Agonist Regulation of Human beta 2-Adrenergic Receptor mRNA Stability Occurs via a Specific AU-rich Element*

Stefan Danner, Monika Frank, and Martin J. LohseDagger

From the Institute of Pharmacology, University of Würzburg, Versbacher Straße 9, 97078 Würzburg, Germany

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Prolonged agonist stimulation of beta 2-adrenergic receptors results in receptor down-regulation, which is closely associated with a reduction of the corresponding mRNA, an effect mediated in part by changes in mRNA stability. Transfection experiments with human beta 2-adrenergic receptor cDNAs bearing or lacking the untranslated regions suggested that the essential agonist sensitivity of the mRNA resides within the 3'-untranslated region. The importance of this region was further confirmed in gel shift experiments; cytosolic preparations from agonist-stimulated DDT1-MF2 smooth muscle cells caused a shift of beta 2-adrenergic receptor mRNAs containing the 3'-untranslated region. Progressive 3'-terminal truncations of the receptor cDNA led to the identification of an AU-rich element at positions 329-337 of the 3'-untranslated region as the responsible cis-acting element. Substitution of this motif by cytosine residues almost completely abolished mRNA down-regulation and inhibited the formation of the RNA-protein complex. Even though the beta 2-adrenergic receptor AU-rich element showed two U right-arrow A transitions compared with the recently proposed AU-rich element consensus sequence, it revealed an almost identical destabilizing potency. Fusion of the beta 2-adrenergic receptor 3'-untranslated region to the beta -globin coding sequence dramatically reduced the half-life of the chimeric transcript in an agonist- and cAMP-dependent manner. This suggests that the agonist-induced beta 2-adrenergic receptor mRNA destabilization is regulated by cAMP-dependent RNA-binding protein(s) via a specific AU-rich element.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Chronic stimulation of the beta 2-adrenergic receptor (beta 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 beta 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 beta 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 beta 2AR mRNA, three binding proteins have been described so far: (i) the beta -adrenergic receptor mRNA-binding protein (beta ARB), a Mr 35,000 cytosolic protein identified in hamster DDT1-MF2 smooth muscle cells (20); (ii) a Mr 85,000 factor mediating beta 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 beta 1AR mRNA (22). Although AU-rich sequence motifs within the beta 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 beta 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 beta 2AR mRNA. However, a sequence alignment revealed no equivalent within the human beta 2AR transcript (26, 27), which in turn suggests that beta 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 beta 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 beta 2AR mRNA down-regulation in vivo.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Plasmid Construction-- The beta 2AR vectors used for transient transfections were constructed based on the plasmid pBC12BI-beta 2 (26), from which a 1.95-kb fragment corresponding to the complete human beta 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-beta 2. beta 2AR 3'-UTR C-terminal truncation mutants were generated by deletion of a PvuII-SmaI (Delta 157) and a PpuMI-SmaI fragment (Delta 313), respectively, from plasmid pBC12BI-beta 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, beta 2AR5' and beta 2AR3', were cloned into pGEM9Zf(-) (Promega) under the control of the T7 promoter. A 130-bp poly(A) stretch was inserted downstream of the beta 2AR cDNAs to obtain polyadenylated mRNAs.

The mutations of the ARE at positions 329-337 of the beta 2AR 3'-UTR were introduced by PCR. 247-bp fragments covering the 3'-halves of the 3'-UTR were amplified using the mutagenesis primers beta 2AR.seq.4, beta 2AR.seq.41, beta 2AR.seq.42, and beta 2AR.seq.43, respectively, and beta 2fus.rev (for all primers see Table I). These fragments were subsequently used as "reverse primers" in a second PCR together with beta 2fus.seq.2. The resulting 571-bp mutated 3'-UTRs were fused to the beta 2AR coding sequence in the vector pcDNA3-beta 2Delta 3'UTR. Additonally, pGEM vectors bearing the mutated AREs were constructed analogous to beta 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 beta 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 beta 2fus.seq.2 and beta 2fus.rev.2, respectively, are underlined.

For the generation of the beta -globin/beta 2AR 3'-UTR fusion plasmid a 2.2-kilobase pair fragment corresponding to the beta -globin primary transcript was excised from pGEM1beta -globin and inserted into pcDNA3. After that, a 540-bp DraI-XbaI fragment comprising the beta -globin 3'-UTR was replaced by the 546-bp beta 2AR 3'-UTR excised from pcDNA3-beta 2Delta 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 beta -galactosidase encoding plasmid, pSVbeta 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 beta 2AR probe were beta 2AR.seq.2 and beta 2AR.rev.4, spanning a 672-bp fragment immediately downstream of the start codon. Probes specific for alpha B-crystallin (468 bp, used as an internal standard) and beta -globin (319 bp) were amplified using the primers cry.seq/cry.rev and hbeta g.seq/hbeta 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).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Mapping of the Agonist-sensitive Region within the beta 2AR mRNA-- Since in many cell types the beta 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 beta 2AR transcript. For this purpose, HEK293 cells were chosen due to their very low level of endogenous beta AR as well as the very high transfection efficiency compared with other cell lines. 48 h after the transfection of vectors bearing human beta 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 beta 2AR mRNA levels were quantified in Northern analyses (Fig. 1A). alpha B-Crystallin, a widely expressed heat-shock protein, was used as an internal standard in all experiments. Cotransfection of a beta -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 beta 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 beta 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 beta 2AR mRNA resides essentially within the 3'-UTR.


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Fig. 1.   Determination of the agonist-sensitive region within the human beta 2AR mRNA. Expression vectors haboring the human beta 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 beta 2AR and alpha 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, beta 2AR coding sequence.

Interaction of the Human beta 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 beta 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 beta 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 beta 2AR mRNA concentrations were quantified in Northern analyses (Fig. 2). The beta 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 beta 2AR mRNA half-life to about 80 min in stimulated cells, which indicates that beta 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 beta 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. beta 2AR mRNA levels were quantified by Northern analyses and presented as percentage of control. The results are mean ± S.E. of three experiments.

To identify the region(s) of the human beta 2AR mRNA that might be interacting with such a protein component, we established a gel shift assay using the human beta 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 beta 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 beta 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 beta 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 beta 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 beta 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.

Identification of an AU-rich Destabilization Motif within the beta 2AR 3'-UTR-- For a more detailed characterization of the human beta 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 beta 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 alpha B-crystallin mRNA remained unchanged in all three cases. These results provide evidence that the region between positions 241 and 397 of the beta 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 beta 2AR 3'-UTR. Two 3'-terminal truncation mutants of the beta 2AR cDNA were generated lacking the last 157 bp (Delta 157) and 313 bp (Delta 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 beta 2AR and alpha B-crystallin were determined by Northern analyses. The wild-type receptor (WT) was used as a control. B, schematic representation of the beta 2AR 3'-UTR. The beta 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 beta 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.

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 beta 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 beta 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 beta 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 beta 2AR mRNA levels, normalized for the respective values for alpha 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 beta 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 right-arrow 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 beta 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 beta 2AR mRNA levels to 75 ± 7% was observed (Fig. 5), demonstrating that at least for the beta 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 beta 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 beta 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. beta 2AR mRNA and alpha 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 beta 2AR 3'-UTR
The mutations were introduced in the beta 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).

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 beta 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 beta 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 beta 2AR 3'-UTR, probably via a specific regulatory mechanism.


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Fig. 6.   Gel shift analysis using the mutated beta 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.

Stability of a Chimeric beta -globin/beta 2AR 3'-UTR Transcript-- To test the hypothesis of a beta 2AR-specific regulation of mRNA stability, we asked whether the elements within the beta 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 beta 2AR 3'-UTR was fused to the coding sequence of the human beta -globin gene, and the half-life of the resulting chimeric transcript was measured in comparison with wild-type beta -globin. To analyze whether the activation of the beta 2AR has an influence on the function of the cis-acting elements within the transcript, a vector haboring the beta 2AR cDNA was cotransfected together with the two beta -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 beta -globin wild-type transcript remained unaffected by agonist stimulation. The beta -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 beta -globin 3'-UTR against the respective region from the beta 2AR dramatically reduced the stability of the chimeric mRNA. In addition, its stability became beta AR agonist-dependent. Upon stimulation of the beta 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 beta 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 beta 2AR mRNA stability is essentially regulated by cAMP.


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Fig. 7.   Fusion of the beta -globin coding sequence with the beta 2AR 3'-UTR. The endogenous 3'-UTR of the beta -globin gene was replaced by the respective region of the beta 2AR (see "Materials and Methods"). Expression vectors encoding the cDNAs for the wild-type beta -globin and the chimeric transcript, respectively, were transfected into HEK293 cells together with plasmid pBC-beta 2wt harboring the complete beta 2AR cDNA. The half-lives of the beta -globin mRNA and the chimeric transcript, respectively, were determined analogous to that of the beta 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 beta -globin and the beta -globin/beta 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.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The beta 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 beta 2AR gene transcription have been identified in the beta 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 beta 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 beta 2AR mRNA-specific proteins identified so far, beta ARB, P85, and AUF1, is selectively competed by poly(U) RNA (20-22). Furthermore, in vitro binding of beta ARB to the hamster beta 2AR mRNA requires both an AUUUA pentamer and U-rich flanking domains (23). However, neither the hamster nor the human beta 2AR mRNAs (26, 27) possess any AU-rich consensus motifs within their 3'-UTR. Therefore, it is tempting to speculate that agonist-induced beta 2AR mRNA destabilization occurs via unique cis-acting elements.

Measurements of the human beta 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 beta 2AR mRNA levels similar to that of the wild-type receptor. Although the human beta 2AR 3'-UTR is sufficient to significantly destabilize the beta -globin mRNA when fused to the beta -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 beta 2AR 3'-UTR was further comfirmed by the observation that a protein, whose synthesis was induced in DDT1-MF2 smooth muscle cells by the beta 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 beta 2AR 3'-UTR identified an AU-rich nonamer, UAAUAUAUU, at positions 329-337 as the critical element for beta 2AR mRNA regulation. Its substitution by a stretch of nine cytosine residues almost completely abolished mRNA down-regulation and inhibited the interaction with the beta 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 beta 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 beta 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 beta 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 beta 2AR mRNA. Therefore, beta 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 beta 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 beta 2AR stimulation does not discriminate between these sequence motifs. Therefore, one may assume that a rather general factor is responsible for beta 2AR transcript destabilization. Two beta 2AR mRNA-binding proteins, beta 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 beta -globin/beta 2AR 3' chimeric transcript confirmed that the regulation of beta 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 beta -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 beta 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 beta 2AR mRNA stability. The biochemical mechanisms mediating this cAMP-dependent regulation remain to be elucidated.

    ACKNOWLEDGEMENTS

We thank Susanne Pippig for participation in the initial phase of the project and for providing the beta 2AR 3'-UTR truncation mutants; Edmund Hoppe for the expression vectors pcDNA3-beta 2cds and pcDNA3-beta 2Delta 5'-UTR; and Horst Domdey for the plasmid pGEM1beta -globin.

    FOOTNOTES

* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB355 and a grant from the Fonds der Chemischen Industrie.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.

Dagger To whom all correspondence should be addressed. Tel.: 49-931-201-5401; Fax: 49-931-201-3539.

1 The abbreviations used are: beta 2AR, beta 2-adrenergic receptor; UTR, untranslated region; ARE, AU-rich element; beta ARB, beta -adrenergic receptor mRNA-binding protein; AUF1, AU-rich element RNA-binding/degradation factor; HEK293 cells, human embryonic kidney cells; DIG, digoxigenin; PCR, polymerase chain reaction; bp, base pair(s).

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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