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Division of Endocrinology and The Ilyssa Center for Molecular and Cellular Endocrinology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA
(Requests for offprints should be addressed to R Salvatori, Division of Endocrinology, Johns Hopkins University School of Medicine, 1830 East Monument Street #333, Baltimore, MD 21287, USA; Email: salvator{at}jhmi.edu)
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-helices, and one intracellular C-terminal domain with multiple potential phosphorylation sites.
Interaction between the GHRH and its receptor leads to activation of the Gs subunit, stimulation of adenylylcyclase, and increase in intracellular cyclic AMP (cAMP), which in turn promotes the phosphorylation of protein-kinase A, thereby causing cellular proliferation and GH secretion. Activation of the GHRH-R also triggers the mitogen-activated protein kinase pathway, which may in part contribute to the proliferation of the somatotroph cells (Pombo et al. 2000).
The first evidence that mutations in the GHRH-R gene cause growth hormone deficiency (GHD) came from the little mouse, a naturally occurring murine model for human isolated GHD (IGHD), characterized by autosomal recessive dwarfism and pituitary hypoplasia (Eicher & Beamer 1976). The little mouse carries a point mutation in the extracellular domain of the receptor that causes a single amino acid substitution (D60G) (Godfrey et al. 1993, Lin et al. 1993). This change impairs the ability of the receptor to bind GHRH (Gaylinn et al. 1999). The first described mutation in the human GHRH-R gene (GHRH-R) was a nonsense mutation in the extracellular domain (codon 72) (Wajnrajch et al. 1996). Since then, several other naturally occurring GHRH-R mutations have been described, including a promoter mutation (Salvatori et al. 2002a), six missense mutations (Salvatori et al. 2001a, Salvatori at al. 2001b, Salvatori et al. 2002a, Salvatori et al. 2002b, Carakushansky et al. 2003), four splice-donor mutations (Salvatori et al. 1999, Roelfsema et al. 2001, Salvatori et al. 2002c, Alba et al. 2004), a second nonsense mutations (Salvatori et al. 2002c), and two small deletions (Salvatori et al. 2001b, Horikawa 2002). All these mutations are transmitted as autosomal recessive traits, with the possible exception of one of the small deletions that has been reported to have an in vitro dominant negative effect (Horikawa 2002). All but the missense mutations are expected to cause the receptor to be prematurely truncated or its translation to be out of frame.
The mechanism by which the six single amino acid substitutions (H137L, L144H, A176V, A222E, F242C, and K329E) alter receptor function is not yet determined. In this study, we have examined the in vitro effect of these mutations on receptor expression and function. We demonstrate that the mutations do not alter proper surface expression of the receptor, but they do alter ligand binding.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using site-directed mutagenesis technique (Kunkel 1985) we introduced each missense mutation (H137L, L144H, A176V, A222E, F242C, K329E) into individual GHRH-R cDNA clones. Plasmids with cDNA’s with different epitopes were used in different experiments:
Cyclic AMP assay
In order to compare the response to GHRH of all six mutated receptors, we measured cAMP response to GHRH in Chinese Hamster Ovary (CHO) cells transiently transfected with either the WT or mutant receptor cDNA. Briefly, cells were trypsinized and seeded in 24-well plates at 2 x 105 cells/well in minimum essential medium (MEM)+10% fetal bovine serum (FBS) (Life Technologies, Gaithersburg, MD, USA) and transfected immediately using FuGENE 6 transfection reagent (Roche, Indianapolis, IN, USA). Not transfected CHO cells (MOCK) and cells transfected with vector alone served as negative controls. Forty-eight h after transfection, cells were washed with serum free MEM containing 0.5 mmol/L isobutylmethylxantine (IBMX) and treated with MEM+IBMX (baseline) or with MEM+IBMX+ forskolin 10–5 mol/L (Sigma-Aldrich, St Louis, MO, USA) or challenged with MEM+IBMX+[Nle27]-GHRH-(1–29) 10–8 M or 10–9 M or 10–10 M (Peninsula Laboratories, Inc, Belmont, CA, USA). After 15 min incubation at 37 °C, total cAMP was extracted by addition of HCL to a final concentration of 0.1 mol/l and a cycle of freeze-thawing. Cellular cAMP in the acid extracts was measured by RIA as previously described (Levine et al. 1983). The results are the mean of three separate experiments, each performed in duplicate wells and each well assayed in duplicate using 100 µl cell extracts. Results have been normalized to the cAMP response to forskolin and are expressed as picomoles of cAMP produced per well.
Immunoprecipitation
CHO cells, which do not express endogenous GHRH-R, were grown in 6 well/plates in MEM+10% FBS (Invitrogen) and transfected at 60–70% confluence with 1 µg of plasmid GFP-tagged GHRH-R cDNA using FuGENE 6 transfection reagent following manufacturer’s recommendations. After 48 h, cells were washed twice with ice-cold PBS, harvested and lysed on ice in RIPA buffer (1 xPBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1%SDS, 10µg/ml PMSF, protease inhibitor cocktail) (Sigma-Aldrich). The lysate was clarified by centrifugation (10 min at 15 000 g at 4 °C) and incubated overnight at 4 °C with 10 µg of mouse anti-GFP agarose-coniugated antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Immunoprecipitates were then washed 3 times with ice cold PBS and resuspended in protein sample buffer. Samples were boiled for 5 min and protein separated on a 10% SDS-PAGE gel, followed by transfer onto polyvinylidene difluoride membrane (PVDF) (Immobilon-P, Millipore Corporation, Billerica, MA, USA). The filter was blocked for 1 h in Tris–buffered saline+0.05% Tween 20 (TBS-T) (Sigma-Aldrich)+3% non-fat dry milk (Bio Rad Lab, Hercules, CA, USA), incubated for 1 h with mouse anti-GFP antibody (1:5000) (Santa Cruz Biotechnology Inc.), washed extensively in TBS-T. After 1 h incubation with HRP-conjugated goat anti mouse IgG (1:3000), and washing in TBS-T, immunoreactivity was detected by enhanced chemiluminescence (ECL kit, Amersham Biosciences, Chalfont St Giles, Buckinghamshire, England) and band size compared with pre-stained protein weight marker (Full Range Rainbow, Amersham Biosciences). All steps were performed at room temperature.
Immunofluorescence
In order to detect whether the missense mutation could alter proper receptor expression on the cell surface we performed immunofluorescence experiments with the goal of detecting the presence of extracellular FLAG epitope in non-permeabilized transfected cells.
CHO cells were grown on glass cover slips and at 50—70% confluence were transfected with FLAG-tagged GHRH-R cDNA using FuGENE 6 transfection reagent. As control, we used CHO cells transfected with WT GHRH-R cDNA with an hemoagglutinin (HA)-tag, kindly donated by Dr Kelly Mayo, in the intracellular C-terminus. At 100% confluence cells were washed three times with PBS and fixed with fresh 2% paraformaldehyde in PBS pH 7.4 for 15 min at room temperature. After four washing steps with PBS, cells were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-FLAG monoclonal antibody (1:100) (Sigma-Aldrich) for 1 h. CHO cells transfected with GHRH-R HA-tagged cDNA were incubated with monoclonal anti-HA FITC-conjugated antibodies (1:200) (Sigma-Aldrich). Cells were then washed four times with PBS at pH 8 and examined by confocal laser scanning microscopy (LSM 410, Carl Zeiss INC, Oberkochen, Germany) using a 40 x objective lens. All slides were scanned for equivalent times using the same contrast and brightness settings.
Binding assay
To determine whether the missense mutations would alter ligand binding, binding studies were performed using radiolabelled GHRH and HEK 239 membrane extracts. HEK 239 cells were chosen because binding experiments in CHO cells had proven to be technically poorly reproducible. Cells were grown in 100 mm dishes and were transfected at 60–70% confluence with GHRH-R cDNA, using FuGENE 6 transfection reagent. After 48 h, cells were washed, harvested by centrifugation, and homogenized in ice cold homogenization buffer (Hepes 50 mM, NaCl 100 mM, EDTA 10 mM, 0.1 mM EGTA, PMSF 1 mM, protease inhibitor cocktail) and 100 µM bacitracin (Sigma-Aldrich). Supernatant was removed by centrifugation and membrane pellets were resuspended in binding buffer (TRIS 50 mM, EGTA 2 mM) adding 5 mM of alamethicin (Sigma-Aldrich) for permeabilization. For each sample, one tube was incubated for 1 h at room temperature with approximately 100 000 c.p.m. of Human (1–44)-NH2 125I-GHRH (Amersham Bio-sciences), and the other with both human (1–44)-NH2 125I-GHRH and excess (1µM) ‘cold’ [Nle27] human GHRH (1–29)-NH2. Following 10 min centrifugation at 15 000 g, supernatant with receptor-bound GHRH was assayed using a gamma counter. Counts represent total binding, and the difference between counts in the absence or presence of cold GHRH represents specific binding. The experiment was repeated three times with three different sets of transfected cells. Results are the means ± S.D. of the three experiments.
Statistical analysis
Data were analyzed using SPSS statistical package (SPSS Inc., Chicago, IL, USA), and considered statistically significant if P < 0.05. ANOVA was used to analyze cAMP data. In the binding experiments, the difference between specific and non-specific binding of each single construct was analyzed by independent sample t-test, while ANOVA was performed to compare the specific binding among all different mutants, mock-transfected and WT.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Only the cells expressing the WT receptor showed a significant cAMP response to GHRH. The cAMP level in cells expressing the WT receptor was significantly higher compared with all other mutants at 10–8 and 10–9 M GHRH concentrations (Fig. 1
). The lack of response to GHRH by the mutant receptors indicates that all the mutations impair the receptor’s ability to transmit GHRH signalling.
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To determine whether the mutated GHRH receptors were translated into mature protein, proteins extracts from CHO cells transiently transfected with either WT or mutant receptors (or with empty plasmid vector) were immunoprecipitated and analysed by Western blotting.
All six mutated receptors were translated into proteins that showed an electrophoretic mobility of about 70–75 kDa (Fig. 2). The shift in the mobility from the displayed size to the expected molecular weight of about 79 kDa (54 kDa for the receptor plus 25 kDa for the GFP-tag), is likely due to the electrophoretic conditions affecting the charge of the glycosylated protein, and no difference in size between the WT and mutants was observed. This experiment was repeated three times, with consistent results. Although immunoprecipitation is not a quantitative assay, the levels of protein expression were similar in all mutants, indicating that the mutations do not negatively affect transcriptional or translational mechanisms and did not cause protein degradation.
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To confirm that the receptors are correctly localized into the cell membrane we performed immunofluorescence studies using non-permealized CHO cells transiently transfected with FLAG-tagged receptor cDNA. Since the FLAG tag is on the extracellular N-terminus of the receptor, only an appropriate localization on the cell membrane would allow interaction with the fluorescent antibody. As shown in Figure 3, using confocal microscopy, no fluorescence was observed either in mock-transfected cells, in cells transfected with vector alone, or in CHO cells transfected with the intracellular C-terminal HA-tagged receptor.
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Binding studies
Since immunoprecipitation and immunofluorescence studies indicated that the protein is translated in its entire sequence and expressed on the cell surface, we wanted to determine whether the inability of the GHRH to trigger the physiological response to GHRH-R was due to an impaired capacity to bind to its receptor.
Crude membrane pellets were obtained by homogenization and permeabilization from cells expressing either the WT or the mutant GHRH-Rs. Permeabilization with alamethicin was performed in order to enhance specific binding, as previous studies shave demonstrated that functional receptors are present also at internal sites of vesicles (Gaylinn et al. 1994, 1999). GHRH binding was measured by competing assay using 125I-human GHRH (Fig. 4). Analysis of specific binding by ANOVA showed that membranes from cells expressing the WT GHRH-R had a significantly higher binding compared with membranes from mock-transfected cells and from cells expressing the mutant receptors (P < 0.05). Interestingly, when we used independent t-test to analyse the response to competitive binding between ‘cold’ and 125 I-GHRH in the WT GHRH-R and in each mutated GHRH-R, we found that some specific binding had also occurred in the mutant K329E (P < 0.002 vs non-specific binding).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The GHRH-R belongs to class family B-III of the GPCR superfamily, together with receptors for glucagon, glucagon like peptide-1, secretin, vasoactive intestinal peptide (VIP), gastric inhibitory peptide, and pituitary adenylate cyclase-activating peptide. Using chimeras of the GHRH-R and VIP and secretin receptors, DeAlemeida and Mayo (1998) have elegantly shown that both N- and C-termini of the GHRH-R are necessary for ligand binding and triggering of signalling. The N-terminal extracellular domain plays a fundamental role in initial interaction with the ligand, while residues in the transmembrane domains, in conjunction with the extra-cellular loops, could have a role in secondary interaction and in determining binding specificity.
All the small deletions, splice mutations or nonsense GHRH mutations described to date either insert a premature stop codon or cause the messenger RNA to be out of frame. Resulting receptors, if translated at all, would lack the C-terminal domain and/or most of the transmembrane domains, possibly preventing the localization of the truncated receptor into the cell membrane or interaction with G proteins. Contrarilly, the GHRH-Rs bearing missense mutations are more likely to be translated into mature protein allowing more extensive studies on how the mutation may interfere with the GHRH signalling pathway. Receptor malfunction could be due to instability and early degradation of the mature protein, improper folding, alterations in intracellular transport or insertion into cell membrane, or in binding of the ligand. Although there is evidence that GPCR’s can exist as dimers, to date no mutation has been found that alters dimerization (Milligan 2004).
Our group has reported six naturally occurring missense mutations that cause familial autosomal recessive IGHD. The six amino acids that are mutated are conserved in all mammalian GHRH-Rs (human, rat, pig, bovine, and ovine) cloned to date (Horikawa et al. 2001), pointing to their importance in receptor function and confirming that these changes are not polymorphisms. Indeed, when expressed in eukaryotic cells, the mutated receptors are not able to activate the intracellular messenger cascade.
To investigate the expression of the mutated proteins, we transiently transfected CHO cells either with WT or mutant GHRH-R cDNA tagged with an intracellular GFP-tag. Receptors were immunoprecipitated and detected by Western blot. All mutant receptors are expressed and are of identical size as the WT. The degree of expression seems not to be altered, suggesting that the mutations do not interfere with posttranscriptional events. The size of band displayed on the Western blot is not in perfect accordance with the expected size of 79 kDa, which could be due to electrophoretic conditions affecting the charge of the proteins and therefore their mobility. Another possible explanation is that the glycosylation of human GHRH-R expressed in HEK 293 is altered (Gaylinn et al. 1994). Nevertheless, the electrophoretic migration of the mutant receptors is identical to the one of the WT protein.
The expression of the mutated receptors is also confirmed by the results of the immunofluorescence studies. All mutant receptors containing an extracellular FLAG-tag display immunofluorescence in non-permeabilized cells, indicating that the receptor is properly transferred through the Golgi apparatus to the cell surface.
Once we determined that the mutated receptors were properly expressed on the cell surface, we investigated their ability to bind to labelled GHRH. Binding studies revealed that all the mutated receptors had reduced ligand binding compared with WT receptor. The K329E mutation seems to retain some binding capacity, as shown by the difference between specific and non-specific binding, but its binding ability remained significantly lower than the wild type. The K329E mutant does not cause any rise in intracellular cAMP. We can only speculate on the reason of this finding. As shown in Table 1 all of the six mutations are located within the transmembrane domains and four (H137L, L144H, A222E, and F242C) produce modifications in the degree of solubility of the amino acid within the lipid layer of the cell membrane. It is conceivable that a change of polarity causes alterations in proper protein folding and receptor function. Since the mutation K329E causes the change from one polar amino acid to another (lysine to glutamic acid) it is possible that the conformation of the protein is conserved to a point that would still allow some degree of ligand binding but not further signalling.
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To determine how the amino acid changes cause the inability of the receptor to bind the ligand would require the creation of three-dimensional models analysing how the single amino acid changes affect the tertiary structure of the protein.
In conclusion, we have shown that the six naturally occurring and disease-causing missense GHRH-R mutations identified to date allow proper surface expression of the receptor. They cause the receptor to be unable to bind to GHRH, impairing its ability to transmit intracellular signalling and ultimately to stimulate GH secretion.
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Acknowledgements |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Carakushansky M, Whatmore AJ, Clayton PE, Shalet SN, Gleeson HK, Price DA, Levine MA & Salvatori R 2003 A new missense mutation in the growth hormone-releasing hormone receptor gene in familial isolated GH deficiency. European Journal of Endocrinology 148 25–30.[Abstract]
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Godfrey P, Rahal JO, Beamer WG, Copeland NG, Jenkins NA & Mayo KE 1993 GHRH receptor of little mice contains a missense mutation in the extracellular domain that disrupts receptor function. Nature Genetics 4 227–232.[CrossRef][Web of Science][Medline]
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Levine MA, Eil C, Downs Jr RW & Spiegel AM 1983 Deficient guanine nucleotide regulatory unit activity in cultured fibroblast membranes from patients with pseudohypoparathyroidism type I. A cause of impaired synthesis of 3', 5'-cyclic AMP by intact and broken cells. Journal of Clinical Investigation 72 316–324.
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Roelfsema F, Biermasz NR, Veldman RG, Veldhuis JD, Frolich M, Stokvis-Brantsma WH & Wit JM 2001 Growth hormone (GH) secretion in patients with an inactivating defect of the GH-releasing hormone (GHRH) receptor is pulsatile: evidence for a role for non-GHRH inputs into the generation of GH pulses. Journal of Clinical Endocrinology and Metabolism 86 2459–2464.
Salvatori R, Hayashida CY, Aguilar-Oliveira MH, Phillips JA III, Souza AH, Gondo RG, Toledo SPA, Conceicao MM, Prince M, Baumann G, Maheshwari H & Levine MA 1999 Familial Dwarfism due to a novel mutation of the growth hormone-releasing hormone receptor gene. Journal of Clinical Endocrinology and Metabolism 84 917–923.
Salvatori R, Fan X, Phillips JA III, Espigares R, Martin de Lara I, Freeman K, Plotnick L, Al-Shawl A & Levine MA 2001a Three new mutations in the gene for growth hormone(GH)-releasing hormone receptor in familial isolated GH deficiency type IB. Journal of Clinical Endocrinology and Metabolism 86 273–279.
Salvatori R, Fan X, Phillips JA III, Prince M & Levine MA 2001b Isolated growth hormone (GH) deficiency due to compound heterozygosity for two new mutations in the GH-releasing hormone receptor gene. Clinical Endocrinology 54 681–687.[CrossRef][Medline]
Salvatori R, Fan X, Mullis PE, Haile A & Levine MA 2002a Decreased expression of the GHRH receptor gene due to a mutation in a Pit-1 binding site. Molecular Endocrinology 16 450–458.
Salvatori R, Aguiar-Oliveira MH, Monte LVB, Hedges L, Santos NL, Pereira RMC & Phillips III JA 2002b Detection of a recurring mutation in the human growth hormone- releasing hormone receptor gene. Clinical Endocrinology 57 77–80.[CrossRef][Medline]
Salvatori R, Fan X, Veldhuis J & Couch R 2002c Serum GH response to pharmacological stimuli and physical exercise in two siblings with two new inactivating mutations in the GH-releasing hormone receptor gene. European Journal of Endocrinology 147 591–597.[Abstract]
Tarasova NI, Stauber RD & Wank SA 2000 Characterization of receptor trafficking with green fluorescent protein. In Regulation of G protein-coupled receptor function and expression. pp 253–272. Ed JL Benovic. New York: John Wiley and Sons.
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Received 16 June 2005
Accepted 28 June 2005
Made available online as an Accepted Preprint 13 July 2005
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