| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208
Address all correspondence and requests for reprints to: Dr. Kelly E. Mayo, Northwestern University, Department of Biochemistry, Hogan Science Building 3–100, 2153 North Campus Drive, Evanston, Illinois 60208. E-mail: k-mayo{at}nwu.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The interaction of GHRH with its receptor stimulates increased
intracellular levels of cAMP (11). Somatotroph hypoplasia and dwarfism
are observed when the GH promoter is used to target a dominant negative
form of the cAMP response element binding protein (CREB) (12) to the
somatotroph cells of the pituitary, whereas chronic stimulation of
adenylyl cyclase in transgenic mice overexpressing GHRH (13) or a
GH-cholera toxin transgene (14) leads to pituitary hyperplasia and
gigantism. Somatotroph proliferation in response to GHRH has been
observed in primary pituitary cultures (15) and in patients with
GHRH-secreting tumors (16, 17) or pituitary tumors expressing a
constitutively active form of the 
-subunit of the stimulatory G
protein (18), suggesting that overstimulation of the GHRH
signaling pathway may lead to inappropriate cell proliferation.
GHRH is structurally related to a family of neurosecretory peptides that includes vasoactive intestinal peptide (VIP), pituitary adenylyl cyclase activating peptide (PACAP), glucagon, glucagon-like peptide 1 (GLP-1), gastric inhibitory polypeptide (GIP) and secretin (19). The receptors for these neuropeptides are also highly related and are grouped into family B (20, 21) of the G protein-coupled receptor (GPCR) superfamily. The GHRH receptor complementary DNA (cDNA) has previously been cloned (11, 22) and the encoded protein found to share several conserved features with other GPCR family B members, such as the presence of one or more N-linked glycosylation sites, six highly conserved cysteine residues, and an absolutely conserved aspartic acid residue within the relatively large aminoterminal extracellular domain (21, 23). The integrity of the conserved aspartic acid residue has been shown to be critical for the interaction of GHRH, VIP, and glucagon with their respective receptors and is the site of receptor mutation in the little mouse (9, 24, 25, 26).
The gene structure of family B GPCRs is highly conserved and seems to be complex, relative to all other GPCRs (27, 28, 29, 30). Partial characterization of the rat GHRH receptor gene suggested there was alternative splicing of an exon coding for an additional 41 amino acids within the third IL of the protein (11). The IL domains of family B GPCRs are involved in G protein coupling, and disruption of these loop regions by alternative splicing alters G protein activation (31, 32). For example, various isoforms of the PACAP receptor are generated by alternative splicing of two exons within the third IL domain, leading to differential coupling to the adenylyl cyclase and phospholipase C signaling pathways (31). This suggests the possibility of alternative effector coupling between the two isoforms of the related GHRH receptor. To understand more fully the regulation of the GHRH receptor gene and the functional differences between the two GHRH receptor isoforms, we isolated and characterized the rat GHRH receptor gene, analyzed the promoter region, and examined the biological activities of the two receptor isoforms. We demonstrate that the coding region of the GHRH receptor gene contains 14 exons, spanning 15 kb of DNA, and that the transcription of the gene is regulated by the POU-homeodomain protein, Pit-1. We also show that the two receptor isoforms are differentially expressed in the pituitary and in a pituitary tumor cell line, and they differ in their ability to stimulate cAMP production in response to GHRH.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
CRG) was provided by Dr. Carolyn Bruzdzinski, University of
Illinois at Chicago (34). The Pit-1 expression constructs were provided
by Dr. Holly Ingraham, University of California at San Francisco (35).
The monoclonal antibody 12CA5 directed against the hemagglutinin (HA)
epitope was provided by Dr. Robert Lamb, Northwestern University; and
the polyclonal antibody against the human GHRH receptor was provided by
Dr. Bruce Gaylinn, University of Virginia, Charlottesville, VA. The
muscarinic M3 receptor clone was provided by Dr. Marlene Hosey,
Northwestern University Medical School.
Animals and cell lines
Tissues were isolated from adult male Sprague Dawley rats
(Harlan Bioproducts for Science, Indianapolis, IN),
housed in facilities approved by the American Association for
Accreditation of Laboratory Animal Care, under a 14-h light, 10-h dark
cycle, with water and food provided ad libitum. The
experimental protocols used in these studies were approved by the
Northwestern University Institutional Animal Care and Use Committee.
Primary rat pituitary cell cultures were isolated and maintained as
previously described (36). COS7 cells were obtained from Dr. Daniel
Linzer, Northwestern University; GH3 cells were obtained from Dr.
Ursala Kaiser, Brigham Women’s Hospital at Harvard Medical School;
GHFT1 cells were obtained from Dr. Pamela Mellon, University of
California at San Diego; and P0 cells were a gift from Dr. Piotr
Chomczynski, Molecular Research Center, Inc. (Cincinnati,
OH). The TG-Pit-5–7 cell line was generated in our laboratory from
dispersed MT-GRF transgenic mouse pituitary tumor cells. MtT/S cells
were purchased from the Riken Cell Bank (Tsukuba, Japan) and maintained
as previously described (37). All other cell lines were maintained in
DMEM (Sigma Chemical Co.) containing 5% (Hela T4 cells)
or 10% (COS7, GH3, P0, GHFT1, and TG-Pit-5–7 cells) FCS (Life Technologies/BRL) plus 100 U/ml penicillin and 100 µg/ml
streptomycin (Life Technologies, Inc.).
Isolation and mapping of GHRH receptor genomic clones
Rat GHRH receptor genomic clones were isolated using a genomic
library derived from rat hepatoma cells (CRG) screened with
radiolabeled probes derived from GHRH receptor cDNA clones RPR64,
RPR11, and RPR20 (11), using standard hybridization methodology (38).
DNA was prepared from isolated plaques and mapped using a variety of
restriction endonucleases. Contiguous overlapping clones of a 25-kb
region, contained within the genomic bacteriophage clones G1, G2 and
G7, were subcloned into the vectors pGEM3Z, pGEM5Z, pGEM7Z; and
selected clones were sequenced using the dideoxy nucleotide
chain-termination method (Amersham Pharmacia Biotech). The
sequences were analyzed with the Geneworks sequence analysis software
package (Intelligenetics, Inc., Mountain View, CA) and
compared with the cDNA to determine the location of the exon/intron
boundaries and the sizes of the introns.
RNA analysis
Total RNA was isolated from tissue or cells using acidic phenol
extraction of guanidine isothiocyanate lysates, as described (39). For
RT-PCR assays, approximately 2.5 µg total RNA from primary pituitary
cells or pituitary cell lines was reverse transcribed using AMV reverse
transcriptase and amplified using PCR, for 26 cycles at an annealing
temperature of 65 C, with AmpliTaq and primers for the GHRH
receptor (RPRJA; 5'-CATCTCCTAGGTCCAAACCAGC-3' and RPRJC;
5'-GAAGTTCAGGGTCATGGCCATA-3'), ribosomal protein L19 (RPL19A;
5'-CTGAAGGTCAAA GGGAATGTG-3' and RPL19B; 5'-GGACAGAGTCTTGATGATCTC-3')
or GH (RMGHA; 5'-CCAT GCCCTTGTCCAGTCTG-3' and RMGHB;
5'-TGCCCTCTTCCAGGTCCTTC-3'), as previously described (36). For Northern
RNA analysis, approximately 20 µg total RNA from rat pituitary or
liver tissue was separated by electrophoresis on denaturing 1.5%
agarose/formaldehyde gels (38). RNAs were transferred to nylon
membranes, covalently cross-linked by UV irradiation, and detected by
hybridization to probes HincII/SacI,
SacI/NcoI, and RPR20 derived from the 5' regions
immediately upstream of the initiation codon of the GHRH receptor gene.
Hybridization to a cDNA probe for ribosomal protein S2 (40) was used to
normalize RNA loading. Autoradiographic exposures were for 24 h
(GHRH receptor) or 2 h (RPS2). For primer extension studies,
oligonucleotide primer 5'-CACAGGTTCAGCAAGCAGAGGACCC-3' (KM2), which is
located 50 bp downstream of the initiation codon in the first coding
exon of the GHRH receptor gene and 143 bp downstream of the longest
known cDNA (22), was synthesized and end-labeled with T4 polynucleotide
kinase and (
-32P)deoxy-ATP, as described (38).
Approximately 20 µg total RNA from rat pituitary or liver tissue were
hybridized for 20 h with 50,000 cpm/ml of the appropriate
end-labeled oligonucleotide in 150 mM KCl, 10
mM Tris-HCl, 1 mM EDTA. Hybridization reactions
were precipitated, and extension products were generated using 5 U of
AMV reverse-transcriptase in 330 mM Tris-HCl (pH 8.3), 150
mM MgCl2, 8 mM dithiothreitol
(DTT), 20 µg/µl actinomycin D, 20 µM nucleotide
triphosphate mix (38). Samples were size separated on 5%
polyacrylamide/8 M urea denaturing gels (38), dried and
exposed to Kodak X-OMAT AR film for 18 h. To confirm
the colinearity of the primer extension products with genomic DNA, 1
µg total RNA was analyzed by RT-PCR for 20 cycles with AmpliTaq, an
annealing temperature of 65 C, and the KM2 primer in combination with
an oligonucleotide corresponding to the 5' end of the longest primer
extension product, 5'-TTCTTGCTGTCAGG TTAGGC-3' (PG2), using conditions
previously described (36). The RT-PCR products were separated on 4%
(3% NuSieve/1% SeaKemGTG) (FMC Bioproducts, Rockland, ME) agarose
gels, transferred to nylon membranes, and hybridized to a radiolabeled
SacI/NcoI fragment located immediately upstream
of the initiation codon. RT-PCR products were cloned into pGEM7Z and
sequenced to verify their identity.
GHRH receptor promoter constructs and cell transfections
An NcoI fragment of the GHRH receptor promoter
(-1672 to +286, relative to the farthest upstream transcriptional
start site) was cloned into the SmaI site of pGEM7Z after
cutting with BstXI and filling in the ends with T4 DNA
polymerase to remove the ATG initiation codon. NcoI,
EcoRI, HindIII, BglII, and
SacI deletion fragments of the GHRH receptor promoter having
identical 3' ends were inserted into the KpnI site of
pA3-Luc (33). The restriction enzyme sites used to generate the
deletion constructs Nco -1672, Eco -1187, Hind -885, Bgl -714, and
Sac +62 are numbered relative to the most 5' transcriptional start site
identified by primer extension. Transfections were performed in GH3 and
COS7 cells using liposomes (41). Briefly, GH3 cells (2 x
105 cells/well) or COS7 cells (1 x 105
cells/well) were grown overnight in 12-well plates, then transfected
for 4–6 h with 2.5 µg of each plasmid DNA in serum-free Opti-MEM
containing lipid reagent, and allowed to recover for 16 h in
complete media. The transfected cells were washed with 1x PBS, and
proteins were extracted in cold lysis buffer [25 mM HEPES
(pH 7.8), 15 mM MgSO4, 1 mM DTT,
0.1% Triton-X 100]. Lysate was mixed with assay buffer [25
mM HEPES (pH 7.8), 15 mM MgSO4, 1
mM DTT, 1 µg/ml BSA, 5 mM ATP, 0.2
mM luciferin] and analyzed for luciferase activity using a
luminometer (Analytical Luminescense Laboratory, San
Diego, CA).
Expression of epitope-tagged GHRH receptor clones
The partial rat GHRH receptor cDNA clones originally described
(11) were recombined into full-length cDNA clones in pGEM5Z, generating
the clones RPRC18 and RPRC2, which are identical in sequence except for
the insertion of the 123 bp derived from exon 11 in the region encoding
the third IL in clone RPRC2. Using PCR, sequences encoding an influenza
virus HA epitope were fused to the C-terminal end of the receptor (42).
A recognition site for the enzyme XbaI was engineered on the
3' side of the HA epitope and contained the stop codon. The products
were digested with EcoRI and XbaI and cloned, in
context with the receptor, into the expression vector pcDNA-3 to
generate full-length clones for the epitope-tagged receptors (RPRC18HA
and RPRC2HA).
All binding and signaling experiments were performed using Hela T4 cells transfected with the epitope-tagged GHRH receptors, RPRC18HA and RPRC2HA, using the Vaccinia Virus-T7 polymerase hybrid expression system, as described (43, 44). The cellular localization of the receptors was determined by indirect immunofluorescence using a human GHRH receptor polyclonal antibody. Hela T4 cells, grown on coverslips and transfected with the various receptors, were fixed with 2% paraformaldehyde and incubated for 4 h at 4 C with a 1:1000 dilution of the polyclonal antiserum in PBS/0.1% BSA, in the presence or absence of 0.1% saponin. After washing, the coverslips were incubated at 4 C for 60 min with 2 µg/ml fluorescein isothiocyanate-conjugated goat antirabbit secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in PBS containing 0.2% whole goat serum, in the presence or absence of saponin. The coverslips were then washed with PBS and mounted using fluorescein isothiocyanate-Guard (Testog Inc., Chicago, IL). The images of cells are optical sections obtained using a confocal laser scanning microscopy with a Carl Zeiss LSM 410 confocal microscope and a 40x objective. All samples were scanned through the central plane of the cell using the same settings of contrast and magnification, and the signal was averaged over 4 frames to reduce background. Expression levels of the two receptor isoforms were assessed by immunoprecipitation of the epitope-tagged receptor proteins from metabolically labeled cells with 12CA5 monoclonal antibody against the HA epitope, as described (44). For glycosylation analysis, immunoprecipitated proteins were digested for 4 h at 37 C with 0.2 U peptide-N-glycosidase F (Roche Molecular Biochemicals, Indianapolis, IN) in a buffer containing 20 mM Na2HPO4 (pH 8.0), 20 mM EDTA, 1% NP40, 1 µg/ml leupeptin, 0.1 µg/ml pepstatin A, and 1 µg/ml aprotinin.
Functional analysis of the rat GHRH receptor proteins
Binding-competition assays were performed using approximately 50
µg of membrane protein per reaction, prepared from transfected cells,
as described (44). The membrane proteins were incubated with
(3-[I125]iodotyrosyl10)hGHRH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44)-amide
present at a concentration of 70 pM, in the absence or
presence of increasing concentrations of the unlabeled hormone. The
nonspecific binding, determined as the percent of input counts bound in
the presence of 1 µM unlabeled hormone, was subtracted
from all raw data to give the specific bound counts. The
binding-competition data were fit to a one-site competition equation,
and ED50 values were determined using the program Prism
(GraphPad Software, Inc., San Diego, CA). For the
measurement of cAMP responses, cells were transfected and treated with
hormones, and the deproteinized lysates were used to assay cAMP as
described (44). The assays were performed with triplicate samples, and
the mean ± SEM of the intracellular cAMP was plotted
as a function of the concentration of the ligand. The dose-response
curves were fit to a sigmoidal dose-response equation and
ED50 values determined using the program Prism
(GraphPad Software, Inc.).
Intracellular calcium mobilization in response to GHRH treatment was measured using fluorimetry. Transfected cells were lightly trypsinized and washed with DMEM containing 5% serum. They were then washed with loading buffer (145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, 1 mM CaCl2, 1% BSA containing 147 µg/ml probenecid) and resuspended in 5 ml loading buffer containing 11.5 µg of the calcium indicator Fluo-3AM (Molecular Probes, Inc., Eugene, OR). After incubation for 30 min, with gentle shaking at room temperature, to load the cells with the dye, the cells were washed twice with loading buffer to remove excess dye; and the concentration of cells was adjusted to 0.5–1 x 106 cells/ml. Fluorometric emission, at 530 nM, was monitored in response to 0.25 µM GHRH and 40 µM digitonin, using an excitation wavelength of 505 nM. Before the addition of GHRH, the basal emission was measured and subtracted from all emission values before plotting.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
.
|
. The length of
the exons and introns of the rat GHRH receptor gene, along with the
splice donor and splice acceptor junction sequences, are summarized in
Table 1. The coding sequence for the
aminoterminal extracellular domain of the rat GHRH receptor is
contained within exons 1–5, whereas exons 5–14 encode the
transmembrane (TM), IL, extracellular loop (EL), and carboxylterminal
domains (Fig. 1B). There are two consensus polyadenylation signals
located 362 bp and 490 bp 3' of the termination codon, both of
which are used based on the analysis of cDNA clones (11, 22). Introns
interrupt the coding sequences of EL1, IL1, EL2, IL3, TM4, TM5, and
TM7. Exon 11, previously identified during the characterization of rat
GHRH receptor cDNA clones (11), codes for amino acids that are
alternatively spliced into IL3. This exon is followed by a nonconsensus
splice donor sequence (Table 1). All of the exons, most of the introns,
and 2 kb of 5' flanking DNA were sequenced on both strands.
|
). Both the RPR20 cDNA clone (III) and
the NcoI/SacI genomic clone (II), which extends
207 bp 5' of RPR20, detected GHRH receptor transcripts in pituitary
RNA; but the HincII/SacI genomic clone (I), which
extends 425 bp upstream of the NcoI/SacI region,
did not detect GHRH receptor transcripts (Fig. 2). Primer extension
analysis was used to define more precisely the sites of GHRH receptor
transcript initiation. Four primer-extended products of 336, 253, 143,
and 134 nucleotides were generated using the primer KM2 (Fig. 3A). To test whether these primer
extension products were colinear with the GHRH receptor genomic DNA,
RT-PCR analysis with the primer KM2 in combination with PG2, an
upstream primer corresponding to the 5' end of the longest primer
extension product (see Fig. 4), was
performed. The expected 336-bp RT-PCR product was detected using the
KM2 and PG2 primers when pituitary RNA, but not liver RNA, was used as
the substrate (Fig. 3B). Sequencing of this product showed it to be
identical to and colinear with the genomic DNA sequence, containing 286
bp upstream of the initiation codon plus the sequence encoding the
first 17 amino acids. The primer extension analysis, therefore,
identified four transcriptional start sites located 286, 203, 93, and
84 bp upstream of the translation initiation codon.
|
|
|
), including potential binding sites for GATA factors, CREB,
C/EBP, Ets-1, Pit-1, and the nuclear receptors for estrogens and
glucocorticoids (ER, GR) (45). The 5' flanking region of the GHRH
receptor gene was analyzed for basal promoter activity by fusing
varying amounts of 5' flanking DNA to the luciferase reporter gene
(Fig. 5A). DNA constructs were
transfected into rat pituitary GH3 cells or monkey kidney COS7 cells,
and cellular proteins were isolated for luciferase assays. Transfection
of 1.7 kb of GHRH receptor promoter DNA (Nco -1672) resulted in low,
but detectable, luciferase activity in GH3 cells, which was 4.5-fold
greater than the activity observed after transfection of the parent
vector pA3-Luc (Fig. 5B). Transfection of Nco -1672 into the
nonpituitary cell line COS7 resulted in equivalent activity to the
parent vector pA3-Luc (Fig. 5C), suggesting no basal promoter activity
in this nonpituitary cell line.
|
HD)
was cotransfected with the Nco -1672 construct.
Cotransfection of the Pit-1+ and Pit-1HD expression constructs with
a series of GHRH receptor promoter deletion constructs (Eco -1187,
Hind -885, Bgl -714, and Sac +62) into GH3 and COS7 cells was used to
more precisely define the regions of the GHRH receptor gene involved in
mediating basal and Pit-1-inducible promoter activity. The GHRH
receptor promoter region deleted to 1.2 kb (Eco -1187) retained
substantial basal activity in GH3 cells (70% of Nco -1672) and gave a
similar 1.6-fold induction upon cotransfection with the Pit-1+
expression construct (Fig. 5B). The GHRH receptor promoter region
deleted to -885 bp (Hind -885) gave reduced basal activity in GH3
cells (50% of Nco -1672) but maintained induction in the presence of
the Pit-1+ expression construct (2.0-fold). Substantially reduced basal
promoter activity (35% of Nco -1672) was detected with the GHRH
receptor promoter deleted to -714 bp (Bgl -714), however Pit-1
induction of promoter activity (1.4-fold) was still maintained.
Significantly, both basal activity and Pit-1 modulation of the promoter
were abolished with the GHRH receptor promoter deleted to a region 62
bp downstream of the most 5' transcriptional start site (Sac +62) (Fig. 5B). No basal activity of any GHRH receptor promoter deletion construct
over that of the pA3-Luc vector was observed in COS7 cells; however,
cotransfection of the Pit-1+ expression construct induced activity of
the Eco -1187 (5.1-fold), Hind -885 (5.2-fold) and Bgl -714
(5.0-fold) GHRH receptor promoter constructs, whereas the Sac +62
promoter construct was not Pit-1 responsive (Fig. 5C).
Functional characterization of GHRH receptor isoforms
The short GHRH receptor isoform is the predominant messenger RNA
(mRNA) detected in primary pituitary cells by RT-PCR with primers that
span exons 7–14 (Fig. 6). This is also
the predominant transcript detected in pituitary tissue (not shown). In
screening pituitary-derived cell lines for expression of the GHRH
receptor, we identified a cell line, MtT/S (37), that expresses both GH
and GHRH receptor mRNA (Fig. 6). These cells express the receptor mRNA
at levels equivalent to (Fig. 6A) or greater than (Fig. 6B) primary
pituitary cells, which are a mixture of somatotroph and other cell
types. The MtT/S cell line is a mixed population of attached cells and
floating cell clusters. Populations enriched for attached cells
(MtT/S-M) express relatively more of the long mRNA isoform, whereas
populations enriched for the floating cells (MtT/S-F) express
relatively more of the short mRNA isoform (Fig. 6B). Cloning and
sequencing of the MtT/S long-isoform PCR product verified that it was
identical to the alternatively spliced long isoform of the receptor
previously identified in pituitary by cDNA cloning (11).
|
The expression levels of both rat GHRH receptor isoforms were
determined by immunoprecipitation of metabolically labeled proteins
with antibodies to the HA-epitope tag (Fig. 7A), and both isoforms were found to be
expressed at similar levels. Treatment of the immunoprecipitated
proteins with peptide-N-glycosidase F decreased the size of both
isoforms, suggesting the presence of N-linked glycosylation.
Immunofluorescence detection of the receptor protein in intact and
permeabilized cells, using a polyclonal antibody against the N terminus
of the GHRH receptor, demonstrated that both receptor isoforms were
localized to the cell surface (Fig. 7B). The ligand-binding properties
of the rat GHRH receptor isoforms were analyzed by measuring binding of
(3-[125I]iodotyrosyl10)GHRH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44)-amide to
cellular membranes derived from cells transiently transfected with
either the long or the short GHRH receptor isoform (Fig. 7C). Both
receptor isoforms showed comparable levels of binding that could be
competed in the presence of increasing amounts of cold GHRH in a
dose-dependent manner, with ED50 values of approximately
1.05 and 1.5 nM for the short and long isoforms of the GHRH
receptor, respectively.
|
). Cells expressing the short isoform
gave a robust accumulation of cAMP, with an ED50 of 6.3
nM. This ligand-mediated cAMP accumulation was not observed
in cells expressing the long isoform of the rat GHRH receptor. Many of
the receptors in family B couple to G proteins that activate
phopholipase C, as well as adenylyl cyclase (31, 32). Studies in
primary pituitary cells have detected Ca2+ mobilization in
response to GHRH treatment (46), suggesting that the GHRH receptor may
activate phospholipase C or Ca2+ channels, in addition to
coupling to adenylyl cyclase. Because the long isoform of the GHRH
receptor did not stimulate cAMP production in response to GHRH, the
ability of the receptor to signal through the phospholipase C pathway
in response to ligand was assessed by measuring Ca2+
mobilization with the calcium dye Fluo-3AM. Neither rat GHRH receptor
isoform stimulated Ca2+ mobilization in transfected cells
in response to GHRH treatment (Fig. 8B), although a control muscarinic
M3 receptor gave a Ca2+ response upon carbachol treatment
(47). The functional integrity of the GHRH receptor in the cells used
for the Ca2+ assay was monitored by assessing cAMP
accumulation in response to GHRH. Both the human GHRH receptor and the
short isoform of the rat GHRH receptor were capable of activating
adenylyl cyclase in response to ligand.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The rat GHRH receptor gene is complex, encompassing 14 exons. Although this organization is atypical for GPCRs in general, it is not unusual for the GPCRs of family B. The genes for the calcitonin, VIP, PTH, GIP, glucagon, PACAP, and CRF receptors have all been characterized and display a similar organization (27, 28, 29, 30). Alignment of the genes for these receptors with the rat GHRH receptor gene suggests that many of the intron/exon junctions and the length of the exons have been conserved, whereas the length of the introns is highly variable. Numerous introns interrupt the coding sequence of these receptor genes, and there seems to be no organization, with regard to particular exons encoding specific functional domains of the protein. The rat GHRH receptor gene contains introns that interrupt the aminoterminal extracellular domain, IL1, IL3, EL1, EL2, TM4, TM5, and TM7. The location of introns within the coding region of the rat GHRH receptor gene is identical to the mouse GHRH receptor gene (10). Two receptor isoforms, differing by 41 amino acids, are generated from the rat GHRH receptor gene by differential splicing of exon 11 within IL3 (11). The large GHRH receptor isoform is much less abundant than the short receptor isoform in normal pituitary, suggesting that the nonconsensus splice donor sequence (gc, rather than gt) following exon 11 may result in less efficient splicing of this exon. The observation that the immortalized somatotrophic cell line MtT/S expresses predominantly the long or short GHRH receptor isoform in different cell population isolates may indicate that splicing of exon 11 is regulated in these cells. Alternatively, because the MtT/S cell line was derived from an estrogen-induced mammotrophic tumor (37), it is possible that this cell line has a mutation or allelic variation in the splicing regulatory regions. A partial structure of the human GHRH receptor gene has recently been reported (52), and it displays a similar organization to that of the rat gene. However, the analysis only extended as far downstream as the exon coding for TM5, precluding any analysis of potential alternatively spliced exons within IL3.
We sequenced 1.9 kb of 5' flanking DNA of the rat GHRH receptor gene and have found it to contain multiple potential transcription factor binding sites. Three major transcription initiation sites were identified 286, 93, and 84 bp upstream of the initiator methionine, whereas one minor start site was observed at 203 bp 5' of the ATG. Only one transcription initiation site was identified for the human GHRH receptor gene, and it is located 40 bp upstream of the translation start site (52). There are no consensus TATA or CAAT box motifs near any of the transcription initiation sites. The absence of TATA and CAAT box motifs is a feature shared with other related family B GPCR genes (27, 28, 29, 30). A 714-bp GHRH receptor gene promoter directed strong basal expression in the rat pituitary tumor cell line GH3.
The GHRH receptor gene is expressed predominately in the anterior pituitary, as assessed by RNA blot hybridization and in situ hybridization (11, 22). The mechanisms that regulate this tissue-specific expression pattern are largely unknown, although the pituitary-specific transcription factor Pit-1 has been implicated (10, 22). Pit-1 is a major modulator of GH gene expression (3, 53) and is necessary for appropriate development of the somatotroph cells of the pituitary (4). In particular, mutations in the Pit-1 gene of both the Snell and Jackson dwarf mice lead to pituitary hypoplasia, which is distinguished by the absence of somatotroph, lactotroph, and thyrotroph cells. Expression of the GH and GHRH receptor genes is not detected in the pituitaries of these dwarf mice (22), suggesting that the expression of Pit-1 is necessary for the expression of both the GH and GHRH receptor genes. The 714-bp GHRH receptor promoter contains two potential Pit-1 binding sites, and it is able to direct Pit-1-dependent expression within pituitary (GH3) and nonpituitary (COS7) cell lines. The higher inducibility, but lower basal activity, observed in COS7 cells is likely caused by the absence of any endogenous Pit-1 in these cells; whereas the higher basal activity, but lower Pit-1 induction, seen in GH3 cells is probably attributable to the presence of endogenous Pit-1 expression in this pituitary cell line. Inclusion of additional sequences upstream of this region, which contain two more potential Pit-1 binding sites, increases basal and Pit-1 inducible expression of the rat GHRH receptor promoter constructs. It was previously reported, using transient transfection assays in COS7 cells, that coexpression of Pit-1 activates a 1.4-kb promoter fragment of the mouse GHRH receptor gene (10). Studies of the human GHRH receptor promoter region (52) have identified four potential binding sites for Pit-1. These authors determined that the minimal region required for basal expression of the human GHRH receptor promoter in GH4 cells was 202 bp upstream of the transcriptional start site. This region of the human GHRH receptor promoter was also sufficient to direct Pit-1-stimulated expression in COS7 cells.
The Pit-1 transcription factor is necessary, but not sufficient, for
the tissue-specific expression of the GH, PRL, and TSH genes,
suggesting that the synergistic interaction of Pit-1 with other
transcriptional regulators may be required. A variety of
trans-acting factors synergize or interfere with Pit-1 to
appropriately regulate gene expression, including Ets-1 (54), C/EBP
(55), GATA (56), and the nuclear receptors ER and GR (57, 58). Pit-1
has been implicated in the regulation of the GH, PRL, and TSH genes in
combination with C/EBP (55), Ets-1 (54), and GATA (56) transcription
factors, respectively. Binding sites for GATA (-1538, -1145, -712,
-632, -221, -16, +126, +215), Ets-1 (-1058), and C/EBP (-1508,
-880) are located within the 5' flanking sequences of the rat GHRH
receptor gene and, therefore, may be involved in the pituitary-specific
expression of the GHRH receptor gene. Conversely, there may be unique
transcription factors interacting with Pit-1 that are required for
tissue specific expression of the GHRH receptor gene.
Expression of the rat GHRH receptor gene is dynamically regulated. Corticosteroids up-regulate GHRH receptor mRNA expression, both in vivo and in cultured anterior pituitary cells (36, 59, 60). Similarly, thyroid hormone treatment of cultured anterior pituitary cells (60) or hypothyroid rats (61) stimulates GHRH receptor mRNA levels. Conversely, estrogens inhibit GHRH receptor mRNA expression (59) and may be involved in the sexually dimorphic regulation of GHRH receptor gene expression that has been observed in the rat (62). An increase in GHRH receptor mRNA levels is observed at the onset of sexual maturation in male rats (63), suggesting a role for testosterone in the higher level of GHRH receptor expression in the male rat (62). Characterization of the human GHRH receptor gene has demonstrated that corticosteroids increase, and estrogens decrease, expression of GHRH receptor promoter constructs (52). The rat GHRH receptor gene contains potential response elements for the nuclear hormone receptors GR (multiple half-sites) and ER, which may be involved in mediating the observed regulatory effects of these hormones. Decreased expression of receptor mRNA is detected after GHRH treatment of primary pituitary cells grown in culture (64) or chronic passive immunization of neonatal rats with GHRH (65). These effects of GHRH are likely to be mediated through cAMP-dependent pathways; and, in that respect, it is interesting that elements that recognize the CREB family of transcription factors are present in the rat GHRH receptor promoter region.
The IL domains and the C-terminal tail of the GPCRs have been
implicated in coupling to the associated G protein, and alterations
within these regions are known to disrupt or augment the signaling
ability of the receptor (66). An activating mutation in IL1 or TM2 of
the PTH receptor leads to ligand-independent stimulation of cAMP but
not inositol phosphate production (67). Conversely, alternative
splicing of 16 amino acids within IL1 of the calcitonin receptor
inhibits receptor coupling to both the adenylyl cyclase and
phospholipase C pathways and inhibits receptor internalization (68, 69). Deletion of residues within IL2 of the glucagon receptor has been
shown to affect agonist activation of adenylyl cyclase (70).
Alternative RNA splicing within IL3 of the PACAP receptor leads to
differential G protein coupling to the adenylyl cyclase and
phospholipase C pathways (31, 71, 72). Significantly, alternative RNA
processing, leading to premature termination of the human GHRH receptor
at IL3, resulting in an inability to transduce any signal, has been
reported in normal pituitary (73) and in pituitary adenomas (74). The
predominant isoform of the rat GHRH receptor contains 25 amino acids in
IL3, and GHRH binding activates the adenylyl cyclase pathway. The
insertion of 41 amino acids into this region, in the long isoform of
the receptor, seems to disrupt receptor signaling through the adenylyl
cyclase pathway, although the receptor is expressed on the cell surface
and binds GHRH. Because alternative RNA processing in this region has
been reported to lead to differential coupling to the adenylyl cyclase
and phospholipase C pathways in the PACAP receptor (31), we tested the
ability of the 2 GHRH receptor isoforms to elicit calcium mobilization
upon ligand stimulation. We were unable to detect any calcium
mobilization from internal stores in response to GHRH treatment of HeLa
T4 cells transiently transfected with either isoform of the rat GHRH
receptor. The N-terminal portion of IL3 adjacent to TM5 is predicted to
form an amphipathic helix in GPCRs that interacts with the G-subunit
(75). Experimentally introduced mutations in this region of the
GLP-1 receptor have been shown to affect signaling (76).
The insertion of 41 amino acids into the C-terminal portion of IL3 in
the long isoform of the rat GHRH receptor may prevent signaling by
disrupting the protein conformation required for productive interaction
of IL3 with the stimulatory G protein.
The identification and characterization of the rat GHRH receptor gene is expected to advance our understanding of GHRH receptor gene regulation and provide insight into the mechanisms of GHRH action on the pituitary somatotroph cell. Given the emergent involvement of the GHRH receptor in diseases of GH secretion, these studies of the receptor gene are also expected to provide an important foundation for the investigation of diseases impacting the GH axis.
|
Acknowledgments |
|---|
CRG genomic library, Dr. Holly
Ingraham (University of California at San Francisco) for the Pit-1
expression vectors, Dr. Bernard Moss (National Institutes of Health,
Bethesda, MD) for the use of the Vaccinia-T7 polymerase system, Dr.
Robert Lamb (Northwestern University) for the 12CA5 monoclonal antibody
against the HA epitope, Dr. Bruce Gaylinn (University of Virginia) for
the polyclonal antibody against the GHRH receptor, Dr. Marlene Hosey
(Northwestern University Medical School) for the muscarinic M3 receptor
clone, and our colleagues for comments on the manuscript. |
Footnotes |
|---|
2 Contributed equally to this work.
3 Present address: Howard Hughes Medical Institute, Department of
Neurobiology, Harvard Medical School, Boston, Massachusetts
02115.
Received January 6, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  K. Bedard, J. Strecko, K. Theriault, J. Bedard, C. Veyrat-Durebex, and P. Gaudreau Effects of a high-glucose environment on the pituitary growth hormone-releasing hormone receptor: type 1 diabetes compared with in vitro glucotoxicity Am J Physiol Endocrinol Metab, April 1, 2008; 294(4): E740 - E751. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Y. Wang, Y. Wang, A. H. Y. Kwok, and F. C Leung Identification of two novel chicken GHRH receptor splice variants: implications for the roles of aspartate 56 in the receptor activation and direct ligand receptor interaction J. Endocrinol., December 1, 2007; 195(3): 525 - 536. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. T. McElvaine, A. I. Korytko, S. M. Kilen, L. Cuttler, and K. E. Mayo Pituitary-Specific Expression and Pit-1 Regulation of the Rat Growth Hormone-Releasing Hormone Receptor Gene Mol. Endocrinol., August 1, 2007; 21(8): 1969 - 1983. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Rovedo and R. Longnecker Epstein-Barr Virus Latent Membrane Protein 2B (LMP2B) Modulates LMP2A Activity J. Virol., January 1, 2007; 81(1): 84 - 94. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. E. Porter, L. E. Ellestad, A. Fay, J. L. Stewart, and I. Bossis Identification of the Chicken Growth Hormone-Releasing Hormone Receptor (GHRH-R) mRNA and Gene: Regulation of Anterior Pituitary GHRH-R mRNA Levels by Homologous and Heterologous Hormones Endocrinology, May 1, 2006; 147(5): 2535 - 2543. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. T. McElvaine and K. E. Mayo A Dominant-Negative Human Growth Hormone-Releasing Hormone (GHRH) Receptor Splice Variant Inhibits GHRH Binding Endocrinology, April 1, 2006; 147(4): 1884 - 1894. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Nogami, Y. Hiraoka, K. Ogasawara, S. Aiso, and S. Hisano The role of pit-1 in the regulation of the rat growth hormone-releasing hormone receptor gene transcription by glucocorticoids J. Mol. Endocrinol., December 1, 2005; 35(3): 477 - 488. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C Mogi, H Goda, K Mogi, A Takaki, K Yokoyama, M Tomida, and K Inoue Multistep differentiation of GH-producing cells from their immature cells J. Endocrinol., January 1, 2005; 184(1): 41 - 50. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Ozawa, F. Han, and M. Kawata Exocytosis sensitivity to growth hormone-releasing hormone in subsets of GH cells in rats under different corticosterone conditions. Ultrastructural study using microwave irradiation for fixation and immunocytochemistry J. Endocrinol., December 1, 2004; 183(3): 507 - 515. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. C. Y. Ip, J. S. Lau, W. L. Au, and F. C. Leung Characterization of the 5'-Flanking Transcriptional Regulatory Region of Chicken Growth Hormone Gene Experimental Biology and Medicine, July 1, 2004; 229(7): 640 - 649. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. M. Luque, R. D. Kineman, S. Park, X.-D. Peng, F. Gracia-Navarro, J. P. Castano, and M. M. Malagon Homologous and Heterologous Regulation of Pituitary Receptors for Ghrelin and Growth Hormone-Releasing Hormone Endocrinology, July 1, 2004; 145(7): 3182 - 3189. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. E. Mayo, L. J. Miller, D. Bataille, S. Dalle, B. Goke, B. Thorens, and D. J. Drucker International Union of Pharmacology. XXXV. The Glucagon Receptor Family Pharmacol. Rev., March 1, 2003; 55(1): 167 - 194. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. R. Cunha and K. E. Mayo Ghrelin and Growth Hormone (GH) Secretagogues Potentiate GH-Releasing Hormone (GHRH)-Induced Cyclic Adenosine 3',5'-Monophosphate Production in Cells Expressing Transfected GHRH and GH Secretagogue Receptors Endocrinology, December 1, 2002; 143(12): 4570 - 4582. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Nogami, Y. Hiraoka, M. Matsubara, E. Nonobe, T. Harigaya, M. Katayama, N. Hemmi, S. Kobayashi, K. Mogi, S. Aiso, et al. A Composite Hormone Response Element Regulates Transcription of the Rat GHRH Receptor Gene Endocrinology, April 1, 2002; 143(4): 1318 - 1326. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Boisvert, C. Pare, C. Veyrat-Durebex, A. Robert, S. Dubuisson, G. Morel, and P. Gaudreau Localization and Regulation of a Functional GHRH Receptor in the Rat Renal Medulla Endocrinology, April 1, 2002; 143(4): 1475 - 1484. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. C. Voss, M. P. Flynn, and D. L. Hurley IGF-I Causes an Ultrasensitive Reduction in GH mRNA Levels via an Extracellular Mechanism Involving IGF Binding Proteins Mol. Endocrinol., September 1, 2001; 15(9): 1549 - 1558. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. J. Lee, T. J. Kotlar, I. Ciric, M. K. Lee, S. K. Lim, H. C. Lee, K. B. Huh, K. E. Mayo, and J. L. Jameson Absence of Constitutively Activating Mutations in the GHRH Receptor in GH-Producing Pituitary Tumors J. Clin. Endocrinol. Metab., August 1, 2001; 86(8): 3989 - 3995. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Obal Jr, J. Fang, P. Taishi, B. Kacsoh, J. Gardi, and J. M. Krueger Deficiency of Growth Hormone-Releasing Hormone Signaling Is Associated with Sleep Alterations in the Dwarf Rat J. Neurosci., April 15, 2001; 21(8): 2912 - 2918. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Andersen and M. G. Rosenfeld POU Domain Factors in the Neuroendocrine System: Lessons from Developmental Biology Provide Insights into Human Disease Endocr. Rev., February 1, 2001; 22(1): 2 - 35. [Abstract] [Full Text]
|
|
|
|
|
|
N. M. Sherwood, S. L. Krueckl, and J. E. McRory The Origin and Function of the Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP)/Glucagon Superfamily Endocr. Rev., December 1, 2000; 21(6): 619 - 670. [Abstract] [Full Text]
|
|
|
|
|
|
H. Nogami, M. Matsubara, T. Harigaya, M. Katayama, and K. Kawamura Retinoic Acids and Thyroid Hormone Act Synergistically with Dexamethasone to Increase Growth Hormone-Releasing Hormone Receptor Messenger Ribonucleic Acid Expression Endocrinology, December 1, 2000; 141(12): 4396 - 4401. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
). The
arrow marks the approximate site of signal sequence
cleavage, and the branched structures represent two consensus sites for
N-linked glycosylation.
