From the Institute of Pharmacology, University of Würzburg,
Versbacher Straße 9, 97078 Würzburg, Germany
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INTRODUCTION |
Chronic stimulation of the
2-adrenergic receptor
(
2AR)1 results
in a decrease of receptor responsiveness, a process called agonist-induced receptor desensitization (1, 2). Long term desensitization often involves a significant reduction of receptor numbers, which is termed receptor down-regulation. Several distinct molecular mechanisms affecting both mRNA and protein levels
contribute to receptor down-regulation (2-4), which appear to be
operative to varying extents in different cell lines. To date there is
evidence that the expression of the
2AR gene can be
regulated at the level of transcription (5, 6), posttranscriptionally
at the level of mRNA stability (7) or at the level of translation
via a short peptide encoded within the 5
-untranslated region (UTR) of
the
2AR gene (8).
Posttranscriptional mechanisms are of particular interest, since they
participate in the stability and turnover of various highly labile
mRNAs, such as granulocyte-macrophage colony-stimulating factor,
interleukin-3, and the oncogenes c-fos and c-myc
(9, 10). AU-rich elements (AREs) are often found in the 3
-UTRs of
these mRNAs and appear to be key determinants of their short half-lives, even if mRNA turnover does not strictly depend on these
motifs. The optimal destabilization motif was recently suggested to be
UUAUUUA(U/A)(U/A) (11, 12), but there is also evidence that an AUUUA
pentamer need not be an integral part of a functional ARE (13). On the
contrary, it appears that each ARE represents a combination of
structurally distinct domains, such as AUUUA motifs, AU nonamers, and
U-rich elements, and that it is the combination of these sequence
elements that determines its ultimate destabilizing function (14). AREs
appear to represent recognition sites for several cytoplasmic and
nucleus-associated RNA-binding proteins, which mediate RNA degradation
(15-19). Some of these proteins have been purified, but their precise
roles in the regulation of mRNA stability remain unclear.
For the
2AR mRNA, three binding proteins have been
described so far: (i) the
-adrenergic receptor mRNA-binding protein
(
ARB), a Mr 35,000 cytosolic protein
identified in hamster DDT1-MF2 smooth muscle cells (20);
(ii) a Mr 85,000 factor mediating
2AR transcript destabilization in adult rat hepatocytes
(21); and (iii) the Mr 37,000 AU-rich element
RNA-binding/degradation factor (AUF1), which has been shown to bind
also
1AR mRNA (22). Although AU-rich sequence motifs
within the
2AR 3
-UTR have been demonstrated to function
as recognition sequences for these proteins in vitro (21-24), the exact nature of the particular cis-acting
elements mediating
2AR mRNA destabilization in
vivo has not been established. During the preparation of this
manuscript, Tholanikunnel and Malbon (25) reported the first
characterization of such an element, a 20-nucleotide AU-rich domain
with an unusual AUUUUA hexamer core, which is obligate for the
destabilization of the hamster
2AR mRNA. However, a
sequence alignment revealed no equivalent within the human
2AR transcript (26, 27), which in turn suggests that
2AR mRNA stability is regulated via species-specific
cis-acting elements.
In this study, we report the identification of a nonconsensus AU-rich
nonamer within the
2AR 3
-UTR as a critical determinant for the agonist-induced destabilization of the human receptor transcript and provide evidence that the participation of a RNA-binding protein and of cAMP are required for
2AR mRNA
down-regulation in vivo.
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MATERIALS AND METHODS |
Plasmid Construction--
The
2AR vectors used
for transient transfections were constructed based on the plasmid
pBC12BI-
2 (26), from which a 1.95-kb fragment
corresponding to the complete human
2AR transcript was excised and inserted into the expression vector pcDNA3
(Invitrogen). Deletion constructs lacking either one or both
untranslated regions were obtained by PCR amplification of the
respective cDNA fragments from pBC12BI-
2.
2AR 3
-UTR C-terminal truncation mutants were generated
by deletion of a PvuII-SmaI (
157) and a
PpuMI-SmaI fragment (
313), respectively, from
plasmid pBC12BI-
2.
The vectors used as templates for in vitro transcription
reactions were constructed by amplification of two ~1-kilobase pair fragments, each comprising one-half of the receptor transcript. These
two fragments,
2AR5
and
2AR3
, were
cloned into pGEM9Zf(
) (Promega) under the control of the T7 promoter.
A 130-bp poly(A) stretch was inserted downstream of the
2AR cDNAs to obtain polyadenylated mRNAs.
The mutations of the ARE at positions 329-337 of the
2AR 3
-UTR were introduced by PCR. 247-bp fragments
covering the 3
-halves of the 3
-UTR were amplified using the
mutagenesis primers
2AR.seq.4,
2AR.seq.41,
2AR.seq.42, and
2AR.seq.43, respectively, and
2fus.rev (for all primers see
Table I). These fragments were
subsequently used as "reverse primers" in a second PCR together
with
2fus.seq.2. The resulting
571-bp mutated 3
-UTRs were fused to the
2AR coding sequence in the vector pcDNA3-
2
3
UTR.
Additonally, pGEM vectors bearing the mutated AREs were constructed
analogous to
2AR3
as templates for in vitro
transcription.
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Table I
List of the primers used in this study
The positions of the corresponding sequences are numbered according to
the respective human cDNAs, with the translational start site
numbered as 1. Positions underlined indicate location within the
2AR 3 -UTR, with the adenosine residue following the stop
codon numbered as 1. Nucleotides differing from the wild-type sequence
as well as the NotI and XbaI sites in
2fus.seq.2 and 2fus.rev.2,
respectively, are underlined.
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For the generation of the
-globin/
2AR 3
-UTR fusion
plasmid a 2.2-kilobase pair fragment corresponding to the
-globin
primary transcript was excised from pGEM1
-globin and inserted into
pcDNA3. After that, a 540-bp DraI-XbaI
fragment comprising the
-globin 3
-UTR was replaced by the 546-bp
2AR 3
-UTR excised from
pcDNA3-
2
5
UTR. The correctness of all constructs
was confirmed by double-stranded DNA sequencing.
Cell Culture and Transfection--
Hamster DDT1-MF2
smooth muscle cells and human embryonic kidney cells (HEK 293) were
grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
supplemented with 10% fetal calf serum, 2 mM glutamine,
100 units/ml penicillin, and 100 µg/ml streptomycin (all purchased
from Life Technologies, Inc.). Monolayer cultures were harvested at
60-70% confluence, and DDT1-MF2 suspension cultures were
maintained at a cell density of about 5 × 105
cells/ml. The calcium phosphate precipitation method (28) was used for
transfection of HEK293 cells. Transfection efficiencies were determined
by cotransfection of a
-galactosidase encoding plasmid,
pSV
gal.
48 h after transfection the cells were stimulated with 10 µM (
)-isoproterenol (Sigma) or 10 µM
forskolin (Sigma) for the times indicated. To block transcription
and/or translation, 5 µg/ml actinomycin D (Roth) and/or 0.5 µg/ml
Pseudomonas exotoxin A (Sigma), respectively, were added 30 min after the begin of agonist stimulation. At the times indicated, the
cells were harvested, washed twice with phosphate-buffered saline, and
subjected to RNA analysis.
RNA Isolation and Northern Analysis--
Total RNA was isolated
by the AGPC extraction method (29), separated on formaldehyde gels, and
subsequently transferred to nylon membranes (Qiagen) by downward
alkaline capillary transfer (30). Single-stranded DNA probes were
prepared in two steps. First, the respective region was amplified in a
"standard" PCR. The resulting double-stranded DNA fragment served
as a template in a second, asymmetric PCR that included only the
3
-primer. 20 µM DIG-11-dUTP (Boehringer Mannheim) was
added for labeling along with 30 µM dNTPs. The primers
used for the preparation of the
2AR probe were
2AR.seq.2 and
2AR.rev.4, spanning a 672-bp fragment immediately downstream of the start codon. Probes specific for
B-crystallin (468 bp, used as an internal standard) and
-globin (319 bp) were amplified using the primers
cry.seq/cry.rev and h
g.seq/h
g.rev, respectively. Hybridization
was done at 37 °C for 24-48 h in 50% formamide, 5 × SSC,
3 × Denhardt's solution, 0.5% SDS, 0.2% sodium
laurylsarcosinate, and 5% dextran sulfate. Chemiluminescent detection
was performed using the DIG Luminescent Detection kit (Boehringer
Mannheim). The signal intensity on the x-ray films was analyzed
densitometrically.
In Vitro Transcription--
Transcripts were generated from 1 µg of linearized template DNA in a total reaction volume of 20 µl
in the presence of 1 unit/µl RNase inhibitor, 1 mM
ATP/CTP/GTP, 0.65 mM UTP, 0.35 mM DIG-UTP, and
2 units/µl T7-RNA-polymerase (all reagents purchased from Boehringer
Mannheim). Cotranscriptional capping was performed by using the cap
analogue m7(5
)Gppp(5
)G (New England Biolabs) in a
concentration 10 times that of GTP. The reaction mixtures were
incubated at 37 °C for 2 h. RNase-free DNase I (Boehringer
Mannheim) was added to remove template DNA. The labeled transcripts
were extracted twice with phenol and then once with chloroform and
precipitated with ethanol.
Gel Shift Assay--
After a 12-h treatment with either
(
)-isoproterenol (10 µM) or vehicle,
DDT1-MF2 smooth muscle cells were washed twice with ice-cold phosphate-buffered saline and scraped into 20 mM
Hepes, pH 7.5, 30 mM KCl, 1 mM dithiothreitol,
2.5 mM EDTA, 2.5 mM EGTA, 20 mg/liter
benzamidine, 20 µM phenylmethylsulfonyl fluoride, 20%
glycerol, and 0.1% Nonidet P-40. Samples were sonicated for 20 s,
incubated on ice for 30 min, and centrifuged at 50,000 × g for 20 min. 20-50 µg of supernatant protein was
incubated for 30 min at room temperature with ~1 µg of DIG-labeled
RNA in a buffer containing 10 mM Hepes, pH 7.5, 20 mM KCl, 1 mM EDTA, 5% glycerol, 0.5 µg/µl
heparin, and 1 unit/µl RNase inhibitor. Samples were electrophoresed
at 20-30 mA for 3 h on nondenaturing 4% polyacrylamide gels and
blotted onto nylon membranes as described above. Signals were
visualized using the DIG Luminescent Detection kit (Boehringer Mannheim).
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RESULTS |
Mapping of the Agonist-sensitive Region within the
2AR mRNA--
Since in many cell types the
2AR mRNA down-regulation appears to be mediated by
several different processes (2-4), we decided to use a transient
transfection system to identify the agonist-sensitive region(s) within
the
2AR transcript. For this purpose, HEK293 cells were
chosen due to their very low level of endogenous
AR as well as the
very high transfection efficiency compared with other cell lines.
48 h after the transfection of vectors bearing human
2AR cDNAs corresponding to the complete receptor
transcript and to mutants lacking either one or both UTRs,
respectively, the cells were stimulated for 12 h with 10 µM isoproterenol, and the
2AR mRNA
levels were quantified in Northern analyses (Fig. 1A).
B-Crystallin, a widely expressed heat-shock protein, was used as an internal standard in all experiments. Cotransfection of a
-galactosidase encoding plasmid and subsequent staining of the cells
revealed transfection efficiencies of about 90% in all samples (not
shown), so that influences resulting from different transfection
efficiencies of various constructs should be minimal. As shown in Fig.
1B, the
2AR wild-type transcript and the
5
-UTR deletion mutant were down-regulated upon agonist stimulation by 60 ± 6% and 68 ± 8%, respectively, compared with
unstimulated controls. The predominant role of elements encoded within
the 3
-UTR for transcript destabilization was further confirmed by the
respective deletion mutant which showed only a small reduction of the
2AR mRNA level by 12 ± 8%. The mRNA
concentration of the transcript covering only the coding sequence
remained almost unchanged. These results suggested that the agonist
sensitivity of the human
2AR mRNA resides
essentially within the 3
-UTR.

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Fig. 1.
Determination of the agonist-sensitive region
within the human 2AR mRNA. Expression vectors
haboring the human 2AR cDNAs corresponding to the
complete receptor transcript and deletion mutants lacking the 5 -UTR,
the 3 -UTR, and both UTRs, respectively, were transiently transfected
into HEK293 cells. 48 h after transfection, the cells were
stimulated for 12 h with either 10 µM
( )-isoproterenol (S) or medium for controls
(C). mRNA levels for the 2AR and
B-crystallin (CRY), which was used as an
internal standard, were determined by Northern analyses as described.
A, representative Northern blot. B, quantitative
analysis. The results are expressed as percentage of control and are
mean ± S.E. of three independent experiments. CDS,
2AR coding sequence.
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Interaction of the Human
2AR mRNA with a Binding
Protein Identified in Hamster DDT1-MF2 Smooth Muscle
Cells--
In a recent study (31), we have demonstrated that the
agonist-induced
2AR mRNA down-regulation occurs in a
cell type-specific manner. In DDT1-MF2 smooth muscle cells,
it is caused predominantly at the posttranscriptional level via
decreased mRNA stability. It has been shown for several highly
labile mRNAs that destabilization motifs may function as
recognition sequences for RNA-binding proteins (15-19). Therefore, we
attempted to prove the existence of such a factor in
DDT1-MF2 cells and to analyze a possible interaction with
the human
2AR transcript. DDT1-MF2 cells
were grown in suspension cultures, and transcription was blocked by
adding actinomycin D to the medium. After various incubation periods,
the
2AR mRNA concentrations were quantified in
Northern analyses (Fig. 2). The
2AR mRNA half-life of untreated control cells was
determined to be about 120 min, whereas in agonist-stimulated cells a
~50% reduction was observed, resulting in a half-life of ~50 min.
These values are similar to those measured under similar conditions (suspension cultures) by Collins et al. (5). Translational blockade by exotoxin A increased the
2AR mRNA
half-life to about 80 min in stimulated cells, which indicates that
2AR stimulation indeed induces the synthesis of a
protein component, which accounts, at least in part, for receptor
mRNA destabilization.

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Fig. 2.
Determination of the 2AR
mRNA half-life in DDT1-MF2 smooth muscle cells.
Control and stimulated cells grown as suspension cultures were treated
with 5 µg/ml actinomycin D (t = 0) 30 min after the
addition of 10 µM ( )-isoproterenol (or medium for
controls) for the times indicated. To inhibit de novo
protein synthesis, some cells were additionally exposed to 0.5 µg/ml
exotoxin A together with actinomycin D. 2AR mRNA
levels were quantified by Northern analyses and presented as percentage
of control. The results are mean ± S.E. of three
experiments.
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To identify the region(s) of the human
2AR mRNA that
might be interacting with such a protein component, we established a gel shift assay using the human
2AR mRNA as a
template. In vitro transcribed, DIG-labeled, capped, and
polyadenylated RNAs corresponding to the 5
-half (189-nucleotide 5
-UTR
plus 806-nucleotide coding sequence) and the 3
-half (441-nucleotide
coding sequence plus 554-nucleotide 3
-UTR) of the
2AR
mRNA, respectively, were incubated with cytosolic extracts prepared
either from DDT1-MF2 control cells or cells stimulated with
isoproterenol for 12 h. The samples were separated on
nondenaturing polyacrylamide gels and transferred onto nylon membranes.
After chemiluminescent detection, a protein-mRNA complex was only
found if cytosolic fractions of stimulated cells were mixed with the
3
-half of the
2AR mRNA (Fig.
3). The addition of exotoxin A to the
cells to block de novo protein synthesis inhibited the
formation of this complex. These observations are a further indication
that the 3
-UTR contains elements critical for the stability of the
human
2AR mRNA. Furthermore, they provide evidence
that binding protein(s) induced in hamster DDT1-MF2 cells can bind to the human transcript and are apparently involved in this
regulation.

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Fig. 3.
Identification of an inducible
2AR mRNA-binding protein in DDT1-MF2
smooth muscle cells by gel shift analysis. 30 µg of cytosolic
protein prepared from unstimulated control cells (C) and
cells after 12 h stimulation with 10 µM
( )-isoproterenol (S) were incubated with about 1 µg of
in vitro transcribed, DIG-labeled mRNA covering either
the 5 - or the 3 -half of the 2AR transcript, respectively. To inhibit protein synthesis, 0.5 µg/ml exotoxin A were
added 30 min after the begin of stimulation (E). The samples were resolved on 4% nondenaturing polyacrylamide gels and blotted onto
nylon membranes by capillary transfer. The signals were visualized on
x-ray films using a chemiluminescent detection protocol.
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Identification of an AU-rich Destabilization Motif within the
2AR 3
-UTR--
For a more detailed characterization of
the human
2AR 3
-UTR, two truncation mutants lacking the
3
-terminal 157 bp and 313 bp of the receptor cDNA, respectively,
were generated, and their degree of agonist-induced
2AR
mRNA down-regulation after transient transfection into HEK293 cells
was determined (Fig. 4A). The
mRNA level changes of the mutant lacking 157 bp were comparable
with those of the wild-type receptor, whereas deletion of 313 bp
completely abolished agonist-mediated down-regulation. The respective
levels of
B-crystallin mRNA remained unchanged in
all three cases. These results provide evidence that the region between
positions 241 and 397 of the
2AR 3
-UTR is critical for
receptor mRNA destabilization (Fig. 4B).

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Fig. 4.
A, mapping of the agonist-sensitive
region within the 2AR 3 -UTR. Two 3 -terminal truncation
mutants of the 2AR cDNA were generated lacking the
last 157 bp ( 157) and 313 bp ( 313) of the 3 -UTR, respectively.
After transfection into HEK293 cells and 12 h stimulation
(S) with 10 µM ( )-isoproterenol (or medium for controls (C)) mRNA levels for the 2AR
and B-crystallin were determined by Northern analyses.
The wild-type receptor (WT) was used as a control.
B, schematic representation of the 2AR
3 -UTR. The 2AR coding sequence (CDS) is
shown in black, and the 3 -UTR is hatched. The
positions of the two truncation mutants are indicated by
triangles. The region between positions 241-397 of the
2AR 3 -UTR critical for agonist-mediated mRNA
destabilization (according to Fig. 4A) is shown as a
rectangle, and the positions of AU-rich destabilization
motifs are marked by arrows and are numbered according to
Table II.
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AU-rich elements have been shown to be key determinants for the
destabilization of several highly regulated mRNAs (9, 10). Their
consensus sequence has recently been proposed to be UUAUUUA(U/A)(U/A) (11, 12). Therfore, we looked for elements consisting of at least nine
consecutive adenosine or uridine residues within the
2AR
3
-UTR. None of the four regions identified (Table
II and Fig. 4B) exactly fits
the proposed consensus sequence. Because of its location in the region
shown to be critical for
2AR mRNA destabilization,
the motif UAAUAUAUU found at positions 329-337 was of special
interest. The sequence differed from the consensus only at positions 2 and 5. To test the importance of this element for
2AR
mRNA stability, the wild-type sequence (Fig.
5, WT) was replaced by a
stretch of nine cytosine residues (M), and the two constructs were transiently transfected into HEK293 cells as before. Upon agonist stimulation, the mutant
2AR mRNA
levels, normalized for the respective values for
B-crystallin, were reduced to only 90 ± 8% of
control levels compared with 35 ± 5% for the wild type (Fig. 5).
Therefore, this element appears to be absolutely essential for the
destabilization of the human
2AR transcript. Three
additional ARE point mutants (M1-3, Table
III) were generated to provide further insights in the minimal sequence requirements of this motif. In mutant
M1, positions 2 and 5 were changed (A
U) so that the resulting ARE
corresponded to the suggested consensus sequence (11, 12). The
respective transcript showed an almost identical degree of
down-regulation in HEK293 cells compared with the wild-type sequence,
with a reduction to 32 ± 8% of the unstimulated control (Fig.
5). The flanking adenosine residues of the consensus sequence have been
shown to be critical for the destabilizing potency of an ARE (11, 12).
In mutant M2, the adenosine residues at positions 2, 3, and 7, respectively, were exchanged for cytosines. The
2AR mRNA levels after agonist stimulation were only slightly reduced to
87 ± 8%, which confirms the importance of these residues.
Finally, in the mutant M3, the three central nucleotides were replaced by cytosines to investigate the function of the ARE core domain. A
small but significant decrease in the respective
2AR
mRNA levels to 75 ± 7% was observed (Fig. 5), demonstrating
that at least for the
2AR ARE the core is not as
essential as proposed for the consensus sequence (11, 12).
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Table II
Putative destabilization motifs within the 3 -UTR of the human
2AR mRNA
In agreement with the proposed ARE consensus sequence (11, 12), only
elements consisting of at least nine consecutive adenosine or uridine
residues are considered. The positions are numbered starting at the
adenosine residue following the stop codon.
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Fig. 5.
Functional analysis of the AU-rich element at
positions 329-337 of the 2AR 3 -UTR. The
respective mutations of the ARE (listed in Table III) were generated as
outlined under "Materials and Methods." The wild-type
(WT) and mutant (M, M1, M2, and M3) receptor cDNAs were
transiently transfected into HEK293 cells. 48 h after
transfection, the cells were stimulated with 10 µM ( )-isoproterenol or vehicle for 12 h. 2AR
mRNA and B-crystallin (CRY) mRNA
levels under control conditions (C) or after agonist stimulation (S) were determined in Northern analyses.
A, representative Northern blot. B, quantitative
analysis of six independent experiments. The results are expressed as
percentage of control and are mean ± S.E.
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Table III
Mutations of the ARE at positions 329-337 of the human
2AR 3 -UTR
The mutations were introduced in the 2AR 3 -UTR by PCR as
described under "Materials and Methods" using the primers given in
Table I. The wild-type sequence (WT) is included for comparison. The
mutant M1 corresponds to the ARE consensus sequence (11, 12).
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Additionally, we performed gel shift experiments to answer the question
whether the protein(s) identified in cytosolic extracts of
agonist-stimulated DDT1-MF2 cells reacts in a similar
manner to mutations of the ARE in the
2AR 3
-UTR. As
shown in Fig. 6, a gel shift was observed
with both the wild-type transcript and the mutant M1, indicating that
the protein does not discriminate between these two sequences. No
interaction was found with the mutants M and M2, supporting the idea
that the flanking adenosine residues are critical for ARE function. For
mutant M3, two signals were identified, one corresponding to the free
mRNA template and a second with a lower intensity comigrating with
the RNA-protein complex (Fig. 6). This agrees with the slight
isoproterenol-induced down-regulation of the M3 mutant mRNA shown
in Fig. 5. Summarizing these results, the agonist-induced
2AR mRNA destabilization appears to be mediated, at
least in part, by an RNA-binding protein recognizing a nonconsensus
AU-rich motif at positions 329-337 of the
2AR 3
-UTR,
probably via a specific regulatory mechanism.

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Fig. 6.
Gel shift analysis using the mutated
2AR mRNAs. In vitro transcribed
DIG-labeled mRNAs corresponding to the 3 -halves of the wild-type
(WT) or the respective mutated receptors (M, M1, M2, and M3)
were incubated with 30 µg of cytosolic protein from
DDT1-MF2 control cells (C) or from cells
stimulated with isoproterenol for 12 h (S),
respectively. The samples were resolved on 4% nondenaturing
polyacrylamide gels and transferred onto nylon membranes and analyzed
as in Fig. 3.
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Stability of a Chimeric
-globin/
2AR 3
-UTR
Transcript--
To test the hypothesis of a
2AR-specific regulation of mRNA stability, we asked
whether the elements within the
2AR 3
-UTR are
sufficient to destabilize a normally stable gene. To consider a
possible participation of sequence motifs beside the ARE, the complete
human
2AR 3
-UTR was fused to the coding sequence of the
human
-globin gene, and the half-life of the resulting chimeric transcript was measured in comparison with wild-type
-globin. To
analyze whether the activation of the
2AR has an
influence on the function of the cis-acting elements within
the transcript, a vector haboring the
2AR cDNA was
cotransfected together with the two
-globin constructs in HEK293
cells. 48 h after transfection, the cells were stimulated either
with 10 µM isoproterenol or with 10 µM
forskolin to directly activate the adenylyl cyclase. The stability of
the
-globin wild-type transcript remained unaffected by agonist
stimulation. The
-globin mRNA half-lives were about 13 h
under all conditions, i.e. in control cells as well as in cells stimulated with either isoproterenol of forskolin (Fig. 7 and Table
IV). The exchange of the endogenous
-globin 3
-UTR against the respective region from the
2AR dramatically reduced the stability of the chimeric
mRNA. In addition, its stability became
AR
agonist-dependent. Upon stimulation of the
2AR with isoproterenol, the half-life of the chimeric
mRNA decreased from about 4 to 2.5 h. The same regulatory
pattern was observed with forskolin, which reduced the half-life of the
chimeric transcript from 4.8 to 3.3 h. Therefore, the elements
encoded within the 3
-UTR of the human
2AR mRNA are
sufficient to regulate mRNA stability in an
agonist-dependent manner in a heterologous system. The
finding that the degree of transcript destabilization is almost the
same using either isoproterenol or forskolin further suggests that
2AR mRNA stability is essentially regulated by
cAMP.

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Fig. 7.
Fusion of the -globin coding sequence with
the 2AR 3 -UTR. The endogenous 3 -UTR of the
-globin gene was replaced by the respective region of the
2AR (see "Materials and Methods"). Expression
vectors encoding the cDNAs for the wild-type -globin and the
chimeric transcript, respectively, were transfected into HEK293 cells
together with plasmid pBC- 2wt harboring the complete 2AR cDNA. The half-lives of the -globin mRNA
and the chimeric transcript, respectively, were determined analogous to
that of the 2AR mRNA (Fig. 2). The results are
expressed as a percentage of control and are the mean ± S.E. of
three independent experiments.
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Table IV
Determination of mRNA half-lives of -globin and the
-globin/ 2AR 3 chimeric transcript
The respective expression vectors were transfected into HEK293 cells.
48 h after transfection, the cells were treated with 5 µg/ml
actinomycin D 30 min after stimulation with either 10 µM
isoproterenol or 10 µM forskolin (or medium for
controls). mRNA levels were determined in Northern analyses. Data
are mean ± S.E. of three independent experiments.
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DISCUSSION |
The
2AR is a prototypical member of the large
family of G-protein-coupled receptors and is subject to a complex
regulation by hormones and other signaling molecules (2-4). Distinct
molecular mechanisms on both mRNA and protein levels, which may be
operative to varying extents in different cell lines and tissues,
contribute to this regulation. Response elements specific for cAMP
(CRE), glucocorticoids (GRE), and thyroid hormones (TRE) regulating
2AR gene transcription have been identified in the
2AR promoter region and the coding sequence (5, 6,
32-34). Here, we report the identification and functional
characterization of a cis-acting element within the 3
-UTR
of the human
2AR mRNA that is sufficient to cause
destabilization of the mRNA.
Many highly labile mRNAs possess AREs within their 3
-UTRs,
although rapid mRNA turnover does not strictly depend on these motifs (9, 10). In cytokine mRNAs, for example, Brown et al. (35) recently identified another class of destabilizing elements, which require at least one stem-loop (hairpin) in the secondary structure. Nevertheless, AREs are considered to be the predominant destabilization determinants.
Analyses using synthetic AU-rich sequences revealed that a nonamer with
an AUUUA pentanucleotide core, UUAUUUA(U/A)(U/A), is a key
destabilization motif (11, 12), although it is not a prerequisite for
ARE function (13). In recent years, evidence has accumulated that it is
the combination of structurally and functionally distinct AU-rich
domains that determines the ultimate destabilizing function of an ARE
(14). Several AU-rich sequences have also been identified in the
3
-UTRs of G-protein-coupled receptors (22, 24), and binding of the
three
2AR mRNA-specific proteins identified so far,
ARB, P85, and AUF1, is selectively competed by poly(U) RNA (20-22).
Furthermore, in vitro binding of
ARB to the hamster
2AR mRNA requires both an AUUUA pentamer and U-rich
flanking domains (23). However, neither the hamster nor the human
2AR mRNAs (26, 27) possess any AU-rich consensus motifs within their 3
-UTR. Therefore, it is tempting to speculate that
agonist-induced
2AR mRNA destabilization occurs via
unique cis-acting elements.
Measurements of the human
2AR mRNA steady-state
levels using mutants lacking either one or both UTRs predicted a
predominant regulatory role for the 3
-UTR. In contrast, deletion of
the 5
-UTR revealed a reduction of
2AR mRNA levels
similar to that of the wild-type receptor. Although the human
2AR 3
-UTR is sufficient to significantly destabilize
the
-globin mRNA when fused to the
-globin coding region, we
cannot exclude the existence of additional determinants within the
5
-UTR that might contribute to mRNA stability. The importance of
the
2AR 3
-UTR was further comfirmed by the observation
that a protein, whose synthesis was induced in DDT1-MF2 smooth muscle cells by the
2AR stimulation, selectively
bound to the 3
-half of the receptor transcript. This supports data from studies (15-19) in which AREs also functioned as recognition motifs for RNA-binding proteins.
A more detailed characterization of the
2AR 3
-UTR
identified an AU-rich nonamer, UAAUAUAUU, at positions 329-337 as the critical element for
2AR mRNA regulation. Its
substitution by a stretch of nine cytosine residues almost completely
abolished mRNA down-regulation and inhibited the interaction with
the
2AR mRNA-binding protein induced in
DDT1-MF2 cells. Therefore, one may conclude that this motif
represents a potent destabilization determinant. This motif differs
from the consensus sequences at positions 2 and 5, which have both been
shown to be important for the destabilizing potency of an ARE (11, 12).
However, mutational analysis of this specific ARE revealed a potency
identical to the consensus element. This shows that a functional ARE
does not have to contain an AUUUA pentamer (13). In accordance with previous reports (11, 12), the flanking adenosine residues appear to
constitute the most critical nucleotides for ARE function, since their
substitution by cytosine residues was sufficient to abolish
2AR transcript destabilization. Surprisingly, the
replacement of the three central nucleotides still allowed mRNA
down-regulation by about 25%. Furthermore, a weak interaction with the
RNA-binding protein induced in DDT1-MF2 cells could be
detected. A possible explanation for this unexpected result is that
this mutant comprises a minimal sequence capable of functioning as an
ARE, at least in the case of the human
2AR mRNA, in
which an intact core domain is not required. Since the region, in which
the ARE is embedded, also does not resemble the U-rich sequences found
in other highly labile mRNAs (9, 10), the human
2AR
mRNA stability appears to be regulated via a potent but
nonconsensus ARE. This parallels a recent study (25), in which a
20-nucleotide AU-rich domain with an unusual AUUUUA hexamer core was
identified as an obligate element for destabilization of the hamster
2AR mRNA. Therefore,
2AR mRNA
stability seems to be regulated via species-specific cis-acting elements. Another possibility is that the
deviations from the consensus can be compensated by other
2AR-specific elements, such as the additional AU-rich
domains within the 3
-UTR or secondary structure elements.
On the other hand, the binding protein(s) observed in
DDT1-MF2 cells upon
2AR stimulation does not
discriminate between these sequence motifs. Therefore, one may assume
that a rather general factor is responsible for
2AR
transcript destabilization. Two
2AR mRNA-binding
proteins,
ARB and AUF1, have been detected in this cell line so far;
the latter one was also identified in the human myocardium (20, 22).
The binding affinity of AUF1 has recently been shown to correlate
directly with the destabilizing potency of the respective ARE in
vitro (36). The biochemical and functional relatedness of the two
proteins initially led to the assumption that they might be identical,
but immunochemical experiments recently suggested that they were
distinct (22).
The analysis of the
-globin/
2AR 3
chimeric
transcript confirmed that the regulation of
2AR mRNA
stability occurs in an agonist-dependent manner and
requires the presence of an RNA-binding protein. Although the stability
of the chimeric mRNA was only about one-third of the
-globin
wild-type transcript, stimulation with either isoproterenol or
forskolin caused a further decrease of the mRNA half-life by almost
a factor of 2. This suggests that the coordinated interplay between
2AR activation and the induction of specific binding
protein(s) is required for efficient destabilization of the receptor
transcript. The almost identical results with isoproterenol and
forskolin show a predominant role for cAMP as a regulator of
2AR mRNA stability. The biochemical mechanisms mediating this cAMP-dependent regulation remain to be
elucidated.
We thank Susanne Pippig for participation in
the initial phase of the project and for providing the
2AR 3
-UTR truncation mutants; Edmund Hoppe for the
expression vectors pcDNA3-
2cds and
pcDNA3-
2
5
-UTR; and Horst Domdey for the plasmid
pGEM1
-globin.