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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-2022
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The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 3 1030-1037
Copyright © 2008 by The Endocrine Society

Growth Hormone (GH) Insensitivity and Insulin-Like Growth Factor-I Deficiency in Inuit Subjects and an Ecuadorian Cohort: Functional Studies of Two Codon 180 GH Receptor Gene Mutations

Peng Fang, Rose Girgis, Brian M. Little, Katherine L. Pratt, Jaime Guevara-Aguirre, Vivian Hwa and Ron G. Rosenfeld

Department of Pediatrics (P.F., B.M.L., K.L.P., V.H., R.G.R.), Oregon Health and Science University, Portland, Oregon 97239-3098; Department of Pediatrics (R.G.), University of Alberta, Edmonton, Canada AB T6G2B7; the Institute of Endocrinology, Metabolism, and Reproduction (J. G.-A.), Quito, Ecuador; Lucile Packard Foundation for Children’s Health (R.G.R.), Palo Alto, California 94304; and Department of Pediatrics, Stanford University (R.G.R), Stanford, California 94305-2038

Address all correspondence and requests for reprints to: Dr. Vivian Hwa, Department of Pediatrics, NRC5, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239-3098. E-mail: hwav{at}ohsu.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 Note Added in Proof
 References
 
Context: Among more than 250 cases of GH insensitivity syndrome (GHIS) reported to date, the largest cohort was identified in southern Ecuador. In the Ecuadorian GHIS cohort, a sense mutation (GAA > GAG) at codon E180 of GH receptor [GHR (E180sp)] results in deletion of codons 181–188. No functional studies of this mutation have been performed, nor have different mutations at codon 180 been reported.

Objective: We now report identification of a novel GHR mutation, also within codon E180, in two distantly related GHIS subjects of Inuit origin and provide mechanistic insights into the defects caused by the Inuit and Ecuadorian GHR mutations.

Patients: The two Inuit subjects, with heights of –5 SD score and –7 SD score, respectively, had elevated circulating levels of GH but low levels of GH-binding protein, IGF-I, and IGF-binding protein-3.

Results: Both Inuit subjects carry the same novel nonsense homozygous GHR mutation at codon E180 (GAA->TAA, E180X). In vitro reconstitution experiments demonstrated that GHR (E180sp), but not GHR (E180X), could be stably expressed. GHR (E180sp), however, could not bind GH and could neither activate signal transducer and activator of transcription-5b nor induce -5b-dependent gene expression on GH treatment. Furthermore, the GHR (E180sp), which has a deletion of eight amino acid residues within the GHR dimerization domain, although retaining the ability to homodimerize, was defective in trafficking to the cell surface.

Conclusions: The E180X mutation identified in two Inuit patients resulted in a truncated, unstably expressed GHR variant, whereas the E180 splicing mutation previously identified in the Ecuadorian cohort, affected both GH binding and GHR trafficking and rendered the abnormal GHR nonfunctional.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 Note Added in Proof
 References
 
The human GH receptor (GHR) is a glycosylated membrane protein encoded by a single gene with 10 exons. In its mature form, the GHR protein comprises 620 amino acid residues and forms three domains, an extracellular domain (encoded by exons 2–7), a single transmembrane domain (encoded by exon 8), and an intracellular domain (encoded by exons 9 and 10). The extracellular domain of the GHR is comprised of two functional subdomains (1): subdomain 1, consisting of residues 1–123 (exons 2–5), is mainly involved in GH-GHR interaction, and subdomain 2, consisting of residues 128–246, is predominantly involved in receptor dimerization and GH-induced rotation (2, 3, 4). In addition, a soluble, proteolytic product of the full-length GHR, corresponding to the extracellular domain, circulates as the GH binding protein (GHBP), serving as a diagnostic marker for assessing the abundance of functional GHR.

Mutations or deletions of the GHR gene affecting the extracellular domain of GHR constitute the etiology of classical GH insensitivity syndrome (GHIS), or Laron syndrome, a pathological condition characterized by severe postnatal growth retardation, and associated with normal-elevated serum concentrations of GH but abnormally low serum levels of IGF-I (5, 6, 7). To date, more than 70 unique GHR mutations, including missense or nonsense mutations, splice-site mutations, and insertions or deletions (5, 6, 7), have been identified in more than 250 GHIS patients. Splice-site mutations, in particular, through eliminating normal splicing sites or creating new splicing sites, may cause an exon or multicodon deletion from the GHR mRNA (8, 9, 10, 11, 12, 13, 14, 15, 16, 17) or, in some cases, a multicodon insertion (as a pseudoexon) into the GHR mRNA (18, 19). When such cases have been shown to have low serum concentrations of GHBP or decreased GH binding to cell membranes, the convenient, but not always proven, explanation has been that the splice defect results in decreased affinity of the mutant receptor for GH.

We now report two female GHIS patients of Inuit origin, with clinical presentations of severe growth retardation [heights below –5 SD score (SDS)], in whom we have identified a novel homozygous nonsense mutation at codon E180 (E180X), within subdomain 2 of the extracellular portion of the GHR. Intriguingly, a mutation within E180 in the largest cohort of GHIS, involving approximately 100 members in Southern Ecuador, had been previously reported (8, 20). The single nucleotide mutation was a sense mutation that altered the coding sequence of E180 from GAA to GAG, and created a cryptic splice site that resulted in an in-frame deletion of residues 181–189 (8). A unique opportunity to compare and contrast the effects(s) of each of these two E180 mutations on GHR expression and function was provided, and we now demonstrate that whereas each mutation renders the GHR nonfunctional, the mechanisms of inactivation are distinct.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 Note Added in Proof
 References
 
Case reports

Patient 1, an Inuit girl from the Northwest Territories, Canada, was born at 37 wk gestation with a normal birth weight (3530 g). At the age of 2 yr 7 months, she had a height of 67 cm (–5 SDS) and weight for length at the 75th percentile for age. Her plasma GH concentration was 42 µg/liter at baseline and 114 µg/liter after stimulation with arginine and clonidine. Her serum levels of GHBP (100 pmol/liter, normal 267-1638 pmol/liter), IGF-I (<20 ng/ml, normal 17–248 ng/ml), and IGF-binding protein (IGFBP)-3 (500 ng/ml, normal 700-5200 ng/ml) were markedly low (Table 1Go). In addition, she had typical features of GHIS, including frontal bossing, upturned nose, and depressed nasal bridge.


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  		TABLE 1. Clinical and biochemical characteristics of the patients

 
Patient 2 was a distantly related female of patient 1 from Nunavut, Canada. She was born at 33 wk gestation with a normal birth weight of 2700 g (above 90th percentile for weight). At the age of 14 yr, she had a height of 111 cm (–7 SDS) and a delayed bone age (7 yr 10 months). She presented with typical GHIS dysmorphic features and was obese (BMI > 95th percentile for age), clinically euthyroid, and prepubertal (Tanner stage I). She had a history of Legg-Calvé-Perthe disease. Her plasma GH concentration was 41 µg/liter after stimulation with arginine and clonidine. Her serum levels of GHBP (180 pmol/liter, normal 431-1892 pmol/liter), IGF-I (<20 ng/ml, normal 75–550 ng/ml), and IGFBP-3 (1000 ng/ml, normal 2700–9500 ng/ml) were also markedly low (Table 1Go).

By report, there are two adult women with similar clinical phenotype from the Nunavut, Canada. The mother of patient 1 reports these women to be her first cousins; the mother of patient 2 reports them to be her second cousins. Detailed genealogies are not available.

Serum assays

The consents from the patients’ parents and assent from patient 2 were obtained before our studies. The serum samples from the patients were analyzed for GHBP by a ligand binding immunoprecipitation assay (Esoterix, Calabasas Hills, CA), for IGF-I and IGFBP-3 by ELISA (IMMULITE; Diagnostic Products Corp., Los Angeles, CA). The serum level of GH for patient 1 was measured by a solid-phase, two-site, chemiluminescent immunometric assay (IMMULITE, Diagnostic Products) or ELISA for patient 2 (Diagnostic Systems Laboratories, Webster, TX).

Genomic DNA

Genomic DNA from whole blood and sequencing of the GHR gene were performed as described previously (21).

Generation of recombinant GHR

The E180X GHR mutant, the E180 splicing GHR mutant, and the N-terminal Flag- or hemagglutinin (HA)-tagged GHR variants were generated with QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA) with full-length GHR (GHRfl) cDNA in the pcDNA1/AMP expression plasmid as template (22). The amino acid sequence for Flag or HA epitope generated in this study was DYLDDDDK or YPYDVPDYA, respectively. The Flag tag or HA tag was inserted immediately after the signal peptide of GHR (between amino acid residues 18 and 19 of the GHR precursor) (3). The primers for mutagenesis were: E180X, 5'-gaacttcaatacaaataagtaaatgaaactaaatgg-3' (sense), 5'-ccatttagtttcatttacttatttgtattgaagttc-3' (antisense); E180splicing: 5'-gaacttcaatacaaagaaatggaccctatattgacaac-3' (sense), 5'-gttgtcaatatagggtccatttctttgtattgaagttc-3' (antisense); Flag-tag: 5'-ggatcaagtgatgctgactacaaggacgacgacgacaagttttctggaagtgag-3' (sense), 5'-ctcacttccagaaaacttgtcgtcgtcgtccttgtagtcagcatcacttgatcc-3' (antisense); HA tag: 5'-gatcaagtgatgcttacccgtacgacgtgcccgactacgcgttttctggaagtg-3' (sense), 5'-cacttccagaaaacgcgtagtcgggcacgtcgtacgggtaagcatcacttgatc-3' (antisense). Resultant GHR variants were confirmed by sequencing.

Cell culture and transfection

Cell culture and plasmid transfection of HEK293 cells were performed as described previously (21). After 24 h transfection, the cells were serum starved in DMEM supplemented with 0.1% BSA for 16 h before treatments with human GH (a generous gift from Genentech, Inc., South San Francisco, CA) as indicated.

GH binding assays

Monolayer GH binding assays were performed as described previously (21). GH binding to GHR on cellular membranes was measured as described (23), with minor modifications. Briefly, HEK293 cells transfected with GHR variants were dislodged from the culture surface with 5 mM EDTA in Hank’s balanced salt solution saline buffer (Hyclone, Logan, UT) supplemented with 0.1% BSA, resuspended in 0.1% BSA-Hank’s balanced salt solution saline containing a cocktail of protease inhibitors (Roche, Indianapolis, IN) and disrupted by sonication on ice. The disrupted cell mixture was centrifuged at 200 g for 5 min to remove unbroken cells. The resulting supernatant was then subjected to centrifugation at 40,000 x g for 1 h to obtain cellular membrane fractions. The membrane fraction was either used for Western immunoblot analyses to estimate the amount of GHR expressed or incubated with I125-GH (NEX-100; GE Healthcare Bio-Sciences Corp., Piscataway, NJ) to measure specific GH binding.

Western immunoblot analysis and immunoprecipitation

Preparation of cell lysates and subsequent Western immunoblot analyses were performed as described previously (21). Immunoprecipitations with anti-HA-agarose beads (Sigma, St. Louis, MO) were performed following the manufacturer’s protocol. For endoglycosidase H (Endo H; New England Biolabs, Inc., Ipswich, MA) treatment, the cell lysates were boiled for 10 min in 1x glycoprotein denaturing buffer (0.5% sodium dodecyl sulfate, 100 mM dithiothreitol) before incubation with/without Endo H (500 U/25 µg protein) in 100 mM sodium acetate (pH 5.5) at 37 C for 3 h. The mixture was then subject to Western immunoblot analyses as described above.

The antibodies used in this study were: rabbit polyclonal IgG against phospho-Tyr694-signal transducer and activator of transcription (STAT)-5 (dilution 1:1000) from Cell Signaling Technology (Beverly, MA); mouse monoclonal IgG against STAT5b (G-2) (dilution 1:1000) from Santa Cruz Biotechnology (Santa Cruz, CA); mouse monoclonal IgG against HA-tag (dilution 1:1000) from Sigma; rabbit polyclonal IgG against human GHR (GHRcyt-AL47) (dilution 1:2000) (24), or mouse monoclonal IgG against rabbit GHR (GHRext-MAB) (dilution 1:1000) (24, 25), both generously provided by Dr. Stuart J. Frank (University of Alabama, Birmingham, AL). Secondary antibodies (antimouse IgG and antirabbit IgG) were obtained from Amersham-Pharmacia Biotech (Uppsala, Sweden). The density of the bands on x-ray film was determined by using IMAGEQUANT 5.1 (Molecular Dynamics, Sunnyvale, CA). All immunoblot data shown are representative of at least three independent experiments.

Luciferase reporter assays

Cell lysates from transfected HEK293 cells were analyzed for reporter activity from an 8x GHRE(Spi2.1)-pGL2 firefly luciferase construct (p8x GHRE-LUC) as previously described (21).

Flow cytometry

HEK293 cells were transfected with vector or vector carrying N-terminal HA-tagged GHR for 42 h and starved in 0.1% BSA-DMEM medium for 4 h. The cells were dislodged from the culture surface with 5 mM EDTA in 0.1% BSA-Dulbecco’s PBS. The dislodged cells (~500,000 cells) were washed, resuspended in 400 µl 1% BSA-Dulbecco’s PBS, and incubated with 0.88 µg anti-HA monoclonal antibody on ice for 30 min. The cells were then washed, resuspended, and incubated with 1.5 µg horse fluorescein antimouse IgG (H+L) antibody (Vector Laboratories, Burlingame, CA) on ice for 20 min. The fluorescein-labeled cells were washed once before flow cytometry analyses with BD FACSCalibur analyzer (BD Biosciences, San Jose, CA). The data were analyzed with BD CellQuest Pro software (BD Biosciences).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 Note Added in Proof
 References
 
A novel homologous nonsense mutation in the GHR gene identified in two Inuit patients

Sequencing of the GHR gene from genomic DNA obtained from whole blood of the two Inuit patients revealed that they both carry a G to T transversion in exon 6, which results in a homologous nonsense mutation at codon E180 (GAA>TAA, E180x) (Fig. 1Go). Analysis of the genomic DNA from the mother of patient 1 revealed that the normal-statured mother was heterozygous for E180X (Fig. 1Go). Interestingly, a sense mutation at codon E180 was previously reported in a cohort of Ecuadorians diagnosed with GHIS (8, 20). The sense mutation, GAA to GAG, did not alter the encoded amino acid residue but created a pseudo-5' splice-donor site, which generated a GHR mRNA lacking the 24 nucleotides that encode residues 181–189 at the 3' end of exon 6 (Fig. 1Go).


Figure 1
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  		FIG. 1. Genetic analyses of the GHR gene. The electropherograms of genomic DNA sequence (sense strand) from normal population, Inuit patient 1, and her mother are shown. The arrows indicate the normal sequence, the homozygous mutation (a G to T conversion), or the heterozygous mutation at codon E180. The schematic depiction of the human GHR gene, the previously identified Ecuadorian E180 splicing mutation, and the currently identified Inuit E180X mutation, as well as the boundary between intron 6 and exon 6 or exon 7, are also shown. The boxed nucleotides indicate the 5' splice donor site, and the underlined nucleotides constitute the 3' splice receptor site.

 
Neither the nonsense mutation nor the splicing mutation at codon E180 renders the resulting GHR functional

The impact of the E180X mutation or the E180 splicing mutation (E180sp) on GHR functionality was assessed in reconstitution systems. The full-length GHRfl(E180X) was not readily immunodetected by our anti-GHR antibodies or the antibodies against the Flag or HA epitopes, when GHRfl(E180X) was N-terminal Flag-tagged or HA-tagged (data not shown), suggesting that the E180X mutation did not result in stable expression of a truncated GHR. On the contrary, the GHRfl(E180sp) was stably expressed and detectable in the cytoplasm (Fig. 2AGo, lane 3). Interestingly, the GHRfl(E180sp) was immunodetectable only by an antibody that recognized the C terminus, or intracellular domain, of the GHR (Fig. 2AGo, lane 3) but not by a conformationally sensitive antibody raised against an epitope located within subdomain 2 of the extracellular domain of the GHR (25) (Fig. 2AGo, lane 6). It was also of note that GHRfl(E180sp), compared with the wild-type, lacked detectable higher molecular weight forms (115–130 kDa) that correspond to the mature, normally glycosylated GHR (Fig. 2AGo, lanes 2 and 5, indicated by an asterisk) (26, 27).


Figure 2
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  		FIG. 2. The E180 splicing mutation renders GHR nonfunctional. A, Expression of GHR(E180sp) in HEK293 cells. The expression of GHR was detected by an antibody against the cytoplasmic domain of the human GHR protein ({alpha}-hGHRcyt) or a conformational sensitive antibody against the extracellular domain of the rabbit GHR protein ({alpha}-rGHRext). The asterisks indicate the mature, normally glycosylated GHR species. IB, Immunoblot. B, I125-GH binding to monolayer HEK293 cells expressing GHRfl or GHR(E180sp). The data are presented as percentage of specific binding (mean ± SE) from four independent experiments performed in duplicates. C, GH-induced STAT5b activation in HEK293 cells expressing GHRfl or GHR(E180sp). The cell lysates were collected 20 min after treatment. Western immunoblots for phospho-STAT5 protein were stripped and reprobed with anti-STAT5b antibody. D, GH-induced luciferase activity in HEK293 cells expressing GHRfl or GHRfl(E180sp). The luciferase activities were normalized to total protein concentration. The results were expressed as relative luciferase activity over control (no exogenous GHR expressed). The results presented (mean ± SE) are from three independent experiments performed in duplicate.

 
Although GHRfl(E180sp) was stably expressed, no significant specific GH binding to the surface of the cells overexpressing GHRfl(E180sp) was detected (Fig. 2BGo). Consistently, GHRfl(E180sp) could neither activate STAT5b in response to 100 ng/ml GH (Fig. 2CGo, lane 6) nor induce STAT5b-dependent luciferase reporter expression in response to 10 or 100 ng/ml GH (Fig. 2DGo). Altogether, these results suggest that the E180 splicing mutation renders the resulting GHR nonfunctional.

GHR(E180sp) can still undergo homodimerization

To determine the effect of the E180 splicing mutation on GHR dimerization, expression plasmids carrying cDNA for the N-terminal Flag-tagged or HA-tagged GHRfl or GHRfl(E180sp) were constructed and coprecipitation of tagged GHR variants determined. Addition of either epitope tag appeared to have minimal effect on the expression of GHRfl(E180sp) (Fig. 3AGo). When coexpressed in HEK293 cells, Flag-tagged mutant GHRfl(E180sp) and HA-tagged GHRfl(E180sp) coimmunoprecipitated, independent of GH treatment (Fig. 3BGo, lanes 4 and 8), suggesting that the ability to homodimerize was retained in GHRfl(E180sp).


Figure 3
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  		FIG. 3. Predimerization of E180 splicing GHR mutant. A, Expression of N-terminal Flag- or HA-tagged GHR in HEK293 cells. The asterisk indicates the mature, normally glycosylated GHR species. IB, Immunoblot. B, Coimmunoprecipitation of N-terminal Flag-tagged GHR with N-terminal HA-tagged GHR. HEK293 cell lysates used for immunoprecipitation (IP) were collected 30 min after treatment.

 
GHR(E180sp) is a trafficking-defective GHR

To investigate whether the GHRfl(E180sp) variant might be defective in protein trafficking, N-terminal HA-tagged GHRfl or GHRfl(E180sp) was expressed in HEK293 cells, and the GHR expression on the cell surface was evaluated by flow cytometry, using a monoclonal antibody against the HA epitope. The results indicate that the fluorescence signal detected on the surface of the cells expressing GHRfl(E180sp) was not significantly different from that detected on the surface of the cells expressing the vector only and was substantially lower than that detected on the surface of the cells expressing GHRfl (Fig. 4AGo).


Figure 4
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  		FIG. 4. GHR(E180sp) is a trafficking-defective GHR. A, Flow cytometry analyses of GHR expression on cell surface. The overlaid histogram and mean fluorescence for a population of HEK293 cells expressing vector, HA-tagged GHRfl, or HA-tagged GHR(E180sp) are shown. The data presented here are representative of at least four independent experiments. B, Endo H treatment of GHRfl or GHR(E180sp). The asterisks indicate the mature, normally glycosylated GHR species that is resistant to Endo H, and the arrows indicate the intermediate glycosylated GHR species that is sensitive to Endo H. IB, Immunoblot.

 
To further confirm that the GHRfl(E180sp) was not fully glycosylated and therefore unable to reach the cell surface, cell extracts containing overexpressed GHRfl(E180sp) or GHRfl were treated with Endo H. Mature, fully glycosylated wild-type GHRfl forms are resistant to Endo H (Fig. 4BGo; compare lane 2 with lane 5, indicated by the asterisk), whereas GHR that is only partially N-glycosylated, is Endo H sensitive (Fig. 4BGo; compare lane 2 with lane 5, indicated by the arrow). GHRfl(E180sp), in contrast to wild-type GHRfl, was completely sensitive to Endo H (Fig. 4BGo; compare lane 3 with lane 6), suggesting that GHRfl(E180sp) does not undergo the entire posttranslational modification process (see Discussion). Altogether these results suggest that the E180 splicing mutation results in abnormal glycosylation and a severe defect in GHR trafficking.

Intracellular GHR(E180sp) cannot bind GH

To determine whether the retained, intracellular GHR(E180sp) variant could bind GH, intracellular membrane fractions from cell lysates overexpressing GHR(E180sp) or GHRfl were purified and used in GH-binding assays. The results (Fig. 5Go) indicate that overall level of GHR(E180sp) detected in the intracellular membrane fraction was 60% less than that of GHRfl and that the total specific GH binding to this amount of GHR(E180sp) was only 4%, compared with GHRfl (Fig. 5Go). Increasing the concentration of GHR(E180sp) 4-fold did not increase specific GH binding (9% relative to GHRfl; Fig. 5Go). Therefore, the E180 splicing mutation, in addition to its effect on GHR trafficking to the cell membrane, also abolishes the ability to bind GH.


Figure 5
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  		FIG. 5. GH binding to GHR on cellular membrane. The cellular membrane fraction was prepared from 2.5 x 105 (one time) or 1 x 106 (four times) HEK293 cells transfected with 0.25 µg (one time) or 1 µg (four times) of vector, vector carrying cDNA for GHRfl or GHR(E180sp) as described in Subjects and Methods. The membrane fraction prepared was either subjected to Western immunoblot (IB) analyses or incubated with 105 cpm (~1 ng/ml) of I125-GH to determine the total nonspecific binding or specific binding (in the presence of 10 µg/ml unlabeled GH). The data are presented as percentage of specific binding (mean ± SE) from three independent experiments performed in duplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
Note Added in Proof
 References
 
Mutations of codon 180 of the GHR gene were originally identified in an Ecuadorian population, in which approximately 100 individuals, derived from an inbred population of apparently full Spanish descent, have been shown to be homozygous for a splicing mutation (8). Here we report two distantly related GH insensitive patients of Inuit origin from a geographically isolated and inbred population of the Queen Elizabeth Islands of northern Canada, who have demonstrated the clinical phenotype of the classical GHIS/Laron syndrome, i.e. severe growth retardation, elevated circulating levels of GH, but low serum concentrations of IGF-I and IGFBP-3. The genetic analyses led to identification of a novel homozygous nonsense mutation at codon E180 of GHR (the E180X mutation), which, like the majority of the genetic aberrancies identified affecting the GHR, occurs in the region encoding the extracellular domain of the receptor. A number of nonsense mutations in close proximity to codon E180 have been identified previously in GHIS patients, including E183X, R217X, or 234X; the latter was caused by a two-nucleotide deletion at codon 230 (13). Although no functional studies have been performed, the nondetectable levels of GHBP found in the serum of these patients (13) implied that the truncated proteins as a consequence of these nonsense mutations were nonfunctional and possibly not stably expressed, despite retention of most of the extracellular domain of the GHR. The low serum levels of GHBP measured in the two Inuit patients (Table 1Go), however, implied that the E180X mutation might generate a truncated GHR with some residual function. Our in vitro reconstitution experiment, on the contrary, indicates that the E180X mutation does not generate a stably expressed GHR. The reason for this discrepancy between the GHBP assay and the reconstitution experiment is unknown but may be due to limitations of the assay systems.

A different mutation at codon E180 of GHR was identified previously in all but one patient within the Ecuadorian cohort, which originally included 79 patients (8). The Ecuadorian mutation, by introducing a 5' splicing donor site, although not changing the amino acid encoded by codon E180, causes an in-frame deletion of eight codons (181–189) from the GHR mRNA (Fig. 1Go). As demonstrated in reconstitution systems, the GHRfl(E180sp) was indeed capable of stable expression (Fig. 2Go), contrary to the previous prediction (28). This observation consequently leaves open the question of why this GHR mutation results in decreased GH binding and GHR activation.

The extracellular domain (ECD) of the GHR consists of two fibronectin type III subdomains, each of which contains seven β-strands (ABCC'EFG), in which sheets ABE form a β-sandwich with sheets CC'FG (1, 29). Residues 181–189, the eight residues missing in GHRfl(E180sp), extend from the linker between strands C and C' into the N terminus part of strand C’ in subdomain 2 of ECD (1, 29, 30). Conceivably, removal of these eight residues would severely disrupt the organization of the subdomain 2.

Subdomain 2 mainly contributes to the GHR dimerization interface. In addition, several residues within subdomain 2, such as W169, K215, also contribute to GHR binding to GH (1, 3, 29, 31). GHR dimerization has been shown to occur in the endoplasmic reticulum (ER), independent of ligand binding (3, 32). Although not conclusive, interaction at the dimerization interface is likely to be required for ligand-independent GHR dimerization (4, 32, 33). According to prior studies, the region containing the deleted eight amino acid residues appears not to be directly involved in the GH binding or dimerization interactions (1, 3, 29, 31). Our in vitro reconstitution experiments, however, revealed that GHRfl(E180sp) lost binding affinity to GH (Fig. 4Go) while still retaining the ability to homodimerize independent of ligand binding (Fig. 3Go). It is of note that the marked reduction in GH binding cannot be attributed entirely to the trafficking defect of the abnormal receptor (see below) because the GHRfl(E180sp) had minimal GH binding, even when present intracellularly. These results thus indicate that although altering the essential ECD structure for GH binding, deletion of residues 181–189 did not significantly disrupt the structural requirement for GHR dimerization. It is also possible, in theory, that this deletion may have removed the original determinants for dimerization but concurrently exposed others that still could facilitate GHR dimerization. Resolving the crystal structure of GHR(E180sp) should provide more precise answers.

Based on our current understanding, the synthesis of GHR should take the following simplified steps: the nascent peptide is synthesized by ribosomes on the rough ER and inserted into the ER membrane; the ECD of the GHR then acquires core glycosylation in the lumen of the ER, and the protein must be correctly folded before being transported from the ER to the Golgi network (cis, medial, or trans Golgi), in which the glycans are further modified; finally, the mature GHR is transported and presented on the plasma membrane (34, 35). Several lines of evidence obtained from our in vitro reconstitution experiments strongly suggest that the E180 splicing mutation causes defective GHR trafficking: 1) Western immunoblot analyses indicated that expression of GHRfl(E180sp) did not generate the mature, properly glycosylated receptor species (Figs. 2AGo and 3AGo); 2) flow cytometry analyses revealed that GHRfl(E180sp) has only minimal presence on the cell surface (Fig. 4AGo); and 3) unlike the wild-type GHR, GHRfl(E180sp) is completely sensitive to Endo H digestion (Fig. 4BGo). As the glycosylated protein acquires resistance to Endo H only after transport to medial Golgi (36), GHRfl(E180sp) is predicted to be trapped inside either cis Golgi or the ER.

It is also of note that among the deleted eight residues, residue N182 is a N-linked glycosylation site (26). Although protein glycosylation has been shown to play an important role in protein quality control in the ER (34, 37), eliminating each individual glycosylation site of the GHR appears not to significantly affect GHR cell surface presentation, GHR binding to GH, GHR internalization, or GH-induced signal transduction (26). Thus, the retention of GHRfl(E180sp) in the ER or cis-Golgi, as discussed above, is unlikely to be a consequence of the loss of a glycosylation site but more likely a result of misfolding of the mutant GHR protein, which, perhaps by exposing protein domain(s) not in the wild-type GHR, prolongs interaction with ER-resident chaperons to cause accumulation in the ER/cis Golgi and/or (eventually) be targeted for the degradation pathway (34, 35). Further investigations are required to illuminate the detailed mechanisms underlying the trafficking deficiency exhibited by GHRfl(E180sp).

In addition to the Ecuadorian E180 splicing mutation, several other mutations identified previously in GHIS patients have also been shown to cause defective GHR trafficking, including mutation F96S (38, 39), I153T, or V155G (40), and an insertion of 36 codons (a pseudoexon) between exons 6 and 7 caused by activation of an intronic 5' pseudosplicing site (18, 19). Unlike the E180 splicing mutation, however, the GHRs generated by these mutations more or less retained the ability to bind GH intracellularly. In addition, the patients carrying F96S, I153T, or V155G displayed classical GHIS clinical phenotypes, with no detectable serum levels of GHBP (38, 40), consistent with the minimal cell surface presentation of the mutated GHR protein. On the other hand, the patients carrying the pseudoexon mutation displayed much milder phenotypes, with apparently normal levels of GHBP (18, 19). Furthermore, the results from reconstitution systems demonstrated that the pseudoexon mutation did not significantly alter the expression profile of the GHR or the ability to transduce signal induced by exogenous GH (23). Therefore, although these two splice site mutations cause an alteration in the same region of the GHR protein, the deletion of eight amino acid residues caused by the E180 splicing mutation has far more detrimental effects on GHR function than the insertion of 36 amino acid residues caused by the pseudoexon mutation.

In conclusion, we have identified a novel mutation of exon 180 of the GHR. Homozygosity for this mutation in two distantly related Inuit patients results in an unstable GHR and classical GHIS. A different and previously reported mutation of exon 180, resulting in a splicing defect and present in homozygous form in approximately 100 patients from southern Ecuador, has now been shown to result in a GHR molecule that is capable of stable expression and homodimerization but abnormal trafficking and GH binding. Thus, although the Inuit and Ecuador populations have mutations affecting the same GHR codon and resulting in classical GHIS phenotypes, the precise molecular bases for their presentation differ.


   Note Added in Proof
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 Note Added in Proof
References
 
During preparation of this manuscript, L. Fassone, et al., also reported identification of the E180X mutation in GHR gene in one GHIS patient originating from Sicily, Italy (42).


   Acknowledgments
 
We thank Dr. Stuart J. Frank (University of Alabama, Birmingham, AL) for providing human GHR antibodies and helpful discussions.


   Footnotes
 
This work was supported by IGF Deficiency Diagnosis Center supported by Tercica Inc. (to R.G.R.).

First Published Online December 11, 2007

Abbreviations: ECD, Extracellular domain; Endo H, endoglycosidase H; ER, endoplasmic reticulum; GHBP, GH binding protein; GHIS, GH insensitivity syndrome; GHR, GH receptor; GHRfl, full-length GHR; HA, hemagglutinin; IGFBP, IGF-binding protein; SDS, SD score; STAT, signal transducer and activator of transcription.

Received September 10, 2007.

Accepted November 30, 2007.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 Note Added in Proof
 References
 

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