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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2003-2133
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 1 542-547
Copyright © 2005 by The Endocrine Society

A Novel C-Terminal Growth Hormone Receptor (GHR) Mutation Results in Impaired GHR-STAT5 But Normal STAT-3 Signaling

Anatoly Tiulpakov, Petr Rubtsov, Ivan Dedov, Valentina Peterkova, Olga Bezlepkina, George P. Chrousos and Ze’ev Hochberg

Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health (A.T., G.P.C.), Bethesda, Maryland 20892; Endocrinological Research Center (A.T., I.D., V.P., O.B.), Moscow 115478, Russia; Engelhardt Institute of Molecular Biology, Russian Academy of Sciences (P.R.), Moscow 117984, Russia; and Meyer Children’s Hospital, Rambam Medical Center (Z.H.), Haifa IL-310961, Israel

Address all correspondence and requests for reprints to: Anatoly Tiulpakov, M.D., Ph.D., Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, 10 Center Drive, Building 10, Suite 9D42, Bethesda, Maryland 20892. E-mail: tiulpaka{at}mail.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
GH insensitivity (GHI) is an autosomal recessive disorder caused by defects in the GH receptor (GHR). In a 17-yr-old female with severe short stature and biochemical features of GHI, sequencing of GHR gene revealed a compound heterozygosity for two novel mutations: C83X and a G deletion at position 1776 (1776del). 1776del is predicted to result in GHR truncation to 581 amino acids with a nonsense sequence of residues 560-581. To clarify the effect of 1776del on GHR function, wild-type GHR, GHR-1776del, and two additional GHR mutants, GHR-L561X (stop codon at site of the 1776del) and GHR-I582X (translation termination in GHR-1776del) were transiently expressed in CHO cells. After incubation with recombinant human GH, GHR-1776del showed lower signal transducer and activator of transcription 5 (STAT5)-mediated transcriptional activation (~50%, P < 0.05), as well as STAT5 Tyr694 phosphorylation (P < 0.05) compared with wild-type GHR, whereas GHR-L561X and GHR-I582X showed normal STAT5 phosphorylation and transcriptional activity. In contrast, all vectors produced similar effects on STAT3-mediated transcriptional activation. In conclusion, this novel GHR-1776del mutation in a classical GHI patient illustrates an important mechanism of impaired GHR-STAT5 but intact GHR-STAT3 signaling. This effect might result from interference of C-terminal nonsense sequence in mutated GHR with STAT5 docking to upstream tyrosine residues.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
GH INSENSITIVITY SYNDROME (GHIS), also known as Laron syndrome (1), is a rare congenital disorder presented with severe postnatal growth failure due to defective GH receptor (GHR) function.

GHR is a 620-residue, single membrane-spanning protein that belongs to the large family of cytokine receptors. Part of GHR corresponding to its extracellular domain is cleaved and present in the circulation as GH binding protein (GHBP) (2). GH signaling is initiated by sequential binding of two distinct sites of the GH molecule to two GHR monomers, which is followed by receptor dimerization and activation of Janus kinase 2 (JAK-2) tyrosine kinase, induction of MAPKs, tyrosine phosphorylation of GHR and other intracellular proteins such as insulin receptor substrate-1 and signal transducers and activators of transcription (STAT1, STAT3, and STAT5) (3). Specific regions in the cytoplasmic domain of GHR have been defined to be associated with some of these signaling steps. A proline-rich region in the intracellular N-terminal domain (residues 279-286, Box 1) is required for JAK2 and MAPK activation, as well as phosphorylation of STAT1 and STAT3 (4), whereas certain tyrosine residues in the C-terminal part are essential for phosphorylation of STAT5 (5).

The GHR gene spans almost 300 kilobase pairs at 5p12 (http://genome.ucsc.edu/) and consists of 10 exons, of which exons 3–10 encode the mature protein (6). More than 50 different defects including complex rearrangements, gross and small deletions, as well as nonsense, missense, and splicing mutations in the GHR gene, have been described (http://archive.uwcm.ac.uk/uwcm/mg/hgmd0.html). The majority of reported mutations are located in exons 3–7, corresponding to the extracellular part of the protein. These defects lead to the failure of the protein translation, as can be predicted in a majority of truncated mutations as a result of nonsense-mediated mRNA decay, lack of GH binding (7), or impaired intracellular trafficking of GHR (8). By contrast, defects in the cytoplasmic domain of GHR are rare, and among them the only functionally characterized cases were reported in patients with familial short stature due to dominant negative splicing mutations resulting in increased expression of truncated GHR1-277 (9, 10). Here, we report for the first time a classical GH insensitivity due to a novel C-terminal mutation resulting in impaired STAT5, but intact STAT3, signaling. This mutation sheds light on the essential role of STAT5 in the growth-promoting effect of GHR.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Patient

A 17-yr-old female presented with severe short stature. She was born at term to nonconsanguineous parents. There is no record of her birth length; her weight was 2800 g; father’s height was 156 cm (–2.87 SD); mother’s height was 156 cm (–1.1 SD); and heights of her elder sister and two brothers were 167 cm (0.8 SD), 168 cm (–1.0 SD), and 170 cm (–0.7 SD), respectively. At presentation, the patient’s height was 130.5 cm (–5.28 SD); sitting height was 70.8 cm (–5.45 SD); and the weight was 28.3 kg. She had the typical Laron syndrome, with moderate truncal obesity, small hands and feet, prominent forehead, and saddle nose. Her puberty staging was Tanner B4P4, and she had regular spontaneous menses since the age of 16 yr. Her bone age was 15 yr.

Her basal GH levels measured on two separate occasions were 10 and 37.6 ng/ml, and during the insulin tolerance test it rose to 165 ng/ml. Basal IGF-I level was 94 ng/ml (–2.6 SD), and after 4 d of GH treatment (0.033 mg/kg·d), it did not change (84.6 ng/ml). Basal IGF binding protein-3 (IGFBP-3) level was 1.5 mg/ml (–3.5 SD), and GHBP was 218 pmol/liter (–2.6 SD). In her mother, basal GH level was 2.9 ng/ml; IGF-I, 83 ng/ml (–1.1 SD); IGFBP-3, 2.4 mg/liter (–0.8 SD); and GHBP, 1000 pmol/liter (–0.5 SD).

All studies were approved by the review board at the Endocrinological Research Center (Moscow, Russia), and written informed consent was obtained from the patient and her parents.

Hormone assays

Serum GH concentrations were measured using a RIA developed in the Laboratory of Protein Hormones of the National Endocrinological Research Center and calibrated to the First International Reference Preparation (IRP 66/217) of human GH (hGH) for Immunoassay provided by the World Health Organization International Laboratory for Biological Standards National Institute for Biological Standards and Control (11). The sensitivity of the assay was 0.2 µg/liter, and the inter- and intraassay coefficients of variation were 8 and 10%, respectively. Serum IGF-I was measured by blocking RIA after acid:alcohol extraction (Esoterix, Calabasas Hills, CA). Serum IGFBP-3 was measured by RIA in dilute serum (Esoterix). Serum GHBP was determined by a ligand-mediated binding assay (Esoterix).

Genetic analysis

Genomic DNA was isolated from peripheral blood leukocytes of the proband and her mother. Exons 3–10 of the GHR gene were individually amplified by PCR using pairs of intronic primers deduced from the published Human Genome sequence (http://genome.ucsc.edu/). The amplification products were purified and directly sequenced using automated DNA sequencer (model 310; Applied Biosystems, Foster City, CA). GenBank GHR cDNA entry with accession number X06562 was used as a reference sequence for analysis of mutations and numbering of nucleotides. Codon numbering is given for mature GHR protein.

Plasmids

The wild-type (WT) full-length cDNA from pUC119-GHR vector (provided by Dr. W. I. Wood, Genentech, Inc., South San Francisco, CA), was digested with restriction enzymes XbaI and HindIII, and resulting 5'-GHR (XbaI-HindIII) and 3'-GHR (HindIII-HindIII) fragments were subcloned in two consecutive steps between NheI-HindIII and then HindIII-HindIII sites into the mammalian expression vector pcDNA3.1(+) (Invitrogen Corp., Carlsbad, CA). Three mutant GHR vectors, pcDNA3.1-GHR1776del, pcDNA3.1-GHRL561X, and pcDNA3.1-GHRI582X, were created by site-directed mutagenesis using the PCR-based "megaprimer" method (12) and pcDNA3.1-GHRWT as a template. The accuracy of all constructs was confirmed by sequencing using automated DNA sequencer (model 310; Applied Biosystems).

Lactogenic hormone response element (LHRE) thymidine kinase-luciferase reporter plasmid (LHREtk-luc) was provided by Dr. P. A. Kelly (Institut National de la Santé et de la Recherche Médicale, Paris, France) (5). pSV40-ß-Galactosidase reporter vector (pSVßG) was purchased from Promega Corp. (Madison, WI). STAT3 expression plasmid (pcDNA3-STAT3) was provided by Dr. J. E. Darnell, Jr. (Rockefeller University, New York, NY) (13). STAT3 reporter vector (pSTAT3-TA-Luc) was purchased from BD Biosciences (Palo Alto, CA).

Cell cultures and transfections

CHO cells were grown in DMEM-Ham’s F-12 medium (Invitrogen Corp.) containing 10% charcoal-stripped fetal bovine serum (HyClone, Logan, UT), 2 mM L-glutamine (Invitrogen Corp.), and 1:100 antibiotic-antimycotic (Invitrogen Corp.) at 37 C in 5% CO2. Transfections were performed at 60% confluence using FuGENE 6 Transfection Reagent (Roche Applied Science, Indianapolis, IN).

Transcription assays

CHO cells were plated in 12-well plates. For STAT5-mediated transcriptional activation assay, cells were transfected with 1.0 µg of different pcDNA3.1-GHR constructs or empty pcDNA3.1(+) vector (control), 1 µg LHREtk-luc, and 0.5 µg pSVßG. For STAT3-mediated transcriptional activation assay, cells were transfected with 1.0 µg of different pcDNA3.1-GHR constructs or empty pcDNA3.1(+) vector (control), 1 µg pcDNA3-STAT3, 1.5 µg pSTAT3-TA-Luc, and 0.5 µg pSVßG.

Twenty-four hours after transfection, cells were incubated with or without 1, 10, 100, 500, and 1000 ng/ml recombinant human GH (rhGH) (provided by BioTechnology General, Rehovot, Israel). In STAT3-mediated transcription assay, 24 h after transfection, cells were incubated in serum-free medium with 250 mM dexamethasone.

Forty-eight hours after transfection, cells were lysed, and extracts were used for determination of luciferase and ß-galactosidase activities. Luciferase activity was normalized to the ß-galactosidase activity, and results are presented as the mean (SEM) of at least three independent experiments performed in duplicate.

GH-induced tyrosine phosphorylation of STAT5 in CHO cells

GH-induced tyrosine phosphorylation of STAT5 was detected by Western blotting as described previously (14). CHO cells were plated in 60-mm culture dishes and transfected with 3.0 µg of different pcDNA3.1-GHR constructs or empty pcDNA3.1(+) vector (control). Twenty-four hours after transfection, cells were incubated with or without 500 ng/ml rhGH for 15 min and lysed with cell lysis buffer (Cell Signaling Technology, Inc., Beverly, MA). STAT5b was detected with STAT5 (C-17) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Detection of tyrosine phosphorylation of STAT5 was performed by two different methods: directly with Phospho-Stat5 (Tyr694) Antibody (Cell Signaling Technology, Inc.), and using antiphosphotyrosine antibody (RC20H; Transduction Laboratories, Lexington, KY) after immunoprecipitation with STAT5 (C-17) agarose-conjugated antibody (Santa Cruz Biotechnology, Inc.). Antibody binding was detected by Phototope-HRP Detection System (Cell Signaling Technology, Inc.). Density of the immunoblotting bands was quantified using an image analyzing system (Eastman Kodak, Rochester, NY).

Binding analysis

Binding analysis was performed using a homologous competitive binding method (15). CHO cells were plated in 12-well plates and transfected with 1.0 µg of different pcDNA3.1-GHR constructs or empty pcDNA3.1(+) vector (control) and 0.5 µg pSVßG. Forty-eight hours after transfection, cells were starved for serum for 2 h, and [125I]hGH (0.4 mCi/ml; NEX-100; PerkinElmer Life Sciences Inc., Boston, MA) was added to the serum-free culture medium containing 0.1% BSA with increasing concentrations of unlabeled hGH and incubated for 90 min. The cells were washed three times with PBS and lysed with Cell Culture Lysis Reagent (Promega Corp.). Cell-associated radioactivity was counted using a gamma-counter (Titertek, Huntsville, AL) and corrected for ß-galactosidase activity. All experiments were performed in triplicate.

Statistical analysis

Differences between groups and between the tests within groups were estimated using ANOVA. All data are recorded as the mean ± SEM. Analysis of binding curves was performed using the Ligand binding program of Sigmaplot 8.0 software package (Systat Software, Inc., Point Richmond, CA).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Genetic analysis

PCR and subsequent direct sequencing of exons 3–7 with adjoining intronic sequences in the patient revealed two heterozygous mutations. One mutation was a C to A substitution at position 346 (exon 5) resulting in cysteine to stop the codon change of residue 83 (C83X) (Fig. 1AGo). The second heterozygous mutation was a G deletion in codon R560 (nucleotide position 1776, exon 10) (Fig. 1BGo). The patient’s mother was shown to have a similar heterozygous 1776del mutation in exon 10, but she showed normal exon 5 sequence (Fig. 1Go, A and B). The father’s DNA was not available for analysis.



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  		FIG. 1. A, Fragment of exon 5 sequence of the GHR gene in the patient and her mother. Mutation position is shown by arrow. B, Fragment of exon 10 sequence of the GHR gene in the patient, her mother, and the control sample. Mutation positions are shown by arrows. C, C-terminal sequences of the WT and mutated GHR distal to residue 560 (tyrosines Y577 and Y609 are underlined).

 
The 1776del mutation is predicted to result in a frameshift with formation of a termination codon at position 582 and a nonsense protein sequence of residues 560 to 581 (Fig. 1CGo).

GHR constructs

To clarify the effect of the GHR1776del mutation on GHR function, in addition to the wild-type (GHRWT) and the mutant (GHR1776del) GHR constructs, two other GHR constructs with truncated WT sequence were created: GHRL561X, with stop codon corresponding to the position of the 1776del mutation; and GHRI582X, with stop codon corresponding to the position of translation termination in GHR1776del (Fig. 2Go).



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  		FIG. 2. Schematic representation of the WT and mutated GHR constructs. Numbers indicate amino acid residues. Box 1 and Box 2 are indicated as solid black lines; the transmembrane domain as a hatched rectangle; and nonsense sequence as a stippled rectangle. Cytoplasmic tyrosines present in different constructs are shown.

 
GH binding to GHRWT and GHR1776del

Binding displacement curves for GHRWT and GHR1776del receptors are presented in Fig. 3Go. The affinity (Kd) of [125I]hGH for GHRWT (3.40 ± 0.53 nM) was comparable with that for GHR1776del (3.10 ± 0.24 nM). The total receptor number (Bmax) values were 12.43 ± 1.65 and 11.34 ± 2.01, respectively.



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  		FIG. 3. GH binding displacement. CHO cells transfected with the pcDNA3.1-GHRWT and pcDNA3.1-GHR1776del constructs were incubated with [125I]hGH and increasing doses of unlabeled rhGH. Data are presented as a percentage of radioactivity measured after transfection with the empty pcDNA3.1(+) vector (control).

 
STAT3-mediated transcriptional activation

Functional activation of STAT3 was studied in transiently transfected CHO cells using STAT3-TA-luc reporter system. When each of the four GHR constructs were cotransfected with the reporter vector alone, GH had little effect (~1.5- to 2-fold activation; data not shown) on STAT3 transcription, indicating that endogenous STAT3 expression in this cell system is relatively low. However, when the experiment was performed in the presence of transfected STAT3 cDNA, there was significant increase in GH-induced STAT3-mediated transcriptional activity (5- to 9-fold) (Fig. 4Go). No differences in functional activation of STAT3 were observed between cells expressing WT and either of the mutant forms of the receptor (P > 0.05, ANOVA). After transfection with empty pcDNA3.1+ vector, there was no significant change of transcription through STAT3 in response to GH.



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  		FIG. 4. Luciferase activity corrected for ß-galactosidase activity after cotransfection of CHO cells with pcDNA3.1 (control), pcDNA3.1-GHRWT, pcDNA3.1–1776del, pcDNA3.1-GHRL561X, or pcDNA3.1-GHRI582X together with pcDNASTAT3, STAT3 reporter vector (pSTAT3-TA-Luc; Clontech, Palo Alto, CA), and pSVßG (Promega Corp.). Data are presented as means of three transfection experiments ± SEM. There were no differences in fold induction between the GHR-expressing vectors (P > 0.05, ANOVA).

 
STAT5-mediated transcriptional

Functional activation of STAT5 was studied in CHO cells transiently transfected with each of four GHR constructs and LHREtk-luc reporter vector (5). The latter consisted of thymidine kinase promoter fused to luciferase gene and LHRE element, a part of a ß-casein promoter originally used for the affinity purification of STAT5 (16). Cells expressing GHRs showed STAT5-mediated transcriptional activity in response to GH in a dose-dependent manner (Fig. 5Go). However, when transfected with GHR1776del, activation of transcription through LHRE at GH concentrations of 100, 500, and 1000 ng/ml was significantly lower (P < 0.05, ANOVA) than for GHRWT, GHRL561X, and GHRI582X. There was no difference in STAT5-mediated transcriptional activity between cells expressing GHRWT, GHRL561X, and GHRI582X. After transfection with empty pcDNA3.1+ vector, no significant change in STAT5-mediated transactivation was observed, indicating negligible expression of endogenous GHR in this system.



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  		FIG. 5. Luciferase activity corrected for ß-galactosidase activity after cotransfection of CHO cells with pcDNA3.1 (control), pcDNA3.1-GHRWT, pcDNA3.1–1776del, pcDNA3.1-GHRL561X, or pcDNA3.1-GHRI582X together with STAT5 reporter vector (LHREtk-luc) and pSVßG (Promega Corp.). Data are presented as means of three transfection experiments ± SEM. Fold induction for pcDNA3.1–1776del is significantly lower compared with all other GHR-expressing vectors (P < 0.05, ANOVA).

 
GH-induced STAT5 tyrosine phosphorylation

We evaluated the ability of GH to induce tyrosine phosphorylation of endogenous STAT5 in CHO cells transiently expressing GHRWT and the three mutant receptors. After GH stimulation for 15 min, cell lysates were either immunoblotted directly with Phospho-STAT5 (Tyr694) antibody or immunoprecipitated with anti-STAT5 antibody and then subjected to Western immunoblotting with antiphosphotyrosine antibody (Fig. 6Go). Relative band densities of tyrosine-phosphorylated STAT5 to total STAT5 after incubation with 500 ng/ml rhGH in CHO cells expressing pcDNA3.1+, pcDNA3.1-GHRWT, pcDNA3.1-GHR1776del, pcDNA3.1-GHRL561X, and pcDNA3.1-GHRI582X constructs were 1 ± 0.23, 32.7 ± 7.6, 1.4 ± 0.4, 13.6 ± 2.3, and 18.7 ± 8.1, respectively. GH-induced phosphorylation of STAT5 in cells expressing GHR1776del vector was close to background levels and significantly lower than after transfection with GHRWT, GHRL561X, or GHRI582X (P < 0.05). There was no significant difference in phosphorylation signal between cells expressing GHRWT and GHRL561X or GHRI582X.



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  		FIG. 6. GH-induced tyrosine phosphorylation of STAT5 in CHO cells transfected with pcDNA3.1 (control), pcDNA3.1-GHRWT, pcDNA3.1-GHR1 776del, pcDNA3.1-GHRL561X, or pcDNA3.1-GHRI582X. Transfected CHO cells were incubated with or without 500 ng/ml rhGH for 15 min and lysed. A, Direct Western blotting using Phospho-STAT5 (Tyr694) antibody (Cell Signaling Technology, Inc.). B and C, Lysis under nondenaturing conditions with subsequent immunoprecipitation with agarose conjugate of anti-STAT5b antibody (STAT5 C-17 AC; Santa Cruz Biotechnology, Inc.) and Western blotting using antiphosphotyrosine antibody (Ptyr; Phosphotyrosine-RC20, Transduction Laboratories) (B) or anti-STAT5b antibody (STAT5 C-17, Santa Cruz Biotechnology, Inc.) (C).

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The case we presented is a young female with severe short stature due to insensitivity to GH. The diagnosis was established on the basis of elevated basal and stimulated serum GH, low basal IGF-I, and IGFBP-3, as well as a lack of IGF-I response after rhGH treatment. Molecular basis of GHIS in this patient was compound heterozygosity for two novel mutations, C83X (exon 5) and a G deletion in exon 10 at position 1776 corresponding to the C-terminal part of the cytoplasmic domain of the receptor. The exon 10 mutation is predicted to cause a frameshift and GHR truncation to 581 amino acids, of which 22 distal residues represent a nonsense sequence, lacking any homology with known protein domains. Because the C83X mutation leads to a lack of GHR expression as a result of mRNA decay or defect in cell membrane anchoring, the C-terminal mutation seems to account for GHR function impairment and severe GHIS phenotype. Clinically, the patient had the typical Laron syndrome.

Although cytoplasmic domain is encoded by almost 60% of the coding sequence of the GHR gene, mutations affecting this part of the receptor are rare. Heterozygous missense mutations in the cytoplasmic domain of GHR were described in patients with idiopathic short stature (17, 18); however, none of them have been proven to be associated with any specific impairment of the receptor function (14). Two splicing mutations were shown to affect function of the intracellular domain. Ayling et al. (10) and then Iida et al. (9) described patients with familial short stature and biochemical features of GH insensitivity caused by heterozygous mutations at splice sites in intron 8 and exon 9, respectively. These mutations resulted in formation of truncated GHR (GHR1-277) with a dominant negative effect on the full-length receptor translated from the unaffected allele (19, 20).

The GHR protein expressed in our patient remains intact in the extracellular, transmembrane, and more than 80% of the cytoplasmic domains. It is expected, therefore, to have normal association with the membrane as well as extracellular dimerization and ligand-binding properties. The latter was confirmed by normal [125I]hGH binding affinity and capacity. Additional evidence of the functional extracellular domain in the patient’s GHR comes from the patient’s low, but detectable, serum GHBP. The cause of the lower than the age reference GHBP concentration is not completely clear. Partly, it could be explained by expression of a single GHR allele. Also, low GHBP may be related to high levels of GH. In patients with acromegaly, GHBP was reported to be low and inversely correlated to GH concentration (21, 22). In agreement with that information are experimental data showing that GHR dimerization inhibits its proteolysis (23).

To clarify the effect of the C-terminal mutation on GH-mediated signal transduction, we studied two pathways known to be associated with different parts of the cytoplasmic domain. The key step in GHR signaling is rapid phosphorylation of JAK-2 (24) associated with the membrane-proximal region of GHR. The N-terminal one-third cytoplasmic region, in particular Box 1, is required and sufficient for interaction between GHR and JAK-2, and tyrosyl phosphorylation of JAK-2 (5, 25). JAK-2 is required for GH-dependent activation of STAT1 and STAT3 (26), whereas C-terminal residues 455–620 did not affect this function (5). GHR truncation in our case did not affect the N-terminal two-thirds of the cytoplasmic domain, including Box 1 and Box 2 motifs; therefore, it was not expected to be associated with impaired JAK-2 and STAT3 activation. Indeed, in our experiment with transient cotransfection of STAT3 cDNA, there was no difference in STAT3-mediated transcriptional activation between GRHWT and any of the three GHR mutants. In contrast, the GHR1776del mutation resulted in significant impairment of STAT5 activation. Activation of STAT5 requires phosphorylated tyrosine residues in the C-terminal part of GHR (5). Residues Y304, Y469, Y516, Y548, and Y609 are phosphorylated in response to GH (27). In the GHR, cytoplasmic residues 455–540, in particular tyrosines 469 and/or 516, are important for STAT5 tyrosine phosphorylation in response to GH stimulation (5). C-terminal tyrosine mutagenesis suggested that any of the three tyrosines, Y516, Y548, and Y609, was sufficient for STAT5 activation (28). Although the precise docking site is not known, there is evidence for direct association between phosphorylated SH2 domain of STAT5 and C-terminal GHR phosphotyrosines. In vitro STAT5 was shown to interact with phosphorylated GHR residues 455–620 fused to glutathione S-transferase (5). In addition, STAT5 was coimmunoprecipitated with GHR from stably expressing GHR cells treated with GH (29). GHR1776del protein lacks the two C-terminal tyrosines, Y577 and Y609, the latter being phosphorylated in response to GH stimulation (27). Yet it retains Y516 and Y548, reported to be sufficient for STAT5 phosphorylation (28). Because 1776del and L561X have the same tyrosines, we speculate that the abnormal folding of the tyrosine-containing domain that is induced by the nonsense 22 C-terminal residues in the GHR-1776del protein interferes with phosphorylation of important upstream tyrosines or with subsequent STAT5-GHR docking. The possibility that the C-terminal sequence itself could trap endogenous STAT5 is another speculative mechanistic assumption.

There is a growing body of evidence pointing to the essential role of STAT5 in GHR signaling. In vitro, STAT5b is the key factor in GH-dependent differentiation of murine 3T3-F442A preadipocytes (30). Mice with targeted STAT5b gene disruption exhibit poor growth, abnormal adipose tissue development, and a loss of sexually dimorphic hepatic expression of several GH-responsive genes (31). Recently, an alanine to proline substitution in the SH2 domain of STAT5b was identified in a patient with an apparent postreceptor form of GHIS (32). The current observation of classical GHIS phenotype associated with GHR-dependent impairment of STAT5 activation provides additional clinical evidence for the essential role of STAT5 in the GH-IGF-I axis.


   Footnotes
 
First Published Online November 9, 2004

Abbreviations: GHBP, GH binding protein; GHIS, GH insensitivity syndrome; GHR, GH receptor; hGH, human GH; IGFBP, IGF binding protein; JAK-2, Janus kinase 2; LHRE, lactogenic hormone response element; LHREtk-luc, LHRE thymidine kinase-luciferase reporter plasmid; pSVßG, pSV40-ß-galactosidase reporter vector; rhGH, recombinant hGH; STAT, signal transducers and activators of transcription; WT, wild type.

Received December 12, 2003.

Accepted October 18, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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  6. Godowski PJ, Leung DW, Meacham LR, Galgani JP, Hellmiss R, Keret R, Rotwein PS, Parks JS, Laron Z, Wood WI 1989 Characterization of the human growth hormone receptor gene and demonstration of a partial gene deletion in two patients with Laron-type dwarfism. Proc Natl Acad Sci USA 86:8083–8087[Abstract/Free Full Text]
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  8. Wojcik J, Berg MA, Esposito N, Geffner ME, Sakati N, Reiter EO, Dower S, Francke U, Postel-Vinay M-C, 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. J Clin Endocrinol Metab 83:4481–4489[Abstract/Free Full Text]
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