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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 7 2554-2561
Copyright © 1998 by The Endocrine Society

Original Studies

A Novel Mutation Affecting the Interdomain Link Region of the Growth Hormone Receptor in a Vietnamese Girl, and Response to Long-Term Treatment with Recombinant Human Insulin-Like Growth Factor-I and Luteinizing Hormone-Releasing Hormone Analogue1

J. L. Walker, P. A. Crock, S. N. Behncken, S. W. Rowlinson, L. M. Nicholson, T. J. C. Boulton and M. J. Waters

School of Paediatrics (J.L.W., L.M.N.), University of New South Wales, Randwick, New South Wales, 2031; John Hunter Children’s Hospital (P.A.C.), Newcastle, New South Wales, 2310; Department of Physiology and Pharmacology and Centre for Molecular Biology (S.N.B., S.W.R, M.J.W.), University of Queensland, St. Lucia, Queensland, 4072; Department of Paediatrics (T.J.C.B.), Nepean Hospital, Penrith, New South Wales, 2751 Australia

Address all correspondence and requests for reprints to: Jan L. Walker, Department of Endocrinology, Sydney Children’s Hospital, High Street, Randwick New South Wales, Australia 2031. E-mail: jan.walker{at}unsw.edu.au


   Abstract
Top
 Abstract
Introduction
 Subject and Methods
 Results
 Discussion
 References
 
A Vietnamese girl with Laron syndrome has been treated with recombinant human insulin-like growth factor-I for 4 yr from age 11.28 yr. Her height SD score increased from -6.3 to -4.7 without acceleration of bone age. Isolated breast development progressed despite pubertal suppression with luteinizing hormone-releasing hormone analogue, which was stopped after 3 yr because of growth deceleration. Facial coarsening was documented with serial photographs.

Sequencing and in vitro analysis identified a homozygous base pair substitution in exon 6 of the proband’s GH receptor (GHR), which changed amino acid 131 from proline to glutamine (P131Q) and disrupted GH binding. Both the P131Q-mutated human GHR and wild-type (wt) hGHR were transiently expressed in COS-1 cells, as demonstrated by Western blotting, but the P131Q-transfected cells did not bind 125I-hGH. Similarly, FDC-P1 cells transfected with wthGHR bound 125I-hGH with high affinity and proliferated in response to GH, whereas the P131Q hGHR cells did neither. In CHO-K1 cells cotransfected with wthGHR and the Egr-1 promotor linked to a luciferase reporter gene, GH evoked a 2.14 ± 0.21-fold increase in luciferase activity, but there was no response in the cells carrying the P131Q hGHR mutation. From examination of the crystal structure of the GHR, we suggest that the P131Q mutation disrupts the interdomain link between the extracellular domains of the GHR, causing a conformational change that results in disruption of the GH binding site.


   Introduction
Top
 Abstract
 Introduction
Subject and Methods
 Results
 Discussion
 References
 
DISRUPTION of GH receptor (GHR) function causes insensitivity to GH (GH insensitivity syndrome: GHIS) and the clinical and biochemical features of Laron syndrome. This rare, autosomal recessive disorder is found most frequently in consanguineous kindreds of Middle Eastern or Mediterranean origin (1); however, it has been reported in diverse and geographically widely separated racial groups, including a few reports in individuals from the Indian subcontinent (1), Japan (1), and Cambodia (2). Treatment with recombinant human insulin-like growth factor-I (rhIGF-I) has been proven to increase the growth rate of affected children (1, 2, 3, 4, 5), with few serious side effects (1, 4), although there has been increasing concern about facial coarsening (6). Puberty occurs spontaneously in untreated individuals with Laron syndrome and is not associated with a growth spurt (1). The short-term use of luteinizing hormone-releasing hormone (LH-RH) analogue together with rhIGF-I, to try to retard pubertal progression and maximize final height, has been reported (7); however, long-term data on growth velocity and pubertal progression are lacking.

Partial gene deletions and rearrangements, and missense, nonsense, splice and frame shift mutations affecting the GHR have been described in GHIS (1). With 2 exceptions (8, 9), all of the 30 described GHR mutations associated with Laron syndrome occur in the extracellular region of the GHR, mostly associated with loss of GH binding activity (1, 10). The extracellular, ligand binding region of the GHR consists of 2 ß sandwich domains (domain 1: residues 1–123; domain 2: residues128–238) linked by 4 amino acids (11). The GH molecule itself has two binding sites (sites 1 and 2) that bind sequentially to essentially the same binding site on two molecules of the receptor. This results in the formation of a GH:[GHR]2 complex, and the resultant dimerization of the receptor initiates signal transduction (11).

Investigation of the missense mutations associated with GHIS has the greatest potential to define the functional significance of different residues within the receptor. In very few of the mutations reported, however, have attempts been made to define how the mutation disrupts GHR function. Investigation of the aspartate (Asp) (D) to histidine (H) mutation at position 152 (D152H) in exon 6 of the GHR, in which GH binding activity was preserved (12, 13), led to the definition of this region as critical for receptor dimerization (12). Conflicting studies of the phenylalanine to serine mutation at position 96, associated with absent serum GH binding activity, have suggested that this mutation interferes with insertion of the GHR into the cell membrane (14) or with GH binding (15).

Study of the crystal structure of the GHR (16) may also elucidate how point mutations disrupt GHR function. Recently, a compound heterozygote with partial GHIS was identified where one of the mutations resulted in a Glu 44 Lys mutation (17). From the crystal structure of the GHR, this mutation would be predicted to disrupt GH site 1 binding, through interference with the hydrogen bond between Glu 44 and the critical residue, tryptophan 169, in receptor 2.

In this study, we describe a Vietnamese girl with Laron syndrome, the effects on her growth and pubertal development of 4 yr of rhIGF-I treatment concurrent with 3 yr of LH-RH analogue treatment, and the functional and structural consequences of the novel missense mutation identified in exon 6 of her GHR gene. By mutational analysis, binding studies, and assay of GH effects in transfected cells, we show that the mutation identified is responsible for GHIS in this kindred. Analysis of the crystal structure of the mutated GHR suggests that the interdomain link region is disrupted, resulting in loss of GH binding and prevention of the cascade of events necessary for signal transduction.


   Subject and Methods
Top
 Abstract
 Introduction
 Subject and Methods
Results
 Discussion
 References
 
Case Study

The child presented was one of three girls and one boy born to apparently unrelated Vietnamese parents. The midparental height SD score (OZGROW, Australian National Growth Database) was -1.8 (153 cm), and her two sisters had height SD scores of -1.5 and -2.2 at chronological ages (CA) 8.3 and 5.9 yr, respectively. In contrast, when our patient arrived in Australia after a period of nutritional deprivation in a refugee camp, her height SD score at 2.4 yr was -5.5 (69.5 cm), weight SD score was -4.9 (6.4 kg), and her head circumference was 44 cm (<2nd percentile). Growth failure on the basis of nutritional deficiency was suspected but did not resolve with hospitalization for nasogastric hyperalimentation. Her subsequent growth (Fig. 1Go) and the investigations performed at the John Hunter Children’s Hospital were typical of Laron syndrome. Some biochemical and clinical details of the girl have been previously reported (18), where she appears as patient 19 in Table 5. The serum GH binding protein activity, measured by high-pressure liquid chromatography, was absent (18).



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  		Figure 1. Growth chart of the proband, showing lack of response to exogenous GH at age 7 yr and response to rhIGF-1 therapy started at 11.3 yr. RhIGF-1 was started at 40 µg/kg twice daily, as shown by the lower hatched bar, and increased to 80 µg/kg twice daily at 11.7 yr (upper and lower bars). There was a transient decrease to 60 µg/kg twice daily for 3 months (indicated by the notch in the upper bar) because of concern about facial coarsening; however, the dose was increased again when height velocity decreased. LH-RH analogue was given between the ages of 11.7 and 14.5 yr. BA, bone age. The growth chart was adapted from P. V. V. Hamill’s NCHS growth curves for children.

 
Treatment with rhIGF-I (Igef, Pharmacia and Upjohn, Stockholm, Sweden) was started at age 11.28 yr, when her height and weight SD scores were -6.3 and -3.4, respectively, and her bone age was 7.8 yr (standards of Greulich and Pyle). The initial dose was 40 µg/kg twice daily, increasing to 80 µg/kg twice daily after 5 months. Her growth rate increased from 4.7 cm/yr pretreatment to 9.2, 7.3, and 7.1 cm/yr during the first, second, and third years of treatment, respectively. At 14.1 yr, her height and weight SD scores had increased to -5.1 and -2.6, respectively, and her bone age of 11 yr had advanced in concert with chronological age. Between the ages of 14.1 and 14.5, her growth rate slowed dramatically (Fig. 1Go), to 2.2 cm/yr, over 5 months.

Despite her delayed bone age, Tanner stage 2 breast development was noted at 10.8 yr. Peak serum concentrations of LH and FSH, in response to an LH-RH stimulation test performed at 11.1 yr, were 4.4 and 12.6 mIU/mL, respectively, and the serum concentrations of estradiol and DHEAS were in the low pubertal range (Table 1Go). LH-RH analogue treatment was started at 11.7 yr, approximately 5 months after the start of rhIGF-I, because of progression of breast development. Intramuscular injections of Lucrin Depot (leuprorelin acetate, 7.5 mg monthly; Abbott Australasia, Kurnell, N.S.W., Australia) were used for 15 months, followed by sc Zoladex implants (goserelin acetate, 3.6 mg monthly; ICI Australia Operations Ltd, East Melbourne, Vic., Australia). Biochemical suppression of LH and FSH secretion was maintained, and serum concentrations of estradiol decreased to the low prepubertal range. Serum concentrations of DHEAS were maintained approximately at the lower limit for pubertal children (Table 1Go). Despite this, breast development had progressed to Tanner stage 4, by 13.8 yr, without any other signs of puberty. A pelvic ultrasound test at 14.2 yr showed a prepubertal uterus of 3.5 cm in length, with ovarian volumes of 2.7 and 3.6 mL.


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  		Table 1. Baseline serum concentrations of estradiol, DHEAS, LH and FSH, and stimulated peak values of LH and FSH before treatment with LHRH analogue (LHRHa) and at the end of 1, 2, and 3 yr of treatment with LHRHa

 
Zoladex was ceased at 14.5 yr (last dose given at 14.4 yr) because of concern that suppression of sex steroids might be contributing to her deceleration in growth. Within 2 months, her growth had improved (Fig. 1Go); and her height velocity was maintained at 5.8 cm/yr over the ensuing 6 months. In the 2 months that followed, however, she again had growth deceleration for no discernible reason, with a height velocity of 1.8 cm/yr over 2 months. At 15.2 yr, her height SD score was -4.7 and her weight SD score was -2.7; she had just started developing pubic hair but had not begun menstruating. Her bone age was 12 yr.

A number of side effects attributable to rhIGF-I were noted, none of which was of sufficient severity to warrant discontinuation of therapy. Because of local discomfort at new injection sites, she would not rotate her injections, and she developed some lipohypertrophy. During the first 3–6 months of treatment, she was noted to have a facial tic, characterized by frequent blinking, especially of her left eye. This resolved spontaneously after 12 months. In the course of treatment, she has developed coarsening of her facial features (Fig. 2Go), most markedly of her nose and lower lip, to which both she and her family are resigned, in the interests of further height gains. The dose of rhIGF-I has not been increased above 80 ug/kg bd (in spite of her recent growth deceleration) because of these side effects.




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  		Figure 2. Facial photographs of the proband before the start of treatment with rhIGF-I (A) and after 1 yr (B and C), 2 yr (D and E), and 4 yr (F and G) of treatment. A photograph of mother and daughter after 1 yr of treatment is included for comparison of facial features (H). Written informed consent was given for publication of these photographs.

 
Methods

Sequence analysis. Genomic DNA was extracted by standard techniques from blood collected from the proband, each member of her family, and a normal statured control. Using primers located close to the intron-exon boundaries, each of the exons of the GHR was amplified by PCR and the size of the products verified by PAGE. The PCR products were isolated with Magic columns (Promega, Anandale, New South Wales, Australia) before [{gamma} 33P]ATP (Amersham, Castle Hill, New South Wales, Australia) end-labeled sequencing (Promega fmol Sequencing System; Promega). Sequencing gels were dried, then autoradiographed for 24–96 h.

Mutation analysis. Mutagenesis.The P131Q [amino acid 131, changed from proline (Pro) to glutamine (Gln)] hGHR mutation identified was recreated using the Altered Sites Mutagenesis system (Promega). Briefly, a 2018-bp fragment was excised from the expression plasmid hGHR-pECE and cloned into p-Alter using the SalI and HindIII restriction sites. The synthetic oligonucleotide 5'-TAGTGCAACC/AAGATCCACC (Oligonucleotide Synthesis Service, Southern Cross University, Lismore, Australia) was annealed to single-stranded hGHR-pAlter DNA, and the mutagenesis reaction was completed in the manner specified by the supplier. After isolation of a clone containing the required mutation, a 346-bp fragment from P131Q hGHR-pAlter, including the P131Q mutation, was subcloned into the hGHR-pECE expression plasmid using the DraIII and EcoRV restriction sites. This 346-bp subcloned region was fully sequenced (Sequenase, USB, Cleveland, OH) to confirm the presence of the P131Q mutation and to check that no other mutations were introduced.

Binding studies and Western blots in COS-1 cells.COS-1 cells were transiently transfected with the P131Q hGHR and wthGHR in 6-well plates, and binding assays were performed on the transfected cells at 4 C to block internalization, as described in Gobius et al.
(19).

To confirm that the hGHR constructs were being expressed, Western blots were performed on COS-1 cells transiently transfected with P131Q or wthGHR-pECE plasmid. Cells were harvested from 6-well plates and processed as described by Lobie et al. (20). Briefly, the cells were extracted in a buffer containing 0.2% Triton X-100 and protease inhibitors. The solubilized GHR was immunoprecipitated with MAb 263, the immunoprecipitate captured with a mix of protein G and A sepharose, and then run on a 7.5% SDS polyacrylamide gel. After electroblotting, the membrane was probed with a rabbit polyclonal antibody raised against a pGEX fusion protein containing the entire rabbit GHR cytoplasmic domain (20). Blots were developed with a peroxidase-labeled second antibody using the Amersham ECL kit.

Binding and proliferation studies in FDC-P1 cells.FDC-P1 cells are an interleukin (IL)-3-dependent murine myeloid cell line. When transfected with the full-length GHR, FDC-P1 cells proliferate in response to GH in the absence of IL-3 (21, 22). The ability of P131Q hGHR to support proliferation of the FDC-P1 cell line in the absence of IL-3 was determined after transfection and G418 selection, as described in Rowlinson et al.
(22). Briefly, stable transfection of FDC-P1 cells with the P131Q hGHR-pECE construct was performed by electroporation of 10 or 20 µg of receptor plasmid in combination with either 1 or 2 µg, respectively, of pCIS-Neo plasmid, as previously described (21, 22). Cells expressing the P131Q hGHR were isolated by GH selection (21, 22) or by selecting with the neomycin-like aminoglycoside G418 at a concentration of 300 µg/mL. GH binding assays on FDC-P1 cells were performed as described by Rowlinson et al. (21, 22). Proliferation assays were based on the 3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide (thiazolyl blue) dye assay, as previously described (21).

Biological responsiveness: GH-mediated activation of the Egr-1 transcription factor in CHO-K1 cells.A number of transcription factors, including Egr-1, show increased DNA binding activity in response to GH activation of the GHR (23). The promotor for Egr-1 has been shown to contain sites similar to those found in the c-fos promotor, known to be responsible for GH-mediated transcriptional activation (23, 24). Activation of the Egr-1 promotor, therefore, is a measure of the ability of the GHR to initiate signal transduction.

We examined the ability of the P131Q hGHR and wthGHR to activate the Egr-1 promoter linked to a luciferase reporter gene cotransfected into CHO-K1 cells. The assay was performed in a manner identical to that described for c-fos activation by Chen et al. (24), except that the c-fos promoter was replaced by the Egr-1 promoter (23). Normalization for transfection efficiency was undertaken by cotransfecting with ß-galactosidase complementary DNA.


   Results
Top
 Abstract
 Introduction
 Subject and Methods
 Results
Discussion
 References
 
The proband was homozygous for a base pair substitution (C -> A) at the beginning of exon 6, resulting in the conversion of a Pro to a Gln at position 131 (Fig. 3Go). Her parents and siblings were heterozygous for the mutation. No other sequence abnormalities were identified.



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  		Figure 3. In the control lane, the four amino acids encoding the beginning of exon 6 are shown: Gln (Q130), Pro (P131), Asp (D132), and Pro (P133). The proband was homozygous for a base pair substitution (C -> A) resulting in the conversion of Pro (CCA) to Gln (CAA) at position 131 (P131Q). Her parents and siblings were heterozygous for the mutation (P/Q: CC/AA; sibling data not shown). No other sequence abnormalities were identified.

 
Unlike COS-1 cells transfected with the wthGHR-pECE plasmid, no binding was detected in COS-1 cells transiently transfected with the P131Q hGHR construct, either to the cell surface or to broken cell membranes. Western blots of COS-1 cells, transfected with either wt- or P131Q hGHR plasmids, showed a receptor-specific band at 120 kDa, which was intense for the receptor mutant (Fig. 4Go), thus confirming the successful expression of both wt- and mutant hGHR constructs.



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  		Figure 4. Western blot showing expression of wthGH receptor and P131Q mutant in COS-1 cells. Solubilized cell extracts were immunoprecipitated with MAb263, and the membrane was probed with an antibody to the receptor cytoplasmic domain, then visualized with the Amersham ECL system, as described in Subject and Methods.

 
No GH binding was detected in G418-selected FDC-P1 cell populations that were cotransfected with pCIS-Neo and P131Q hGHR-pECE plasmid. Moreover, these cells did not survive in medium with GH substituted for IL-3, whereas cells transfected with the wthGHR proliferated effectively.

CHO-K1 cells transiently transfected with wt receptor and a construct containing the Egr promoter linked to a luciferase reporter gene showed a 2.14 ± 0.21-fold (mean ± SD, n = 3) increase in luciferase activity, in response to the addition of GH. Luciferase activity did not increase significantly in cells transfected with P131Q hGHR when GH was added (1.11 ± 0.02-fold induction, mean ± SD, n = 3).


   Discussion
Top
 Abstract
 Introduction
 Subject and Methods
 Results
 Discussion
References
 
The identification of a novel missense mutation, associated with Laron Syndrome, in a Vietnamese family adds to the racial and genetic diversity associated with this syndrome. Despite exhaustive questioning, there seems to be no consanguinity within this kindred. That the parents were heterozygous for the same novel defect suggests a founder effect and also suggests that there are likely to be other, similarly affected individuals within this population.

The girl described in this study had a vigorous growth response to treatment with rhIGF-I, sustained over 3 yr. Periodic decelerations in growth, such as that noted in our patient at the end of 3 and 4 yr of treatment with rhIGF-I, have been attributed to excessive sensitivity of individuals with GHIS to alterations in diet or to intercurrent illness (5); however, there was no evidence for this in our patient. Deceleration of growth velocity has been described during LH-RH analogue treatment of short pubertal children, despite concurrent treatment with biosynthetic GH (25, 26). The almost immediate increase in our patient’s height velocity after cessation of LH-RH analogue treatment at 14.5 yr suggests that suppression of sex steroids may have been contributing to her poor growth. The improvement in her growth velocity, however, was not accompanied by further pubertal development. The poor growth of an adolescent with Laron syndrome, after 9 months of concurrent treatment with LH-RH analogue and rhIGF-I, was attributed to injecting into hypertrophied injection sites (7). This also may have contributed to our patient’s periods of growth failure.

The progression of isolated breast development, despite biochemical suppression of puberty, is curious. Puberty in Laron syndrome is delayed in only 50% of individuals (1), so the occurrence of breast development at age 10.8 yr was not unusual. Serum concentrations of estrogen, albeit measured by a standard rather than supersensitive assay, decreased into the low prepubertal range during LH-RH analogue therapy (Table 1Go), suggesting that the progression of breast development occurred independently of central gonadal stimulation. Although fluctuations in serum estrogen concentrations may have occurred, there was no concurrent maturation of the uterus during analogue therapy, which would be expected if undetected pubertal levels of estrogen were responsible for her breast development. Gynecomastia is a recognized complication of treatment with GH (27); and in adults, this correlates with a rise in serum concentrations of IGF-I to greater than 1 U/mL (28). In keeping with this, gynecomastia also has been reported in association with rhIGF-I treatment (29). We postulate that our patient’s progressive but isolated breast development, in the face of pubertal suppression, was caused by treatment with rhIGF-I in the presence of low levels of estrogen derived from peripheral aromatization of adrenal androgens.

Apart from pain at the injection sites, treatment was well tolerated. Bell’s palsy has been reported to be a complication of treatment with rhIGF-I (30, 31). The transient facial tic, observed in our patient during the first year of treatment, was attributed by her family to shyness; however, it may have been caused by irritation of the facial nerve. Coarsening of facial features and an increase in head circumference have been noted in other studies during treatment with rhIGF-I (4, 6, 32). Facial morphology is abnormal in children with GHIS, possibly indicating a developmental role for GH or IGF-I in utero (32, 33). It has been suggested that the changes observed in the faces of children treated with IGF-I may represent maturation and catch-up growth, rather than deformity (32); however, a recent study suggests that there is relative overgrowth in the mandible (6). Similar changes are seen in acromegaly and may represent an endocrine effect of IGF-I vs. the regulated and targeted autocrine/paracrine effects of IGF-I associated with normal GH secretion and sensitivity. The potentially disfiguring effect of continuing treatment, therefore, must be balanced against the potentially beneficial effect on final height. Untreated, final height has been reported to lie between -12 and -3.4 SD for Laron syndrome (1). Our patient has already achieved a height SD score of -5.2 for adult women with approximately 2 yr of growth remaining, according to her bone age. The family’s judgment is that continued growth is more important than her current degree of facial coarsening.

Homozygosity for the P131Q base pair substitution strongly suggested that this mutation was responsible for the clinical manifestations of GHIS in the child we have described. This was confirmed by in vitro studies using mutational analysis and transfection of wt- and mutated GHRs into three different cell types. Consistent with the absence of GH binding activity in serum, COS-1 and FDC-P1 cells transfected with the mutant receptor failed to bind GH. The successful expression of the mutant receptor in COS-1 cells, detected by Western blot, indicates that the P131Q mutation disrupts GH binding rather than biosynthesis. The failure of the FDC-P1 and the CHO-K1 cells to respond to GH by proliferation (FDC-P1 cells) or activation of the cotransfected Egr promoter (CHO-K1 cells) further indicates that the absence of GH binding observed is followed by disruption of signal transduction.

The key to the loss of function associated with mutation of Pro 131 to a Gln probably resides in its proximity to the interdomain link, thought to be critical for mediation of the biological effects of GH (21, 34). The importance of the link region and domain orientation for hormone binding is evident from analysis of mutations of other class 1 cytokine receptors. Thus, for the IL-6 receptor, serine 247, phenylalanine 248 and arginine (Arg) 250 within the link region are critical for ligand binding (35). One type of severe combined immunodeficiency is caused by conversion of alanine 156 to a valine in the linker region of the IL-2 receptor {gamma} chain (36). The interdomain angle of the Prolactin and GHRs differs because of the substitution of a tyrosine in the Prolactin receptor for a Gln in the GHR at position 127 in the link region (37). The difference in the domain angle between the two receptors contributes to the specificity of ligand binding of each receptor (37).

An examination of the crystal structure of the GH:[GHR]2 dimer (Fig. 5aGo) provides two possible mechanisms for the disruption of GH binding associated with the P131Q mutation through alteration of the conformation of the interdomain link. Pro 131 sits between Gln 130 and Asp 132 within domain 2 of the receptor, just carboxy terminal to the interdomain link sequence (residues 124–127). Asp 132, in particular, has an important role in stabilizing the angle between domains 1 and 2 of the GHR through a salt bridge to Arg 39, and Gln 130 also appears to form a hydrogen bond with the inner guanido nitrogen of Arg 39 (Fig. 5bGo). The importance of Arg 39 and Asp 132 was shown by charge reversal of Arg 39 to Gln, resulting in a 20-fold decrease in GH binding (19) and, similarly, alanine conversion of Asp 132, resulting in a 6-fold decrease in affinity for GH (38). The mutation of Pro to Gln at position 131 would result in loss of the rigidity in the positioning of Gln 130 and Asp 132 conferred by Pro 131, with potential disruption to their interactions with Arg 39. A second means of disrupting hormone binding may be interference in the positioning of the important B’-C’ loop containing tryptophan (Trp) 169, known to be critical for GH:GHR binding. Insertion of a Gln at 131 would position the head group of the Gln adjacent to methionine 171, which may allow it to hydrogen bond and reposition Trp 169.



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  		Figure 5. A, The crystal structure of hGH (yellow ribbon) bound to the extracellular domains of two hGH receptors (hGHR1 in white ribbon, hGHR2 in light blue ribbon). Pro 131, Pro 133 (both purple), Gln 130 (yellow), and Asp 132 (red) are part of the membrane proximal domain (domain 2), whereas Arg 39 (dark blue) is part of the membrane distal domain (domain 1). B, Close-up view of Arg 39 (dark blue) interacting with Asp 132 (red) in receptor 1 of the complex. Certain residues are not displayed, for clarity. Pro 131 and Pro 133 (both purple) may be important in orientating Asp 132 so that it can make a salt bridge to Arg 39 in Domain 1, thus fixing the angle between Domain 1 and Domain 2. Also, Gln 130 (yellow) could form a hydrogen bond with Arg 39, aiding in stabilization of the interaction. Similar interactions also exist in receptor 2 (not shown). Distances (in angstroms) between relevant groups are as follows: receptor 1: D132 O{delta}1 - R39 N{eta}1 = 3.62, D132 O{delta}1 - R39 N{eta}2 = 2.93, and Q130 N{epsilon}2 - R39 N{epsilon} = 3.04; receptor 2: D132 O{delta}1 - R39 N{eta}1 = 4.03, D132 O{delta}1 - R39 N{eta}2 = 2.98, and Q130 N{epsilon}2 - R39 N{epsilon} = 3.81.

 
In conclusion, we have identified a new GHR mutation, which causes Laron syndrome by disruption of GH binding to the receptor. We propose that this is the result of destabilization of domain-orientation interactions between Arg 39 (domain 1) and Gln 130 and Asp 132 (domain 2). It is also possible that the new Gln 131 disrupts the orientation of the important binding residue Trp 169. Together or separately, these disruptions could account for the total loss of hormone binding that we observed, resulting in the clinical consequence of insensitivity to GH.


   Acknowledgments
 
We wish to thank Ms Elizabeth Nunn, Clinical Nurse Consultant (Pediatric Endocrinology) for her dedication to the care of this family over many years. Recombinant human IGF-I (Igef) was supplied free of charge by Pharmacia-Upjohn, Sweden, and in particular the support of Dr. Rolf Gunnarsson and Ms. Sharon Hammond is gratefully acknowledged. We are grateful to Mr. Stephen McInally for his photographic expertise and to Mr. Bruce Turnbull for the computer graphics.


   Footnotes
 
1 Grant support for the work presented was received from the Prince Henry Hospital Centenary Research Fund (J.L.W.) and the NHMRC (M.J.W.). Back

Received October 8, 1997.

Revised March 18, 1998.

Accepted April 3, 1998.


   References
Top
 Abstract
 Introduction
 Subject and Methods
 Results
 Discussion
 References
 

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