s.e.m. and a P<0.05 was considered to indicate statistically significant differences. For the detection of GH-induced gene transcription in GHR expressing cells, the data was analysed using a 2-way factorial ANOVA with blocking structure. Comparisons were made between grand means using appropriate contrasts, and P-values were calculated by reference to t-distributions. Again, P<0.05 was considered to be statistically significant.

Results

Binding of GH to cell surface GHR

GHR1–656 and GHRwt were transfected into HEK293 cells and binding at the cell surface detected using iodinated human GH (I125-GH) (Fig. 1). GH binding to GHR1–656 was consistently markedly reduced compared with GHRwt (P=0.01). The same results were obtained in Chinese ovary cell line and human hepatoma cell line HuH7 cells (data not shown).

Flow cytometry detection of GHR expressed at the cell surface

GHR1–656 binds less GH at the cell surface than GH-Rwt. To further clarify the protein expression at the cell surface, flow cytometry detection was carried out using the 2C8 antibody against the extracellular domain of GHR (Fig. 2). This antibody recognises a site distant from the pseudoexon insertion. Both GHRwt and GHR1–656 exhibited higher fluorescence than untransfected cells. Mean fluorescence levels for expressed GHR1–656 were much lower than for GHRwt.

Binding of GH to total cellular GHR

GHR1–656 and GHRwt were transfected into HEK293 cells and total cell membranes prepared. Binding was performed using iodinated human GH (I125-GH) (Fig. 3). No significant binding was detected in cells transfected with control plasmids. Transfection of GHR gave a significant increase in GH binding to membranes (ANOVA, P<0.002). In cells transfected with GHR1–656, consistently higher levels of GH binding to membranes was detected compared with GHRwt (P<0.03). The same results were obtained in Chinese ovary cell line and human hepatoma cell line HuH7 cells (data not shown).

Subcellular localisation of GHR by immunofluorescence

To visualize the receptors independently of their ability to bind GH, we performed immunofluorescence studies with the monoclonal antibody 2C8 recognizing an epitope in the extracellular domain of the receptor. Cells were fixed and incubated directly or after permeabilization with Triton to visualize receptor present at the cell surface (Fig. 4a) and in intracellular compartments(Fig. 4b). In non-permeabilized cells, no fluorescence was detected in cells not transfected with GHR. A bright cell surface fluorescent signal was detected in cells expressing the GHRwt. In contrast, only a faint signal was detected on cells expressing the mutant receptor GHR 1–656. When intracellular staining was performed, comparable levels of immunofluorescence were observed in intracellular compartments of cells expressing GHRwt and GHR1–656. However, the pattern of fluorescence localisation was different. GHRwt was localized in cytoplasmic vesicles and in a region adjacent to the nucleus consistent with the Golgi apparatus. In contrast, the mutant GHR was mainly circumferential to the nucleus and only a few vesicles were detected in the cytoplasm consistent with the endoplasmic reticulum (ER) and compatible with the Golgi apparatus in 293 cells as described previously (Maamra et al. 1999, Wojcik et al. 1998) (Fig. 4b). The same results of intracellular pattern were obtained with the antibody 12 directed against the intracellular region of GHR (Fig. 4c).

Detection of total GHR by Western blot

To further determine the level of protein expression of the different GHRs, Western blotting was performed (Fig. 5). The antibody used was directed against the intracellular domain of GHR. The GHRwt exhibited several bands, consistent with different glycosylation states and degradation by GHR turnover. The two lower bands of GHRwt were around 60 and 65 kDa and the same bands were detected for GHR1–656. Two bands migrated at around 110 kDa and 130 kDa consistent with previous reports for GHRwt. In GHR 1–656, the two corresponding bands were slightly higher consistent with the insertion of 36 amino acids in the mutant.

GH-induced Jak2 phosphorylation

293 cells were transfected with plasmids expressing GHR1–656 or GHRwt and stimulated with 100 ng/ml GH for the times indicated. No phosphorylation of Jak2 was detected in cells transfected with mock control plasmid (data not shown). Phosphorylated Jak2 was detected in both cells expressing GHRwt and GHR1–656 when stimulated with GH (Fig. 6). However, the level of activation was lower in GHR1–656 than in GHRwt expressing cells.

GH-induced Stat5 phosphorylation

Cells were stimulated with ‘low’ (25 ng/ml) and ‘high’(100 ng/ml) dose GH (Fig. 7a) for 15 min. No activation of Stat5 was detected in cells transfected with empty vector. In cells expressing GHRwt, phosphorylation of Stat5 was detected after stimulation with 25 ng/ml of GH for 15 min. At the higher concentration of GH (100 ng/ml), the level of phosphorylated Stat5 was increased. At both concentrations, GHR 1–656 expressing cells showed phosphorylation of Stat5, but to a lesser extent than on GHRwt expressing cells. A time response was performed. In GHRwt expressing cells, Stat5 phosphorylation was detected at 15 min after GH stimulation and was sustained for 60 min (Fig. 7b). The same pattern was observed in GHR1–656 expressing cells, although GH seemed to induce Stat5 phosphorylation to a lesser extent.

GH-induced gene expression through LHRE

To further quantify the difference in signaling between GHRwt and the mutant GHR, cells were transfected with 1.5 μg per 12 well plate of receptor expression plasmid and with the Stat5 responsive reporter LHRE-TK-Luc (Fig. 8). Cells were stimulated with varying doses of GH in the presence (b) or absence (a) of dexamethasone. Dexamethasone has been shown to exert a synergetic effect on GH-induced gene expression through LHRE. In cells transfected with control plasmid, no activation of transcription through LHRE was detected (Fig. 8a). When GHRwt was transfected, a six hours exposure to GH induces a dose-dependent increase in luciferase activity compared with unstimulated cells (P<0.001; GHRwt vs control), with activation visible at doses as low as 6.25 ng/ml GH. In cells expressing GHR1–656, GH also induces a dose-dependent LHRE activation, although at a lower level than in GHRwt expressing cells (P<0.001; GHR1–656 vs GHRwt). Similar results were obtained when cells were transfected with lower levels of GHR (data not shown). When the experiments were repeated in the presence of dexamethasone, similar results were obtained, with lower activation of LHRE by GHR1–656 compared with GHRwt (P=0.022 GHR1–656 vs control; P<0.001 GHRwt vs control; P<0.001 GHR1–656 vs GHRwt; Fig. 8b). All P values quoted are for differences between grand means of each GHR type.

Discussion

This manuscript reports in vitro studies of a GHR mutant identified in siblings with GHIS. The patients presented with atypical GHIS and our goal was to investigate the functional capacity of the mutant GHR.

An important first-step in GH signaling is the formation of a trimer complex with one molecule of ligand and two molecules of receptors. Recent studies suggest that GHR pre-exists as a dimer when synthesised (Ross et al. 2001, Gent et al. 2002), and that GH binding results in a conformational change (Ross et al. 2001) and the formation of a cysteine bond (Zhang et al. 1999). As the pseudo-exon insertion in the GHR mutant lies between exons 6 and 7 (Metherell et al. 2001), a region previously described as critically involved in receptor dimerization (Cunningham et al. 1991), it had been suggested that the mutation resulted in a dimerization defect resulting in GHI. However, our studies indicate that the primary cause underlying the GHIS was related to a trafficking defect in the mutant receptor.

Binding studies on cell monolayers indicated that GHR1–656 binds less GH than GHRwt at the cell surface. We considered that GHR1–656 could have a reduced affinity for GH, however, our flow cytometry analysis confirmed that the levels of cell surface GHR1–656 protein were lower than for GHRwt. Some mutations in the GHR result in a defect in expression (Esposito et al. 1998), but this is not the case with GHR1–656 as RT-PCR from the patient’s fibroblasts to construct the expression vector confirmed the previous studies that the mutant GHR mRNA was expressed at nominal levels (Metherell et al. 2001). Although cell surface binding was reduced for the mutant the overall binding for the mutant from total membranes was if anything increased. As the total cellular GHR binding of GH was not reduced in GHR mutant compared with GHRwt, this suggests that the affinity for GH is not a major defect for this mutation.

The signaling capacity of GHR 1–656 was evaluated by Western blotting and transcription assays. Both GHRwt and GHR1–656 were able to activate the Jak-Stat pathway, but activation was lower in cells transfected with the GHR1–656. GH-induced Stat5 and Jak2 phosphorylation seemed to be lower in cells transfected with the mutant receptor. Although at 60 min, Stat5 phosphorylation at 60 min appears to be comparable for GHRwt and mutant, this is more likely due to antibody maximal binding capacity and detection sensitivity. This was confirmed quantitatively in the LHRE transcription assay. In this assay, GHR1–656 showed at least 2-fold reduction in signaling capacities compared with GHRwt. At low concentrations of GH, the mutant receptor appeared to induce a similar level of transcription to the wt receptor, however, we suspect this is an artifact of the assay. It maybe that at low levels of GH, the assay is unable to differentiate different levels of signaling. Low total cellular expression of the mutant is not a likely explanation for this defect, as by Western blot, the levels of GHRwt and GHR1–656 were similar. The explanation for reduced signaling probably relates to lower cell surface expression of the mutant GHR

Glycosylation occurs with the GHR and is thought to play a role in protection of the protein. According to previous reports on GHR, glycosylation and ubiquitination are likely to account for an important part of the GHR size in mammalian cells (Harding et al. 1994, Alves dos Santos et al. 2001). Given the sizes detected in the Western blots, it is unlikely that glycosylation is affected in the GHR mutant.

Stat5 has been shown to be a major player in GH action. Generation of Stat5a/b knockout mice has been shown to result in mice significantly reduced in size in both sexes and size correlated with reduced levels of insulin-like growth factor (IGF-1) (Udy et al. 1997, Teglund et al. 1998). Before Stat5 can be phosphorylated by GH, it must first bind via its SH2 domain to specific phospho-tyrosyl residues in the cytoplasmic domain of the GHR. The GHR intracellular domain undergoes constitutive and GH-induced degradation, leading to the detection of remnant GHR fragments in the cell (Zhang et al. 2001). One could potentially hypothesise that the mutation in GHR 1–656 could influence GHR degradation and thereby alter Stat5 binding. However, the Western blotting confirmed the presence of a full size GHR, and the remnant GHR at lower size (50–75 kDa) in both GHR 1–656 and GHRwt, making abnormal internal degradation of the mutant GHR unlikely. Both WT and mutant appear to have similar relative abundances of mature and precursor GHR which, is reflected by the two higher forms of the full-length receptor (Fig. 5). This would suggest that the mature (fully glycosylated and therefore post-Golgi) mutant receptor is less likely to be on the surface than the mature WT receptor. The lower cell surface expression of the mutant at the cell surface is translated into the lower signalling capacities. To determine exactly if the trafficking differs during biogenesis (processing ER to Golgi to surface) and/or after GH-independent endocytosis (lower cell surface half-life) and will require further investigation.

Our immunofluorescence data on fixed cells confirmed the low level of mutant receptor at the cell surface. More interestingly, it revealed a different fluorescent pattern for the mutant GHR when compared with GHRwt. Whereas the GHRwt is present in the Golgi Apparatus and in numerous small vesicles in the cytoplasm and at the plasma membrane, GHR 1–656 seemed to be trapped around the nucleus in a circumferential pattern of fluorescence. The pattern of fluorescence is at compatible with the Golgi apparatus in 293 cells as described previously (Wojcik et al. 1998, Maamra et al. 1999). This is consistent with the hypothesis that the mutant GHR is retained inside the cells, because of a failure of proper protein folding. This is the most likely cause of the disease observed in the patients. These patients have an atypical GHIS phenotype. This appears to be explained by the fact that (1) low levels of GHRwt are expressed from a minor wild type GHR transcript and (2) because the mutant GHR retains the capacity for apparently normal signaling, despite significantly reduced expression at the cell surface. Both the wt and mutant GHRs may give rise to circulating GHBP explaining the normal serum levels of this marker found in these patients.

Figure 1
Figure 1

Cell monolayer binding by GHRs. Radioligand binding was carried out on HEK293 cells expressing either GHR1–656 or GHRwt, using I125-hGH in the absence (total binding) or presence (non-specific binding) of excess cold GH. Non-transfected cells were used as a negative control. Results of 3 experiments are expressed as percent specific binding of labelled GH (mean ± s.e.m).

Citation: Journal of Endocrinology 188, 2; 10.1677/joe.1.06252

Figure 2
Figure 2

Flow cytometry analysis on GHR1–656 and GHRwt expressing cells. Untransfected HEK293 cells and HEK293 cells transfected with either GHR1–656 or GHRwt were incubated with 2C8 (mouse anti-human GHR). Biotinylated goat anti-mouse and phycoerythrin linked to streptavidin were used for detection. 2C8 recognises an epitope in the extracellular domain of GHR. Flow cytometry analysis results are expressed as mean fluorescence of cells with 2C8.

Citation: Journal of Endocrinology 188, 2; 10.1677/joe.1.06252

Figure 3
Figure 3

Total cell membranes binding by GHRs. Radioligand binding was carried out on total membranes preparations from HEK293 cells expressing either GHR1–656 or GHRwt, using I125-hGH in the absence (total binding) or presence (non-specific binding) of excess cold GH. Non-transfected cells were used as a negative control. Results of 3 experiments are expressed as percent specific binding of labelled GH (mean ± s.e.m).

Citation: Journal of Endocrinology 188, 2; 10.1677/joe.1.06252

Figure 4
Figure 4

Immunofluorescence cellular localization of GHRs. Cells transfected with control plasmid pcDNA3, GHRwt, or mutated GHR1–656 were grown on coverslips. Cells were fixed with 2% PFA and then labelled with 10ug/ml anti-GHR antibody for which the epitope is in the extracellular domain of the receptor and biotinylated anti-mouse and streptavidin labelled Alexa 488 (a). Cells were also permeabilized with 0.1% Triton X-100 prior to staining (b). Alternatively, permeabilized cells were then labelled with a rabbit polyclonal antibody against the intracellular domain of the receptor (c) and anti-rabbit antibody labelled with Alexa 594.

Citation: Journal of Endocrinology 188, 2; 10.1677/joe.1.06252

Figure 5
Figure 5

Detection of GHR cell expression by Western blot. Cells transfected with control plasmid pcDNA3, GHRwt, or mutated GHR1–656 were lysed and crude lysates run on SDS–PAGE gel and blotted onto PVDF membranes. Membranes were probed with a rabbit polyclonal antibody against the intracellular domain of the receptor and antibody binding detected by ECL.

Citation: Journal of Endocrinology 188, 2; 10.1677/joe.1.06252

Figure 6
Figure 6

Detection of Jak2 phosphorylation in GHR expressing cells by Western blot. Cells transfected with control plasmid pcDNA3, GHRwt, or mutated GHR1–656 were stimulated with 100 ng/ml GH for the time indicated. Cells were lysed and lysates immunoprecipitated with an anti-Jak2 antibody. Samples were run on SDS–PAGE gel and blotted onto PVDF membranes. Membranes were probed with the anti-phoshotyrosine antibody 4 G10 and antibody binding detected by ECL. Blots were reprobed for total Jak2.

Citation: Journal of Endocrinology 188, 2; 10.1677/joe.1.06252

Figure 7
Figure 7

Detection of Stat5 phosphorylation in GHR expressing cells by Western blot. (a) Cells transfected with control plasmid pcDNA3, GHRwt, or mutated GHR1–656 were stimulated for 15 min with GH at the concentration indicated. Cells were lysed and lysates immunoprecipitated with an anti-Stat5 antibody. Samples were run on SDS–PAGE gel and blotted onto PVDF membranes. Membranes were probed with the anti-phoshotyrosine antibody 4 G10 and antibody binding detected by ECL. Blots were reprobed for total Stat5. (b) Cells transfected with control plasmid pcDNA3, GHRwt, or mutated GHR1–656 were stimulated with 100 ng/ml GH for the time indicated. Samples were analysed as in (a).

Citation: Journal of Endocrinology 188, 2; 10.1677/joe.1.06252

Figure 8
Figure 8

Detection of GH-induced gene transcription in GHR expressing cells. Cells were transfected with control plasmid pcDNA3, GHRwt, or mutated GHR1–656 plus 1.5ug LHRE/TK-luc and an internal transfection control CMV-renilla luciferase expression vector. Cells were incubated without (a) or with (b) dexamethasone (0.5μM) in the presence of increasing amounts of GH and cell lysates were used for determination of luciferase activity. Values of 3 experiments are expressed as fold induction (mean ± s.e.m).

Citation: Journal of Endocrinology 188, 2; 10.1677/joe.1.06252

We are grateful to Prof. CJ Strasburger and Dr J Finidori for supplying anti-GHR antibodies.

Funding

This work was supported by Novo Nordisk. We are grateful to Novo Nordisk for supporting M M and supplying iodinated GH. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

References

  • Alves dos SantosCM ten Broeke T & Strous GJ 2001 Growth hormone receptor ubiquitination endocytosis and degradation are independent of signal transduction via Janus kinase 2. Journal of Biological Chemistry27632635–32641.

    • Search Google Scholar
    • Export Citation
  • ArgetsingerLS Campbell GS Yang X Witthuhn BA Silvennoinen O Ihle JN & Carter-Su C 1993 Identification of JAK2 as a growth hormone receptor-associated tyrosine kinase. Cell74237–244.

    • Search Google Scholar
    • Export Citation
  • AylingRM Ross R Towner P Von Laue S Finidori J Moutoussamy S Buchanan CR Clayton PE & Norman MR 1997 A dominant-negative mutation of the growth hormone receptor causes familial short stature. Nature Genetics1613–14.

    • Search Google Scholar
    • Export Citation
  • AylingRM Ross RJM Towner P Von L Finidori J Moutoussamy S Buchanan CR Clayton PE & Norman MR 1999 New growth hormone receptor exon 9 mutation causes genetic short stature. Acta Paediatrica88168–172.

    • Search Google Scholar
    • Export Citation
  • Behncken & Waters 1999 SN & Waters MJ 1999 Molecular recognition events involved in the activation of the growth hormone receptor by growth hormone. Journal of Molecular Recognition12355–362.

    • Search Google Scholar
    • Export Citation
  • CampbellGS Meyer DJ Raz R Levy DE Schwartz J & Carter-Su C 1995 Activation of acute phase response factor (APRF)/Stat3 transcription factor by growth hormone. Journal of Biological Chemistry2703974–3979.

    • Search Google Scholar
    • Export Citation
  • ColosiP Wong K Leong SR & Wood WI 1993 Mutational analysis of the intracellular domain of the human growth hormone receptor. Journal of Biological Chemistry26812617–12623.

    • Search Google Scholar
    • Export Citation
  • CunninghamBC Ultsch M de Vos AM Mulkerrin MG Clauser KR & Wells JA 1991 Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule. Science254821–825.

    • Search Google Scholar
    • Export Citation
  • EspositoN Wojcik J Chomilier J Martini JF Kelly PA Finidori J & Postel-Vinay MC 1998 The D152H mutation found in growth hormone insensitivity syndrome impairs expression and function of human growth hormone receptor but is silent in rat receptor. Journal of Molecular Endocrinology2161–72.

    • Search Google Scholar
    • Export Citation
  • GastierJM Berg MA Vesterhus P Reiter EO & Francke U 2000 Diverse deletions in the growth hormone receptor gene cause growth hormone insensitivity syndrome. Human Mutations16323–333.

    • Search Google Scholar
    • Export Citation
  • GentJ van Kerkhof P Roza M Bu G & Strous GJ 2002 Ligand-independent growth hormone receptor dimerization occurs in the endoplasmic reticulum and is required for ubiquitin system-dependent endocytosis. PNAS999858–9863.

    • Search Google Scholar
    • Export Citation
  • HardingPA Wang XZ Kelder B Souza S Okada S & Kopchick JJ 1994 In vitro mutagenesis of growth hormone receptor Asn-linked glycosylation sites. Molecular and Cellular Endocrinology106171–180.

    • Search Google Scholar
    • Export Citation
  • IidaK Takahashi Y Kaji H Nose O Okimura Y Abe H & Chihara K 1998 Growth hormone (GH) insensitivity syndrome with high serum GH-binding protein levels caused by a heterozygous splice site mutation of the GH receptor gene producing a lack of intracellular domain. Journal of Clinical Endocrinology and Metabolism83531–537.

    • Search Google Scholar
    • Export Citation
  • IidaK Takahashi Y Kaji H Onodera N Takahashi MO Okimura Y Abe H & Chihara K 1999 The C422F mutation of the growth hormone receptor gene is not responsible for short stature. Journal of Clinical Endocrinology and Metabolism844214–4219.

    • Search Google Scholar
    • Export Citation
  • KajiH Nose O Tajiri H Takahashi Y Iida K Takahashi T Okimura Y Abe H & Chihara K 1997 Novel compound heterozygous mutations of growth hormone (GH) receptor gene in a patient with GH insensitivity syndrome. Journal of Clinical Endocrinology and Metabolism823705–3709.

    • Search Google Scholar
    • Export Citation
  • LeungDW Spencer SA Cachianes G Hammonds RG Collins C Henzel WJ Barnard R Waters MJ & Wood WI 1987 Growth hormone receptor and serum binding protein: purification cloning and expression. Nature330537–543.

    • Search Google Scholar
    • Export Citation
  • MaamraM Finidori J Von Laue S Simon S Justice S Webster J Dower & Ross R 1999 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. Journal of Biological Chemistry27414791–14798.

    • Search Google Scholar
    • Export Citation
  • MetherellLA Akker SA Munroe PB Rose SJ Caulfield M Savage MO Chew SL & Clark AJ 2001 Pseudoexon activation as a novel mechanism for disease resulting in atypical growth-hormone insensitivity. American Journal of Human Genetics69641–646.

    • Search Google Scholar
    • Export Citation
  • MeyerDJ Campbell GS Cochran BH Argetsinger LS Larner AC Finbloom DS Carter-Su C & Schwartz J 1994 Growth hormone induces a DNA binding factor related to the interferon-stimulated 91-kDa transcription factor. Journal of Biological Chemistry2694701–4704.

    • Search Google Scholar
    • Export Citation
  • RosenfeldRG Rosenbloom AL & Guevara-Aguirre J 1994 Growth hormone (GH) insensitivity due to primary GH receptor deficiency. Endocrine Reviews15369–390.

    • Search Google Scholar
    • Export Citation
  • RosenfeldRG Kofoed E Little B Woods K Buckway C Pratt K & Hwa V 2004 Growth hormone insensitivity resulting from post-GH receptor defects. Growth Hormone and IGF Research14 (Suppl A) S35–S38.

    • Search Google Scholar
    • Export Citation
  • RossRJ Esposito N Shen XY Von Laue S Chew SL Dobson PR Postel-Vinay MC & Finidori J 1997 A short isoform of the human growth hormone receptor functions as a dominant negative inhibitor of the full-length receptor and generates large amounts of binding protein. Molecular Endocrinology11265–273.

    • Search Google Scholar
    • Export Citation
  • RossRJ Leung KC Maamra M Bennett W Doyle N Waters MJ & Ho KK 2001 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. Journal of Clinical Endocrinology and Metabolism861716–1723.

    • Search Google Scholar
    • Export Citation
  • RowlinsonSW Behncken SN Rowland JE Clarkson RW Strasburger CJ Wu Z Baumbach W & Waters MJ 1998 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 signaling. Journal of Biological Chemistry2735307–5314.

    • Search Google Scholar
    • Export Citation
  • TeglundS McKay C Schuetz E van Deursen JM Stravopodis D Wang D Brown M Bodner S Grosveld G & Ihle JN 1998 Stat5a and Stat5b proteins have essential and nonessential or redundant roles in cytokine responses. Cell93841–850.

    • Search Google Scholar
    • Export Citation
  • UdyGB Towers RP Snell RG Wilkins RJ Park SH Ram PA Waxman DJ & Davey HW 1997 Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression. PNAS947239–7244.

    • Search Google Scholar
    • Export Citation
  • WangYD & Wood WI 1995 Amino acids of the human growth hormone receptor that are required for proliferation and Jak-STAT signaling. Molecular Endocrinology9303–311.

    • Search Google Scholar
    • Export Citation
  • WinstonLA & Bertics PJ 1992 Growth hormone stimulates the tyrosine phosphorylation of 42- and 45-kDa ERK-related proteins. Journal of Biological Chemistry2674747–4751.

    • Search Google Scholar
    • Export Citation
  • WojcikJ Berg MA Esposito N Geffner ME Sakati N Reiter EO Dower S Francke U Postel-Vinay MC & Finidori J 1998 Four contiguous amino acid substitutions identified in patients with Laron syndrome differently affect the binding affinity and intracellular trafficking of the growth hormone receptor. Journal of Clinical Endocrinology and Metabolism834481–4489.

    • Search Google Scholar
    • Export Citation
  • WoodTJ Sliva D Lobie PE Pircher TJ Gouilleux F Wakao H Gustafsson JA Groner B Norstedt G & Haldosen LA 1995 Mediation of growth hormone-dependent transcriptional activation by mammary gland factor/Stat 5. Journal of Biological Chemistry2709448–9453.

    • Search Google Scholar
    • Export Citation
  • WoodsKA Fraser NC Postel-Vinay MC Savage MO & Clark AJ 1996 A homozygous splice site mutation affecting the intracellular domain of the growth hormone (GH) receptor resulting in Laron syndrome with elevated GH-binding protein. Journal of Clinical Endocrinology and Metabolism811686–1690.

    • Search Google Scholar
    • Export Citation
  • WoodsKA Dastot F Preece MA Clark AJ Postel-Vinay MC Chatelain PG Ranke MB Rosenfeld RG Amselem S & Savage MO 1997 Phenotype: genotype relationships in growth hormone insensitivity syndrome. Journal of Clinical Endocrinology and Metabolism823529–3535.

    • Search Google Scholar
    • Export Citation
  • ZhangY Jiang J Kopchick JJ & Frank SJ 1999 Disulfide linkage of growth hormone (GH) receptors (GHR) reflects GH-induced GHR dimerization. Association of JAK2 with the GHR is enhanced by receptor dimerization. Journal of Biological Chemistry27433072–33084.

    • Search Google Scholar
    • Export Citation
  • ZhangY Guan R Jiang J Kopchick JJ Black RA Baumann G & Frank SJ 2001 Growth hormone (GH)-induced dimerization inhibits phorbol ester-stimulated GH receptor proteolysis. Journal of Biological Chemistry27624565–24573.

    • Search Google Scholar
    • Export Citation
  • ZhuT & Lobie PE 2000 Janus kinase 2-dependent activation of p38 mitogen-activated protein kinase by growth hormone. Resultant transcriptional activation of ATF-2 and CHOP cytoskeletal re-organization and mitogenesis. Journal of Biological Chemistry2752103–2114.

    • Search Google Scholar
    • Export Citation
  • ZhuT Goh EL LeRoith D & Lobie PE 1998 Growth hormone stimulates the formation of a multiprotein signaling complex involving p130(Cas) and CrkII. Resultant activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK). Journal of Biological Chemistry27333864–33875.

    • Search Google Scholar
    • Export Citation

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    Cell monolayer binding by GHRs. Radioligand binding was carried out on HEK293 cells expressing either GHR1–656 or GHRwt, using I125-hGH in the absence (total binding) or presence (non-specific binding) of excess cold GH. Non-transfected cells were used as a negative control. Results of 3 experiments are expressed as percent specific binding of labelled GH (mean ± s.e.m).

  • View in gallery

    Flow cytometry analysis on GHR1–656 and GHRwt expressing cells. Untransfected HEK293 cells and HEK293 cells transfected with either GHR1–656 or GHRwt were incubated with 2C8 (mouse anti-human GHR). Biotinylated goat anti-mouse and phycoerythrin linked to streptavidin were used for detection. 2C8 recognises an epitope in the extracellular domain of GHR. Flow cytometry analysis results are expressed as mean fluorescence of cells with 2C8.

  • View in gallery

    Total cell membranes binding by GHRs. Radioligand binding was carried out on total membranes preparations from HEK293 cells expressing either GHR1–656 or GHRwt, using I125-hGH in the absence (total binding) or presence (non-specific binding) of excess cold GH. Non-transfected cells were used as a negative control. Results of 3 experiments are expressed as percent specific binding of labelled GH (mean ± s.e.m).

  • View in gallery

    Immunofluorescence cellular localization of GHRs. Cells transfected with control plasmid pcDNA3, GHRwt, or mutated GHR1–656 were grown on coverslips. Cells were fixed with 2% PFA and then labelled with 10ug/ml anti-GHR antibody for which the epitope is in the extracellular domain of the receptor and biotinylated anti-mouse and streptavidin labelled Alexa 488 (a). Cells were also permeabilized with 0.1% Triton X-100 prior to staining (b). Alternatively, permeabilized cells were then labelled with a rabbit polyclonal antibody against the intracellular domain of the receptor (c) and anti-rabbit antibody labelled with Alexa 594.

  • View in gallery

    Detection of GHR cell expression by Western blot. Cells transfected with control plasmid pcDNA3, GHRwt, or mutated GHR1–656 were lysed and crude lysates run on SDS–PAGE gel and blotted onto PVDF membranes. Membranes were probed with a rabbit polyclonal antibody against the intracellular domain of the receptor and antibody binding detected by ECL.

  • View in gallery

    Detection of Jak2 phosphorylation in GHR expressing cells by Western blot. Cells transfected with control plasmid pcDNA3, GHRwt, or mutated GHR1–656 were stimulated with 100 ng/ml GH for the time indicated. Cells were lysed and lysates immunoprecipitated with an anti-Jak2 antibody. Samples were run on SDS–PAGE gel and blotted onto PVDF membranes. Membranes were probed with the anti-phoshotyrosine antibody 4 G10 and antibody binding detected by ECL. Blots were reprobed for total Jak2.

  • View in gallery

    Detection of Stat5 phosphorylation in GHR expressing cells by Western blot. (a) Cells transfected with control plasmid pcDNA3, GHRwt, or mutated GHR1–656 were stimulated for 15 min with GH at the concentration indicated. Cells were lysed and lysates immunoprecipitated with an anti-Stat5 antibody. Samples were run on SDS–PAGE gel and blotted onto PVDF membranes. Membranes were probed with the anti-phoshotyrosine antibody 4 G10 and antibody binding detected by ECL. Blots were reprobed for total Stat5. (b) Cells transfected with control plasmid pcDNA3, GHRwt, or mutated GHR1–656 were stimulated with 100 ng/ml GH for the time indicated. Samples were analysed as in (a).

  • View in gallery

    Detection of GH-induced gene transcription in GHR expressing cells. Cells were transfected with control plasmid pcDNA3, GHRwt, or mutated GHR1–656 plus 1.5ug LHRE/TK-luc and an internal transfection control CMV-renilla luciferase expression vector. Cells were incubated without (a) or with (b) dexamethasone (0.5μM) in the presence of increasing amounts of GH and cell lysates were used for determination of luciferase activity. Values of 3 experiments are expressed as fold induction (mean ± s.e.m).

  • Alves dos SantosCM ten Broeke T & Strous GJ 2001 Growth hormone receptor ubiquitination endocytosis and degradation are independent of signal transduction via Janus kinase 2. Journal of Biological Chemistry27632635–32641.

    • Search Google Scholar
    • Export Citation
  • ArgetsingerLS Campbell GS Yang X Witthuhn BA Silvennoinen O Ihle JN & Carter-Su C 1993 Identification of JAK2 as a growth hormone receptor-associated tyrosine kinase. Cell74237–244.

    • Search Google Scholar
    • Export Citation
  • AylingRM Ross R Towner P Von Laue S Finidori J Moutoussamy S Buchanan CR Clayton PE & Norman MR 1997 A dominant-negative mutation of the growth hormone receptor causes familial short stature. Nature Genetics1613–14.

    • Search Google Scholar
    • Export Citation
  • AylingRM Ross RJM Towner P Von L Finidori J Moutoussamy S Buchanan CR Clayton PE & Norman MR 1999 New growth hormone receptor exon 9 mutation causes genetic short stature. Acta Paediatrica88168–172.

    • Search Google Scholar
    • Export Citation
  • Behncken & Waters 1999 SN & Waters MJ 1999 Molecular recognition events involved in the activation of the growth hormone receptor by growth hormone. Journal of Molecular Recognition12355–362.

    • Search Google Scholar
    • Export Citation
  • CampbellGS Meyer DJ Raz R Levy DE Schwartz J & Carter-Su C 1995 Activation of acute phase response factor (APRF)/Stat3 transcription factor by growth hormone. Journal of Biological Chemistry2703974–3979.

    • Search Google Scholar
    • Export Citation
  • ColosiP Wong K Leong SR & Wood WI 1993 Mutational analysis of the intracellular domain of the human growth hormone receptor. Journal of Biological Chemistry26812617–12623.

    • Search Google Scholar
    • Export Citation
  • CunninghamBC Ultsch M de Vos AM Mulkerrin MG Clauser KR & Wells JA 1991 Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule. Science254821–825.

    • Search Google Scholar
    • Export Citation
  • EspositoN Wojcik J Chomilier J Martini JF Kelly PA Finidori J & Postel-Vinay MC 1998 The D152H mutation found in growth hormone insensitivity syndrome impairs expression and function of human growth hormone receptor but is silent in rat receptor. Journal of Molecular Endocrinology2161–72.

    • Search Google Scholar
    • Export Citation
  • GastierJM Berg MA Vesterhus P Reiter EO & Francke U 2000 Diverse deletions in the growth hormone receptor gene cause growth hormone insensitivity syndrome. Human Mutations16323–333.

    • Search Google Scholar
    • Export Citation
  • GentJ van Kerkhof P Roza M Bu G & Strous GJ 2002 Ligand-independent growth hormone receptor dimerization occurs in the endoplasmic reticulum and is required for ubiquitin system-dependent endocytosis. PNAS999858–9863.

    • Search Google Scholar
    • Export Citation
  • HardingPA Wang XZ Kelder B Souza S Okada S & Kopchick JJ 1994 In vitro mutagenesis of growth hormone receptor Asn-linked glycosylation sites. Molecular and Cellular Endocrinology106171–180.

    • Search Google Scholar
    • Export Citation
  • IidaK Takahashi Y Kaji H Nose O Okimura Y Abe H & Chihara K 1998 Growth hormone (GH) insensitivity syndrome with high serum GH-binding protein levels caused by a heterozygous splice site mutation of the GH receptor gene producing a lack of intracellular domain. Journal of Clinical Endocrinology and Metabolism83531–537.

    • Search Google Scholar
    • Export Citation
  • IidaK Takahashi Y Kaji H Onodera N Takahashi MO Okimura Y Abe H & Chihara K 1999 The C422F mutation of the growth hormone receptor gene is not responsible for short stature. Journal of Clinical Endocrinology and Metabolism844214–4219.

    • Search Google Scholar
    • Export Citation
  • KajiH Nose O Tajiri H Takahashi Y Iida K Takahashi T Okimura Y Abe H & Chihara K 1997 Novel compound heterozygous mutations of growth hormone (GH) receptor gene in a patient with GH insensitivity syndrome. Journal of Clinical Endocrinology and Metabolism823705–3709.

    • Search Google Scholar
    • Export Citation
  • LeungDW Spencer SA Cachianes G Hammonds RG Collins C Henzel WJ Barnard R Waters MJ & Wood WI 1987 Growth hormone receptor and serum binding protein: purification cloning and expression. Nature330537–543.

    • Search Google Scholar
    • Export Citation
  • MaamraM Finidori J Von Laue S Simon S Justice S Webster J Dower & Ross R 1999 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. Journal of Biological Chemistry27414791–14798.

    • Search Google Scholar
    • Export Citation
  • MetherellLA Akker SA Munroe PB Rose SJ Caulfield M Savage MO Chew SL & Clark AJ 2001 Pseudoexon activation as a novel mechanism for disease resulting in atypical growth-hormone insensitivity. American Journal of Human Genetics69641–646.

    • Search Google Scholar
    • Export Citation
  • MeyerDJ Campbell GS Cochran BH Argetsinger LS Larner AC Finbloom DS Carter-Su C & Schwartz J 1994 Growth hormone induces a DNA binding factor related to the interferon-stimulated 91-kDa transcription factor. Journal of Biological Chemistry2694701–4704.

    • Search Google Scholar
    • Export Citation
  • RosenfeldRG Rosenbloom AL & Guevara-Aguirre J 1994 Growth hormone (GH) insensitivity due to primary GH receptor deficiency. Endocrine Reviews15369–390.

    • Search Google Scholar
    • Export Citation
  • RosenfeldRG Kofoed E Little B Woods K Buckway C Pratt K & Hwa V 2004 Growth hormone insensitivity resulting from post-GH receptor defects. Growth Hormone and IGF Research14 (Suppl A) S35–S38.

    • Search Google Scholar
    • Export Citation
  • RossRJ Esposito N Shen XY Von Laue S Chew SL Dobson PR Postel-Vinay MC & Finidori J 1997 A short isoform of the human growth hormone receptor functions as a dominant negative inhibitor of the full-length receptor and generates large amounts of binding protein. Molecular Endocrinology11265–273.

    • Search Google Scholar
    • Export Citation
  • RossRJ Leung KC Maamra M Bennett W Doyle N Waters MJ & Ho KK 2001 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. Journal of Clinical Endocrinology and Metabolism861716–1723.

    • Search Google Scholar
    • Export Citation
  • RowlinsonSW Behncken SN Rowland JE Clarkson RW Strasburger CJ Wu Z Baumbach W & Waters MJ 1998 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 signaling. Journal of Biological Chemistry2735307–5314.

    • Search Google Scholar
    • Export Citation
  • TeglundS McKay C Schuetz E van Deursen JM Stravopodis D Wang D Brown M Bodner S Grosveld G & Ihle JN 1998 Stat5a and Stat5b proteins have essential and nonessential or redundant roles in cytokine responses. Cell93841–850.

    • Search Google Scholar
    • Export Citation
  • UdyGB Towers RP Snell RG Wilkins RJ Park SH Ram PA Waxman DJ & Davey HW 1997 Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression. PNAS947239–7244.

    • Search Google Scholar
    • Export Citation
  • WangYD & Wood WI 1995 Amino acids of the human growth hormone receptor that are required for proliferation and Jak-STAT signaling. Molecular Endocrinology9303–311.

    • Search Google Scholar
    • Export Citation
  • WinstonLA & Bertics PJ 1992 Growth hormone stimulates the tyrosine phosphorylation of 42- and 45-kDa ERK-related proteins. Journal of Biological Chemistry2674747–4751.

    • Search Google Scholar
    • Export Citation
  • WojcikJ Berg MA Esposito N Geffner ME Sakati N Reiter EO Dower S Francke U Postel-Vinay MC & Finidori J 1998 Four contiguous amino acid substitutions identified in patients with Laron syndrome differently affect the binding affinity and intracellular trafficking of the growth hormone receptor. Journal of Clinical Endocrinology and Metabolism834481–4489.

    • Search Google Scholar
    • Export Citation
  • WoodTJ Sliva D Lobie PE Pircher TJ Gouilleux F Wakao H Gustafsson JA Groner B Norstedt G & Haldosen LA 1995 Mediation of growth hormone-dependent transcriptional activation by mammary gland factor/Stat 5. Journal of Biological Chemistry2709448–9453.

    • Search Google Scholar
    • Export Citation
  • WoodsKA Fraser NC Postel-Vinay MC Savage MO & Clark AJ 1996 A homozygous splice site mutation affecting the intracellular domain of the growth hormone (GH) receptor resulting in Laron syndrome with elevated GH-binding protein. Journal of Clinical Endocrinology and Metabolism811686–1690.

    • Search Google Scholar
    • Export Citation
  • WoodsKA Dastot F Preece MA Clark AJ Postel-Vinay MC Chatelain PG Ranke MB Rosenfeld RG Amselem S & Savage MO 1997 Phenotype: genotype relationships in growth hormone insensitivity syndrome. Journal of Clinical Endocrinology and Metabolism823529–3535.

    • Search Google Scholar
    • Export Citation
  • ZhangY Jiang J Kopchick JJ & Frank SJ 1999 Disulfide linkage of growth hormone (GH) receptors (GHR) reflects GH-induced GHR dimerization. Association of JAK2 with the GHR is enhanced by receptor dimerization. Journal of Biological Chemistry27433072–33084.

    • Search Google Scholar
    • Export Citation
  • ZhangY Guan R Jiang J Kopchick JJ Black RA Baumann G & Frank SJ 2001 Growth hormone (GH)-induced dimerization inhibits phorbol ester-stimulated GH receptor proteolysis. Journal of Biological Chemistry27624565–24573.

    • Search Google Scholar
    • Export Citation
  • ZhuT & Lobie PE 2000 Janus kinase 2-dependent activation of p38 mitogen-activated protein kinase by growth hormone. Resultant transcriptional activation of ATF-2 and CHOP cytoskeletal re-organization and mitogenesis. Journal of Biological Chemistry2752103–2114.

    • Search Google Scholar
    • Export Citation
  • ZhuT Goh EL LeRoith D & Lobie PE 1998 Growth hormone stimulates the formation of a multiprotein signaling complex involving p130(Cas) and CrkII. Resultant activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK). Journal of Biological Chemistry27333864–33875.

    • Search Google Scholar
    • Export Citation