Involvement of upstream open reading frames in regulation of
rat V1b vasopressin receptor expression
Atsushi
Nomura1,
Yasumasa
Iwasaki2,
Masayuki
Saito3,
Yoshiaki
Aoki1,
Etsuko
Yamamori1,
Nobuaki
Ozaki1,
Kazushige
Tachikawa1,
Noriko
Mutsuga1,
Minako
Morishita1,
Masanori
Yoshida1,
Masato
Asai1,
Yutaka
Oiso1, and
Hidehiko
Saito1
1 First Department of Internal Medicine and
2 Department of Clinical Laboratory Medicine, Nagoya University
School of Medicine and Hospital, Nagoya 466-8560; and
3 Molecular Medicine Research Laboratories, Yamanouchi Institute
for Drug Discovery Research, Tsukuba 305-0841, Japan
 |
ABSTRACT |
The
V1b vasopressin receptor, expressed mainly in the
corticotroph of the anterior pituitary, mediates the stimulatory effect of vasopressin on ACTH release. To clarify the regulation of receptor expression, we cloned, sequenced (up to ~5 kb from the translation start site), and characterized the 5'-flanking region of the rat V1b receptor gene. We identified the transcription start
site by amplification of cDNA ends and found a new intron within the 5'-untranslated region (5'-UTR) by comparing the sequence with that of
cDNA. We then confirmed that the obtained promoter indeed has
transcriptional activity by use of the luciferase reporter in AtT-20
mouse corticotroph cells. Interestingly, there were five short upstream
open reading frames (uORFs) located within the 5'-UTR that were found
to suppress V1b expression. Subsequent mutational analyses
showed that the two downstream uORFs have an inhibitory effect on
expression in both homologous and heterologous contexts. Furthermore,
the inhibition did not accompany a parallel decrease in mRNA,
suggesting that the suppressive effect occurs at a level downstream of
transcription. Taken together, our data strongly suggest that the
expression of the V1b receptor is regulated at the
posttranscriptional as well as transcriptional level through uORFs
within the 5'-UTR region of the mRNA. Whether the uORF-mediated regulation of V1b expression is functionally linked to any
intracellular and/or extracellular factor(s) awaits further research.
vasopressin; receptor; pituitary
 |
INTRODUCTION |
ADRENOCORTICOTROPIN
(ACTH), a key regulator of the hypothalamo-pituitary-adrenal axis, is
expressed in the corticotroph cells of the anterior pituitary, the
release of which is stimulated by two hypothalamic factors,
corticotropin-releasing hormone (CRH) and vasopressin (9).
Recently, various subtypes of receptors for CRH and vasopressin have
been identified, with subsequent cloning of the genes being almost
completed. Among the receptor subtypes, CRH-R1 and
V1b vasopressin receptors are known to be expressed in the
corticotroph cells of the pituitary (14, 19, 25). It has
also been shown that both hormones are involved in the synthesis and
secretion of ACTH through specific receptors (17).
The expression of ACTH, however, is not solely determined by the
extracellular levels of CRH and/or vasopressin. It is well known that
chronic stimulation of a hormone, in general, alters the expression of
its own or related receptors, and indeed CRH has been shown to decrease
the expression of CRH-R1 receptor mRNA (26).
In this sense, it is also important to clarify the regulation of the
V1b vasopressin receptor for complete understanding of the
regulation of ACTH at the corticotroph level.
For this purpose, in this study we cloned, sequenced, and characterized
the 5'-flanking region of the rat V1b vasopressin receptor
gene. The DNA we cloned showed substantial transcriptional activity in
the AtT-20 cell, indicating that the region is the 5'-promoter of the
V1b receptor gene. More interestingly, multiple upstream
open reading frames (uORFs) were found to be located in the
5'-untranslated region (5'-UTR) of the gene, and some of these were
shown to have potent inhibitory effects on V1b receptor expression.
 |
MATERIALS AND METHODS |
Cloning and sequencing.
An ~5 kb of the upstream region of the rat V1b
vasopressin receptor gene (25) was subcloned into the
pBluescript plasmid vector (Stratagene, La Jolla, CA) and sequenced by
an ABI PRISM 310 genetic analyzer (Perkin-Elmer, Foster City, CA) with
a BigDye Terminator Cycle Sequencing Reaction kit (Perkin-Elmer).
5'-Amplification of cDNA ends.
The transcription start site(s) of the rat V1b vasopressin
receptor gene was determined by 5'-amplification of cDNA ends (RACE) by
use of a Marathon cDNA amplification kit (Clontech, Palo Alto, CA).
Total RNA was extracted from the Sprague-Dawley rat pituitary gland
with TRIzol reagent (Life Technologies, Grand Island, NY), and
poly(A)+ RNA was isolated using the poly(A) quick mRNA
isolation kit (Stratagene). Then double-stranded cDNA synthesis and
adapter ligation were performed, and the product was used as a template
for the RACE analysis. PCR was performed using the AP1 primer
(provided by the supplier) and the gene-specific antisense primer
(5'-CAGTAGCTAGGATACCGATCTCC-3') complementary to the published
nucleotide sequence of the rat V1b receptor cDNA
(25). Finally, the PCR product was cloned into pCR2.1
using the TA Cloning kit (Invitrogen, Carlsbad, CA), and >10 clones
were isolated for nucleotide sequencing. If multiple clones had the
same sequence starting at the most upstream region of the 5'-UTR, the
5'-end was recognized as the transcription start site.
Plasmid construction.
Various lengths of the promoter region of the V1b
vasopressin receptor gene were obtained by digestion with appropriate
restriction enzymes and subcloned into the plasmid containing
luciferase reporter gene, pA3Luc (15). Inactivation of one
or more translation start site(s) of the uORFs was performed using
PCR-based site-directed mutagenesis (7), in which ATG was
changed to TTG, ACG, ATT, and CTG for uORF-1, -2, -3, and -4, respectively. To see the translational activity of each uORF, the
coding sequence of the luciferase protein was directly linked to the
initiation codon of each uORF. For testing the function of each uORF in
the heterologous context, the region containing an individual uORF
(nucleotide positions between
472 and
345 for uORF-1 and -2, between
364 and
213 for uORF-3, and between
230 and
46 for
uORF-4) was isolated and inserted between the Raus sarcoma virus (RSV)
long terminal repeat promoter and luciferase reporter gene in pA3Luc.
To check the sequence dependency of the function of uORFs, a construct in which the nucleotide sequences of the uORF-3 were replaced with the
partial sequence of
-lactamase was made and used as a representative
one. Finally, constructs containing 5'-UTR with uORF-1 to -4 (between
472 and
1), in which all or all but one (uORF-3) of the ATGs were
inactivated, were used in the mRNA expression experiment.
Cell culture and transfection.
The murine corticotroph tumor cell line AtT-20/D16v (AtT-20) was
maintained in a T75 culture flask with Dulbecco's modified Eagle's medium (DMEM; Life Technologies) supplemented with 10% fetal
bovine serum (FBS; Life Technologies) and antibiotics (50 µU/ml
penicillin and 50 µg/ml streptomycin; Life Technologies) under 5%
CO2-95% atmosphere at 37°C. Culture medium was changed twice a week, and the cells were subcultured once a week.
In each experiment, the cells were plated in 3.5-cm-diameter culture
dishes, and transient transfection was performed using TransIT
polyamine transfection reagent (Pan Vera, Madison, WI). The
-galactosidase (
-gal) or secreted placental alkaline phosphatase (SEAP) (pSEAP2-control, Clontech) expression plasmids were used as an
internal control. At the end of each experiment, cells were harvested
and applied for luciferase assay. The difference in transfection
efficiency among the constructs was corrected by
-gal or SEAP activity.
Northern blot analysis.
Northern blot analysis was performed essentially as previously
described (27). Briefly, total RNA was isolated from the cells transfected with V1b-luciferase (RSVORF
or RSVORF3+) and pSEAP2-control plasmids by use of the
RNeasy RNA extraction kit (Qiagen, Hilden, Germany). Fifteen micrograms
of the RNA were denatured and electrophoresed with 1% agarose gel and
then transferred to a nylon membrane (GeneScreen Plus, NEN, Boston,
MA), followed by prehybridization at 42°C for 3 h. Antisense
oligonucleotide probes complementary to the coding sequences of the
luciferase or of the rat glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) mRNA were labeled with [
-32P]dCTP by use of
the Nick Translation System (Promega, Madison, WI). Hybridization was
performed at 42°C for 20 h in a rotating bottle of the
hybridization oven. Membranes were then washed, and the signals were
densitometrically quantified using a Bioimage analyzer (BAS-2000, Fuji
Photo Film, Tokyo, Japan), with subsequent normalization against a
corresponding relative amount of GAPDH mRNA in each sample. The values
were also normalized with transfection efficiency determined by the
SEAP activity.
Reporter assays.
Luciferase assay was performed as previously described
(1). SEAP activity was determined using the SEAP reporter
assay kit (Toyobo, Osaka, Japan). The
-gal activity was determined by Galacto-Light Plus
-gal assay kit (Tropix, Bedford, MA).
Data analysis.
Samples in each group of the experiments were in triplicate or
quadruplicate. All data were expressed as means ± SE. When statistical analyses were performed, data were compared by one-way ANOVA with Fisher's protected least significant difference test, and
P values <0.05 were considered significant.
 |
RESULTS |
Determination of the nucleotide sequences and gene structure of the
5'-flanking region and 5'-UTR of the rat V1b vasopressin
receptor gene.
Approximately 5 kb of the upstream region of the rat V1b
vasopressin receptor gene were sequenced and deposited in the GenBank database (accession number AB042197). Figure
1 shows the nucleotide sequences of the
downstream region (
1000 to +3; +1 designates the translation start
site of the open reading frame encoding the V1b receptor
protein). 5'-RACE identified the major transcriptional start site 801 bp upstream from the authentic translation start site. There was no
TATA box, but stretches of CT or CA repeats were recognized near the
transcription start site. In addition, comparison of the sequences with
the sequence of cDNA revealed a new intron within the 5'-UTR (
769 to
608). Furthermore, analysis of the sequences of the 5'-UTR showed
five uORFs that precede the major ORF encoding the V1b
receptor protein.

View larger version (80K):
[in this window]
[in a new window]
|
Fig. 1.
Nucleotide sequences of the rat V1b vasopressin
receptor gene 5'-promoter and 5'-untranslated region (UTR). Italic,
bold, and lower case letters designate the 5'-promoter, 5'-UTR, and
intron, respectively. The major transcription start site is circled.
Boxes and underlines indicate the initiation and termination codon,
respectively. +1 designates the first nucleotide of the translation
start site of the major open reading frame (ORF) for V1b
protein.
|
|
Serial deletion analysis of the transcriptional activity of the rat
vasopressin V1b receptor gene 5'-promoter.
The transcriptional activities of the various lengths of the
5'-promoter region of the cloned gene were examined using the AtT-20
mouse corticotroph cell line. As shown in Fig.
2, substantial luciferase activity was
obtained in all of the constructs compared with the promoterless
vector. Serial deletion revealed that the promoter activity was highest
in the second shortest construct examined (
1641-
444) and was still
maintained in the shortest construct examined (
1173-
444). These
results indicate that the region examined is indeed a 5'-promoter of
the V1b vasopressin receptor gene and that the area between
1173 and
444 appears to be enough for the basal promoter activity.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 2.
Effects of serial deletion of the rat V1b vasopressin
receptor 5'-promoter ( 4894, 3366, 2663, 1641, 1173) on basal
transcriptional activity in corticotroph cells. AtT-20 cells were
transfected transiently with various lengths of the rat V1b
vasopressin receptor 5'-promoter luciferase (Luc) fusion gene. Values
were normalized by -galactosidase ( -gal) activity used as an
internal control.
|
|
Characterization of the function of the uORFs in the 5'-UTR of the
V1b vasopressin receptor mRNA.
As mentioned above, nucleotide sequences of the 5'-UTR showed the
presence of multiple uORFs (designated as uORF-0 to -5), and thus we
tried to see whether any of the uORFs would influence the expression of
the V1b receptor. We focused on uORFs-1 to -4, because the
first one (uORF-0) is very close to the transcription start site. When
the 5'-flanking region with or without the uORFs was subcloned into the
luciferase vector and expressed in AtT-20 cells, luciferase activity
was much lower in the construct with uORFs (WT) than in that without
them (
ORFs) (Fig. 3), indicating the
inhibitory nature of some of the uORF(s).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 3.
A: schematic representation of the upstream ORFs (uORFs)
in the 5'-UTR of the V1b vasopressin receptor gene.
Wild-type (WT) construct contains the uORFs (uORF-1 to -4), whereas the
ORF construct does not. B: luciferase expression of the
WT and ORFs in AtT-20 cells. Values were normalized by -gal
activity. *P < 0.05 vs. ORFs.
|
|
We then examined which uORF was responsible for the inhibitory effect
by using mutated constructs in which the initiation codon of each uORF
was individually inactivated by introducing a point mutation within
ATG. As shown in Fig. 4, abrogation of either of the ATGs did not restore the luciferase expression, indicating that more than one uORF is responsible for the suppression. Further investigations using different series of mutated constructs in
which all but one uORF was inactivated or deleted showed that uORF-1
had no effect and that uORF-2 had a mild inhibitory effect, although it
did not reach statistical significance (Fig.
5A). In contrast, constructs
with either uORF-3 or uORF-4 showed inhibitory effects (Fig.
5B).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 4.
Effects of inactivation of the initiation codon of each uORF on
luciferase expression. WT or mutated ( ORFs, M1, M2, M3, M4)
constructs were transfected into the AtT-20 cells, and luciferase
activity was determined. Values were normalized by -gal activity.
Circles and crosses designate the intact and inactivated initiation
codons, respectively. *P < 0.05 vs. ORFs.
|
|

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of individual uORFs on luciferase expression in AtT-20
cells. A: comparison of luciferase expression among
constructs in which none ( 1/2), only uORF-1 (ORF1), or only uORF-2
(ORF2) is intact. uORF-3 and -4 are deleted in all constructs.
B: comparison of luciferase expression among constructs in
which none ( ORFs), only uORF-3 (ORF3), or only uORF-4 (ORF4) is
intact. C: translational activity of the uORF-3 and -4. The
initiation codon of either uORF-3 or -4 was directly linked to
luciferase protein (ORF3AUG, ORF4AUG), and expression of luciferase
protein was compared with that of ORFs. *P < 0.05 vs. ORFs.
|
|
When the nucleotide sequence contexts around the initiation codons of
each uORF were compared, those of uORF-3 and -4 were in preferable
context with Kozak's consensus sequence (Table
1) (11). Therefore, we tried
to see whether the uORF-3 and -4 functioned as translation initiation
sites by directly linking the initiation codon of each uORF with
luciferase protein. The results showed that uORF-3 and -4 actually were
translationally active, although weaker than
ORFs (Fig.
5C). These results suggest that the two downstream uORFs are
mainly responsible for the inhibitory effect.
Effect of the uORFs in the context of a heterologous promoter.
To see whether the negative effect of the uORFs is exhibited only in
the V1b vasopressin receptor gene or is still functional in
a heterologous context, a series of constructs in which the DNA
fragments containing each uORF were individually located downstream of
the RSV promoter were expressed in AtT-20 cells. The results showed a
pattern virtually similar to that of the V1b vasopressin gene: mild suppressive effect with uORF-2, and potent inhibition with
both uORF-3 and -4 (Fig. 6, A
and B). Thus the suppression is valid in a heterologous
context as well, suggesting a common mechanism in the inhibitory effect
of the uORFs.

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of an individual uORF in a heterologous promoter on
luciferase expression in AtT-20 cells. A: the 5'-UTR of the
V1b vasopressin receptor gene containing uORF-1 and -2 was
isolated and inserted between the Raus sarcoma virus (RSV) promoter and
luciferase reporter gene. Because uORF-1 and -2 overlap, the insert
section contains both uORFs, but the initiation codon of either
uORF-1 (RSVORF1) or uORF-2 (RSVORF2) is made intact. The
luciferase expression of the constructs was compared with that without
uORF (RSV ORFs). B: the 5'-UTR of the V1b
vasopressin receptor containing uORF-3 or -4 was isolated and inserted
between the RSV promoter and the luciferase reporter gene. Luciferase
expression of constructs was compared with that without uORF
(RSV ORFs). C: the coding sequence of uORF-3 except
initiation and stop codons was replaced with an unrelated one (partial
coding sequence of -lactamase) (RSVORF3R), and the suppressive
effect was compared with the authentic one (RSVORF3A) or the
construct without uORFs (RSV ORFs). *P < 0.05 vs. RSV ORFs.
|
|
In addition, we also examined whether the suppression depends on the
nucleotide sequence within the ORFs. We chose uORF-3 as a
representative one and replaced the sequence with a completely different one (partial coding sequence of
-lactamase) (RSVORF3R). The results showed that the uORF-3 with an altered coding sequence still had an inhibitory effect (Fig. 6C), although it was
less than the authentic uORF-3. Thus the inhibition is caused, at least partly, by a sequence-independent mechanism (i.e., occurrence of
translation initiation itself).
To clarify the mechanism of inhibition more precisely, we tried to
determine whether the effect of the uORFs is transcriptional or
posttranscriptional. We used two new constructs in which the initiation
codons of all 4 uORFs (RSVORF
) or all but uORF3 (which
showed the most potent inhibitory effect) were inactivated
(RSVORF3+). When the two constructs were expressed in the
AtT-20 cells, the level of luciferase mRNA determined by Northern blot
analysis was comparable (Fig. 7). In
contrast, the expression of luciferase protein determined by its
activity was still lower in the construct with uORF-3. The results
strongly suggest that the inhibition occurs not at the transcriptional
but rather at the posttranscriptional, probably translational, level.

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of uORF-3 on the expression of luciferase mRNA and
protein. Constructs containing no uORF (RSVORF ) or only
uORF-3 (RSVORF3+) were transfected into AtT-20 cells, and
both mRNA and protein levels were determined by Northern blot analysis
and luciferase assay, respectively. Photograph shows representative
Northern blot of luciferase mRNA. *P < 0.05 vs.
RSVORF .
|
|
Possible regulation of V1b receptor expression by
intracellular calcium through the uORFs.
Finally, to see whether the uORF-mediated inhibition is associated with
a specific regulatory process, we examined the responsiveness of
V1b promoter-luciferase protein expression to increased
intracellular calcium by thapsigargin, which mimics the stimulation of
corticotroph with vasopressin through the V1b receptor. In
the construct with uORFs (WT), treatment of the cells with thapsigargin
significantly increased the V1b-luciferase expression (Fig.
8), whereas no response was
found in that without uORFs (
ORFs). This suggests that an intracellular calcium-mediated signaling pathway might regulate the
expression of the V1b receptor posttranscriptionally
through the uORFs.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 8.
Effect of increase in intracellular calcium on expression of
luciferase protein. Constructs with (WT) or without uORFs ( ORFs)
were treated with thapsigargin (2 µM) or vehicle for 5 h. Open
and closed bars represent cells treated with vehicle or thapsigargin,
respectively. *P < 0.05 vs. vehicle-treated group.
|
|
 |
DISCUSSION |
In this paper, we cloned the 5'-promoter and 5'-UTR of the rat
V1b vasopressin receptor gene and determined the nucleotide sequences up to 5 kb from the translation start site. Using the cloned
gene, we confirmed that the 5'-promoter region actually has
transcriptional activity in the corticotroph cell line AtT-20. Interestingly, the 5'-UTR of the gene has multiple uORFs, some of which
are found to exert inhibitory effects on protein synthesis. Our results
raise the possibility that the expression of the V1b receptor is regulated not only at the transcriptional but also at the
posttranscriptional level.
The vasopressin receptor consists of three subtypes, i.e.,
V1a, V1b, and V2 (28).
The V2 receptor plays a key role in the regulation of water
reabsorption at the collecting duct of the kidney (10),
whereas the V1a receptor is thought to be involved in
cardiovascular regulation (6). The V1b
receptor, on the other hand, is expressed mainly in the corticotroph of
the anterior pituitary, where it mediates the effect of vasopressin on
ACTH secretion (21). In this study, our data clearly
indicate by use of the luciferase reporter system that the area we
examined indeed has promoter activity in corticotroph cells. It is a
TATA-less promoter, and ~0.35 kb from the transcription start site
(between
1173 and
801) seems to be enough to maintain the basal
promoter activity. Moreover, repressor and enhancer elements were
suggested to be located in the regions
2663-
1641 and
1641-
1173,
respectively. Interestingly, Rabadan-Diehl et al. (22)
failed to find any transcriptional activity of the same promoter in
AtT-20 cells.
The most interesting feature of the 5'-UTR sequences of the
V1b vasopressin receptor gene is the location of five uORFs
preceding the initiation codon of the major ORF, which is relatively
uncommon in mRNA of vertebrate cells. Initially, we tried to
characterize the transcriptional regulation of the V1b
promoter region by using the constructs with the uORFs, and we found
very low promoter activity. Subsequently, however, when the area
containing uORFs (uORF-1 to uORF-4) was eliminated from the construct,
high basal activity was consistently obtained, suggesting that some of
the uORFs have an inhibitory effect on protein expression. Extensive examinations by mutating the translation start sites of each uORF revealed that the two downstream uORFs (uORF-3 and -4) seemed to
mediate the suppressive effect in both homologous and heterologous contexts. In fact, when the sequences around the initiation codon of
each uORF were compared with Kozak's consensus (11),
those of the two downstream uORFs were in favorable context (Table 1). Furthermore, the expression of the protein product (luciferase) in the
construct with intact uORF-3 was much decreased compared with that with
the inactivated one despite the comparable levels of mRNAs, suggesting
strongly that the suppression by the uORF is occurring at the
posttranscriptional level.
The mechanism(s) whereby these uORFs are involved in the suppressive
effect is not entirely clear. In general, one mRNA harbors a unique
ORF, and <10% of the vertebrate mRNA has uORF(s) (12). However, it is reported that uORFs are found with a relatively high
frequency in the 5'-UTR of the G protein-coupled receptor (GPCR) genes
such as CRH, angiotensin II, and
2-adrenergic receptors (3, 13, 16, 18, 29). If an mRNA has uORF(s), it is expected that the uORF(s) somehow interferes with the function of the
major ORF, because re-initiation of translation is not common in
eukaryocytes. Two major possible mechanisms regarding the function of
uORF(s) are proposed. The first possibility is that the product of the
uORF(s) (a small peptide) interferes with the initiation of the
authentic ORF (4, 24). The second one is that the
initiation at the uORF(s) by itself inhibits the re-initiation at
downstream ORF (2, 3, 8). Our data show that the
inhibition occurred mainly at the uORFs with Kozak's consensus
(11), and actually uORF-3 and -4 were functional in terms
of translation. We also found that replacement of the coding sequence
of uORF3 with a completely different one (RSVORF3R) still had a
significant suppressive effect, supporting the second possibility
(sequence-independent event). However, the sequence-dependent mechanism
cannot completely be ruled out, because the degree of inhibition was
significantly less in RSVORF3R than in RSVORF3A.
Another interesting point is whether any kind of regulatory
mechanism(s) involves the function of uORF(s). In some cases, such as
carbamoyl-phosphate synthetase or S-adenosylmethionine decarboxylase mRNAs, the inhibition through uORF(s) is shown to be
regulated by the metabolic milieu within the cell (4, 24). Because uORFs are sometimes found in the GPCR or other types of receptor genes (13), including our case, it may be
possible that the preceding uORF(s) is regulating the expression of the major ORF by switching its translation on and off. In our study, the
expression of the V1b vasopressin receptor constructs
containing the uORFs was potently suppressed at the posttranscriptional
level in AtT-20 cells. However, because the V1b receptor
mRNA containing the same uORFs is normally expressed in the
corticotroph cells in vivo, there should be some mechanism(s) for
releasing the inhibition by uORFs. In fact, a discrepancy between the
levels of mRNA and binding capacity in vivo has been suggested, further
indicating the additional posttranscriptional regulatory step(s)
(20, 23). Our data showed the increase in
V1b-luciferase expression by thapsigargin, suggesting that
vasopressin-induced rise in intracellular calcium through the
V1b receptor might regulate the expression of the receptor
itself through an uORF-mediated posttranscriptional mechanism. We could
not confirm this hypothesis in this study, because AtT20 cells did not
express a functional V1b vasopressin receptor (data not shown).
CRH and vasopressin, the two major positive regulators of ACTH
secretion, exert their effects through different intracellular signaling pathways, i.e., the cAMP/protein kinase A and the
phosphoinositide/Ca2+ pathways, respectively. Furthermore,
the cross talk between the two pathways has also been shown
(5). In this sense, it is possible that one ligand
influences the expression of the receptor of the other ligand(s), and
indeed the administration of CRH is shown to decrease the
V1b receptor mRNA as well as that of the CRH-R1
receptor (26). Interestingly, the mRNA of the
CRH-R1 receptor also has uORFs (29), and
therefore we suppose that this kind of cross talk may be occurring
through the uORFs. Investigation of the regulation of the
CRH-R1 and V1b vasopressin receptors at both
transcriptional and posttranscriptional levels will be necessary for
the complete understanding of the molecular mechanism of the
CRH-vasopressin interaction.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: Y. Iwasaki, Dept. of Clinical Laboratory Medicine, Nagoya Univ. School of
Medicine and Hospital, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8560, Japan (E-mail: iwasakiy{at}med.nagoya-u.ac.jp).
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.
Received 8 June 2000; accepted in final form 22 January 2001.
 |
REFERENCES |
1.
Aoki, Y,
Iwasaki Y,
Katahira M,
Oiso Y,
and
Saito H.
Regulation of the rat proopiomelanocortin gene expression in AtT-20 cells. I. Effects of the common secretagogues.
Endocrinology
138:
1923-1929,
1997[Abstract/Free Full Text].
2.
Child, SJ,
Miller MK,
and
Gaballe AP.
Translational control by an upstream open reading frame in the HER-2/neu transcript.
J Biol Chem
274:
24335-24341,
1999[Abstract/Free Full Text].
3.
Curnow, KM,
Pascoe L,
Davies E,
White PC,
Corvol P,
and
Clauser E.
Alternatively spliced human type 1 angiotensin II receptor mRNAs are translated at different efficiencies and encode two receptor isoforms.
Mol Endocrinol
9:
1250-1262,
1995[Abstract].
4.
Delbecq, P,
Werner M,
Feller A,
Filipkowski RK,
Messenguy F,
and
Pierard A.
A segment of mRNA encoding the leader peptide of the CPA1 gene confers repression by arginine on a heterologous yeast gene transcript.
Mol Cell Biol
14:
2378-2390,
1994[Abstract].
5.
Gillies, GE,
Linton EA,
and
Lowry PJ.
Corticotropin releasing activity of the new CRF is potentiated several times by vasopressin.
Nature
299:
355-357,
1982[ISI][Medline].
6.
Hasser, EM,
Bishop VS,
and
Hay M.
Interactions between vasopressin and baroreflex control of the sympathetic nervous system.
Clin Exp Pharmacol Physiol
24:
102-108,
1997[ISI][Medline].
7.
Higuchi, R,
Krummel B,
and
Saiki K.
A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions.
Nucleic Acids Res
16:
7351-7367,
1988[Abstract].
8.
Hinnebusch, AG.
Gene-specific translational control of the yeast GCN4 gene by phosphorylation of eukaryotic initiation factor 2.
Mol Microbiol
10:
215-223,
1993[ISI][Medline].
9.
Holm, IA,
and
Majzoub JA.
Adrenocorticotropin.
In: The Pituitary, edited by Melmed S.. Cambridge, UK: Brackwell, 1995, p. 45-97.
10.
Knepper, MA,
Kim GH,
Fernandez-Llama P,
and
Ecelbarger CA.
Regulation of thick ascending limb transport by vasopressin.
J Am Soc Nephrol
10:
628-634,
1999[Free Full Text].
11.
Kozak, M.
Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes.
Cell
44:
283-292,
1986[ISI][Medline].
12.
Kozak, M.
An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs.
Nucleic Acids Res
15:
8125-8148,
1987[Abstract].
13.
Kozak, M.
An analysis of vertebrate mRNA sequences: intimations of translational control.
J Cell Biol
115:
887-903,
1991[Abstract].
14.
Lolait, SJ,
O'Carroll AM,
Mahan LC,
Felder CC,
Button DC,
Young WS, III,
Mezey E,
and
Brownstein MJ.
Extrapituitary expression of the rat V1b vasopressin receptor gene.
Proc Natl Acad Sci USA
92:
6783-6787,
1995[Abstract].
15.
Maxwell, IH,
Harrison GS,
Wood WM,
and
Maxwell F.
A DNA cassette containing a trimerized SV40 polyadenylation signal which efficiently blocks spurious plasmid-initiated transcription.
Biotechniques
7:
276-280,
1989[ISI][Medline].
16.
McGraw, DW,
Forbes SL,
Kramer LA,
and
Liggett SB.
Polymorphisms of the 5' leader cistron of the human beta2-adrenergic receptor regulate receptor expression.
J Clin Invest
102:
1927-1932,
1998[Abstract/Free Full Text].
17.
Muglia, LJ,
Jacobson L,
Luedke C,
Vogt SK,
Schaefer ML,
Dikkes P,
Fukuda S,
Sakai Y,
Suda T,
and
Majzoub JA.
Corticotropin-releasing hormone links pituitary adrenocorticotropin gene expression and release during adrenal insufficiency.
J Clin Invest
105:
1269-1277,
2000[Abstract/Free Full Text].
18.
Parola, AL,
and
Kobilka BK.
The peptide product of a 5' leader cistron in the beta 2 adrenergic receptor mRNA inhibits receptor synthesis.
J Biol Chem
269:
4497-4505,
1994[Abstract/Free Full Text].
19.
Perrin, MH,
Donaldson CJ,
Chen R,
Lewis KA,
and
Vale WW.
Cloning and functional expression of a rat brain corticotropin releasing factor (CRF) receptor.
Endocrinology
133:
3058-3061,
1993[Abstract].
20.
Rabadan-Diehl, C,
and
Aguilera G.
Glucocorticoids increase vasopressin V1b receptor coupling to phospholipase C.
Endocrinology
139:
3220-3226,
1998[Abstract/Free Full Text].
21.
Rabadan-Diehl, C,
Lolait SJ,
and
Aguilera G.
Regulation of pituitary V1b vasopressin receptor mRNA during stress in the rat.
J Neuroendocrinol
7:
903-910,
1995[ISI][Medline].
22.
Rabadan-Diehl, C,
Lolait S,
and
Aguilera G.
Isolation and characterization of the promoter region of the rat vasopressin V1b receptor gene.
J Neuroendocrinol
12:
437-444,
2000[ISI][Medline].
23.
Rabadan-Diehl, C,
Makara G,
Kiss A,
Lolait S,
Zelena D,
Ochedalski T,
and
Aguilera G.
Regulation of pituitary V1b vasopressin receptor messenger ribonucleic acid by adrenalectomy and glucocorticoid administration.
Endocrinology
138:
5189-5194,
1997[Abstract/Free Full Text].
24.
Ruan, H,
Shantz LM,
Pegg AE,
and
Morris DR.
The upstream open reading frame of the mRNA encoding S-adenosylmethionine decarboxylase is a polyamine-responsive translational control element.
J Biol Chem
271:
29576-29582,
1996[Abstract/Free Full Text].
25.
Saito, M,
Sugimoto T,
Tahara A,
and
Kawashima H.
Molecular cloning and characterization of rat V1b vasopressin receptor: evidence for its expression in extra-pituitary tissues.
Biochem Biophys Res Commun
212:
751-757,
1995[ISI][Medline].
26.
Sakai, K,
Horiba N,
Sakai Y,
Tozawa F,
Demura H,
and
Suda T.
Regulation of corticotropin-releasing factor receptor messenger ribonucleic acid in rat anterior pituitary.
Endocrinology
137:
1758-1763,
1996[Abstract].
27.
Terashima, Y,
Kondo K,
Inagaki A,
Yokoi H,
Arima H,
Murase T,
Iwasaki Y,
and
Oiso Y.
Age-associated decrease in response of rat aquaporin-2 gene expression to dehydration.
Life Sci
62:
873-882,
1998[ISI][Medline].
28.
Thibonnier, M,
Berti-Mattera LN,
Dulin N,
Conarty DM,
and
Mattera R.
Signal transduction pathways of the human V1-vascular, V2-renal, V3-pituitary vasopressin and oxytocin receptors.
Prog Brain Res
119:
147-161,
1998[ISI][Medline].
29.
Tsai-Morris, CH,
Buczko E,
Geng Y,
Gamboa-Pinto A,
and
Dufau ML.
The genomic structure of the rat corticotropin releasing factor receptor. A member of the class II G protein-coupled receptors.
J Biol Chem
271:
14519-14525,
1996[Abstract/Free Full Text].
Am J Physiol Endocrinol Metab 280(5):E780-E787
0193-1849/01 $5.00
Copyright © 2001 the American Physiological Society