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Special Feature |
Section of Endocrinology (M.D.L., T.E.E., M.F.S.), Department of Medicine, Institute of Medical Genetics (M.H., D.S.M., V.N., A.M.P., D.N.C.), Department of Child Health (J.W.G.), University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, United Kingdom; Pharmacia AB (L.F.), Lindhagensgatan 98, SE-112 87, Stockholm, Sweden; Biovitrum AB (M.N.), Nordenflychtsvagen 62:6, SE-112 87, Stockholm, Sweden; Paediatric Endocrinology Unit (C.-J.D.V.), Hospital Virgen del Rocío, and Paediatric Endocrinology Unit (L.F.L.-C.), Hospital Militar Universitario, 41013 Sevilla, Spain; Paediatric Endocrinology Unit (J.P.L.-S.), Hospital Materno-Infantil Carlos Haya, 29011 Málaga, Spain; Paediatric Endocrinology Unit (R.C.), Hospital Reina Sofía, 14004 Córdoba, Spain; Paediatric Endocrinology Unit (N.D.-T.), Hospital Infanta Elena, 21007 Huelva, Spain; Paediatric Endocrinology Unit (R.E.), Hospital Universitario de Valme, 41014 Sevilla, Spain; and Medical Department (A.U.), Pharmacia Spain, Barcelona, Spain
Address all correspondence and requests for reprints to: Dr. Mark Lewis, Section of Endocrinology, Department of Medicine, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom. E-mail: lewismd{at}cardiff.ac.uk.
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Abstract |
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G promoter variant. The Ile179Met variant was shown to exhibit a similar degree of resistance to proteolysis as wild-type GH, indicating that the introduction of Met does not cause significant misfolding. Secretion of Ile179Met GH from rat pituitary cells was also similar to that of wild type. Although receptor binding studies failed to show any difference in binding characteristics, molecular modeling studies suggested that the Ile179Met substitution might nevertheless perturb interactions between GH and the GH receptor loop containing the hotspot residue Trp169, thereby affecting signal transduction. The ability of the Ile179Met variant to activate a signal transducer and activator of transcription (STAT) 5-responsive luciferase reporter gene and induce phosphorylation of STAT 5 and ERK was therefore studied. In contrast to its ability to activate STAT 5 normally, activation of ERK by the Ile179Met variant was reduced to half that observed with wild type. Although differential effects on the activation of distinct signaling pathways by a mutant receptor agonist are unprecedented, these findings also suggest that the ERK pathway could play a role in mediating the action of GH. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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GH is a pleiotropic cytokine that promotes the postnatal growth of skeletal and soft tissue. GH also acts as an important regulator of bone turnover, muscle mass, and immune function. The biological actions of GH are mediated through the activation of a cell surface receptor (GHR). Each GH molecule has two binding sites, ensuring that the binding of GH to the GHR results in the formation of a receptor homodimer (4). The intracellular tyrosine kinase, Janus tyrosine kinase (JAK) 2, is associated with the cytoplasmic tail of the GHR. After GH binding, two JAK 2 molecules are brought into close proximity resulting in cross-phosphorylation of both each other and tyrosine residues on the cytoplasmic tail of the GHR. These phosphotyrosines act as docking points for cell signaling intermediates such as signal transducer and activator of transcription 5 (STAT 5) (5). STAT 5 binding to the phosphorylated receptor tail then brings it into close proximity to JAK 2, resulting in its own phosphorylation by JAK 2. Phospho-STAT 5 dimerizes and translocates to the nucleus in which it transactivates GH-responsive genes leading to the observed biological effects of GH (6, 7). Until recently it had been assumed that GH signaling was mediated primarily by the JAK/STAT pathway. However, it is now known that GH can also activate the phosphatidylinositol 3'-kinase (8) and p42/44 ERK pathways (9). Activation of STAT 5 and the phosphatidylinositol 3'-kinase pathway can induce hepatic IGF-1 production (10, 11), but the ERK pathway does not appear to do so (12).
The pituitary-expressed GH1 gene is located on chromosome 17q23 within a cluster of five highly homologous genes: two chorionic somatomammotropin genes (CSH1 and CSH2), a second GH-related gene (GH2), and a chorionic somatomammotropin pseudogene (CSHP1). We have previously shown that polymorphic variation in both the locus control region and the proximal promoter region of the GH1 gene influences GH1 gene expression and may contribute to the genetic variance of human stature (13). We have further demonstrated that GH1 gene lesions occur not only in individuals with short stature and idiopathic GH deficiency but also in individuals selected for short stature, reduced height velocity, and significant bone age delay (14). In an attempt to extend this work, a group of individuals with familial short stature was screened for mutations in their GH1 genes.
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Subjects and Methods |
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10 ng/ml). Pharmacological tests used were clonidine (34 patients), propanolol (25 patients), and insulin (15 patients). Ethical approval for genetic studies was obtained from each participating center and the Multi-Regional Ethics Committee. Written informed consent was obtained from each participating individual.
The SDS were calculated for height, body mass index, paternal and maternal heights, midparental height, IGF-1 and IGFBP-3 levels, peak GH secretion in nanograms per milliliter, and GH binding protein (as a percentage). These data are presented in Table 1
for the two individuals (B4 and B49) in whom a novel GH1 gene lesion was found and as group means for the cohort of individuals studied.
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Genomic DNA was extracted from patient lymphocytes by standard procedures. PCR amplification of a 3.2-kb GH1-specific fragment was performed as described (13).
Cloning and sequencing of GH1 gene-specific PCR fragments
GH1 gene-specific (3.2 kb) PCR fragments were sequenced directly with BigDye v3.0 (Applied Biosystems, Foster City, CA) and analyzed on an ABI 3100 DNA sequencer (Applied Biosystems) as described (13). Additional primers used for sequencing in the reverse direction were GHBFR (5'-TGGGTGCCCTCTGGCC-3'; -262 to -278), GHSEQ1R (5'-AGATTGGCCAAATACTGG-3'; +215 to +198), GHSEQ2R (5'-GGAATAGACTCTGAGAAAC-3'; +785 to +767), GHSEQ3R (5'-TCCCTTTCTCATTCATTC-3'; +1281 to +1264), GHSEQ4R (5'-CCCGAATAGACCCCGC-3'; +1745 to +1730) (numbering relative to the transcriptional initiation site at +1; GenBank accession no. J03071). Samples containing sequence variants were cloned into pGEM-T (Promega, Madison, WI) followed by sequencing of a minimum of four clones per individual.
Characterization of promoter variant by luciferase reporter gene assay
Luciferase reporter gene expression vectors were constructed and tested as described (13).
In vitro expression of GH variants
A cloned wild-type GH1 cDNA incorporating a His tag on the carboxy terminal was modified using site-directed mutagenesis as previously described (14) to generate the Ile179Met variant. This vector was then transfected into High Five insect cells (Invitrogen, Carlsbad, CA) as previously described (14) and human GH in the culture supernatants quantified by ELISA (DRG Diagnostics, Marburg, Germany). The cross-reactivity in the ELISA of the Ile179Met GH variant, and insect cell-expressed wild-type GH was confirmed by dilutional analysis to be equal to that of the assay reference preparation (calibrated against the MRC first IRP 80/505 reference preparation). For Western blotting studies of the activation of the ERK pathway, the His-tagged forms of wild-type GH and the Ile179Met variant were harvested and purified on nickel columns (Invitrogen) according to the manufacturer’s instructions. GH was eluted from the column with 350 mM imidazole. A buffer change step was performed at this stage by concentrating the GH from the imidazole eluate by ultrafiltration (Centriplus YM-10, Millipore, Bedford, MA), washing in PBS, and finally resuspending in PBS. The identity and purity (>95%) of the purified His-tagged GH was confirmed by SDS-PAGE with silver staining, and Western blotting. The purified forms of GH were quantified as described above.
Molecular modeling
The Ile179Met variant was structurally analyzed by inspection of the appropriate amino acid residue in the x-ray crystallographic structure of human GH (PDB: 3HHR) (16). The wild-type and mutant GH structures were compared with respect to electrostatic interactions, hydrogen bonding, hydrophobic interactions, and surface exposure. Molecular graphics were performed using the ICM molecular modeling software suite (Molsoft, San Diego, CA).
Receptor binding studies
Receptor binding studies were performed using HK293hi cells transfected with the full-length human GHR and selected on the basis of elevated GHR expression (HK293hi cells) (17, 18). Two micrograms GH (human pituitary iodination grade, Calbiochem, San Diego, CA) were labeled with 37 MBq iodine-125 (Amersham Biosciences, Little Chalfont, UK) to a specific activity of 87 MBq/nmol using chloramine T (0.7 mM) for 45 sec and purified using a Sephadex G-10 column. Cells were plated into 12-well plates (300,000/well) and cultured overnight in DMEM/F-12 (1:1) containing 10% fetal calf serum. Cultures were washed once in serum-free DMEM/F-12, preincubated in serum-free medium for 3 h at 37 C, and then washed twice with PBS containing 1% BSA (PBS-BSA), and incubated with labeled GH (200,000 cpm/well) in 1 ml PBS-BSA with varying amounts of either wild-type or Ile179Met GH for a further 3 h at room temperature. At the end of the incubation period, cells were washed twice with PBS-BSA and solubilized in 1 M NaOH for quantification of bound 125I-GH. Experiments (n = 4) were performed in duplicate wells and dissociation constant (Kd) values calculated by Scatchard analysis of the data.
Proteolytic digestion of the GH variant
Trypsin, chymotrypsin, or proteinase K (all Sigma, Poole, UK) were added to a final concentration of 0.1 µg/ml to 100 µl culture medium harvested from insect cells expressing either wild-type GH or the Ile179Met variant (60 nM) and then incubated at 37 C for 1 h. Previous dose-dependent studies on wild-type GH had shown that 0.1 µg/ml was the lowest concentration at which GH degradation was detectable by all three enzymes. After the 1-h treatment period, 10 µl trypsin-chymotrypsin inhibitor (500 µg/ml) were added to stop the trypsin and chymotrypsin digests and 1 µl phenylmethylsulfonyl fluoride (0.1 M) was added to stop the proteinase K digest. Each reaction was then incubated for a further 15 min at 37 C. Samples were analyzed by SDS-PAGE on a 12% gel using a minigel apparatus (Bio-Rad Laboratories, Hercules, CA). Equivalent amounts of undigested wild-type GH and Ile179Met variant that had been incubated for 1 h at 37 C were also run on the gel. The gel was electroblotted onto polyvinyl difluoride membrane as previously described (19), probed with a mouse monoclonal antihuman GH antibody (Lab Vision, Fremont, CA), diluted 1:500, detected using an antimouse IgG-horseradish peroxidase conjugate (1:5000, Amersham Biosciences) and visualized by enhanced chemiluminescence (ECL Plus, Amersham Biosciences). Films were analyzed using the Alpha Imager 1200 digital imaging system (Alpha Innotech Corp, San Leandro, CA) and the results expressed as the amount of GH remaining after enzyme digestion as a percentage of undigested GH. The experiments were repeated three times and assessed statistically by a two-tailed t test.
GH secretion studies in mammalian cells
Rat pituitary (GC) cells were transfected with a pGEM-T plasmid containing a 3.2-kb gene fragment spanning the entire wild-type GH1 gene or the equivalent construct for the Ile179Met variant and assayed as previously described (14).
Luciferase reporter gene assay of STAT 5 activation
HK293 cells were used to assay STAT 5 activation (17, 18). HK293hi cells were transfected with a STAT 5-responsive luciferase reporter gene construct (17, 18), treated with GH (wild-type and variant) for 6 h and luciferase expression measured as described previously (14).
Activation of the ERK pathway
Activation of the ERK signal transduction pathway by wild-type and Ile179Met GH was investigated by stimulating murine 3T3-F442A preadipocytes (20). Cells (250,000) were plated into 10-cm culture dishes and cultured in DMEM containing 10% calf serum for 3 d before the experiment. The plates were washed with PBS and the cells incubated in serum-free DMEM for two successive 2 h washout periods. GH was spiked directly into the serum-free DMEM at the end of the second washout period and the cells incubated for the appropriate time. After this period, the medium was removed, and the cells were washed with ice-cold PBS containing 1 mM sodium orthovanadate, lysed in 0.5 ml Laemmli buffer containing 1 mM orthovanadate, and 1 mM phenylmethylsulfonyl fluoride and analyzed by SDS-PAGE on a 10% gel as described above. The gel was blotted onto polyvinyl difluoride membrane and probed using monoclonal antibodies that detect the activated forms of ERK 1/2 phosphorylated on residues Thr202/Tyr204 (Cell Signaling Technology, Beverly, MA) and STAT 5 phosphorylated on residues Tyr694/Tyr699 (Upstate Biotechnology, Lake Placid, NY). Western blots were processed, visualized using ECL Plus, and the images analyzed as described above. To ensure equal protein loading between lanes, blots were stripped, and reprobed with rabbit antibodies that recognize total ERK or STAT 5 (Santa Cruz Biotechnology, Santa Cruz, CA) as appropriate. Both phospho-specific and total STAT 5 antibodies cross-react equally with STAT 5a and 5b. Second antibodies were either antimouse or antirabbit IgG-horseradish peroxidase conjugates depending on the primary antibody used (1:5000, Amersham Biosciences). Films were analyzed by imaging densitometry as described above. Results for phospho-ERK and phospho-STAT 5 were normalized with respect to total ERK or STAT 5 in the same sample.
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Results |
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A group of 74 individuals with familial short stature was screened for GH1 gene lesions by sequencing the promoter, exons, introns, and untranslated regions of the gene. Five heterozygous GH1 gene lesions were identified in different individuals. Four of these mutations were different: three missense mutations and a single base pair substitution in the promoter region (Table 2). Two of these lesions (Ile179Met and -360 AG) were novel. The Mendelian segregation of both the Ile179Met and -360 AG mutations was confirmed by detection of the underlying substitutions in the fathers of the two probands. Both novel lesions could in principle have been templated by gene conversion, the donor sequence being one or other of the four paralogous genes in the GH cluster. The Thr-24Ala variant has been previously reported by Miyata et al. (21) and appears to be a neutral polymorphism (14). By contrast, the Val110Ile variant represents a previously reported functional polymorphism associated with reduced GH secretion in vitro (14). The authenticity of the novel lesions was verified by sequencing the opposite strand from independently PCR-amplified material. Neither lesion was noted in a total of 154 normal Caucasian controls (14). Evidence for or against the functional significance of the lesions was then sought from the analysis of orthologous sequence data, molecular modeling, the in vitro assay of either the biological activity, structure, and secretion of the protein, or, in the case of the -360 AG variant, the promoter strength.
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The evolutionary conservation of the hydrophobic residue Ile179 was examined by ClustalW multiple sequence alignment of orthologous GH proteins from 19 vertebrates (22). This residue is a hydrophobic valine in all vertebrates examined except turtle, indicating that the substitution by Ile in the human lineage was conservative. Comparison with the paralogous genes of the human GH cluster revealed that the residue analogous to Ile179 is Met in CSH1, CSH2, and the CSH pseudogene (CSHP1). This is consistent with the conservative Ile179Met substitution having been templated by gene conversion.
The Ile179Met substitution was then modeled by replacement of the residue in the x-ray crystallographic structure of human GH (Fig. 1). Ile179 is situated in helix 4 in which it is partially exposed, allowing hydrophobic interactions with the side chain of the hot spot GHR residue Trp169 (23, 24). Further interactions with the GHR occur between the side chain and backbone atoms of Ile179 and the backbone atoms of GHR residues Lys167 and Gly168. The replacement of the Ile179 residue with methionine in the model introduced unfavorable van der Waals (i.e. steric) interactions with the side chain of the Trp169 residue. Receptor binding studies were performed to determine the affinity of wild-type GH and the Ile179Met variant for the GHR. The introduction of a methionine residue failed to alter the receptor binding (Fig. 2); the Kd values of the wild-type and Ile179Met GH molecules were found to be 1.99 nM and 2.04 nM, respectively.
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) when compared with wild-type GH at a concentration of 1 nM, the approximate ED50 value for GH in this assay system. Whereas this variant could simply have failed to manifest its detrimental effects in a static in vitro system, the possibility was also considered that it might have exerted a deleterious effect on a signal transduction pathway other than JAK/STAT.
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). A 10-min GH treatment time was therefore selected for use in subsequent studies because this was the time of maximal ERK activation and was on the plateau phase of maximal STAT 5 activation. Cells were treated with a range of concentrations of wild-type and variant GH (0.5–20 nM) for 10 min and activation of ERK and STAT 5 analyzed. The Ile179Met variant exhibited a reduced ability to activate ERK throughout the concentration range studied, whereas there was no difference in STAT 5 activation (Fig. 5). Analysis of the data from multiple experiments using a single maximal dose of GH (20 nM) indicated that the Ile179Met variant had a significantly reduced ability to activate ERK to only 45% of the level produced by wild-type GH (4.35 ± 0.66 times basal level of activation for the Ile179Met variant as compared with 9.76 ± 2.52 times basal level of activation produced by wild-type GH; mean ± SEM, n = 5 separate experiments, P < 0.05, Student’s t test, Fig. 5). This contrasted with the variant’s ability to activate STAT 5 to the same level as wild-type GH at a concentration of 20 nM (36.50 times basal level for the wild-type vs. 38.62 times for the Ile179Met variant). These latter data served to provide confirmation of the results obtained from the STAT 5- responsive luciferase reporter gene assay that yielded similar levels of activity for both wild-type GH and the Ile179Met variant.
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The -360 AG transition detected in the GH1 gene promoter of individual B4 could in principle have been templated by gene conversion, the donor sequence being any one of the four paralogous genes in the GH cluster (all four sequences possess a G nucleotide at the analogous location). The -360 AG lesion was assessed in terms of its ability to drive luciferase gene expression in a reporter gene assay. To be meaningful, the luciferase activity of the promoter variant must be normalized with respect to the specific proximal promoter haplotype (13) associated in cis with that variant. However, the promoter haplotype noted in individual B4 has not been noted before. We therefore termed this specific combination of 16 SNP alleles (GGGTTGTGGGGTGAAT) haplotype 41 to distinguish it from the other 40 previously described haplotypes (13). The luciferase activity of the -360 AG variant was normalized with respect to the expression level associated with promoter haplotype 41. This yielded a near normal value (110 ± 7% wild-type) for promoter activity that was not indicative of the -360 AG variant being either of functional or phenotypic significance.
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Discussion |
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G) in separate individuals. Two other lesions, a Val110Ile functional polymorphism and the Thr-24Ala neutral polymorphism, have been reported previously (14). The phenotypic significance of both novel lesions was then explored by functional assay. Both evolutionary conservation data and evidence from molecular modeling suggested that the Ile179Met variant was a conservative substitution. This conclusion was underscored by the similar degree of resistance manifested by the Ile179Met GH variant to proteolytic cleavage as compared with wild-type Ile179 GH, consistent with the view that the introduction of Met at position 179 does not cause significant misfolding of the GH molecule. This should be considered within the context that some 67% of our previously identified GH variants (14) manifested increased susceptibility to proteolysis, compared with wild-type GH (Easter, T. E., and M. D. Lewis, unpublished observations). Consistent with the absence of misfolding, the level of secretion of the Ile179Met variant from rat pituitary cells was indistinguishable from that of wild-type GH.
Ile179 is located in the C-terminal portion of helix 4, which is involved in site 1 binding. Molecular modeling studies have suggested that although the Ile179Met is a conservative substitution, it may perturb interactions between the GH molecule and the GHR loop containing the hot spot residue Trp169 (23, 24). We found no difference in the overall binding affinities (Kd) of wild-type and Ile179Met GH variant, suggesting that if any differences exist between the binding characteristics of wild-type and variant GH, they are subtle and do not alter the Kd as measured in a static system. Alanine-scanning mutagenesis has previously identified Ile179 as contributing to a patch of residues that determine receptor affinity; a nonconservative alanine substitution of this residue resulted in a marked decrease in GHR binding (Kd increased from 0.34 to 0.92 nM) (25). In view of the fact that Ile179 contributes to receptor binding affinity, it was considered possible that the Ile179Met variant could exert a subtle effect on GHR binding. We therefore investigated whether the putatively perturbed interaction between the Ile179Met GH variant and GHR could lead to reduced activation of the STAT 5 and ERK pathways. STAT 5 activation was studied directly by determining the level of activated phospho-STAT 5 by Western blotting and indirectly using a luciferase reporter gene assay. Both approaches indicated identical levels of activation in response to wild-type GH and the Ile179Met variant. By contrast, activation of the ERK pathway by the Ile179Met variant (as determined by Western blotting analysis of phospho-ERK levels) occurred at only half the level elicited by wild-type GH. To our knowledge, the differential activation of cell signaling pathways by a mutant receptor agonist is quite unprecedented. In contrast to the Ile179Met variant, all GH variants previously identified in studies from our laboratory were either associated with reduced secretion or a reduced ability to activate the JAK/STAT pathway or both (14).
Activation of JAK 2 and ERK are dependent on different regions of the cytoplasmic domain of the GHR from those involved in STAT 5 activation. STAT 5 activation requires JAK 2-mediated phosphorylation of tyrosine residues 534, 566, and 627, located toward the C-terminal end of the cytoplasmic domain of the GHR that are not required for GH-induced ERK activation (26). By contrast, activation of JAK 2 and the ERK pathway is dependent on a 46-amino acid stretch containing a proline-rich (box 1) domain located adjacent to the cell membrane (27). Activation of ERK after GHR activation appears to be complex, involving multiple mechanisms. One of these mechanisms is mediated by JAK 2- dependent activation of the Shc-Grb2-Sos-Ras pathway (28, 29), possibly involving multiple docking proteins such as insulin receptor substrate-1 (30), Gab-1 (31), and the epithelial growth factor receptor (32). An alternative JAK 2-independent mechanism of ERK activation via Src-dependent activation of Ral and phospholipase D has recently been reported (33). Full ERK activation by GH requires activation of both JAK 2 and Src, although Src activation alone is sufficient for partial ERK activation (33). It is thus possible that GHR activation by the Ile179Met GH variant could activate JAK 2 normally but not Src, resulting in complete STAT 5 activation but only partial activation of ERK.
The identification of a GH variant manifesting normal activation of STAT 5 but reduced activation of ERK in a child exhibiting short stature raises some interesting questions regarding the role that ERK might play in mediating the actions of GH. Previous studies have suggested that the ERK pathway is not involved in the GH induction of IGF1 gene expression (12, 34). It may be noted that the Ile179Met variant did not cosegregate with the short stature phenotype in the family under study, strongly suggesting that this variant is on its own insufficient to fully account for the observed clinical phenotype. However, it remains possible that this variant may nevertheless still have contributed to the short stature manifested by the proband while acting in concert with variants at unlinked loci encoding other GH axis proteins. In support of this view is the recently reported finding that a mutant GHR (containing a pseudoexon insertion resulting in inclusion of an extra 36 amino acids) found in a family with marked short stature (35) shows increased ability to activate STAT 5 with only minimal activation of the ERK pathway, compared with wild-type GHR after GH stimulation (36). Stimulation of wild-type GHR by the Ile179Met GH variant described in this report would yield a similar profile of signaling pathway activation to that produced by the mutant GHR containing the pseudoexon insertion. This leads one to speculate that the ERK pathway may play a role, as yet unidentified, in regulating the growth-promoting effects of GH.
A recent report has described a patient with GH insensitivity and a homozygous missense mutation in the STAT 5b gene (37) but with normal STAT 5a. This might suggest that STAT 5b plays a crucial role in mediating the growth- promoting effects of GH but that STAT 5a is not as important; supporting this view are the previous studies demonstrating that STAT 5b is required for the GH induction of liver IGF1 expression (10, 11). It remains possible, although not likely, that the Ile179Met GH variant described in this study exerted differential effects on STAT 5a and STAT 5b, which were not identified during the course of the study because the assays of STAT 5 activation employed (phospho-STAT 5 antibody and LHRE-luciferase reporter gene) did not discriminate between the two forms of STAT 5.
During the course of this study, a -360 AG transition was also detected in the GH1 gene promoter. However, luciferase reporter gene assays provided no evidence for a difference in expression between mutant and wild-type promoters. This notwithstanding, we cannot entirely exclude the possibility that in human pituitary cells in vivo, in the context of an extended promoter including the locus control region, this mutation could exert an effect on expression of the downstream GH1 gene.
We have noted before that the GH1 gene manifests a remarkably high level of mutation with a continuum of genetic change from neutral polymorphisms to dysfunctional variants (14). The results of this study serve to indicate that the GH1 gene may also harbor rare variants such as Ile179Met that exhibit a reduced ability to activate the ERK pathway while retaining normal ability to activate STAT 5. Since this variant was identified in a child with short stature, it may be that the ERK pathway plays a role in mediating some of the growth-promoting or other effects of GH.
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Acknowledgments |
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Footnotes |
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Abbreviations: GHR, GH receptor; JAK, Janus tyrosine kinase; SDS, SD score; STAT, signal transducer and activator of transcription.
Received April 15, 2003.
Accepted November 14, 2003.
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