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Division of Endocrinology, Metabolism, and Molecular Medicine (E.J.L., T.J.K., J.L.J.), Department of Neurosurgery (I.C.), and Department of Biochemistry, Molecular Biology and Cell Biology (K.E.M.), Northwestern University Medical School, Chicago, Illinois 60611; and Department of Pathology (M.K.L.), Division of Endocrinology (H.C.L., K.B.H.), Department of Medicine, Yonsei University College of Medicine, Seoul, Korea 120-752
Address all correspondence and requests for reprints to: J. Larry Jameson, M.D., Ph.D., Division of Endocrinology, Metabolism and Molecular Medicine, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago, Illinois 60611.
Abstract
The molecular events leading to the development of GH-producing pituitary tumors remain largely unknown. We hypothesized that activating mutations of the GHRH receptor might occur in a subset of GH-producing pituitary tumors. Genomic DNA samples from 54 GH-producing pituitary tumor tissues were screened for mutations of the GHRH receptor. Eleven homozygous or heterozygous nucleotide substitutions [169G > A (A57T), 338C > T (P113L), 363G > T (E121D), 409C > T (H137Y), 547G > A (D183N), 673G > A (V225I), 749G > A (W250X), 760G > A (V254M), 785G > A (S262N), 880G > A (G294R), 1268G > A (C423Y)] were found in 12 patients (22.2%). The 169G > A substitution (A57T) appears to be a polymorphism (4 patients, 7.4%). E121D and V225I were each found in 2 patients. In 1 patient with the V225I sequence, the substitution was not found in genomic DNA from peripheral leukocytes, suggesting a somatic mutation. A patient with a heterozygous W250X mutation was homozygous for the C423Y substitution.
These variant GHRH receptors were studied in transfected TSA-201 cells to evaluate the functional consequences of the amino acid changes. None of the GHRH receptor variants was associated with basal elevation of intracellular cAMP. GHRH induced variable cAMP responses. With the W250X and G294R variants, there was no cAMP stimulation by GHRH, indicating that the mutations are inactivating. Expression of the W250X GHRH receptor on the cell membrane was severely decreased and GHRH binding to the G294R GHRH receptor was impaired.
Although GHRH receptor variants are common in GH- producing pituitary adenomas, constitutively activating mutations, as a mechanism for GH-producing pituitary tumors appear to be rare.
HYPOTHALAMIC GHRH stimulates the synthesis
and secretion of GH from the anterior pituitary. GHRH binds to
its stimulatory G protein-coupled receptor at the cell membrane,
activating adenylyl cyclase and increasing intracellular cAMP levels.
Somatic mutations that inhibit GTPase activity of the G protein 
chain (Gs) are a well established molecular cause of GH-producing
pituitary tumors. These mutations have been identified in 30–40% of
GH-producing pituitary tumors (1, 2). Activating mutations
of G protein-coupled-coupled receptors (GPCRs) have also been found in
many endocrine diseases, such as hyperfunctioning thyroid adenomas (TSH
receptor) (3), familial male-limited precocious puberty
(LH receptor) (4), and Jansen’s metaphyseal
chondrodysplasia (PTHrP receptor) (5). These mutations
confer constitutive activation to the receptors and result in cellular
proliferation and increased hormone synthesis and secretion. Based on
these observations, we hypothesized that activating mutation of GHRH
receptor might occur in a subset of GH-producing pituitary adenomas. In
this study, we analyzed the GHRH receptor gene in 54 GH-producing
pituitary tumors. Several GHRH receptor variants were identified and
their functional characteristics are described.
Materials and Methods
Subjects and isolation of DNA
Fifty-four patients with GH-producing pituitary tumors were included in this study. The diagnosis of a GH-secreting tumor was established by measurement of GH and IGF-1 and confirmed in all cases by positive immunohistochemical staining of tumor tissue for GH. The study protocol was approved by the hospital committees of Yonsei Medical Center and Northwestern University Medical School, and written consent was obtained from all patients.
Genomic DNA was extracted from paraffin-embedded pituitary tumor specimens. Five to seven adjacent 5-µm sections were cut from each paraffin block and mounted on slides. The middle section was stained with hematoxylin-eosin and served to distinguish tumor tissue from normal surrounding tissue. DNA was isolated from tumors sections as previously described (6).
Sequence analysis
Pairs of primers flanking each exon of the GHRH receptor (Table 1
) were designed based on recently
identified sequence information. A 127-kb fragment from the human
p14-p15 region of chromosome 7 containing the complete GHRH receptor
with its introns and promoter region has been sequenced as part of the
Human Genome Project (7). PCR primers were designed with
additional 5' sequences for universal primers (-21 M13 or
M13 reverse) to facilitate sequencing. The PCR (50 µl) included 300
ng DNA, 10 pmol of each primer, 50 µM deoxynucleoside
triphosphate (deoxy-NTPs), 5 mM MgCl2
in PCR buffer (50 mM KCl, 10 mM Tris-HCl (pH
9.0), 0.1% Triton X-100) containing 2.5 U Taq DNA
polymerase (Promega Corp., Madison, WI). Cycle conditions
were: 2 min hot start at 96 C, followed by 35 cycles of 1 min at 94 C,
45 sec at 58 C, and 45 sec at 72 C, followed by extension at 72 C for
15 min. The PCR products were resolved on 1.5% agarose gels and
purified as described previously (8). Direct DNA
sequencing was performed using the ABI Prism dRhodamine Terminator
Cycle Sequencing Ready Reaction kits with AmpliTaq DNA Polymerase FS
(Perkin-Elmer Corp., Foster City, CA) and the ABI PRISM
377 DNA automated Sequencer (Perkin-Elmer Corp.).
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exon 8
(codon 201) and exon 9 (codon 227). Amplified products were purified
and sequenced as described above. Construction of GHRHR variants and generation of HA epitope-tagged receptor constructs
The wild-type GHRH receptor cDNA (1617 bp) was inserted into the pcDNA-3 vector (Invitrogen, Carlsbad, CA) downstream of the T7 RNA polymerase promoter and flanked by BamHI and XhoI sites at the 5' and 3' ends, respectively. Using site-directed mutagenesis, GHRH receptor variants were generated. The wild-type and variant GHRH receptors were subcloned into the pSVL plasmid (Pharmacia Biotech, Piscataway, NJ) at BamHI and XhoI sites (created by oligonucleotide annealing).
Fusion constructs were created by incorporating an N-terminal influenza virus HA epitope (YPYDVPDYA) in frame with the GHRH receptor (wild-type and variants) in the pSVL vector. All the final constructs were verified by DNA sequencing.
Immunocytochemical analysis of GHRH receptor expression
TSA-201 (hereafter referred to TSA) cells, derived from human embryonic kidney 293 cells were maintained in DMEM supplemented with 10% FBS and transfected using the calcium phosphate method. Forty-eight hours after transfection of the GHRH receptor or HA-epitope tagged GHRH receptors, TSA cells were collected, washed twice with PBS, mounted on glass slides, and air-dried for 10 min. Slides were fixed for 5 min with 4% paraformaldehyde in sodium phosphate (pH 7.2) and treated with 3% hydrogen peroxide in PBS for 10 min. After preincubation with serum blocking solution (Zymed Laboratories, Inc., San Francisco, CA), specimens were incubated with rat monoclonal anti-HA high affinity antibody (2.5 µg/ml, Roche Molecular Biochemicals, Indianapolis, IN) for 2 h at room temperature. After washing with TBS/0.025% Tween, slides were incubated with antirat Ig-biotin, F(ab')2 fragment (5 µg/ml, Roche Molecular Biochemicals) for 20 min at room temperature. Streptavidin-peroxidase (Zymed Laboratories, Inc.) was used with the 3-amino-9-ethylcarbazole chromogen (DAKO Corp., Carpinteria, CA). For permeabilized cells transfected with the HA-tagged W250X GHRH receptor, slides were fixed in ice cold methanol (5 min) and acetone (5 min) and boiled in 0.02% NP40/sodium citrate buffer (pH 6.0) for 10 min using a microwave oven. Slides were then treated with 3% hydrogen peroxide in methanol for 10 min. After preincubation with serum blocking solution, the subsequent steps were performed as described above.
Immunofluorescent studies were also performed in TSA cells transfected with GHRH receptors. A rabbit antihuman GHRH-receptor antibody (1: 1000, provided by Dr. Bruce Gaylinn, University of Virginia, Charlottesville, Virginia) was used to detect the GHRH receptor. After incubation for 1 h with the primary antibody, staining was performed using biotinylated secondary antibodies (ABC kit, Vector Laboratories, Inc., Burlingame, CA) and streptavidin-FITC (1:100, Vector Laboratories, Inc.). Cell images were analyzed using a Carl Zeiss microscope (Axioskop, Carl Zeiss Inc., Oberkochen, Germany).
RNA isolation and RT-PCR for the GHRH receptor
To verify expression of the GHRH receptor constructs, total RNA was isolated from transfected TSA cells using TRIZOL reagent (Life Technologies, Inc., Gaithersburg, MD) as described by the manufacturer. Twenty micrograms of RNA was treated with DNase-I for 30 min at room temperature. Random hexamers were used to synthesize single-strand cDNA using 10 µg of DNase-I treated RNA. A portion (1/40) of the cDNA solution was used for amplification of the human GHRH receptor or GAPDH.
Cycle conditions were: 2 min hot start at 96 C, followed by 30 cycles of 1 min at 94 C, 45 sec at 55 C, followed by 1 min at 72 C, and extension at 72 C for 15 min. An aliquot (20%) of each PCR was resolved by electrophoresis on 1.75% agarose gels and DNA products were visualized with ethidium bromide.
Measurement of ligand binding
TSA cells grown to 50% confluency in DMEM/F12 on 6-well plates
were transfected by calcium phosphate precipitation with 1 µg of DNA
per well. After 36 h, cells were rinsed with 1.0 ml of binding
buffer: HEPES 50 mM, sucrose 100 mM,
CaCl2. 6H2O 5
mM, ovalbumin 0.1% (wt/vol). 125I
-labeled human GHRH 1–44 amide (50,000 dpm) (Amersham Pharmacia Biotech, Piscataway, NJ) was added to each well along with
10-12 or
10-8 M hGHRH
1–44 amide (Sigma, St. Louis, MO). Incubation was
continued at room temperature for 3 h and the cells were rinsed
twice with 1.0 ml of binding buffer followed by the addition of 0.5 ml
of 1.0 N NaOH. After 1 h, the wells were scraped, and
the contents transferred to a glass tube. The wells were washed two
additional times with 0.25 ml 1.0 N NaOH, and the solution
was transferred to the collecting tubes. Radioactivity was measured in
a 
counter (United Technologies Packard, Downers Grove, IL). Data
are expressed as the percent of counts bound by the wild-type GHRH
receptor.
Measurement of intracellular cAMP levels
Triplicate wells of TSA cells, grown to 50% confluency, were transfected with 0.1 µg or 1.0 µg GHRH receptor plasmid DNA per well using the calcium phosphate method. Transfected cells were maintained in DMEM/F12 with 5% dialyzed FBS (10 kDa molecular cut-off, Sigma, St. Louis, MO). Forty-eight hours after transfection, the cells were treated with 0.1 mM isobutylmethylxanthine for 20 min at 37 C. hGHRH 1–44 amide (1 nM) was added in fresh 37 C media, and the incubation was continued for another 30 min at 37 C. The medium was removed and 0.5 ml cold 0.1 M HCl was added to each well. Cell lysates were centrifuged for 10 min at 4 C to remove protein, and the supernatants were neutralized with an equal volume of 150 mM Tris-HCl (pH 8.0) containing 4 mM EDTA. cAMP levels were measured with an RIA Kit (Biomedical Technologies Inc., Stoughton, MA) according to the manufacturer’s instructions.
Results
Sequence analysis of GHRH receptor and Gs
Based on the hypothesis that activating mutations of GHRH receptor
might occur independent of Gs mutations, tumors were first screened
for activating mutation of the Gs subunits. Activating Gs
mutations are located in exons 8 or 9. Therefore, we screened exons 8
and 9 of Gs and found 15 (27.8%) mutations in 54 acromegalic
tumors. Thirteen mutations were located in codon 201 (twelve R201C and
one R201H) and two in codon 227 (Q227L), one of which was
homozygous.
The nucleotide sequence of the entire coding region of the GHRH
receptor gene was analyzed in all 54 tumors. Eleven nucleotide
substitutions (169G > A, 338C > T, 363G > T,
409C > T, 547G > A, 673G > A, 749G > A,
760G > A, 785G > A, 880G > A, 1268G > A) were
identified in 12 patients (22.2%) of 54 patients (Fig. 1). The resulting amino acid changes are
A57T, P113L, E121D, H137Y, D183N, V225I, W250X, V254M, S262N, G294R,
and C423Y, respectively. A schematic diagram of the variant GHRH
receptors is shown in Fig. 2. Four
patients have homozygous variants (A57T and E121D, P113L, D183N, and
C423Y). The A57T form of the GHRH receptor was found in 4 patients
(7.4%), one of whom also had a Gs mutation. To determine whether
these variations were polymorphisms, we screened 40 genomic DNA samples
from peripheral leukocytes of normal persons, 3 of whom (7.5%) were
found to have the A57T GHRH receptor. Based on this result, the
169G > A (A57T) substitution is a polymorphism. The E121D
substitution was found in two patients who had A57T. One of these
individuals was homozygous for both variants. The V225I substitution
was also found in two patients, one of whom had A57T. A patient with
the W250X termination mutation was also homozygous for C423Y. We
obtained peripheral leukocyte DNA from one patient whose tumor had the
V225I GHRH receptor. Sequence analysis showed the wild-type genotype of
the receptor in peripheral blood, suggesting that the V225I
substitution is a somatic mutation.
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Immunocytochemistry of aminoterminally HA-tagged GHRH receptors
was performed using a monoclonal antibody directed against the HA
epitope to assess the membrane expression of variant GHRH receptors.
Indirect immunofluorescence was also performed using a rabbit
polyclonal antibody directed against the N terminus of the GHRH
receptor. The two detection methods gave similar results (data not
shown). The wild-type and variant GHRH receptors were expressed on the
cell surface (Fig. 3, A and B). All of
the variant GHRH receptors showed a similar level of expression with
the exception of W250X, which was minimally (2% of normal) expressed
(Fig. 3, A-c). Immunocytochemistry of permeabilized cells (data not
shown) revealed that the expression level of W250X was similar to that
of nonpermeabilized cells. RT-PCR analyses confirmed that the mRNA
expression of W250X was similar to that of the wild-type GHRH receptor
(Fig. 4). These findings suggest that the
W250X protein is unstable and may be degraded in the cytoplasm.
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Ligand competition studies were carried out to assess GHRH binding
to GHRH receptor variants. With the exception of the G294R, all
receptor variants bound GHRH specifically (Fig. 5). Maximum binding (compared with
wild-type GHRH receptor) varied from 107% (A57T) to 44% (W250X), and
unlabeled ligand competed with radiolabeled GHRH.
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The wild-type and variant GHRH receptors were expressed in TSA
cells to assess their activities in the absence and presence of GHRH.
Basal and GHRH-induced activities were assessed by measuring cAMP, the
major second messenger for the GHRH receptor. The antisense orientation
of wild-type receptor was used as a negative control. To investigate
basal cAMP production, two different doses (0.1 µg and 1.0 µg/well)
of transfected receptor were analyzed. The basal levels of
intracellular cAMP were similar for both doses of wild-type and variant
GHRH receptors, and not different from mock transfected cells (data not
shown). The addition of GHRH (1 nM) induced variable
degrees of cAMP elevation. No cAMP elevation was detected with the
W250X and G294R receptor variants (Fig. 6), indicating that these mutations
impair signal transduction by GHRH.
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Pituitary tumors are known to be monoclonal in origin
(9), suggesting that somatic mutations in progenitor cells
are initiating events in tumorigenesis. In GH-producing pituitary
adenomas, LOH of chromosome 11q13 (10, 11), and activating
mutations of Gs (1, 2, 12) have been identified as
pathogenetic mechanisms. The activating mutations of Gs lead to
constitutive activation of adenylyl cyclase with a consequent high rate
of cAMP production. Increased cAMP levels activate protein kinase A,
which in turn phosphorylatesthe transcription factor CREB. Because this
signaling pathway is activated by GHRH, we hypothesized that activating
mutations of the GHRH receptor might provide a potential mechanism for
initiating or enhancing the growth of GH-producing pituitary tumors.
Our efforts were initially focused on possible mutations in the third
intracytoplasmic loop (IL) and the sixth transmembrane (TM) domain of
the GHRH receptor given the high frequency of activating mutations in
these regions in other GPCRs. However, no mutations were found in these
regions. Recently, the entire genomic sequence of the human GHRH
receptor has been published (7), leading us to assess the
entire coding sequence of the GHRH receptor. In 54 acromegalic tumor
samples, we identified 11 different nucleotide substitutions. No
constitutive activation was found as a result of these alterations,
suggesting that activating mutations of the GHRH are probably not a
common cause of GH-producing pituitary adenomas.
Recently, two activating mutations (H223R and T410P) in PTH/PTHrP receptor were identified in a patient with Jansen’s metaphyseal chondrodysplasia (5, 13). The wild-type amino acids at these sites are highly conserved among same GPCR family B members. H223R and T410P are located at the junction between IL1 and TM2, and IL3 and TM6, respectively. Of the amino acids in variant GHRH receptors in this study, only two, D183N and W250X, are conserved in other GPCR family B members (14, 15). D183N is located at the junction between TM2 and the first extracellular loop (EL1), which is close to the location of H223R PTH/PTHrP receptor. However, the D183N GHRH receptor diminished the cAMP response (40–50% of wild-type) to GHRH, without evidence of basal constitutive activity.
Several lines of evidence suggest that the IL domains and the C-terminal tail of the GPCRs are involved in G protein coupling. Alternative splicing within IL1 of the calcitonin receptor (16, 17) and IL3 of the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor (18, 19, 20), affects receptor coupling to the adenylyl cyclase and PLC pathways. Recently, two alternatively spliced isoforms of the rat GHRH receptor have been identified (21). The long isoform, which has an additional 41 amino acids in IL3, has reduced receptor signaling through the adenylyl cyclase pathway, although the receptor is expressed on the cell surface and binds GHRH. In the W250X variant, which is truncated downstream of EL2, the binding of GHRH was partially maintained, but the cAMP response to GHRH is absent, suggesting that IL3 and the C-terminal tail are important for G protein coupling to the GHRH receptor.
The expression level of G294R was equivalent to the wild-type GHRH receptor, but the single amino acid change in TM5 abolished the receptor’s ability to bind GHRH. In several diseases, single point mutations of GPCRs have been shown to decrease the expression of the receptor on the cell surface as well as its affinity for the ligand (22, 23). The G294R mutation decreased ligand binding without affecting the cell surface localization of the receptor, suggesting that that G294R is an inactivating mutation. These characteristics are similar to the features of the mutant GHRH receptor of the little mouse (24, 25).
Interestingly, four patients in this study appeared to be homozygous for GHRH receptor variants. Possibly, these patients are actually hemizygous due to LOH in this region of the genome. LOH has been described in many tumors; adrenocortical tumors (chromosome 18p11.2: ACTH receptor gene) (26), sporadic parathyroid tumors (chromosome 1) (27), pancreatic islet cell tumors (chromosome 3p14.2–3p21) (28), hepatocellular carcinomas (chromosome 8p23) (29), and colorectal carcinomas (chromosome 3p23) (30). These findings suggest that the deletion of specific regions of a chromosome can result in loss of normal differentiation and clonal expansion of malignant cell clones. LOH could not be formally assessed in this study because normal tissue was not available. However, it might be of interest to investigate LOH at the GHRH receptor locus in association with the tumorigenesis of acromegaly.
The lack of obvious gain or loss of function associated with most of the variants described here suggests that the nucleotide substitutions in these variant GHRH receptors may be polymorphisms. The A57T GHRH receptor was found in 4 cases (7.4%) in acromegalic tumor tissue, but it was also found in genomic DNA of 3 (7.5%) of 40 normal subjects. This is consistent with another report demonstrating that A57T is a polymorphism (31). V225I was not found in genomic DNA from the patient’s leukocytes, suggesting that V225I is a somatic mutation in the tumor cell.
The pathogenesis of a large subset of pituitary tumors causing acromegaly is not well understood. In this study, genetic abnormalities of the GHRH receptor were investigated and several variant GHRH receptors were identified in human acromegalic pituitary tumor tissues. None of them was associated with constitutive activation of the cAMP pathway. Although some of these mutations may affect tumorigenesis by other mechanisms, activating mutations of the GHRH receptor are probably not a common cause of human GH-producing pituitary tumors.
Acknowledgments
We are grateful to Dr. Bruce Gaylinn for providing an antibody against human GHRH receptors, and to Drs. Peter Kopp and Jeffrey Weiss for critical reading and helpful discussions. We also thank Dr. Su-Youn Nam and Dr. Jae Hyun Nam for their help in obtaining genomic DNA samples from patients.
Footnotes
This work was supported by a grant from the Northwestern Memorial Foundation and by NIH Grant DK-48071 (to K.E.M.). E.J.L. is a recipient of a grant of Yonsei Research Foundation.
Abbreviations: GPCRs, G protein-coupled-coupled receptors;
Gs, G protein chain; IL, intracytoplasmic loop; PACAP, pituitary
adenylate cyclase-activating polypeptide; TM, transmembrane.
Received December 27, 2000.
Accepted April 17, 2001.
References
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of Gs and stimulate adenylyl cyclase in human pituitary tumours. Nature 340:692–696[CrossRef][Medline]
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