(GH) Mutant (R77C) - Semantic Scholar

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The Journal of Clinical Endocrinology & Metabolism 92(8):2893–2901 Copyright © 2007 by The Endocrine Society doi: 10.1210/jc.2006-2238

Evaluation of the Biological Activity of a Growth Hormone (GH) Mutant (R77C) and Its Impact on GH Responsiveness and Stature Vibor Petkovic, Ame´lie Besson, Mario Thevis, Didier Lochmatter, Andre´e Eble´, Christa E. Flu¨ck, and Primus E. Mullis Department of Pediatric Endocrinology, Diabetology, and Metabolism (V.P., A.B., D.L., A.E., C.E.F., P.E.M.), University Children’s Hospital, Inselspital, CH-3010 Bern, Switzerland; and Center for Preventive Doping Research-Institute of Biochemistry (M.T.), German Sport University Cologne, 50933 Cologne, Germany Context and Objective: A single missense mutation in the GH-1 gene converting codon 77 from arginine (R) to cysteine (C) yields a mutant GH-R77C peptide, which was described as natural GH antagonist. Design, Setting, and Patients: Heterozygosity for GH-R77C/ wt-GH was identified in a Syrian family. The index patient, a boy, was referred for assessment of his short stature (⫺2.5 SD score) and partial GH insensitivity was diagnosed. His mother and grandfather were also carrying the same mutation and showed partial GH insensitivity with modest short stature. Interventions and Results: Functional characterization of the GHR77C was performed through studies of GH receptor binding and activation of Janus kinase 2/Stat5 pathway. No differences in the binding affinity and bioactivity between wt-GH and GH-R77C were

G

ROWTH DISORDERS AND short stature are a common clinical problem. Although most cases are sporadic and result from environmental insults or developmental abnormalities, there are estimates of the frequency of genetic causes, including GH deficiency, GH insensitivity (Laron syndrome), and/or reduced bioactivity of the GH (Kowarski syndrome), ranging from 1 in 4,000 to 1 in 10,000 live births (1– 4). GH plays a major role in postnatal growth. The gene is mainly expressed in the pituitary gland as a protein, which is characterized by an arrangement of four antiparallel ␣-helices separated by connecting loops (5). A single GH molecule contains two GH receptor (GHR)-binding sites, site 1 (the high affinity site) composed mainly of the long extended loop between helices 1 and 2 and site 2 located mainly on helix 1 (6). GHR exists as an inactive dimer at the cell surface, and its activation involves a hormone-induced relative rotation of subunits, bringing the Janus kinase (Jak)-2 in close proximity First Published Online May 22, 2007 Abbreviations: C, Cysteine; CHO, Chinese hamster ovary; ER, endoplasmic reticulum; FCS, fetal calf serum; GHBP, GH-binding protein; GHR, GH receptor; HEK, human embryonic kidney; IGF-BP, IGF-binding protein; Jak, Janus kinase; R, arginine; rh, recombinant human; Stat, signal transducer and activator of transcription. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

found. Similarly, cell viability and proliferation after expression of both GH peptides in AtT-20 cells were identical. Quantitative confocal microscopy analysis revealed no significant difference in the extent of subcellular colocalization between wt-GH and GH-R77C with endoplasmic reticulum, Golgi, or secretory vesicles. Furthermore studies demonstrated a reduced capability of GH-R77C to induce GHR/ GHBP gene transcription rate when compared with wt-GH. Conclusion: Reduced GH receptor/GH-binding protein expression might be a possible cause for the partial GH insensitivity with delay in growth and pubertal development found in our patients. In addition, this group of patients deserves further attention because they could represent a distinct clinical entity underlining that an altered GH peptide may also have a direct impact on GHR/GHBP gene expression causing partial GH insensitivity. (J Clin Endocrinol Metab 92: 2893–2901, 2007)

(7, 8). The kinases phosphorylate the receptor, cross-phosphorylate themselves, and start signal transduction by phosphorylating downstream signal transduction molecules, including signal transducer and activator of transcription (Stat)-5 (9, 10). Activated Stat5 is translocated in the nucleus in which it transactivates a series of GH-responsive genes (11). Short stature associated with bioinactive GH is clinically characterized by a normal or slightly increased GH secretion, pathologically low IGF-I levels, and normal catch-up growth on GH replacement therapy (12). Although overall incidence of short stature caused by bioinactive GH is low, several reports suggested the presence of Kowarski’s syndrome (13– 17). Six GH variants were reported to be bioinactive by Millar et al. (18). Takahashi and colleagues (19, 20) described severe short stature in a boy due to a single mutation converting codon 77 from arginine (R) to cysteine (C; GH-R77C). However, the patient’s father was of normal stature, even though he was carrying the same heterozygous mutation. Chihara and colleagues (19, 20) reported that the affinity of this mutant GH for the GH-binding protein (GHBP) was 6 times higher than that of wt-GH and that mutant GH not only failed to stimulate tyrosine phosphorylation by itself but also inhibited the activity of the wt-GH. Recombinant GH therapy used in this study was not sufficiently effective in stimulating somatic growth of this patient. These data together with the data of Takahashi and colleagues (19, 20) led the authors to

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conclude that the effect that GH-R77C mutant exerts is more of an antagonistic nature. Furthermore, Deladoey et al. (21) studied the effect of GH-R77C on GHR/GHBP gene transcription and clearly showed, compared with wt-GH, a reduced capability of GH-R77C in stimulation of GHR/GHBP gene transcription. In this study, we describe a pedigree over three generations of a Syrian family in which all affected subjects carried the same heterozygous GH-R77C mutation. Our patient presented with short stature (⫺2.5 sd score) at the age of 6 yr caused by partial GH insensitivity. Because all these abnormalities were also present in the other affected family members carrying the GH-R77C allele but reached a normal final height within the parental target height after a reported delay of growth and pubertal development in childhood, we focused on the functional analysis of this GH-R77C mutant form. Subjects and Methods Patient and family The patient, of Syrian (Kurd) ancestry, immigrated to Switzerland at the age of 6 yr. He presented at this time with short stature (⫺2.5 sd score for age and sex) (22). The pedigree of his nonconsanguineous family is presented in Fig. 1A. Standard auxological assessment was performed (23). He had no evidence of an organic disease, psychological deprivation, or any eating disorder, and renal and hepatic functions were normal. A standard pharmacological stimulation test (insulin-induced hypoglycemia) was performed (24). Auxological and laboratory data are depicted and presented in Fig. 2 and Table 1, respectively. Genetic analysis revealed a normal GHR gene (all the exons, exon/intron bound-

FIG. 1. A, A three-generation pedigree of the patient’s family is shown, with affected individuals indicated by open symbols containing smaller solid symbols. The adult height SD score is reported. DNA sequencing revealed that the affected subjects presented heterozygosity for the R77C transition mutation of the GH-1 gene. The R77C mutation was screened for, but was not detected, in 100 alleles from unrelated normal individuals. B, Isoelectric focusing of GH in serum from the patient III:3. The serum fractions were pooled separately and assayed for GH immunoreactivity. The pH gradient formed during isoelectric focusing is indicated. The peaks for wild-type and mutant GH are indicated by the open and solid arrows, respectively. C, Isoelectric focusing of GH in serum from the patient II:3.

Petkovic et al. • Impact of GH-R77C on GH Sensitivity

aries were sequenced), but a familial R77C heterozygous mutation of the GH-1 gene was found. Combined with the clinical findings as well as laboratory data, an autosomal dominant growth disorder was diagnosed. However, because the other genetically affected family members were of normal adult stature and, at follow-up of this affected boy, the height velocity remained in the normal range for his family, no GH treatment was initiated, but he was followed up through puberty, which was delayed, allowing at that time the clinical diagnosis of delay of growth and pubertal development. Finally, at the age of 20 yr, he reached the final height of 167.2 cm (⫺1.9 sd score), was in good health, and his school performance was normal. Informed written consent was obtained from the patient, subjects examined, and the parents of the patients enrolled in this study.

Isoelectric focusing Isoelectric focusing was performed as described previously (25). Serum samples (200 –300 ␮l) were electrofocused in a buffer containing 1% hydroxypropyl methylcellulose and 4% ampholine (pH gradient, 3.5– 8.0) at 200 V for 12 h and then at 500 V for 12 h. The fractions were collected and assayed for immunoreactive GH. Pooled serum samples from seven normal subjects were used as the control.

Cell culture and treatment Mouse pituitary (AtT-20/D16v-F2) cells were purchased from American Type Culture Collection (Manassas, VA) and cultured in DMEM (4.5 g/liter glucose) supplemented with 10% heat-inactivated fetal calf serum (FCS; Life Technologies, Invitrogen AG, Basel, Switzerland) and 100 U/liter penicillin/streptomycin. This F2 subclone was developed from the original AtT-20 cells, an ACTH-secreting cell line established from murine pituitary tumor. Chinese hamster ovary (CHO-K1) cells were a gift from Professor U. Wiesmann (Inselspital, Bern, Switzerland) and were cultured in Ham’s F12 medium (Biochrom AG Seromed, Berlin, Germany) supplemented

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termined by the DSL-10 – 48100 Active* human GHBP ELISA kit (Diagnostic Systems Laboratories).

Receptor binding assay Receptor binding assays were performed using 293HEK cells stably expressing human GHR (293GHR) (12, 27–29). Three independent experiments were performed in triplicate wells, and IC50 values for the different GH peptides were determined by nonlinear regression, using a single-site competition model (GraphPad Prism software, version 2.0; GraphPad, San Diego, CA). Identical experiments were performed using IM-9 as well as HuH7 cells, cells expressing endogenous GHR (19, 21).

Luciferase reporter gene assay of Stat5 activation

FIG. 2. Growth chart of the affected boy (III:3). Adult height of mother and father as well as adult target height (arrows: mean value; 2 SD; extreme right) are given. Percentiles are shown on the extreme right. The solid circles indicate the height measurements, the open circles, the bone ages. The arrows pointing up indicate P2 (pubic hair stage 2) and T:4 ml (testicle volume of 4 ml), respectively. with 10% FCS, 100 U/liter penicillin/streptomycin (Biochrom AG), and 2 mm l-glutamine (Life Technologies, Inc.-BRL, Basel, Switzerland). Human embryonic kidney (HEK) 293 cells stably expressing human GHR (293GHR) were grown in DMEM-nutrient mix F12 (DMEM Nut F12; Life Technologies), supplemented with 10% FCS, 100 U/liter penicillin/streptomycin, 2 mm l-glutamine, and 400 ␮g/ml Geneticin G418 (Promega Corp., Madison, WI). HuH7 is a human hepatoma cell line that is reported to retain differentiated functions in culture and therefore allows functional studies of GHR. The cells were cultured as previously described (21). IM-9 cells, human lymphoblasts, generously provided by Professor U. Wiesmann were maintained at 37 C. in Eagle’s MEM supplemented with 10% FCS.

Expression vectors Wild-type GH was cloned in pcDNA3.1 (⫺) neo (Invitrogen, La Jolla, CA) vector as described (26). To generate the GH mutant studied (GHR77C), site-directed mutagenesis was performed using the QuickChange site-directed mutagenesis kit from Stratagene (Basel, Switzerland).

Production of GH peptides To produce GH variants, wt-GH, and GH-R77C, stable clones were generated in Chinese hamster ovary cells (CHO-K1) by transfection with FuGene 6 (Roche Diagnostics AG, Rotkreuz, Switzerland). The concentration of the GH produced by the CHO cells during 3 d in Ham’s F12 plus 2% FCS media was measured by the DSL-GH ELISA kit (Diagnostic Systems Laboratories, Webster, TX). To confirm that the mutation R77C does not affect the affinity of the antibody used in the DSL-ELISA, additionally two different GH assays were performed on two samples of CHO supernatant and the results were compared (12).

Hormonal measurement The serum GH was measured by the DSL-10 –1900 Active* human GH ELISA kit (Diagnostic Systems Laboratories), IGF-I and IGF-binding protein (IGF-BP)-3 measurements were performed using the IGF-I immunoradiometric assay (radioisotopic assay kit; Nichols Institute Diagnostics, San Juan Capistrano, CA) and the IGF-binding protein-3 RIA kit (Nichols Institute Diagnostics), respectively. Serum GHBP was de-

293GHR cells were used to assay Stat5 activation as described before (12, 28, 29). Briefly, cells were transfected with a Stat5-responsive luciferase reporter gene construct (30, 31) and treated with increasing amounts of GH (rhGH, wt-GH, and GH-R77C) for 6 h. Luciferase expression was then measured with a luminometer (Mediators PhL; Aureon Biosystems, Vienna, Austria). EC50 values were obtained from sigmoidal dose-response curves generated using GraphPad Prism software 2.0. Recombinant human (rh) GHBP was a gift from Terri Smith (Genentech, San Francisco, CA). Identical experiments were performed using IM-9 as well as HuH7 cells.

Antibodies Polyclonal rabbit antihuman GH antibodies were purchased from ICN Pharmaceuticals, Inc. (Eschwege, Germany). Rat monoclonal antiGrp94 [endoplasmic reticulum (ER)] antibodies were purchased from MBL through LabForce AG (Nunningen, Switzerland). Mouse monoclonal anti-Golgi (anti-␤COP) and mouse monoclonal anti-Rab3a (secretory granules) antibodies were purchased from Sigma Aldrich (St. Louis, MO) and Synaptic Systems (Go¨ttingen, Germany), respectively. All conjugated secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. and Molecular Probes (West Grove, PA).

Cell preparation and immunofluorescence staining AtT-20 cells were grown on slide flask Nunc-␦, article Nunc170920 (Falcon; Nalge Nunc International, Naperville, IL), and were transfected with 1 ␮g of the wt-GH or GH-R77C using FuGene 6 (Roche Diagnostics AG). Twenty-four hours later immunofluorescence staining was performed after quantitative colocalization analysis as described (32). The mean fluorescence intensity of 20 cells was measured for each organelle and GH.

Nucleofection and AtT-20 cell proliferation assay The impact of expression of the wt-GH or GH-R77C on AtT-20 cell viability was assessed by cell proliferation assays after transient transfection as described by Salemi et al. (32).

Structure of GH-R77C To compare and study the chemical structure of GH-R77C, wt-GH was extracted and purified from somatrope cell material (Anti-Doping Laboratory, Cologne, Germany) and GH-R77C from the cell culture supernatant as described. The extracts were subjected to solid-phase extraction using prepacked Oasis HLB C-18 cartridges (Waters, Milford, MA) and subsequently hydrolyzed using sequencing grade porcine trypsin (Promega). The samples were applied to a capillary liquid chromatograph (Agilent Technologies, Waldbronn, Germany) interfaced to an LTQ-Orbitrap mass analyzer (Thermo Electron, Bremen, Germany). After gradient elution, full-scan mass spectrometry was performed using the LTQ-Orbitrap at a resolution of 30,000, and product ion spectra were recorded using the LTQ linear ion trap only. In addition, GH-R77C was derivatized using acrylamide under nonreducing conditions and subsequently trypsinized, and additional alkylation studies were performed and the disulfide bonds were structurally analyzed.

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TABLE 1. Auxological and laboratory data Basal values Age (yr)

Ht-vel (cm/yr) 关SDS兴

IGF-I (ng/ml) 关SDS兴 (control: range)

IGF-BP3 (mg/liter) 关SDS兴 (control: range)

GHBP (pmol/liter) (control: range)

6

389 关0.1兴 (163–584)

5.2 关0.2兴 (3.1–7.9)

3790 (534 –5785)

28

45 关⫺2.2兴 (52–297) 51 关⫺2.1兴 (57–316) 39 关⫺2.5兴 (57–316) 189 关⫺3.1兴 (226 –902) 127 关⫺2.7兴 (193–731)

2.4 关⫺0.5兴 (1.3–5.6) 2.6 关⫺0.4兴 (1.4 – 6.1) 2.2 关⫺0.9兴 (1.4 – 6.1) 2.4 关⫺1.0兴 (1.7– 6.2) 4.8 关⫺0.8兴 (3.2– 8.7)

ITT Peak GH (ng/ml)

IGF-I (ng/ml) (control: range*)

GHBP (pmol/liter)

253

124* (205–758)

724*

317 (267–935)

1215

Ht (cm) 关SDS兴

Wt (kg) 关SDS兴

157.8 关⫺1.2兴

51 ⫺0.9

107.1 关⫺2.5兴

15.9 关⫺2.3兴

6.5

109.8 关⫺2.6兴

16.4 关⫺2.6兴

5.4 关⫺1.3兴

7

112.0 关⫺2.3兴

17.8 关⫺2.8兴

4.4 关⫺3.3兴

5.75

36

16

152.1 关⫺3.3兴

32.2 关⫺3.8兴

6.1 关⫹1.8兴

12.5

39

20

167.2 关⫺1.9兴

58.2 关⫺0.9兴

0.5

168.2 关⫺1.5兴

68.0 关0.5兴

4

180 关0.0兴 (109 –284)

4.6 关⫺0.2兴 (3.4 – 6.7)

2341 (556 –3944)

156.1 关⫺1.4兴

57.8 关0.3兴

32

261

189*/322 (201– 873)

803*/2140

71 关0.4兴

26

3.6 关⫺1.9兴 (3.5–7.6) 1.5 关⫺1.7兴 1.4 –3.2

461 (750 – 4868)

167.7 关⫺1.7兴

108 关⫺2.1兴 (115–307) 37 关⫺2.3兴 (49 –200)

316 (560 –2559)

242

96*/254 (186 –714)

741*/1896

III:1 18 III:3 6

II:4 36 II:3 32 I:1 64

Bone age (yr)

After IGF-I generation test

GH (ng/ml)

5.0

30

35

129 (320 –3820) 132 (320 –3820)

Two IGF-I generation tests (a, daily injection of rhGH of 0.1 IU/kg/d and 0.2 IU/kg for 4 and 3 d; b, rhGH of 0.2 IU/kg/d for 7 d) were performed. Asterisks indicate values obtained with test a, and the IGF-I control values stated were standardized to test a as well. The normative data of the IGF-I generation test stated were obtained by the analysis of n:6 (males and females) of various age groups: less than 10 yr, 10 –17 yr, 18 – 40 yr, and more than 40 yr. SDS, SD score; Ht, height; Wt, weight; vel, velocity.

Statistical analysis The statistical significance in bioassay (testing bioactivity through GHR binding and activation of Jak2/Stat5 pathway) was assessed using the nonparametric Mann-Whitney test, whereas statistical analyses for confocal studies were performed using ANOVA one-way test plus Dunnett’s multiple comparison tests comparing GH-R77C to wt-GH.

Results Clinical diagnosis and follow-up

Because this family entered Switzerland as refugees, no exact data on birth weight and length are available. However, the parents reported no abnormalities in his psychomotor development. Because the clinical phenotype as well as the laboratory finding of rather high basal GH concentration, high peak GH level after provocation test, low basal IGF-I concentration in this 7-yr-old boy suggested partial GHinsensitivity (Table 1), the GHR gene was sequenced, and an IGF-I generation test was performed (33). This detailed assessment, however, presented a normal sequence of the GHR gene (exons, exon/intron boundaries) but only a subnormal IGF-I (124 ng/ml) response after an IGF-I generation test (daily injection of rhGH of 0.1 IU/kg䡠d and 0.2 IU/kg for 4 and 3 d, respectively), and therefore the possibility of a func-

tionally altered GH affecting GHR binding/signaling was hypothesized. The sequencing of the GH-1 gene revealed a heterozygous R77C transition mutation, which previously was reported to have some GH antagonistic effect (19, 20). Therefore, the whole family, including his unaffected father and sister and affected mother as well as grandfather, was clinically studied in more detail (Fig. 1A and Table 1). Importantly, isoelectric focusing revealed the presence of an abnormal GH peak in addition to a normal peak (Fig. 1B, III:3; Fig. 1C, II:3) in the boy as well as the mother. In this family the presence of GH-R77C correlated with the delay in growth and pubertal development (mother menarche at the age of 18 yr) but did not present a clear phenotype in terms of short stature/final height. In contrast, at the hormonal level, a slight GH insensitivity was hypothesized, and therefore, further studies were performed to define the effect of GH-R77C at the cellular level in more detail. In addition, in the boy (III:3) at the age of 16 yr, the IGF-I generation test was repeated. However, the dose of rhGH was increased to 0.2 IU/kg for 7 d. The IGF-I response could be significantly increased from a subnormal value (189 ng/ml) to 317 ng/ml, which is accepted as normal (Table 1) (33). Importantly, under these circumstances GHBP increased as well (Table 1).

Petkovic et al. • Impact of GH-R77C on GH Sensitivity

In addition, in affected subjects (II:3; I:1), two IGF-I generations tests were performed [1) daily injection of rhGH of 0.1 IU/kg䡠d and 0.2 IU/kg for 4 and 3 d; and 2) rhGH of 0.2 IU/kg䡠d for 7 d], confirming the results obtained in III:3 (Table 1). Functional characterization of GH-R77C through GHR binding and activation of Jak/Stat pathway

Receptor binding studies were performed to determine the affinity of wt-GH, rhGH, and GH-R77C for the GHR (Fig. 3). The IC50 values for the wt-GH, rhGH, and GH-R77C were found to be 21.2, 12.2, and 14.4 ng/ml, respectively. No significant difference was observed between the IC50 values from wt-GH and rhGH and from wt-GH and GH-R77C. To investigate in more detail the bioactivity of GH-R77C, 293GHR cells stably expressing GHR were used. Furthermore, a Stat5-responsive luciferase reporter gene assay system (28, 29) performed in these cells, which requires all stages of the Jak2/Stat5 signaling pathway to be functional, was used to assay and quantify signal transduction activity of GH-R77C. The ability of the mutant to activate Jak2/Stat5 pathway was comparable with that of wt-GH and rhGH (Fig. 4A). EC50 values of wt-GH, rhGH, and GH-R77C were 44.4, 31.9, and 24.1 ng/ml, respectively. As depicted in Fig. 4B, the effect of 50 ng/ml wt-GH (wt-GH 50) on activation of the Jak2/Stat5 pathway is similar to 50 ng/ml rhGH (rhGH 50) as well 50 ng/ml GH-R77C (R77C 50). The stimulation of the 293GHR cells with 25 ng/ml of rhGH (rhGH 25) led to a significant decrease (P ⬍ 0.01) of the Jak2/Stat5 pathway activation, compared with stimulation with rhGH 50 and wt-GH 50. This decrease can be entirely reestablished with the addition of 25 ng/ml GHR77C (R77C25/wt-GH25). Furthermore, the costimulation of wt-GH and GH-R77C, both at 50 ng/ml (R77C50/wt-GH50) showed no statistical difference when compared with the stimulation with rhGH 100 ng/ml (rhGH 100), which showed, as expected, a significant increase in Jak2/Stat5

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activation, compared with wt-GH 50 ng/ml. Three negative controls were used in this experiment: PSA, corresponding to the supernatant of CHO cells transfected with pSecPSA; empty, representing the supernatant of CHO cells transfected with an empty pSec plasmid; and NT, which is the supernatant of nontransfected CHO cells. To investigate the possible impact of GHBP on activation of Jak2/Stat5 signaling pathway evoked by GH-R77C or wt-GH, variable concentrations of recombinant GHBP (20, 50, and 100 ng/ml) were added to the GH-R77C (R77C/ GHBP 20, R77C/GHBP 50, R77C/GHBP 100) or the wt-GH (wt-GH/GHBP 20, wt-GH/GHBP 50, wt-GH/GHBP 100), both GH peptides at concentration of 50 ng/ml. As shown in Fig. 4C, the activation of the Jak2/Stat5 pathway was decreased in a dose-dependent manner after adding increased amounts of GHBP (r ⫽ 0.755 for wt-GH and GHBP, r ⫽ 0.805 for GH-R77C and GHBP), and no significant differences were observed between GH-R77C and wt-GH at any of the different GHBP concentrations used. Of note is that Western blots performed with the supernatant from CHO cells containing either wt-GH or GH-R77C confirmed that the sizes of both GH variants were the same, 22 kDa (data not shown). Taken together, these functional assays indicate that wt-GH and GH-R77C bind to the GHR with almost equal affinity and that they are equally potent in activation of the Jak2/Stat5 signaling pathway. Identical experiments were performed using IM-9 and HuH7 cells, naturally expressing GHR. Importantly, these data confirmed the results obtained using 293GHR cells in all the experiments, suggesting the direct effect of GH-R77C on GHR expression (21). Bearing in mind that the expression of GHR in IM-9 and HuH7 cells, compared with HEK293 cells stably transfected with GHR, was rather low, and therefore, the differences obtained (mean ⫾ sd) were small, GH-R77C decreased GHR binding insignificantly by 8 ⫾ 1.2% (IM-9) and 10 ⫾ 1.1% (HuH7) but signaling significantly by 12 ⫾ 1.0% (IM-9, P ⬍ 0.05) and 11 ⫾ 0.8% (HuH7, P ⬍ 0.05) at the dose of 50 ng/ml when compared with wt-GH. Furthermore, after GH-R77C addition, expression of GHR/GHBP was significantly decreased by 14 ⫾ 1.2% (IM-9, P ⬍ 0.05) and 17 ⫾ 1.1% (HuH7, P ⬍ 0.01) when 50 ng/ml of either GH-R77C or wt-GH were added. Structural analysis of GH-R77C

FIG. 3. Competitive displacement of 125I-GH binding to the GHR by rhGH (positive control), wt-GH, and GH-R77C. Results are normalized to the amount of 125I-GH bound in the absence of any unlabeled GH. Each point is the mean of three separate experiments performed in triplicate ⫾ SD.

Because the additional cysteine at position 77 may affect the various disulfide bonds and therefore change the structural conformation, the structural analysis of the GH-R77C was performed. Besides the mutated amino acid at codon 77 (arginine to cysteine), the structural analysis of GH-R77C revealed to be similar to wt-GH. Furthermore, the peptide comprising the modified position (C77) was found to be normally alkylated, indicating that the free sulfydryl residue is not involved in other intramolecular disulfide bonds, although it has been reported that the alkylation of cysteines involved in disulfide bonds may occur up to a very minor extend (34).

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FIG. 4. A, Jak2/Stat5 signaling capacity of rhGH, wt-GH, and GH-R77C. Results are expressed as x-fold induction relative to the basic activity of unstimulated cells and represent the means ⫾ SD of three separate experiments performed in duplicate. B, Synergistic effect of the costimulation of the 293GHR cells with wt-GH and GH-R77C [n ⫽ 34 (wt-GH 50, R77C 50), n ⫽ 14 (rhGH 50, rhGH 100), n ⫽ 12 (R77C 25/wt-GH25, R77C 50/wtGH50, PSA, empty, NT), n ⫽ 6 (rhGH 25)]. PSA, empty, and NT are all negative controls. Results are expressed as percentage of wt-GH activity. The concentrations are in nanograms per milliliter. **, P ⬍ 0.01; *, P ⬍ 0.05. Statistical significance was assessed using the nonparametric test Mann-Whitney. C, Relative ability of GH-R77C to activate GHRmediated Jak2/Stat5 pathway in the presence of recombinant GHBP. The GH concentration for both wt-GH and GHR77C is 50 ng/ml, and the results are given as percentage of wt-GH activity. [n ⫽ 34 (wt-GH and GH-R77C), n ⫽ 16 (wt-GH/GHBP 20, R77C/GHBP 20, wtGH/GHBP 100, R77C/GHBP 100), n ⫽ 12 (wt-GH/GHBP 50, R77C/GHBP 50)]. No statistical differences were found when comparing wt-GH and GH-R77C at the same concentration of GHBP added. wtGH, Wild-type GH; PSA, pSecPSA plasmid; empty, empty pSec plasmid; NT, nontransfected CHO cells.

Intracellular localization of wt-GH and GH-R77C analyzed by confocal microscopy

Although the structural analysis of the GH-R77C secreted from mammalian cells (CHO cells) did not reveal any obvious conformational alteration, which is not unexpected in contrast to the data reported using Escherichia coli (20, 35, 36), it does not demonstrate that a conformationally altered GHR77C present at the intracellular level may not affect the regulated secretory pathway. Therefore, to analyze the impact of GH-R77C on the secretory pathway, colocalization of the wt-GH or GH-R77C with different compartments within the secretory pathway was studied in AtT-20 cells, mouse pituitary gland, applying quantitative confocal microscopy analysis (Fig. 5). AtT-20 cells were transiently transfected with either wt-GH or GH-R77C, and the expression was examined 24 h after transfection. The transfection efficiency was checked by EGFP-N1 (enhanced green fluorescent plasmid) and was found to be consistent among different experiments. Figure 5 (row I) shows the overall distribution of wt-GH and GH-R77C, with a perinuclear region having the heaviest staining, which is consistent with accumulation in the ER and Golgi complex and in a punctuate staining near the plasma membrane, being consistent with accumulation of GH peptide in secretory granules. Costaining with antibodies against markers for ER (anti-Grp94), Golgi (anti␤COP), and/or granules (anti-Rab3a) were performed, and confocal microscopy images of independent cells were taken. Fluorescent intensities of colocalized areas were measured for wt-GH and GH-R77C with each organelle. The degree of colocalization of two fluorochrome signals was measured

from a single cell. The average Pearson correlation coefficients did not reveal any significant difference in the extent of subcellular colocalization between wt-GH and GH-R77C in any of the organelles analyzed. These results suggest that these two GH peptides pass through the very complex secretory machinery in almost identical manner. Cell proliferation assay

To support and complement the secretion studies, which showed no difference in secretion between wt-GH and GH-R77C, the cell proliferation assay was performed. In AtT-20 cells transiently transfected with either wt-GH or cotransfected with wt-GH and GH-R77C, cell proliferation was compared 24, 48, 72, and 96 h after transfection (Fig. 6). Using EGFP-N1 as a positive control, the transfection efficiency in viable AtT-20 cells was checked. Throughout the 4-d time course, the proliferation rate of cells expressing wt-GH was equivalent to that of cells expressing wt-GH and GH-R77C (Fig. 6). As expected and in line with the secretion data, when expressed in AtT-20 cells, GH-R77C did not exert any effect different from that of the wt-GH in terms of cell viability and/or proliferation. In addition to cell proliferation of AtT-20 cells, we checked whether the expression GH-R77C accelerated the apoptosis of these cells, compared with wt-GH. After investigating DNA fragmentation by propidium iodide staining, we did not observe any significant difference in AtT-20 cells transfected either with wt-GH or cotransfected with wt-GH and GH-R77C (data not shown).

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FIG. 5. Subcellular colocalization of wt-GH and GH-R77C with different organelle markers in fixed cells. A, GH and Grp94 (ER marker) colocalization. B, GH and ␤COP (Golgi marker). C, GH and Rab3a (secretory granules). Row I, Specific staining for wt-GH or GH-R77C is indicated by fluorescent green color (FITC labeled). Row II, Positive staining for cell organelles is shown by fluorescent red color (Cy3 labeled) in the same microscopic field as in row I. Row III, Merged images. Row IV, Masked areas, in which the intensity of the green and red fluorescence was measured in the colocalized area. Below each set of confocal images, the degree of colocalization between GH and that given organelle is presented as the average of Pearson correlation coefficients for 20 independent cells.

Discussion

A heterozygous missense mutation in the GH-1 gene converting codon 77 from R to C was identified in our patient. Apart from the affected patient, his mother and grandfather were also carrying the same mutation. However, they did not differ in terms of phenotype (final height) but in terms of laboratory values (GH insensitivity) from the other unaffected family members (Fig. 1A and Table 1). The presence of both wt-GH and GH-R77C in the patient’s as well as

FIG. 6. Cell proliferation was assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. AtT-20 cells were transiently transfected with either wt-GH or GH-R77C and empty pcDNA 3.1 vector as control. Proliferation was estimated at 24, 48, 72, and 96 h after transfection. Results are expressed as mean OD of four independent experiments in triplicate (means ⫾ SEM).

affected family members’ sera have been confirmed by isoelectric focusing (Fig. 1, B and C). Therefore, our clinical findings do not support the previously reported hypothesis that the presence of GH-R77C in circulation correlates with the severe short stature (19, 20). The competition binding experiments performed in cells, stably expressing GHR on their surface (293GHR), revealed no difference in binding affinity for the GHR between wt-GH and GH-R77C. These results are in line with previously published data analyzing binding of [125I]hGH to GHR without the addition of GHBP in IM-9 cells, lymphoblasts expressing GHR, which was inhibited by wt-GH and GH-R77C in not only a dose-dependent manner but also a similar way (19, 20). Furthermore, functional characterization of this mutant was pursued by bioassay. The efficiency of this assay focusing on the GH Jak2/Stat5 signaling pathway was assessed first by stimulation of the 293GHR cells with increasing amounts of rhGH. Effectively, the production of Firefly luciferase increased proportionally to the concentrations of rhGH added in the system (data not shown). Moreover, the activity of the wt-GH produced by the CHO cells was comparable with that of the rhGH, both at the same concentration (Fig. 4). The bioactivity of the GH-R77C showed no significant difference to that of the wt-GH. Moreover, the costimulation of wt-GH and GH-R77C underlined their synergistic effect because the decrease in activation of the Jak2/Stat5 pathway evoked by 25 ng/ml wt-GH could be perfectly reestablished after the addition of 25 ng/ml of GH-R77C. In addition, no statistical difference was found between the costimulation of the 293GHR cells with wt-GH and GHR77C, both at 50 ng/ml (R77C50/wt-GH50) and the stimu-

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lation with rhGH 100 ng/ml. These results demonstrate a similar bioactivity of the GH-R77C, compared with wt-GH. The addition of rhGHBP to GH-R77C or wt-GH could not underline increased affinity of the mutant GH for GHBP, compared with wt-GH. The activation of the Jak2/Stat5 pathway was decreased in a dose-dependent manner after adding increasing amounts of GHBP to the GH-R77C and the wt-GH, both being present at the same concentration (Fig. 4C). This diminution observed was the same for both wt-GH and GHR77C. These findings are in contrast to the previously reported data showing approximately 6-fold higher affinity of GH-R77C to GHBP, compared with wt-GH (19, 20). These differences in the binding affinity of GH-R77C for GHBP between our and previous studies may be explained by the different cell lines used (IM-9 cells: lymphoblasts endogenously expressing GHR vs. 293HEK cells: stably GHR transfected cells) as well in the sensitivity of the methods applied (luciferase reporter gene assay of Jak2/Stat5 signaling activity vs. immunoprecipitation). Of note is to mention that the other functional studies we performed, especially the measurement of GH-R77C bioactivity, complement and clearly support the competition binding data of GH-R77C we obtained. In a previous study reported by our group, Deladoey et al. (21) analyzed the impact of the different GH mutants, including GH-R77C, on GHR/GHBP gene transcription in a human hepatoma cell line (HuH7 cells). It was observed for the GHR transcription rate to be significantly reduced by GH-R77C when compared with that of the wt-GH but still significantly higher than that of the genetically engineered GHR antagonist, pegvisomant. In addition, these data were confirmed by run-on experiments using cyclohexamide, the inhibitor of protein synthesis, which did not effect these changes, supporting the notion that the GHR/GHBP gene transcription was directly stimulated and depended, at least partially, on some preexisting factors. To study the suggested direct effect of GH-R77C on signaling and binding to GHR in a cell system in which GHR expression could be induced, similar binding and Jak2/Stat5 signaling studies were performed in IM-9 and HuH7 cells, endogenously expressing GHR, as described in the 293HEK cells, stably transfected with GHR. Although, in comparison with 293GHR cells, the GHR expression rate was low in IM-9 and HuH7 cells, 50 ng/ml GH-R77C decreased GHR/GHBP expression by 14% (IM-9) and 17% (HuH7) and subsequently signaling by 12% (IM-9) and 11% (HuH7), compared with wt-GH at the same dose. The production of abundant quantities of peptide hormones is one of the major tasks of peptide-secreting neuroendocrine cells. The secretory pathway consists of a series of membrane-bound compartments through which proteins targeted for secretion are bound (37–39). Although we found that the structure of GH-R77C secreted was similar to the wt-GH and that the possibility of an additional disulfide bond involving cysteine 77 is rather unlikely in mammalian cells, a structurally altered GH peptide might affect intracellularly its secretory pathway. Therefore, we compared the association of either wt-GH or GH-R77C with different compartments within the secretory pathway, namely ER, Golgi, and secretory vesicles by costaining with antibodies against

Petkovic et al. • Impact of GH-R77C on GH Sensitivity

GH and for one of the organelles mentioned. Our results revealed no significant difference in colocalization of either wt-GH or GH-R77C with ER, Golgi, or secretory vesicles. The secretion studies were complemented with cell proliferation experiments showing that the viability and proliferation of AtT-20 cells did not change upon transfection with either wt-GH or cotransfection with wt-GH and GH-R77C. Importantly, these molecular data may help to explain the clinical findings. On a clinical and the protein basis, in the affected subjects the GHBP was significantly decreased (P ⬍ 0.001), compared with the normal controls. Furthermore, because the GHR was sequenced and found to be normal, the reduced GHR/GHBP expression caused by the GH-R77C mutant might be responsible for causing GH insensitivity. This possible direct impact of GH-R77C at the GHR/GHBP level, also still unclear in its mechanism, is underlined by the normalization of IGF-I response when rhGH dose was increased from 0.1 IU/kg䡠d to 0.2 IU/kg during the period of 7 d. It is well known that under normal physiological conditions, GHBP is bound to almost half of GH in human plasma (40, 41), acting as a reservoir of GH and thereby prolonging its half-life. GHBP is generated by proteolytic cleavage of GHR at the cell surface and modulation of GHR turnover/ internalization might impact the level of GHR available for proteolysis. Therefore, one could hypothesize that the reduced GHR/GHBP gene transcription rate evoked by GHR77C, compared with the wt-GH, may lead to a reduced amount of GHR, which are going to be produced and exported at the cell surface. Consequently, that might affect the level of GHBP generated by proteolytic cleavage of GHR and might explain the low levels of GHBP found in our patient carrying GH-R77C mutation. In conclusion, we found a heterozygous missense mutation, R77C in the GH molecule in a patient with growth retardation and delayed pubertal development. However, no differences between wt-GH and GH-R77C were found by functional characterization of the GH-R77C through GHR binding, activation of Jak2/Stat5 pathway, and additional secretion studies together with cell proliferation when stably GHR transfected cells (293GHR) were used. On the other hand, reduced capability of GH-R77C to directly induce GHR/GHBP gene transcription rate could indirectly affect the levels of GHBP in the circulation of our patient. In addition, this group of patients deserve further attention because they could represent a distinct clinical entity underlining that an altered GH peptide may cause partial GH insensitivity through direct impact on GHR/GHBP gene expression, leading to the delay of growth and pubertal development. Finally, GH-R77C is not invariably associated with short stature, although the serum IGF-I levels are low, the GH is elevated, and the GHBP levels are somewhat low, consistent with some degree of GH insensitivity, which is, presumably, compensated for by excess of GH production. Whether this is due to GHR transcription defects remains unclear. Acknowledgments We thank Dr. Terri Smith from Genentech Inc. (San Francisco, CA) for generously providing us with recombinant human GHBP. Furthermore,

Petkovic et al. • Impact of GH-R77C on GH Sensitivity

we are very grateful to Ron G. Rosenfeld, M.D., for his constructive comments and suggestions. Received October 13, 2006. Accepted May 15, 2007. Address all correspondence and requests for reprints to: Professor Dr. Primus E. Mullis, University Children’s Hospital, Pediatric Endocrinology, Inselspital, CH-3010 Bern, Switzerland. E-mail: [email protected]. This work was supported by Swiss National Science Foundation Grant 3200B0-105853/1 (to P.E.M.). Disclosure Statement: The authors have nothing to disclose.

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22. 23.

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