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J Biol Chem, Vol. 273, Issue 51, 33885-33888, December 18, 1998
,From the Third Division, Department of Medicine, Kobe University School of Medicine, Kobe 650-0017, Japan
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ABSTRACT |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Recently, the growth hormone secretagogue
receptor (GHS-R) cDNA has been isolated from the pituitary and
hypothalamus. To evaluate the regulation of human (h) GHS-R gene
expression, we cloned the hGHS-R gene containing the 5'-flanking region
of 0.6-2.9 kilobase pairs. Analysis of the hGHS-R transcripts with
5'-rapid amplification of cDNA ends suggested that the putative
transcription initiation site was approximately Growth hormone
(GH)1 secretion
is regulated mainly by the hypothalamic stimulatory factor,
GH-releasing hormone, and the inhibitory factor, somatostatin. On the
other hand, GH secretagogues have been developed as a small synthetic
peptide, GH-releasing peptide (1), and non-peptides, L-692,429 (2) and
MK-0677 (3), with potent GH-secreting activity, especially in
vivo and in humans. The recent cloning of the human, porcine (4),
and rat (5) GHS receptor (GHS-R) cDNA has suggested an additional
physiological regulation for GH release. GHS-R mRNA is expressed
not only in the pituitary and hypothalamus but also in the hippocampus,
pancreas (6), and neuroendocrine tumors (7), including human
somatotropinomas and rat GH3 cells (8). There are still
only a few reports about the regulation of GHS-R expression. Bennett
et al. (9) have recently reported that GHS-R expression in
the hypothalamus was markedly increased in dw/dw dwarf rats and was
down-regulated in dw/dw rats treated with GH. They have also reported
that GHS-R mRNA expression in the ventromedial nucleus of the
hypothalamus was lower in male than in female rats.
To understand the transcriptional regulation of the human GHS-R
(hGHS-R) gene expression, we have cloned and characterized the
5'-flanking region of the hGHS-R gene.
Materials and Cell Culture--
All chemicals were obtained from
Sigma. Fetal calf serum (FCS), horse serum, Ham's F-10, and
Dulbecco's modified Eagle's medium were obtained from Life
Technologies, Inc. Radionucleotides were obtained from Amersham
Pharmacia Biotech (Tokyo, Japan). The rat GH- and prolactin
(PRL)-producing pituitary tumor cell line GH3 purchased
from American Type Culture Collection (ATCC) was grown in Ham's F-10
medium with 15% horse serum and 2.5% FCS at 37 °C in a humidified
atmosphere of 5% CO2 and 95% air. HeLa and EP-1 cells
obtained from ATCC were grown in Dulbecco's modified Eagle's medium
with 10% FCS in the same atmospheric conditions.
Cloning of the 5'-Flanking Region of the hGHS-R Gene--
We
have cloned the 5'-flanking region of the hGHS-R gene with the
PCR-based gene walking method (Human Genome Walker kit; CLONTECH, Palo Alto, CA). The 5'-flanking region of
the hGHS-R gene was amplified from the five Human Genome Walker genomic
libraries with two consecutive rounds of PCR using the adaptor
primers AP1 and AP2 and the gene-specific reverse primers R1
(5'-GGCACTCGTTGGTGTCCCAAGGGTC-3', nucleotides at 571-595) and R2
(5'-AAGCATCCCAGTCCAGGTCGGCCAG-3', at 46-70) (the translation
start site was set at +1) (see Fig. 1A). The gene-specific
oligonucleotide primers were synthesized based on the sequences of the
hGHS-R cDNA (GenBank accession no. U60179). The first amplification
was performed using AP1 and R1. The reaction involved 7 cycles
consisting of 25 s of denaturation at 94 °C and 4 min of
annealing and extension at 72 °C, followed by 32 cycles consisting
of 25 s of denaturation at 94 °C and 4 min of annealing and
extension at 67 °C, with a final extension step at 67 °C for 4 min. The PCR products were diluted to one-fiftieth and then subjected
to the secondary PCR with the nested primers AP2 and R2 using the same
protocol except that the first and second steps consisted of 5 and 22 cycles, respectively. The PCR products were subcloned into the pT7 blue
vector (Novagen, Madison, WI) and were sequenced with a DNA
autosequencer (ABI Prism377A; Perkin-Elmer).
Rapid Amplification of cDNA Ends--
The 5'-end of the
hGHS-R cDNA was determined with the rapid amplification of cDNA
ends (5'-RACE) method (see Fig. 3A). Human pituitary gland
Marathon-Ready cDNA (CLONTECH) was amplified
with PCR using the adaptor primer AP1' and cDNA-specific primer R1. The reaction involved 1 min of denaturation at 94 °C, followed by 5 cycles consisting of 30 s of denaturation at 94 °C and 4 min of
annealing and extension at 72 °C, 5 cycles consisting of 30 s
of denaturation at 94 °C and 4 min of annealing and extension at
70 °C, and 25 cycles consisting of 20 s of denaturation at 94 °C and 4 min of annealing and extension at 68 °C. The PCR
products were diluted to one-fiftieth and then subjected to the
secondary PCR with the nested primers AP2' and R2 using the same
protocol. The PCR products were sequenced directly with a DNA
autosequencer or after being subcloned into the pT7 blue vector. The
secondary PCR was also performed using AP2' and a 30-bp
oligonucleotide primer R3 complementary to Primer Extension Analysis--
We attempted to determine the
transcription initiation site of the hGHS-R gene using a primer R3
end-labeled with [ Transient Transfection and Luciferase Assay--
A fragment of
the 5'-flanking region of the hGHS-R gene ( Cloning of the 5'-Flanking Region of the hGHS-R Gene--
We
obtained four different sizes of PCR products extending to the first
PvuII, EcoRV, DraI, and
SspI sites (approximately 0.6, 1.2, 2.7, and 2.9 kb in each
size) upstream of the translation initiation site of the hGHS-R
cDNA (Fig. 1B). The
sequence of the 1.2-kb fragment (Fig. 2)
was the same as the downstream sequence of the 2.7- or 2.9-kb fragments
and included in its downstream region the same sequence as the known +1
to +46 sequence of hGHS-R cDNA (the translation start site was set
at +1). When this sequence from
The 5'-end of cDNA of the hGHS-R gene was determined by the 5'-RACE
as well as the primer extension method. The secondary PCR product
obtained with 5'-RACE using primers AP2' and R2 appeared as a broad
band on agarose gel electrophoresis (Fig.
3B). The direct sequencing of
this band has shown the major 5'-end as
Extended products could not be seen in primer extension analysis when
we used up to 1 µg of poly(A)+ RNA from the human
pituitary gland and primer R3, although 87 bases of extended bands
could be detected in parallel lanes when we used 10 ng of kanamycin RNA
and its primer as a positive control (data not shown).
The proximal sequence of the 5'-flanking region did not contain
potential elements that are usually required for transcription initiation, including the TATA, CAAT, or GC box, but there was a 7/8
match TdT-initiator (Inr) sequence (10)(at
The 5'-flanking region contains a number of putative response elements
(Fig. 2), including complete sequence matches for activator protein-2
(AP2) (at Functional Analysis of the hGHS-R 5'-Flanking Region--
To
identify the important regulatory regions for the expression of the
hGHS-R gene, the region between The present study is the first report of a cloning and
characterization of a human genomic DNA fragment containing the
5'-flanking region of the hGHS-R gene. When the sequence of the
5'-flanking region of the hGHS-R gene was compared with the upstream
sequence of the hGHS-R cDNA, a single exon upstream of the coding
exon was identified as shown for other human G protein-coupled receptor genes such as those for the M2 muscarinic acetylcholine,
adrenocorticotropin, endothelin-A, and thyrotropin-releasing hormone
(11-14).
Because primer extension analysis could not detect the expression of
the hGHS-R gene in the pituitary as expected by its extremely low
expression (4, 6, 9), 5'-RACE analysis was conducted to determine the
transcription initiation site. The direct sequencing of the 5'-RACE
products suggests that the major 5'-end of the hGHS-R cDNA is In addition to the Inr-like sequence, there are several putative
sites for binding transcription factors as shown in Fig. 2. Of
particular interest is the presence of consensus sequences for the
POU-domain transcription factors, Pit-1, Oct-1, and Ptx1, which have
been shown to be involved in pituitary-specific expression (25-27).
The octamer binding protein Oct-1 is expressed ubiquitously and
activates certain eukaryotic TATA-less promoters (26). Ptx1 is
expressed in all pituitary cell types and is essential for transcription of the glycoprotein hormone The promoter activity of the hGHS-R 5'-flanking region was assessed
after insertion upstream of the luciferase coding sequence in the
pGL3-Basic vector. The activity of the GHS-R promoter was detected in
GH3 cells but not in HeLa human epithelioid carcinoma of
cervix cells and EP1 human neuroblastoma cells, suggesting that this
DNA fragment is necessary and sufficient to drive expression of a
heterologous gene in GH3. This finding is consistent with the recent report demonstrating the expression of GHS-R mRNA in GH3 cells (8). Deletion studies allowed us to define
positive regulatory elements for the basal promoter activity located
between In summary, the 5'-flanking region of the hGHS-R gene contains a
TATA-less promoter with cell-specific activity and putative binding
sites for several transcription factors in the regions required for the
basal activity. These initial characterizations should facilitate
further study of the mechanisms involved in the transcriptional
regulation of the hGHS-R gene expression in human health and disease.
453 base pairs
upstream of the translation initiation site (+1). There is no typical
TATA, CAAT, or GC box but an initiator-like sequence and putative
binding sites for several transcription factors around the putative
transcription start site. The 5'-flanking region inserted into a
luciferase reporter vector had promoter activity in
GH3 cells but had activity indistinguishable from
background in HeLa or EP1 cells. The hGHS-R promoter activity in
GH3 cells increased by deletion of nucleotides from 1224
to 734, whereas it was decreased by further deletion from 734 to
608. Knowledge of the promoter region of the hGHS-R gene will
facilitate elucidation of its transcriptional control.
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
358 to 329 of the
hGHS-R gene (5'-CTGTCACCAGCCCTGCCTCGCATTTGCGTT-3').
-32P]ATP and T4 nucleotide kinase
(Promega, Madison, WI). The primer was hybridized with
poly(A)+ RNA from the human pituitary gland
(CLONTECH) at 58 °C for 1 h or longer and
extended with avian myeloblastosis virus reverse transcriptase for 30 min at 42 °C. The resulting products were analyzed on an 8%
polyacrylamide, 7 M urea gel in parallel with 32P-labeled markers and kanamycin RNA primer extension as a
positive control.
1224 to 121) or PRL gene
(1500 bp) as a control was subcloned into a reporter plasmid,
pGL3-Basic vector (Promega), to be fused to the luciferase gene
(1224/121 GHSR/Luc). Deletion mutant plasmids were generated by PCR
(961, 734, 669, 608, 531, and 475/121 GHSR/Luc). The
correct sequence of these deletion mutant plasmids was confirmed by DNA
sequencing. After transfection with LipofectoACE (Life Technologies,
Inc.), the cells were grown in a normal growth medium for 24 h.
Luciferase activity was determined in a Turner design luminometer
TD-20/20 (Promega, Tokyo, Japan) using the dual luciferase assay system
(Promega) and was normalized with luciferase activity of co-transfected
pRL-CMV containing the cDNA encoding Renilla luciferase (Promega).
Values are expressed as -fold induction relative to the activity of the
promoterless construct PGL3-Basic and represent the mean ± S.E.
of at least three determinations.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1234 to +3 was scanned against the
data base, it had no significant relatedness to the already identified
genes.
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Fig. 1.
Cloning of the 5'-flanking region of the
hGHS-R gene. A, schematic diagram representing the
positions of primers used for the PCR-based gene walking method. The
5'-flanking region of the hGHS-R gene was amplified from the five Human
Genome Walker genomic libraries with two consecutive rounds of PCR
using the adaptor primers and the gene-specific reverse primers.
B, results of agarose gel (1%) electrophoresis and ethidium
bromide staining of amplification products are shown. We obtained four
different sizes of PCR products extending to the first PvuII
(0.6 kb) (lane 5), EcoRV (1.2 kb) (lane
2), DraI (2.7 kb) (lane 4), and
SspI sites (2.9 kb) (lane 6) upstream of the
translation initiation site of the hGHS-R cDNA. Molecular size
marker (DNA molecular marker III) was run in lane 1.
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Fig. 2.
The nucleotide sequence of the 5'-flanking
region of the human GHS-R gene. The translation start site was set
at +1. The major 5'-ends of the 5'-RACE products are denoted by an
asterisk. The sequences shown as bold
italic letters indicate exons.
Underlined sequences indicate the putative
binding sites for transcription factors or the initiator-like
sequences. Closed triangles ( 
) indicate 5'-
(1224, 961, 734, 669, 608, 531, and 475) and 3'- (121)
ends of deletion constructs.
453 and did not include the
sequence between 328 and 133, suggesting that this sequence
corresponds to the first intron (Fig. 2). When the human pituitary
gland cDNA was amplified by the second PCR using AP2' and primer R3
(Fig. 3A), the sequences of the PCR products subcloned into
the pT7 blue vector showed multiple 5'-ends of the cDNA between
508 and 402 as one of the sequences shown in Fig. 3C.
The direct sequencing of the PCR products in the 3' to 5' direction
resulted in a dramatic decrease of sequence signals upstream from
453, again indicating that the major 5'-end of the hGHS-R cDNA
was presumed to be 453.
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Fig. 3.
5'-RACE of the human GHS receptor.
A, schematic diagram representing the positions of primers
used for 5'-RACE. B, human pituitary gland Marathon cDNA
was subjected to two sets of PCR. Results of agarose gel (1%)
electrophoresis and ethidium bromide staining of amplification products
are shown (lane 2). Molecular size marker (DNA molecular
marker V) was run in lane 1. C, amplified
products were subcloned and sequenced, and representative data are
shown.
444 to 437, CTCACGCT; underline indicates nucleotides match
to the consensus sequence) (Fig. 2).
1125, CCCAAGGG; at 648, CCCTCCCC;
at 519, CCCCACCC; at 469, TCGCCCAGGG),
basic helix-loop-helix (bHLH) (at 1024, CACTTG; at 594
and 560, CAGCTG), PEA-3 (at 1066, TTTCCT;
at 577, AGGAAG), Myb (at 1088, CAGTTA; at
897, TAACCG), NF-IL6 (at 580, TGGAGGAAG),
and half-site for the estrogen response element (at 502,
TGACCT). Furthermore, there were several putative binding
sites for POU-homeodomain factors, Oct-1 (at 939,
GTTTGCAT), pituitary homeobox 1 (Ptx1) (at 628,
CAAGCT), and two 6/7 match Pit-1 sites (at 710,
TATTCTT; and 690,
TATGAAT).
1224 and 121 as well as a series of
5'-deletion constructs of the promoter were subcloned into the
pGL3-Basic and then cotransfected with pRL-CMV into GH3 cells. Fig. 4A summarizes the
effects of these deletions on luciferase reporter activity in
GH3 cells. The luciferase activity was increased by
deletion from 1224 to 734 and decreased by further deletion from
734 to 608. Luciferase activity of 734/121 GHS-R was extremely
low or indistinguishable from background in EP-1 or HeLa cells when
compared with those in GH3 cells (Fig. 4B).
Luciferase activity of 1500 PRL, highly expressed hormone in this
cell, was expectedly much higher (215 ± 20) than those for GHS-R
in GH3 cells (data not shown).
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Fig. 4.
The human GHS-R promoter activity.
A, deletion analysis of the hGHS-R promoter. The
schematic diagram on the left
represents each deletion construct of the hGHS-R gene (black
box represents exon 1 and part of exon 2) fused into the
upstream region of the luciferase gene (white
box), with variable 5'-ends to the same MluI
cleavage site at 121 relative to the translation start site. Each
construct was transiently cotransfected with pRL-CMV into
GH3. Promoter activity is normalized with Renilla
luciferase activity and expressed as -fold induction relative to the
activity of promoterless PGL3-Basic. The data are the mean ± S.E.
of triplicate determinations. Similar results were obtained in 2-3
independent experiments. B, cell specificity of the GHS-R
promoter activity. Plasmids PGL3-Basic containing 734/121GHS-R were
transiently transfected into GH3, EP-1, and HeLa cells.
Promoter activity is normalized with Renilla luciferase activity and
expressed as -fold induction relative to the activity of PGL3-Basic.
The data are the mean ± S.E. of triplicate determinations.
Similar results were obtained in 2-3 independent experiments.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
453
bp relative to the translation initiation site. However, we obtained at
least 13 RACE products with different 5'-termini. Thus, we cannot
exclude the possibility that the hGHS-R gene has multiple initiation
sites like other G protein-coupled receptors such as those for the
dopamine D4 (15), thyroid-stimulating hormone (16),
1B-adrenergic hormone (17), 
1-adrenergic
hormone (18), and luteinizing hormone (LH) (19). Sequence analysis of
the 5'-untranslated flanking region of the hGHS-R gene indicated that
the region upstream of the putative transcriptional start site does not
have any of the typical characteristics of promoter regions such as a
TATA box, CAAT box, or the GC-rich region. The TATA-less promoter,
found primarily in housekeeping genes, is characteristic of G
protein-coupled receptors such as those for -adrenergic hormone,
1B-adrenergic hormone, dopamine D4, LH, thyroid-stimulating hormone, gonadotropin-releasing hormone,
thyrotropin-releasing hormone, and GH-releasing hormone (13-24). As
there is a TdT-Inr family-like element that overlaps with the putative
transcription start site, initiator binding protein other than
TATA-binding protein may be a rate-limiting factor for the hGHS-R gene
transcription initiation.
-subunit gene and Lim3/hk3 and activates transcription of the GH or PRL gene in synergy with Pit-1
in somatolactotrophs, the LH gene with SF-1 in gonadotrophs (27),
and the POMC gene with NeuroD1, one of bHLH factors, in corticotrophs
(28). Putative binding sites for several transcription factors, such as
bHLH factors and AP2, were also identified on the hGHS-R gene, some of
which may be responsible for the basal activity of the hGHS-R gene
promoter. The presence of multiple putative sites for AP2 binding
suggests their involvement in mediating transcriptional activation by
phorbol esters and cAMP (29). There were no GH response elements such
as the signal transducers and activators of transcription (STAT)3 and
STAT5, cis-inducible elements, or serum-response elements. The reported
inhibition of rat GHS-R mRNA expression in the hypothalamus by GH
(9) may be caused by an indirect action of GH.
734 and 608, where there are putative binding sites for
Pit-1, PEA-3, AP2, and Ptx1. However, the small decrease of promoter activity by deleting from 734 to 669, where two Pit-1 binding like
elements located, did not support strong Pit-1 dependence. Furthermore,
deletion of the upstream fragments from 1224 to 734 led to a
significant increase in the promoter activity, suggesting that this
region works as a negative regulatory element such as a repressor in
the hGHS-R gene. Further study is required to determine which
transcription factors play a key role in the hGHS-R gene transcription.
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ACKNOWLEDGEMENT |
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We thank Chikako Ogata for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by Grants-in-aid for Scientific Research from the Japanese Ministry of Education, Science, and Culture, from Growth Science Foundation in Japan, and from Kaken Pharmaceutical Co.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF099083.
To whom correspondence should be addressed. Fax: 81-78-371-6468;
E-mail: hkaji{at}med.kobe-u.ac.jp.
The abbreviations used are: GH, growth hormone; GHS-R, GHS receptor; hGHS-R, human GHS-R; FCS, fetal calf serum; PRL, prolactin; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; bp, base pair(s); kb, kilobase pair(s); Inr, initiator; bHLH, basic helix-loop-helix; Ptx1, pituitary homeobox 1; LH, luteinizing hormone; STAT, signal transducers and activators of transcription.
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REFERENCES |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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