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Original Studies |
Third Division, Department of Medicine, Kobe University School of Medicine (K.I., Y.T., H.K., M.O.T., Y.O., H.A., K.C.), 7–5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017; and the Department of Pediatrics, Iwate Prefectural Kitakami Hospital (N.O.), Iwate, 024-0063, Japan
Address all correspondence and requests for reprints to: Dr. K. Iida, Third Division, Department of Medicine, Kobe University School of Medicine, 7–5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan. 960d640 m{at}mailgate.kobe-u.ac.jp
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Abstract |
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Introduction |
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The GHR belongs to the superfamily of cytokine receptors and consists of the extracellular, transmembrane, and intracellular domains (6). The GHR gene includes nine coding exons from exon 2 to exon 10 (6, 7). Exon 2 encodes a signal sequence, and exons 3–7 encode the extracellular domain of GHR, which includes the sequences necessary for the ligand binding (8) and dimerization of GHR (9) in response to a single GH molecule. Exon 8 encodes the transmembrane domain and first four amino acids of the intracellular domain. Exons 9 and 10 encode the intracellular domain including the conserved box 1 region, which is critical for Janus kinase 2 (JAK2) association and signal transduction (10).
To date, more than 30 mutations in the GHR gene have been reported, most of which were located at the region encoding for the extracellular domain and caused an impaired binding of GH to GHR (2). Recently, splice site mutations were reported to result in skipping of exon 8 (11, 12) or exon 9 (13, 14), thereby producing truncated GHR that lack the transmembrane (11, 12) or intracellular (13, 14) domain, respectively. In contrast, there were a few mutations found in the region encoding for the intracellular domain (15, 16, 17). Kou et al. (15) reported a short child with double heterozygous missense mutations, C422F and P561T, both located in the intracellular domain of GHR. It was controversial, however, that these mutations were responsible for short stature, because the body height of his mother with the same mutations was normal (15), and some populations in normal height possessed the same mutations (16). The function of mutant GHR, C422F or P561T, has not been characterized to date. We previously reported that the heterozygous missense mutation, P561T, which was identical to one of the mutations found in the case reported by Kou et al. (15), was not correlated with body height (18).
In this study, we have identified the C422F mutation in a Japanese family, which was identical to another mutation found by Kou et al. (15), and examined the function of mutant GHR to clarify whether this single amino acid substitution in the intracellular domain of GHR is responsible for short stature.
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Subjects and Methods |
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The proband was born at 39 weeks gestation to nonconsanguineous parents. He was 49.5 cm in height and 3510 g in weight at birth. At the age of 5.7 yr, his height was 92.4 cm (4.2 SD below the mean for age and sex), and his weight was 13.3 kg. His bone age was 4.5 yr (79% of chronological age). His serum GH level was increased from 3.9 to 19.0 µg/L by arginine (0.5 g/kg, iv infusion for 30 min), from 3.9 to 9.8 µg/L by clonidine (0.15 mg, oral ingestion), and from 2.3 to 83.0 µg/L by GHRH (1 µg/kg, iv injection). The serum insulin like growth factor I (IGF-I) level was 73 µg/L at the basal level (normal range for age, 37–411 µg/L) and increased to 95 µg/L after daily sc injections of 0.1 U/kg recombinant human (rh) GH for 3 days. The serum IGF binding protein-3 (IGFBP-3) level was 4.30 µg/mL (normal range for age, 1.78–4.43 µg/mL), and the GH-binding protein (GHBP) level was 72.1 pmol/L (normal range, 65–408 pmol/L).
Hormone assays
Serum GH was measured by an immunoradiometric assay (Daiichi Pharmaceutical Co. Ltd., Tokyo, Japan). Serum IGF-I was measured by an immunoradiometric assay after extraction of the binding proteins (SRL, Inc., Tokyo, Japan). Serum IGFBP-3 was measured by immunoradiometric assay (Mitsubishi Kagaku Bio-Clinical Laboratories, Tokyo, Japan). Serum GHBP was determined by a ligand-mediated immunofunctional assay (Mitsubishi Kagaku Bio-Clinical Laboratories, Tokyo, Japan).
Genetic analysis
Genomic DNA was isolated from peripheral blood leukocytes of the proband, his parents, brother, sister, maternal grandparents, and normal subjects. Each exon of the GHR gene was individually amplified by PCR, as previously described (14). The amplification products were purified and were analyzed by direct sequencing using a DNA sequencer (model 310, Perkin Elmer Corp., PE Applied Biosystems, Foster City, CA).
Construction of wild-type (GHR-wt) and mutant GHR (GHR-C422F) expression vectors
The pUC119 vector containing the wild-type GHR complementary DNA (cDNA; pUC119-GHR), provided by Dr. W. I. Wood (Genentech, Inc. South San Francisco, CA), was subcloned into the expression vector pcDNAI (Invitrogen, Leek, The Netherlands) using the BamHI and SphI restriction sites (pcDNA1/GHR-wt). The amplification fragment including a part of exon 10 of the GHR gene of the proband by PCR was subcloned into the pT7 Blue T-vector (Novagen, Inc., Madison, WI) and digested with restriction enzymes BspMI and EcoRI, then inserted into the pcDNAI vector using the BspMI-EcoRI sites (pcDNA1/GHR-C422F). The accuracy of construction of GHR-C422F cDNA was confirmed by sequencing.
Cell cultures and transfections
CHO cells were grown in DMEM-Ham’s F-12 (Life Technologies, Inc., Grand Island, N.Y) containing 10% FBS (BioWhittaker, Inc., Walkersville, MD), penicillin, and kanamycin at 37 C in 5% CO2. Transfections were performed at 60% confluence in 35-mm culture dishes using LipofectAce reagent (Life Technologies, Inc.) with 2.0 µg pcDNA1/GHR-wt or 2.0 µg pcDNA1/GHR-C422F, 1.5 µg lactogenic hormone response element (LHRE)/thymidine kinase (TK)-luciferase reporter gene (provided by Dr. P. A. Kelly, INSERM, Paris, France) (19), which was STAT5 binding reporter gene, and 2.0 µg pSV-ß galactosidase control vector (Promega Corp., Madison, WI). The ß-galactosidase activities were measured as an internal control of transfections using an enzyme assay system kit (Promega Corp.) according to the manufacturer’s instructions. The cells transfected with empty pcDNA1 were used as control experiments.
Scatchard plots of hGH binding to GHR-wt and GHR-C422F
Forty-eight hours after transfection, binding analysis was
performed as described previously (20) in GHR-wt and GHR-C422F
expressing-CHO cells. Briefly, 2 h after starvation for serum,
[125I]hGH (0.4 µCi/mL; NEX-100, DuPont,
Wilmington, DE) 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 lysed with 0.1% NaOH and the
cell-associated radioactivity was counted using a 
-counter
(Pharmacia Biotech, Piscataway, NJ).
STAT5-mediated transcriptional activation in GHR-expressing CHO cells
Twenty four hours after transfection, cells were incubated without or with 1, 10, 50, 100 and 500 ng/mL rhGH. After another 24 hours, luciferase activities were calculated using a luciferase reporter assay system kit (Promega Corp.) according to the manufactures’ instructions and corrected for ß galactosidase activities.
GH-dependent tyrosine phosphorylation of STAT5b in GHR-expressing CHO cells
CHO cells were transfected with 3.0 µg pcDNA1/GHR-wt or 3.0 µg pcDNA1/GHR-C422F in 60-mm culture dishes. The cells transfected with empty pcDNA1 were used as controls. The transfected cells were incubated without or with 1, 10, 50, 100, and 500 ng/mL rhGH for 15 min and lysed with detergent buffer (1% deoxycholic acid, 1% Triton X-100, 0.15 mol/L NaCl, 50 mmol/L Tris, 10 mg/mL aprotinin, 1 mmol/L phenylmethylsulfonylfluoride, and 1 mmol/L orthovanadate). GH-induced tyrosine phosphorylation of STAT5b in the cells expressing GHR-wt or GHR-C422F was determined by Western blotting as described previously (21). Specific antibody for STAT5b (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and antiphosphotyrosine antibody (RC20H, Transduction Laboratories, Lexington, KY) were used for immunoprecipitation and immunoblotting, respectively. Antibody binding was detected using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Arlington Heights, IL). The densities of the immunoblotting bands were quantified using an image analyzing system (Lane Analyzer ver 2.0, Atto, Co., Tokyo, Japan). All experiments were performed in triplicate.
Statistical analysis
The values were expressed as the mean ± SE. Statistical significance between the different values was determined using Student’s t test.
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Results |
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Sequencing of the GHR gene of the proband revealed a heterozygous
G to T transversion at the second base of codon 422 (Fig. 1
), which was determined by direct
sequencing in both directions using sense and antisense primers. This
mutation predicted to convert codon 422 from cysteine to phenylalanine.
No additional abnormalities were detected in his GHR gene. The father
of the proband had normal genotype, but his mother, whose height was
normal, possessed homozygous mutations in both alleles (Fig. 1). The
genotypes and body heights of the other family members were shown in
Fig. 2, indicating that body heights of
his family members with heterozygous and homozygous mutations at the
second base of codon 422 were normal.
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Scatchard analysis revealed that the binding sites and binding
affinity to GH of GHR-C422F-expressing cells were comparable to those
of GHR-wt expressing cells. The representative data are shown in Fig. 3. Triplicate experiments revealed that
the association constant (Ka) for GHR-wt is
0.94 ± 0.11 x 109 mol/L, and that for
GHR-C422F is 0.90 ± 0.08 x 109mol/L.
The binding sites of cells expressing GHR-wt and GHR-C422F were 39
± 8 and 36 ± 10 fmol/106 cells,
respectively, indicating that GHR-wt and GHR-C422F were expressed at
equivalent levels in CHO cells.
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GH-induced tyrosine phosphorylation of STAT5b was compared between
GHR-C422F- and GHR-wt-expressing CHO cells. Representative data are
shown in Fig. 4a. Relative band densities
of tyrosine-phosphorylated STAT5b to total STAT5b after stimulation of
1, 10, 50, 100, and 500 ng/mL rhGH were 0-, 1.0-, 2.8 ± 0.50-,
4.2 ± 0.25-, and 4.5 ± 0.42-fold, respectively, in
GHR-wt-expressing cells and 0-, 1.0 ± 0.29-, 2.5 ± 0.53-,
4.6 ± 0.35-, and 4.5 ± 0.63-fold, respectively, in
GHR-C422F-expressing cells (Fig. 4b). There were no significant
differences between the values. Parallel experiments with the empty
expression plasmid revealed that tyrosine-phosphorylated STAT5b was not
detected after GH stimulation despite the presence of endogenous STAT5b
in CHO cells, indicating that the influence of endogenous GH receptor
activation is negligible in our system.
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As shown in Fig. 5, rhGH
dose-relatedly stimulated the expression of the reporter gene through
GHR-wt (25 ± 6.5, 46 ± 5.2, 60 ± 5.8, 78 ±
8.06, 110 ± 13.0, and 105 ± 15.0 light units stimulated by
0, 1, 10, 50, 100, and 500 ng/mL rhGH, respectively) and through
GHR-C422F (21 ± 5.2, 51 ± 6.1, 65 ± 7.0, 72 ±
6.1, 105 ± 8.2, and 110 ± 8.0 light units stimulated by 0,
1, 10, 50, 100, and 500 ng/mL rhGH, respectively). There were no
statistical differences between GHR-wt- and GHR-C422F-expressing cells.
Luciferase activities remained unchanged after rhGH stimulation in the
control study with the empty expression plasmid, indicating that
endogenous GH receptor did not affect to activate STAT5-mediated
transcription.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Most of molecular abnormalities of GHR in GHIS were located at the region encoding for the extracellular domain, some of which were characterized in transfected cells and elucidated due to the lack of hormone-binding sites (22, 23), the impaired GHR expression (24, 25) and the impaired homodimerization of GHR (26). A few reports were documented about the mutations affecting the intracellular domain (13, 14, 15, 16, 17). We previously reported two families with mutations affecting the intracellular domain of GHR (14, 17). The intracellular mutation of the GHR gene in our patient with GHIS, which was one of the compound heterozygous mutations, was a C deletion at position 981 in exon 10, resulting in a frame shift to cause a truncation of carboxyl-terminus of GHR (17). Interestingly, the messenger ribonucleic acid (mRNA) transcribed from the mutated allele could not be detected by RT-PCR methods, indicating that this one-point deletion might affect the expression or stability of mRNA coding for GHR. Another mutation in a different patient was located in a donor splice site of exon 9, resulting in exon 9 skipping of the GHR mRNA and production of truncated GHR lacking most of the intracellular domain, which acts as a dominant negative inhibitor to wild-type GHR (14, 20). In contrast, a single missense mutation responsible for GHIS in the region coding the intracellular domain has not yet identified. Although Goddard et al. (16) reported a missense mutation (A478T) in the intracellular domain in a patient who showed short stature with normal serum GH, low IGF-I, and normal GHBP levels, the functional characterization of this mutant GHR was not determined. Other missense mutations in the intracellular domain were identified (P561T, P477T, and I526L), but the frequency between controls and short statured patients was not different, and these were considered to be polymorphisms (16, 18). Again, it remains unclear whether these mutations affected the function of GHR. In this study, we focussed on determining the function of GHR with a C422F mutation alone.
GH binding to GHR induces dimerization of GHR and activation of tyrosine kinase JAK2 (27), followed by tyrosine phosphorylation of JAK2 itself and GHR, and activation of a variety of signaling molecules, including signal transducer and activator of transcription (STAT) members, mitogen-activated protein (MAP) kinase, phosphatidylinositol 3-phosphate kinase, etc. (28). The importance of the JAK-STAT pathway to body length in mice was clearly demonstrated by the experiment using STAT5b knockout mice (29). Mutational analysis in the intracellular domain of GHR revealed that two distinct regions, that is a region adjacent to the membrane including boxes 1 and 2 and a carboxyl-terminal region, were necessary for activation of JAK2 and for transcriptional activation, respectively (10, 30, 31). Moreover, deletion of the midportion of the intracellular domain did not affect the activation of JAK2 and MAP kinase (30). The codon 422 was located in the midportion of the intracellular domain of GHR, suggesting that the C422F mutation would not affect the function of GHR. The cysteine, however, could be an important amino acid for the structure of the GHR molecule because of the formation of the disulfide bonds (8). A substitution of phenylalanine for cysteine might cause the conformational change in the intracellular domain of GHR and affect the signal transduction of GH.
To elucidate the effects of the C422F mutation on GH-induced JAK-STAT
pathway, we examined GH-induced tyrosine phosphorylation of STAT5b,
which is the major pathway in GH signal transduction. As shown in Fig. 4
, no significant difference was observed in the amounts of
tyrosine-phosphorylated STAT5b between GHR-wt- and GHR-C422F-expressing
cells. Moreover, we examined the function of STAT5-mediated
transcriptional activation of GHR-C422F by means of the luciferase
reporter gene linked with LHRE, which is found in the ß-casein gene
promoter (19). Added GH dose dependently and equally increased the
luciferase activities in GHR-wt- and in GHR-C422F-expressing cells,
indicating that the C422F mutation did not affect the transcriptional
activation. The possibilities cannot be ruled out that the C422F
mutation might affect the still unknown pathway in a GH signal
transduction system. However, these in vitro results were
comparable to the results of the analysis of genotypes of family
members as shown in Fig. 2. The height of the proband’s mother was
normal despite the genotype of homozygous C422F mutation. The
proband’s brother, sister, and mother’s parents, all of whom were
normal in height, possessed the heterozygous mutation identical to the
genotype of the proband. Our findings of genotype analysis of his
family are also compatible with the previous report (16), in which the
frequency of C422F and P561T double mutations was not different between
normal subjects and short children with GHIS (2% in normal subjects
vs. 3% in patients). Taken together, these results suggest
that the C422F mutation of the GHR gene is not the cause of short
stature. The cause of short stature of the proband remains unknown.
In conclusion, we have identified heterozygosity and homozygosity for the C422F missense mutation in a Japanese family and provided clinical and in vitro evidence that this mutation is a polymorphism of the GHR gene.
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Acknowledgments |
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Footnotes |
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Received March 11, 1999.
Revised June 15, 1999.
Accepted July 7, 1999.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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