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Divisions of Endocrinology (R.S., X.F., A.H.) and Pediatric Endocrinology (M.A.L.), and the Ilyssa Center for Molecular and Cellular Endocrinology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287; and Department of Pediatrics (P.E.M.), Inselspital, University of Bern, Bern, 3010 Switzerland
Address all correspondence and requests for reprints to: Roberto Salvatori, M.D., Division of Endocrinology, Johns Hopkins University School of Medicine, 1830 East Monument Street, no. 333, Baltimore, Maryland 21287. E-mail: salvator{at}jhmi.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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C transversion (position -124) in one of the two sites of the promoter region that binds the pituitary-specific transcription factor Pit-1, which is required for GHRHR expression. Chinese hamster ovary cells that were transfected with a cDNA encoding the K329E GHRHR expressed the receptor but failed to show a cAMP response after treatment with GHRH, confirming the lack of functionality. To test the effect of the AC mutation at position -124 of the promoter, we transfected rat GH3 pituitary cells, which express endogenous Pit-1, with plasmids in which the luciferase reporter gene was under the control of either the wild-type or the mutant promoter. GH3 cells expressing the mutant promoter showed significantly less luciferase activity than cells expressing the wild-type promoter. DNA-binding studies confirmed that the AC base change markedly reduces DNA binding to the Pit-1 protein. These results demonstrate that mutations in the GHRHR are not limited to the coding sequence and that promoter mutations that impair Pit-1 binding can reduce expression of the GHRHR gene. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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C transversion in the promoter region of the gene that inhibits binding of the transcription factor Pit-1 and which prevents transcriptional activity. |
RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Analysis of the GHRHR using peripheral blood genomic DNA revealed three heterozygous nucleotide changes compared with published coding and promoter sequences (4, 11, 12). One was a AG transition in codon 329, which replaces lysine (AAG) with glutamic acid (GAG) (K329E) at the junction of the third intracellular loop and the sixth transmembrane domain of the receptor (Fig. 1
). The second change is an AC transversion in position -124 of the promoter region of the gene (Fig. 2). A third CT heterozygous change from the published sequence at -235 of the promoter was present in the patient (data not shown) but was also present in homozygous pattern in a normal, unrelated subject, and in 8 of 26 (30%) chromosomes of the Polymorphism Discovery Resource panel (13), making it a rather common polymorphism.
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Expression and Function of the K329E GHRHR
Because the ability of the GHRHR to activate adenylyl cyclase is directly related to the number of receptors expressed on the cell surface, we first assessed cellular expression of wild-type and mutant receptors by confocal microscopy. We used site-directed mutagenesis to introduce the K329E mutation into a wild-type GHRHR cDNA that contained the sequence for the Aeqorea victoria green fluorescent protein (GFP) (14) in its 3'-region and which is fully bioactive (data not shown). The wild-type and mutant recombinant receptor proteins were transiently expressed in Chinese hamster ovary (CHO) cells, which do not express endogenous GHRHRs. Figure 3 indicates that cells that had been transfected with no plasmid (panel 1) had no fluorescence. On the other hand, cells transfected with wild type (panel 2) and the K329E mutant GHRHR cDNAs (panel 3) showed similar pattern of fluorescence, indicating similar receptor expression.
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, the size of the recombinant K329E protein is similar to the size of the wild-type receptor when expressed in CHO cells.
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), thus excluding the possibility that the K329E mutation has a dominant negative effect.
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, GH3 cells that were transfected with a plasmid in which the expression of the luciferase reporter gene is under the control of a minimal promoter containing both the -124 AC mutation and the -235 CT polymorphism (MUT) demonstrated a significant reduction in promoter activity when compared with GH3 cells that had been transfected with the construct containing the wild-type allele sequence (WT) or the polymorphism alone (POL). The activity of the promoter containing the -235 polymorphism is not significantly different from the activity of the wild-type promoter.
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, the intensity of the band produced by interactions between purified Pit-1 protein and a radiolabeled oligonucleotide containing the AC mutation in the Pit-1 binding site is markedly weaker than the band generated by the wild-type oligonucleotide at two different concentrations of Pit-1, indicating that the point mutation impairs binding of Pit-1 to the promoter. Both bands essentially disappear with the addition of excess unlabeled probe.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The transcription factor Pit-1 is a pituitary-specific POU domain factor that is necessary for development of somatotroph, lactotroph, and thyrotroph cells. Genetic mutations that lead to loss of Pit-1 activity cause combined GH, PRL, and TSH deficiency in animals and humans (21). Indeed, studies of the Pit-1-defective Snell mouse revealed that pituitary cells lack GHRHR, demonstrating that Pit-1 is required for the proper expression of the GHRHR gene (3). Pit-1 activates transcription of a GHRHR reporter gene in vitro by binding to two areas of the minimal GHRHR promoter, one located between -129 and -123 (P1) and a second one located between -171 and -160 (P2) (4). Artificially introduced mutations in either one of these two sites reduce GHRHR promoter function when tested in vitro in the Pit-1-expressing rat GH3 pituitary cell line (4). Therefore, it is conceivable that lack of Pit-1 binding to the GHRHR promoter in vivo may also reduce GHRHR expression and consequently lead to somatotroph cell hypoplasia.
In this report we describe a patient with IGHD IB who has all the clinical, biochemical, and radiological features described in other patients with GHRHR defects: severe growth failure, low but measurable serum GH, lack of serum GH response to a variety of stimuli, MRI evidence of pituitary hypoplasia, and sustained growth response to exogenous GH (5, 6, 7, 8, 9, 10, 18, 20). In addition, he failed to show an increase in growth velocity after a 6-month treatment with parenteral GHRH. He is heterozygous for a previously undescribed coding sequence mutation in codon 329 that causes replacement of lysine with glutamic acid (K329E). This change was not found in 88 normal chromosomes, indicating that it is not a common polymorphism. Moreover, CHO cells transfected with the K329E GHRHR cDNA expressed the recombinant receptor (although our experimental approach does not allow an accurate quantification of the amount of protein expressed), but failed to show an increase in intracellular cAMP after treatment with GHRH, indicating that the mutant receptor is nonfunctional. The K329 residue is part of a highly conserved amino acid sequence (QYWRLSKSTL) that is present in all the mammalian GHRHRs cloned so far (human, mouse, rat, pig, bovine, and ovine) (11, 12, 22, 23, 24, 25), and which spans the third intracellular loop and the sixth transmembrane domain. Immunoprecipitation and confocal microscopy experiments showed that the recombinant mutant receptor is expressed, although our experimental approach (i.e. using a GFP tag in the C terminus of the receptor) did not allow us to definitively exclude defective targeting of the receptor to the cell surface. Therefore, the K329E mutation may alter proper cell membrane expression, ligand binding, or coupling with G protein.
The patient’s phenotype, however, cannot be solely explained by the heterozygous K329E mutation, as this mutation lacks dominant inhibitory action, and haploinsufficiency of the GHRHR is insufficient to reduce GH secretion in mice (17) and humans (5, 6, 7, 8, 9, 10, 20). Therefore, we reasoned that another mutation must be present on the opposite allele, despite the fact that direct sequencing of the entire coding sequence and of the exon-intron boundaries of the GHRHR failed to reveal other changes from the published sequence. However, sequencing of the promoter region revealed a unique AC change in one of the two Pit-1 binding sites necessary for promoter activity in vitro (4). Characterization of the minimal GHRHR promoter carrying this mutation and a common polymorphism at -235 showed significantly reduced activity in GH3 cells. A promoter carrying only the -235 polymorphism was as active as the wild type, proving that the reduced activity is due solely to the -124 AC mutation. The observation that treatment of GH3 cells with Pit-1 antisense oligonucleotides inhibits transcription of a reporter gene construct containing the somatostatin receptor 1 promoter (which contains a Pit-1 binding site) and blocks the expression of the endogenous somatostatin receptor 1 gene indicates that GH3 cells express functional Pit-1 protein and are therefore a suitable model to study the effects of the Pit-1 binding site mutation we identified in the GHRHR gene (26). To confirm this effect directly, we performed DNA binding experiments, which demonstrated markedly reduced binding of the mutant sequence to Pit-1 protein, and provided a basis for the reduced promoter activity observed in GH3 cells. Finally, previous studies in which a 3-bp mutation was introduced in this area (129–123: TATGCAATGTACGA), including the A nucleotide replaced in our patient, have shown that alterations in this sequence reduce both promoter activity and Pit-1 binding in a manner similar to the effects observed in our experiments (4). Thus, this GHRHR promoter mutation can be added to the growing list of mutations in the promoter area of other genes that impair binding of transcription factors and can decrease gene expression (27, 28, 29).
Regrettably, DNA from the patient’s normal-statured parents was not available to demonstrate that the two mutations (the coding region and the promoter) originated from different heterozygous parents and therefore are located on opposite alleles. In addition, despite numerous attempts with three different long-range PCR methods (Herculase Enhanced DNA Polymerase, Stratagene, San Diego, CA; Expand Long Template, Roche Diagnostic, Mannheim, Germany; LA PCR, Takara Shuzo, Otsu, Japan), we were unable to amplify the 12.5-kb portion of genomic DNA that includes the two mutations to subclone DNA from each allele. This segment of the gene contains the extremely GC-rich 4.5-kb intron 1, which has consistently resisted PCR amplification by others (Baumann, G., personal communication) and us. Although we considered the possibility of using recombination targeting to clone large DNA fragments directly from genomic DNA (30), we do not have sufficient amounts of DNA to perform these techniques, and, as the patient is no longer available, we are unable to obtain additional DNA. Although the clinical and endocrine findings are more consistent with the presence of the two mutations on opposite alleles, we are unable to prove this experimentally.
This report, the third describing a patient with IGHD IB who carries two different GHRHR mutations (5, 9), emphasizes that novel mutations in this gene may arise frequently; thus, GHRHR mutations are likely to be common and should be considered in all patients with IGHD IB, even those whose parents are not consanguineous. More importantly, this is the first description of a naturally occurring mutation in the promoter region of the GHRHR and demonstrates that defective binding of the transcription factor Pit-1 can impair promoter activity. These results confirm the importance of Pit-1 in the expression of the GHRHR and, consequently, on the development and function of somatotroph cells in humans.
In conclusion, we have identified two new GHRHR mutations in a patient with IGHD IB. Our data indicate that mutations in the promoter of the GHRHR can contribute to the IGHD IB phenotype and emphasize that future analysis of the GHRHR in patients with IGHD will have to include the promoter as well as the coding region of the gene.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The patient’s parental guardians gave informed consent for the genetic studies. Parents were not available for genetic studies.
Amplification of the GHRHR and Mutation Detection
Genomic DNA was extracted from peripheral blood leukocytes by standard techniques. The 13 exons and the corresponding intron-exon boundaries of the GHRHR were individually amplified via PCR using oligonucleotide primers corresponding to intronic sequences that are located 9–41 bases from intron-exon junctions (5, 8). The exon 1 sense primer annealed to the promoter region, and the exon 13 antisense primer annealed to the untranslated region of exon 13. The primers used to amplify the promoter region amplify the proximal 285 bases (from -327 to-42) of the promoter region, and the amplification product overlaps the exon 1 amplicon, therefore including in the screening the proximal 327 bp of the promoter region (4). The fragment of promoter from -310 to -19 has been shown in vitro to be sufficient to confer maximal promoter activity (4).
PCR products were analyzed by PAGE, and, after isolation from gels, DNA fragments were sequenced directly using the Thermo-Sequenase Cycle Sequencing Kit (Amersham Pharmacia Biotech, Piscataway, NJ). To determine the prevalence of the two newly identified mutations, DNA was amplified with a set of oligonucleotides primers in which one primer of each pair was synthesized with a 40-nucleotide GC-rich 5'-extension to increase sensitivity of detection via DGGE (34). For the K329E mutation prevalence studies, the primers used were the same used for exon 11 amplification. The GC-rich extension was attached to the sense primer. For the promoter area, a new sense primer containing a GC-rich extension was designed, which anneals to sequences from -212 to -192 (5'-TCAGGGTCAATGCTGAGCTGG-3'), only 68 bp upstream of the identified mutation, and thus avoiding position -235 (where the polymorphism was identified). After confirmation that the nucleotide changes identified in the patient altered the migration patterns of the amplicons when analyzed by DGGE, the prevalence of the two newly discovered changes in the GHRHR was determined using a commercial panel of genomic DNAs from 44 unrelated normal subjects (88 chromosomes) (DNA Polymorphism Discovery Resource) of diverse ethnicity (13). The prevalence of the -235 polymorphism was analyzed in 26 chromosomes of the panel by direct sequencing.
Transient Expression of GHRHR
To determine whether the K329E missense mutation alters receptor expression or function, we used site-directed mutagenesis (35) to introduce the amino acid change into a wild-type GHRHR cDNA containing either the Aeqorea victoria green fluorescent protein (GFP) (14) or the Haemophilus influenzae HA epitope tag in the intracellular C-terminal domain (15). The GFP-tagged receptor and the HA-tagged receptor retain full bioactivity. Mutagenesis was confirmed by direct sequencing of the cDNA clones. Wild-type and mutant HA-tagged GHRHR cDNAs were cloned in the pcDNA1.0 Amp expression plasmid (Invitrogen, Carlsbad, CA), whereas the GFP-tagged cDNAs were cloned into GFP-N2 expression plasmid (CLONTECH Laboratories, Inc., Palo Alto, CA). These plasmids were transiently expressed in CHO cells.
For expression studies CHO cells were cultured on glass coverslips to 70–80% confluence and transfected with 1 µg of wild-type or mutant GHRHR cDNAs using LipofectAMINE PLUS (Life Technologies, Inc., Gaithersburg, MD). Forty-eight hours after transfection the cells were washed twice with PBS and fixed with 0.4% acetic acid in methanol for 15 min at -20 C. Cells were then washed twice with PBS and examined for fluorescence by confocal laser scanning microscopy (LSM 410, Carl Zeiss Inc., Oberkochen, Germany) using a 40x objective. Samples were scanned for equivalent times with the same contrast and brightness settings.
To verify the size of the K329E mutant GHRHR protein, immunoprecipitation studies were performed. CHO cells at 70–80% confluence were transfected with 8 µg of plasmid DNA in T-75 flasks using LipofecAMINE Plus (Life Technologies, Inc.). After 48 h, cells were scraped in ice-cold lysis buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, 4 mM 4-nytrophenyl phosphate, 10 mM EDTA, 40 mM NaF, 100 µg/ml leupeptin, 50 µg/ml trypsin inhibitor, and 1.5 µg/ml aprotinin), protein content in the extracts was measured, and 500 µg of protein extract were used for immunoprecipitation. After preclearance of lysate with protein A/Staphylococcus aureus suspension, the GFP-tagged GHRHR was reacted with the 4 µg of mouse anti-GFP monoclonal antibody (CLONTECH Laboratories, Inc.) and extracted from solution with agarose-immobilized protein A. The beads were then washed with ice-cold PBS, boiled with SDS sample buffer, and separated via SDS-PAGE and blotted onto polyvinylidene difluoride membranes. The membranes were then blocked with 5% dry milk in washing buffer (10 mM Tris, pH 7, 4, 150 mM NaCl, 0.1% Tween 20) overnight at 4 C, incubated with 1 µg of mouse anti-GFP monoclonal antibody (CLONTECH Laboratories, Inc.), and then exposed to the secondary antibody (goat antimouse) conjugated with horseradish peroxidase for 1 h. Antibody binding was detected by enhanced chemiluminescence (ECL kit, Amersham Pharmacia Biotech).
To assess the ability of the K329E GHRHR to activate Gs and increase adenylyl cyclase activity, CHO cells at 70–80% confluence were transfected with 8 µg of plasmid DNA in T-75 flasks using LipofectAMINE PLUS (Life Technologies, Inc.) and 24 h later the cells were harvested by gentle trypsinization and seeded into 24-well plates at 2 x 105 cells per well. The cells were cultured for an additional 24 h. The culture media were then replaced with serum-free media containing 0.5 mM isobutyl-methylxanthine and various concentrations of (His1, Nle27)-GHRH 1–32 (Peninsula Laboratories, Inc., Belmont, CA) or forskolin (10-5 M). After 15 min incubation at 37 C, total cAMP was extracted by addition of HCl to a final concentration of 0.1 N and a cycle of freeze-thawing. Cellular cAMP in the acid extracts was measured by RIA as previously described (36). Results were normalized to the cAMP response to forskolin and were expressed as picomoles of cAMP produced per well. Data are presented as the mean ± SE of three independent experiments, each performed in triplicate. Results were analyzed by ANOVA using experimental means.
Analysis of GHRHR Promoter Activity
A fragment of the GHRHR promoter (from -348 to +5) was amplified by PCR from the patient’s DNA and from the DNA of a normal subject who is homozygous for the -235 CT polymorphism (see above) using the sense primer previously described (5) and the antisense primer 5'-TCCATGGTGAGCCCAGCAGTGGC-3'. The promoter area between -310 and -19 has been shown to be sufficient for maximal promoter activity in GH3 cells (4). The amplification products were then digested with NlaIII restriction enzyme, to release a fragment corresponding to -313 and -19 of the promoter region, which was ligated into a SmaI site of the promoterless plasmid vector (poLUC) containing the luciferase reporter gene (37). Ligation products were transformed into competent DH5
E. coli, and individual clones were screened for the presence of insert by restriction digestion. Clones containing the expected size insert were subjected to direct sequencing to verify orientation of the insert. Three clones were finally identified, with inserts in the correct orientation, one containing the wild-type sequence, one containing the AC mutation in position -124 and the CT polymorphism at -235, and one containing only the -235 polymorphism.
Transient Transfections and Luciferase Measurement.
To determine the activity of the mutant minimal promoter we used rat GH3 pituitary cells (American Type Culture Collection, Manassas, VA), that normally express endogenous Pit-1 (26, 38). GH3 cells were grown in DMEM containing 10% FBS and penicillin and streptomycin to approximately 50% confluence in six-well dishes and transfected with 10 µg of plasmid DNA using lipofectin (Life Technologies, Inc.) following the manufacturer’s instructions. To assess transfection efficiency, GH3 cells were cotransfected with 5 µg of the pxGH5 plasmid containing the human GH reporter gene under the control of the mouse metallothionein promoter. Forty-eight hours after transfection, media were collected, and human GH concentration was measured by a RIA that does not cross-react with rat GH (Allegro HGH Transient Gene Expression Assay system, Nichols Institute Diagnostics, San Juan Capistrano, CA). Cells were harvested as described (37), and luciferase activity was determined using a luminometer (Analytical Luminescence Laboratory, San Diego CA). The experiments were performed in duplicate wells, each assayed in duplicate and were repeated four times. The luminometer results were normalized for transfection efficiency determined by human GH concentration in the media. Data are the mean ± SE of four independent experiments. Activities of wild-type and mutant promoter were compared using unpaired t test performed on experimental means, and statistical significance was assumed for P < 0.05.
Gel Mobility Shift Analysis.
As the promoter region of the GHRHR containing the mutation has been shown to bind the transcription factor Pit-1, we determined whether the mutation reduces Pit-1 binding using the method of Prywes and Roeder (39), with minor modifications. Sense and antisense 32-base oligonucleotides corresponding to the mutant or wild-type sequence (-141 to -108) were synthesized and purified by PAGE. Each oligonucleotide was annealed to its complement, and double-stranded oligonucleotides were labeled with [32P]-dATP with the Klenow fragment of DNA polymerase. Labeled probes were then separated from unincorporated [32P]-dATP via ProbeQuant G-50 Microcolumns (Amersham Pharmacia Biotech), and specific activity was measured. Binding was performed using 0.02 or 0.04 µg of purified Pit-1 for 30 min at room temperature with either wild-type or mutant probe and equal amounts of radioactivity at equivalent specific activity (105 cpm/ng) in 20-µl reactions containing 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 5% glycerol, and 0.25 mg/ml BSA. Binding mixtures were then separated by electrophoresis through a nondenaturing 5% acrylamide gel, which was then dried and exposed to x-ray film. Purified recombinant full-length rat Pit-1 protein (amino acids 2–291) was a generous gift of Drs. D. F. Gordon and E. C. Ridgway (University of Colorado Health Science, Denver, CO) (40). Oligonucleotides used were: wild-type sense: 5'-CTAGCTCCTGCCTATGCAAACAGCCACCTGAG-3'; wild-type antisense: 5'-TTCTCAGGTGGCTGTTTGCATAGGCAGGAGCT-3'; mutant sense: 5'-CTAGCTCCTGCCTATGCCAACAGCCACCTGAG-3'; mutant antisense: 5'-TTCTCAGGTGGCTGTTGGCATAGGCAGGAGCT-3'.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: CHO, Chinese hamster ovary; DGGE, denaturing gradient gel electrophoresis; GFP, green fluorescent protein; GHRHR, GHRH receptor; HA, hemagglutinin antigen; IGHD IB, isolated GH deficiency type IB.
Received for publication February 8, 2001. Accepted for publication November 19, 2001.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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