Decreased Expression of the GHRH Receptor Gene Due to a Mutation in a Pit-1 Binding Site
Roberto Salvatori,
Xiaoguang Fan,
Primus E. Mullis,
Azeb Haile and
Michael A. Levine
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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A variety of mutations in the gene encoding the GHRH receptor (GHRHR) that are predicted to alter protein structure or function have been recently described in patients with isolated GH deficiency type IB. In the present report we describe a patient with isolated GH deficiency type IB who was heterozygous for two novel mutations in this gene: a missense mutation in codon 329 that replaces lysine with glutamic acid (K329E) and an A
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 A
C 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 A
C 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.
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INTRODUCTION
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GHRH IS A hypothalamic polypeptide essential for the proliferation of somatotroph cells and for secretion of GH from mature somatotrophs (1). GHRH acts on the pituitary by binding to a seven-transmembrane domain receptor that is a member of the group III-B family of G protein-coupled receptors. Activation of the GHRH receptor (GHRHR) leads to increased adenylyl cyclase activity and accumulation of intracellular cAMP (2). The expression of the GHRHR gene (GHRHR) requires the presence of the pituitary POU domain factor Pit-1 (3, 4). Mutations in the coding region of the GHRHR are present in approximately 10% of patients with familial isolated GH deficiency (IGHD) type IB, characterized by autosomal recessive transmission, low but measurable serum GH levels, and a favorable response to GH therapy (5). Mutations in this gene described to date include one nonsense mutation, one splicing mutation, four missense mutations, and two small deletions (5, 6, 7, 8, 9, 10), all of which are predicted to generate severely truncated receptor protein or to impair receptor structure or function. In the present study we describe the analysis of the GHRHR in a patient with IGHD IB who did not increase his growth velocity during a therapeutic trial of parenteral GHRH. The patient has two heterozygous mutations in the gene: in addition to a novel missense mutation that leads to loss of receptor function, he has an A
C transversion in the promoter region of the gene that inhibits binding of the transcription factor Pit-1 and which prevents transcriptional activity.
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RESULTS
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Identification of Mutations
The patient was a 4-yr-old boy with severe growth retardation due to IGHD IB. His parents were of normal height.
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 A
G 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 A
C transversion in position -124 of the promoter region of the gene (Fig. 2
). A third C
T 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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Figure 1. Sequence Analysis of a Portion of Exon 11 of the GHRHR from Genomic DNA of the Patient (left)
Normal sequence (Control) is from an unrelated normal subject. The heterozygous A G transition is indicated by the arrow.
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Figure 2. Sequence Analysis of a Portion of Promoter Region of the GHRHR from Genomic DNA of the Patient (Left)
Normal sequence (Control) is from an unrelated normal subject. The heterozygous A C transversion is indicated by the arrow.
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Neither the K329E nor the -124 mutation was detected in 88 chromosomes of the Polymorphism Discovery Resource screened via denaturing gradient gel electrophoresis (DGGE).
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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Figure 3. Localization of the GFP-Tagged GHRHR
CHO cells were transiently transfected with LipofectAMINE only (1), wild type GFP-tagged GHRHR cDNA (2), and K329E mutant GFP-tagged GHRHR cDNA (3). All images were scanned with a confocal microscope for equivalent times and are representative of two separate experiments.
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To rule out the possibility that the K329E mutation causes a change in protein size, we performed immunoprecipitation studies using anti-GFP antibodies. As shown in Fig. 4
, 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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Figure 4. Immunoprecipitation of CHO Cells Mock Transfected (M) or Expressing Either the Wild-Type (WT) or Mutant (MUT) GHRHR Tagged with a GFP Tag
The GFP-labeled GHRHR (GFP-GHRHR) band is indicated by the arrow (expected size: 80 kDa, 55 kDa for the receptor plus 25 kDa for the GFP tag). The lower band present in all three lanes is nonspecific and likely represents immunoglobulins.
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We then tested the ability of the mutant receptor to generate cAMP in response to different concentrations (10-7 to 10-10 M) of GHRH. For these experiments, we used an hemagglutinin antigen (HA)-tagged GHRHR cDNA, which was previously shown to have biological activity that is equivalent to that of the native receptor (15). Cells treated with LipofectAMINE without DNA (Mock) did not show a significant cAMP response to GHRH. GHRH-stimulated cAMP production was significantly less in cells expressing the mutant receptor than in cells that were expressing wild-type GHRHR (WT), at hormone concentrations ranging from 10-7 to 10-9 M, demonstrating that the K329E amino acid change impairs the ability of the receptor to transmit GHRH signaling (Fig. 5
).

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Figure 5. Basal (Bas) and GHRH-Stimulated (10-10 to10-7) Intracellular cAMP Levels in CHO Cells Transiently Transfected with LipofectAMINE Alone (Mock), Wild-Type (WT), or with the K329E Mutant GHRHR (MUT) cDNAs
The results are the means of three separate experiments, each one carried in triplicate wells. Each well was assayed in duplicate. Values are normalized according to cAMP response to forskolin. GHRH caused a statistically significant increase in cAMP, compared with basal, in cells transfected with the wild-type receptor at concentrations ranging from 10-7 to 10-9. *, P < 0.05 compared with wild type.
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To test whether the K329E mutant GHRHR has dominant inhibitory properties, we coexpressed wild-type and mutant receptors in CHO cells. Cells that were transfected with 4 µg of wild-type cDNA plus 4 µg of vector DNA [pcDNA 1.0 (WT+VEC)] showed a cAMP response to GHRH that was similar to the response of cells transfected with 4 µg of wild-type cDNA and 4 µg of the K329E mutant cDNA (WT+K329E) (Fig. 6
), thus excluding the possibility that the K329E mutation has a dominant negative effect.

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Figure 6. Basal (Bas) and GHRH-Stimulated (10-10 to 10-7) Intracellular cAMP Levels in CHO Cells Transiently Transfected with LipofectAMINE Alone (Mock), a Combination Of Wild-Type GHRHR cDNA and pcDNA (WT+VEC), or a Combination Of Wild-Type and K329E Mutant GHRHR cDNAs (WT+K329E)
The results are the mean of three separate experiments, each one performed in triplicate wells. Each well was assayed in duplicate. No statistically significant differences between WT+VEC and WT+K329E were present at any of the GHRH concentrations. Both WT+VEC and WT+k329E were statistically significant from basal at GHRH concentrations 10-9, 10-8, and 10-7.
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Promoter Activity
As shown in Fig. 7
, 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 A
C mutation and the -235 C
T 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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Figure 7. GHRHR Promoter Activity in GH3 Cells
Cells were transiently transfected with plasmid constructs containing either no promoter (VECT) or GHRHR promoter containing the wild-type (WT) or mutant (MUT) or -235 polymorphism (POLY) sequences linked to the reporter gene firefly luciferase. Cells were then lysed and luciferase activity was assayed with a luminometer. The results [expressed in relative light units (RLU)] are the mean of four separate experiments, each one carried out in duplicate wells. Each well was assayed in duplicate. Values are normalized according to transfection efficiency. *, P < 0.05 compared with wild type.
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Gel Mobility Shift Assay
As shown in Fig. 8
, the intensity of the band produced by interactions between purified Pit-1 protein and a radiolabeled oligonucleotide containing the A
C 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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Figure 8. Gel Mobility Shift Analysis Using Wild-Type or -124 A C Mutant Oligonucleotide Probes Derived from the Region Between -141 and -108 of the GHRHR and Purified Rat Pit-1 Protein
Lanes 1, 3, 5, and 7, wild-type probe. Lanes 2, 4, 6, and 8, mutant probe. Lanes 3 and 4, Pit-1= 0.02 µg. Lanes 58, Pit-1 = 0.04 µg. Lanes 7 and 8 contain excess unlabeled probe.
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DISCUSSION
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Proliferation of pituitary somatotroph cells and secretion of GH require the activation of signal pathways by the hypothalamic hormone GHRH. In both mice and humans, the lack of GHRH action, due to null mutations in the GHRHR gene, results in severe GH deficiency and pituitary hypoplasia (16, 17, 18). In the little mouse, a murine model of dwarfism due to lack of functional GHRHR (19), the pituitary is hypoplastic and contains only 10% of the somatotroph cells that are present in normal animals (17). Although clusters of somatotroph cells can be identified in the anterolateral part of the little mouse pituitary, they fail to colonize the postero-caudal area of the gland, proving that GHRH action is required for proliferation but not for differentiation of somatotrophs (17). Although histological studies have not been performed on pituitaries from humans who have GHRHR mutations, it is likely that similar findings would occur in patients with nonfunctional GHRHR. Indeed, all patients reported so far with GHRHR mutations in whom magnetic resonance imaging (MRI) studies have been performed have shown evidence of a hypoplastic pituitary (5, 18, 20).
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 patients 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 A
C 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 A
C 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 (129123: TATGCAA
TGTACGA), 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 patients 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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Patient
The patient was a 4-yr-old boy referred to the endocrine clinic for short stature. He was the first born of parents who were both of normal stature [father, 173.5 cm (-0.6 SD), mother, 169.8 cm (+0.5 SD)] (31). The second and third children of this couple showed normal growth and development. There was no known parental consanguinity. The patient was the product of an uncomplicated full-term pregnancy. Birth weight was 3,200 g, and Apgar scores were 9 and 10, 5 and 10 min after birth, respectively. No neonatal hypoglycemia was observed. When initially evaluated for short stature, his height was 88 cm (-4.6 SD). His growth velocity during the 2 yr before referral was 3 cm/yr (-4.4 SD). Bone age (calculated by the method of Greulich and Pyle) was 2 and 3/12 yr with a chronological age of 4 yr (32). The child had no dysmorphic features and, except for proportionate short stature, the physical examination was normal. He had normal serum electrolytes, blood hemoglobin level, thyroid function tests, and 46XY karyotype. The patient underwent two GH stimulation tests. Administration of insulin (4 U/m2 of regular insulin iv bolus) produced appropriate hypoglycemia (glucose nadir 1.44 mmol/liter), but peak serum GH concentration was only 1.2 µg/liter. Peak serum GH concentration after administration of GHRH [GHRH (129)-NH2 (GRF Ferring Pharmaceuticals Ltd., Dübendorf, Switzerland) 1 µg/kg iv bolus] was only 1.6 µg/liter. Serum GH was measured by a commercial immunoradiometric assay (HGH Maia Clone, Biodata, Bologna, Italy). MRI of the sellar region showed a hypoplastic pituitary gland without any abnormal masses or other defects. Mutations in the gene encoding for GH (GH1) were excluded by direct sequencing, as previously reported (33). The patient was treated for 6 months with exogenous GHRH (25 µg/kg/d by twice daily sc injections) but did not show a significant increase in growth velocity. He was then treated with exogenous recombinant GH (Novo-Nordisk, Gentofle, Denmark) from age 5.5 yr to 14.5 yr with marked acceleration of growth velocity (9.0 cm/yr during the first 2 yr of therapy). He underwent spontaneous puberty, and his final adult height at age 18 yr was 175 cm (-0.1 SD).
The patients 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 941 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 7080% 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 7080% 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 7080% 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 132 (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 patients DNA and from the DNA of a normal subject who is homozygous for the -235 C
T 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 A
C mutation in position -124 and the C
T 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 manufacturers 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 2291) 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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We thank Drs. E. Chester Ridgway and Dr. David Gordon (University of Colorado Health Science Center, Denver, CO) for generously providing purified Pit-1 protein, Dr. Kelly Mayo (Northwestern University, Chicago, IL) for generously providing the HA-tagged GHRHR cDNA, and Dr. Mark Kronenberg (University of Connecticut Health Center, Farmington, CT) for advice on DNA binding studies.
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FOOTNOTES
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This work was supported by NIH-NCRR GCRC-CAP award 3 M01 RR000052-38S1 (to R.S.), by a grant from the Genentech Foundation for Growth and Development (R.S.), by NIH Grants DK-34281 (to M.A.L.), NIH-NCRR GCRC 5 M01RR00052, and by a grant from the Swiss National Science Foundation SNF 32-53714.98 (to P.E.M.).
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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