©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of the mRNA-binding Protein AUF1 by Activation of the -Adrenergic Receptor Signal Transduction Pathway (*)

(Received for publication, August 1, 1995; and in revised form, January 3, 1996)

Aldo Pende (1)(§) Kelli D. Tremmel (1) Christine T. DeMaria (3)(¶) Burns C. Blaxall (2)(**) Wayne A. Minobe (1) Jonathan A. Sherman (1) John D. Bisognano (1)(§§) Michael R. Bristow (1) Gary Brewer (3) J. David Port (1) (2)(¶¶)

From the  (1)University of Colorado Health Sciences Center, Division of Cardiology, and (2)Department of Pharmacology 80262, (3)Bowman Gray School of Medicine of Wake Forest University, Department of Microbiology and Immunology, Winston-Salem, North Carolina 27157-1064

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

In both cell culture based model systems and in the failing human heart, beta-adrenergic receptors (beta-AR) undergo agonist-mediated down-regulation. This decrease correlates closely with down-regulation of its mRNA, an effect regulated in part by changes in mRNA stability. Regulation of mRNA stability has been associated with mRNA-binding proteins that recognize A + U-rich elements within the 3`-untranslated regions of many mRNAs encoding proto-oncogene and cytokine mRNAs. We demonstrate here that the mRNA-binding protein, AUF1, is present in both human heart and in hamster DDT(1)-MF2 smooth muscle cells and that its abundance is regulated by beta-AR agonist stimulation. In human heart, AUF1 mRNA and protein was significantly increased in individuals with myocardial failure, a condition associated with increases in the beta-adrenergic receptor agonist norepinephrine. In the same hearts, there was a significant decrease (50%) in the abundance of beta(1)-AR mRNA and protein. In DDT(1)-MF2 cells, where agonist-mediated destabilization of beta(2)-AR mRNA was first described, exposure to beta-AR agonist resulted in a significant increase in AUF1 mRNA and protein (100%). Conversely, agonist exposure significantly decreased (40%) beta(2)-adrenergic receptor mRNA abundance. Last, we demonstrate that AUF1 can be immunoprecipitated from polysome-derived proteins following UV cross-linking to the 3`-untranslated region of the human beta(1)-AR mRNA and that purified, recombinant p37 protein also binds to beta(1)-AR 3`-untranslated region mRNA.


INTRODUCTION

The condition of heart failure is associated with heightened activity of the adrenergic nervous system(1) , the severity of failure correlating with increases in circulating and cardiac concentrations of the catecholamine, norepinephrine(2) . As a consequence of this increased ``adrenergic drive'' the cardiac beta-AR(^1)/G-protein/adenylyl cyclase pathway can become markedly desensitized. One major component of the desensitization is selective down-regulation of the dominant adrenergic receptor subtype within the human myocardium, the beta(1)-AR (1, 3, 4, 5) . Recently, we (6) and others (7) have demonstrated that the observed decrease in beta(1)-adrenergic receptors in failing human heart is closely associated with a corresponding down-regulation of beta(1)AR mRNA. Therefore, it is of interest to better define potential mechanisms responsible for down-regulation of beta-AR mRNA.

Experiments performed using hamster DDT(1)-MF2 smooth muscle cells (8, 9) suggest that down-regulation of the endogenously expressed beta(2)-AR mRNA does not appear to be caused by a decrease in the rate of transcription; rather, it appears that agonist exposure decreases the half-life of beta(2)-AR mRNA from approximately 12 to 5 h(8) . This regulatory mechanism has been demonstrated previously to be important for numerous mRNAs encoding proto-oncogenes, lymphokines, and cytokines(10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) . For these gene products regulation of mRNA stability has also been associated with the interaction of the mRNA with a family of cytosolic proteins (M(r) 30,000-40,000) that often bind to A + U-rich elements (AREs) commonly located within the 3`-untranslated region (3`-UTR) of the mRNA(28, 29, 30, 31, 32, 33, 34) . This interaction induces mRNA degradation by mechanisms only partially understood. However, for some mRNAs including those containing AREs(35, 36) , the degradation of mRNA may be associated with the process of translation. The cytosolic A + U-rich mRNA-binding proteins are in general considered to be distinct from other mRNA-binding proteins such as the heterogenous nuclear ribonucleoprotein particles(37, 38) , however, the role of heterogenous nuclear ribonucleoprotein particles A1 and C proteins as cytoplasmic factors regulating mRNA stability is currently undergoing reassessment(39, 40) .

From previous studies (41, 42, 43) using cytosolic extracts produced from DDT(1)-MF2 hamster smooth muscle cells, the properties of a beta-AR mRNA-binding protein (beta-ARB), which binds to hamster beta(2)-AR and human beta(1)-AR mRNAs, has undergone preliminary characterization. Binding of beta-ARB to mRNA was determined to involve regions of the 3`-UTR of the hamster beta(2)-AR mRNA containing an ARE(41, 42) . In addition, agonist stimulation of the beta-AR pathway or protein kinase A activation by a cAMP analogue resulted in significant up-regulation (3-4-fold) of beta-ARB protein as detected by UV cross-linking. Conversely, treatment of DDT(1)-MF2 cells with dexamethasone, which up-regulates beta(2)-AR mRNA, down-regulated beta-ARB by 50%. Therefore, agents that regulate hamster beta(2)-AR mRNA stability and abundance appear to affect reciprocally the abundance of beta-ARB protein. Among the family of G-protein-coupled receptors, the mRNAs of the hamster beta(2)-AR, the human beta(1)- and beta(2)-AR, and the thrombin receptor have all been demonstrated to interact with beta-ARB(41, 42, 43) . To date, the identity of beta-ARB has remained unresolved. However, beta-ARB does share characteristics in common with several described A + U-rich mRNA-binding proteins(41) , including AUF1(31) .

The cytoplasmic RNA-binding protein, AUF1 (A + U-rich element RNA-binding/degradation factor), has recently been cloned and characterized(31) . AUF1 binds to the 3`-UTRs of several highly regulated mRNAs including c-myc, GM-CSF, and c-fos. Furthermore, there is evidence of ``cause and effect'' between AUF1 and regulation of mRNA stability in that partially purified AUF1 can selectively accelerate the degradation of c-myc mRNA in an in vitro mRNA decay system(34) . Based on these findings, we endeavored to determine if AUF1 was expressed in human heart and in DDT(1)-MF2 cells, and if so, if AUF1 abundance was regulated by stimulation of the beta-AR pathway. Here we report that the mRNA encoding AUF1 protein is expressed in both human heart and DDT(1)-MF2 cells. Furthermore, exposure of DDT(1)-MF2 cells to beta-AR agonist, or high adrenergic drive, as manifest in the failing human heart, results in up-regulation of the AUF1 gene product. In addition, we show that purified, recombinant p37 protein binds to an ARE within the 3`-UTR of the human beta(1)-AR mRNA, and that cellular AUF1 can be immunoprecipitated from polysome-derived proteins following UV cross-linking to the 3`-UTR of the human beta(1)-AR mRNA. These data link for the first time a specific mRNA-binding protein known to be associated with the regulation of mRNA stability, with the mRNA of a G-protein-coupled receptor. In addition, they demonstrate that the abundance of this mRNA-binding protein is up-regulated by adrenergic stimulation, an effect known to destabilize beta-AR mRNA.


MATERIALS AND METHODS

Tissue Procurement

Human ventricular myocardium was obtained from two categories of adult subjects. Failing hearts were obtained from patients undergoing heart transplantation for end stage heart failure (n = 20) due exclusively to idiopathic dilated cardiomyopathy. These individuals had not received intravenous beta-AR agonists, phosphodiesterase inhibitors, or beta-blocking drugs prior to transplantation. Nonfailing hearts were obtained from adult organ donors whose hearts were unsuitable for cardiac transplantation due to blood type or size incompatibility (n = 14). Organ donors' hearts had normal left ventricular function, as determined by echocardiography. Left ventricular aliquots were removed from the heart immediately upon explantation, and either immersed in liquid nitrogen for mRNA and protein quantification or placed in ice-cold, oxygenated Tyrode's solution for preparation of material for radioligand binding assays, as described previously(3) .

Cell Culture

DDT(1)-MF2 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum (HyClone, Logan UT), penicillin (60 µg/ml), and streptomycin (100 µg/ml) as described previously(41) . Cells were treated with either beta-AR agonist 1 µM(-)-isoproterenol, or vehicle (1 mM ascorbic acid) as described in each individual protocol.

AUF1 mRNA Measurement

A 233-base pair fragment of p37 cDNA (31) was cloned from human heart DNA by the use of reverse transcription-PCR. Primers utilized for this reaction spanned a segment of the human p37 coding region cDNA sequence from nucleotides 471 to 702 (31) and incorporated restriction enzyme recognition sites at the 5` ends (SmaI for the forward primer and XbaI for the reverse primer). Primer sequences were: 5`-CCCGGGAAGCTTGGGAAAATGTTTATAGGAGGCC-3` for the forward primer, and 5`-GATCTCTAGAGCTTTGGCCCTTTTAGGATC-3` for the reverse primer. The PCR product was subcloned into pBluescript II KS (Stratagene, Inc., La Jolla, CA) and sequenced using the dideoxy method (Sequenase Version 2, U. S. Biochemical Corp.). Radiolabeled antisense riboprobes were transcribed from the HindIII digested p37 cDNA fragment using T7 DNA-dependent RNA polymerase, [alpha-P]UTP (800 Ci/mmol, DuPont NEN), and the Maxiscript kit (Ambion, Inc., Austin, TX). Total cellular RNA from human ventricular myocardium or from DDT(1)-MF2 cells was extracted by the method of Chomczynski and Sacchi (44) using RNA Stat-60 (Tel Test, Inc., Friendswood, TX), and quantified by absorbance at A. In each ribonuclease protection assay (RPA), 10 µg of RNA were hybridized overnight with 10^6 cpm of radiolabeled AUF1 riboprobe and a low specific activity 18 S rRNA riboprobe (Ambion, Inc.) using the RPA II kit (Ambion Inc.). Since 18 S rRNA abundance is in excess of mRNAs, 18 S probe was produced at a low specific activity to assure molar excess of probe to target without producing a signal beyond the linear range when measured simultaneously with AUF1. The hybridization reaction was digested with RNase A and RNase T1. RNA-RNA hybrids were resolved by electrophoresis in an 8% polyacrylamide, 8 M urea gel. Protected fragments corresponding to AUF1 and 18 S rRNA signals were quantified using a PhosphorImager (Bio-Rad).

beta(2)-AR mRNA Measurement

The abundance of hamster beta(2)-AR mRNA from DDT(1)-MF2 cells was measured by RPA using a specific riboprobe. All measurements were made as described above for AUF1 including normalization of the beta(2)-AR mRNA signal to the signal for 18 S rRNA. The riboprobe (311 nucleotides) was generated from plasmid DNA encoding the hamster beta(2)-AR using PCR primers corresponding to nucleotides 1201 to 1511 (45) and including restriction sites for XhoI and EcoRI. The forward primer was 5`-GATCCTCGAGGATTTCAGGATTGCCTTCCA-3`, and the reverse primer was 5`-GATCGAATTCTAGTGTCCTGTCAGGGAGGG-3`. The PCR product, corresponding to the 3` end of the coding region and the 5` end of the 3`-UTR, was subcloned into pBluescript II KS, and the nucleotide sequence was verified by DNA sequence analysis. Antisense RNA probe was generated using T7 RNA polymerase from riboprobe construct linearized with XhoI as described above.

beta(1)-AR mRNA Measurement

Human beta(1)-AR mRNA abundance from human ventricular myocardium was measured by quantitative reverse transcription-PCR as described in detail previously(6) . Briefly, poly(A)-enriched RNA was extracted from samples of human ventricular myocardium using oligo(dT)-cellulose (Micro-Fast Track mRNA Isolation Kit Version 1.2, Invitrogen Corp., San Diego, CA). mRNA was subjected to a reverse transcriptase reaction in the presence of a fixed amount of synthetic (84mer) RNA ``internal standard'' such that target mRNA (beta(1)AR) and internal standard were amplified colinearly. PCR primers were end-labeled with [-P]ATP and the absolute amounts of beta(1)-AR and internal standard PCR products were determined for each heart by linear modeling of at least 3 points on the linear portion of the amplification curves.

beta-AR Quantification

beta-AR density from human ventricular myocardium was determined in a crude membrane fraction as described previously(3) . Briefly, the total population of beta(1)- plus beta(2)-AR was measured by the nonselective radioligand [I]iodocyanopindolol with and without the use of 1 µML-propranolol to determine total and nonspecific binding, respectively. Maximum binding (B(max)) and [I]iodocyanopindolol dissociation constant (K(d)) were determined by nonlinear least-squares computer modeling of the specific binding curve. beta(1)- and beta(2)-AR subtype proportions were determined using the beta(1)-AR selective ligand CGP-20712A(3) . Protein concentrations were determined by the Peterson modification of the method of Lowry(46) .

Immunoblot Analysis of AUF1

Abundance of AUF1 polypeptides was determined in extracts of DDT(1)-MF2 cells and in human heart tissues using a polyclonal antibody described previously(31) . In human K562 cells, this antibody recognizes p37,p40 an apparent splice form of p37, and 45-kDa protein, an immunologically related but uncharacterized protein(31) . DDT(1)-MF2 cells were either untreated or treated with 10 µM(-)-isoproterenol for 24 or 48 h. Cells were harvested with ice-cold phosphate-buffered saline containing 1 mM EDTA, centrifuged for 5 min at 1000 times g, and resuspended in lysis buffer (20 mM Tris-HCl, pH 7.5, 0.1% Triton X-100, 5 mM beta-mercaptoethanol, 5 µg/ml aprotinin, and 5 µg/ml leupeptine). Cell lysates were subjected to eight rounds of freeze/thaw (dry ice/ethanol for 1 min, 37 °C water bath for 1 min). The samples were centrifuged at 16,250 times g for 10 min, and the supernatant was collected. Total protein concentrations were measured using the BCA reagent kit (Pierce, Rockford, IL). Equal amounts of protein were resuspended in Laemmli loading buffer(47) , boiled for 5 min, and separated by SDS-PAGE (10% resolution phase and 5% stacking phase). Western blotting was performed as described previously(48) . Proteins were transferred to 0.1-µm nitrocellulose membranes (Schleicher and Schuell) for 2 h at 40 V. Blots were blocked overnight in phosphate-buffered saline with 5% nonfat dry milk, washed 5 times 5 min with 0.5% phosphate-buffered saline, incubated for 1 h with 1:1000 anti-AUF1 antibody(31) , washed again in phosphate-buffered saline, and incubated with 1:1000 GAR (Jackson Immunoresearch Laboratories, Inc., Westgrave, PA). Signal was visualized using ECL detection using the manufacturer's protocol (Amersham) and Kodak X-Omat AR film. Signal intensity was determined using an Alpha Innotech IS-1000 Digital Imaging System (San Leandro, CA). Linear range of the protein signal for AUF1 was determined by comparison of increasing amounts of protein (1-25 µg) on the immunoblot. All subsequent quantification was performed using 2.5 µg of total cellular protein, a concentration at the lower end of the linear range.

For human heart tissues, approximately 100 mg of tissue frozen in liquid N(2) was placed in 200 µl of lysis buffer (20 mM Tris-HCl, 0.1% Triton X-100, 5 mM beta-mercaptoethanol, aprotinin 10 µg/ml, and leupeptin 10 µg/ml). The tissue was homogenized using 25 strokes of a Teflon pestle and a T-liner at 4 °C. The homogenate was subjected to 8 rounds of freeze/thaw and centrifuged at 16,250 times g for 15 min. The supernatant was transferred to a fresh tube and the protein concentration determined as above. To perform Western analysis, 100 µg of total cellular protein was subjected to SDS-PAGE as described above and transferred to a polyvinylidine difluoride membrane (Millipore, Marlborough MA) for 2.5 h at 40 V. Subsequent immunodetection methods were as outlined above.

Sequencing of the cDNA Encoding the Human beta(1)-Adrenergic Receptor 3`-UTR

The 2.4-kilobase cDNA encoding the human beta(1)-AR (49) was subcloned into pBluescript II KS at the EcoRI site and its orientation confirmed by DNA sequencing. Nucleotide sequence was determined from purified, double stranded plasmid DNA by the dideoxy method (Sequenase Version 2). Sequencing primers corresponding to the published sequence of the beta(1)-AR coding region and to the T3 primer were used initially. Internal primers were used once additional sequence had been established. TAQ-uense DNA sequencing kit (U. S. Biochemical Corp.) was used to sequence the T-rich portion of the cDNA. Each DNA strand was sequenced at least twice to insure accuracy. The cDNA sequence of the 3`-UTR of the human beta(1)-AR has been submitted to GenBank (U29690).

In Vitro Transcription of RNA for UV Cross-linking

A 919-base pair cDNA fragment corresponding to the beta(1)-AR 3`-UTR was synthesized by PCR and subcloned into pcDNA3 (Invitrogen) utilizing the XhoI and XbaI restriction endonuclease sites. The resulting vector was linearized with XbaI, and in vitro transcription was performed as described previously(41) . Briefly, radiolabeled RNA was synthesized using T7 DNA-directed RNA polymerase and [alpha-P]UTP (800 Ci/mmol, DuPont New England Nuclear) to produce uniformly labeled, 5`-capped RNA. After transcription, RNase-free DNase I was added to the mixture to remove template DNA. The labeled transcript was extracted with phenol/chloroform, then chloroform only, precipitated with ethanol and washed with 70% ethanol, resuspended in RNase-free water, and maintained at -80 °C until use.

Preparation of Ribosomal Salt Wash

A 0.3 M KCl ribosomal salt wash (RSW) was produced from DDT(1)-MF2 cells using the method of Brewer and Ross(50) .

Purification of Recombinant p37 Protein

The coding region of p37 resides on a 910-base pair BsmAI fragment spanning nucleotides 236 to 1146 of the cDNA(31) . This fragment was blunted and inserted into the SmaI site of the pGEM7Z(+) vector (Promega) to yield the pGEM7Z/P37CR plasmid. To generate the corresponding His(6)-AUF1 fusion peptide expression vector, an Asp718-HindIII fragment from pGEM/P37CR was inserted into Asp718-HindIII digested pTrcHisB (Invitrogen) resulting in pTrcHisB/P37CR. The reading frame of the His(6)-AUF1 fusion polypeptide was confirmed by both dideoxy sequencing and reactivity of the fusion polypeptide with polyclonal AUF1 antiserum.

An Escherichia coli TOP10 (Invitrogen) clone containing pTrcHisB/P37CR was induced to express plasmid-encoded protein by culturing with 1 mM isopropyl-beta-D-thiogalactopyranoside (U. S. Biochemical). His(6)-AUF1 fusion polypeptide was purified using the Xpress System (Invitrogen) under native conditions as described by the manufacturer. Selected fractions were electrophoresed, and the protein profile assessed by Coomassie staining. Fractions 4-11 were pooled, and human alpha-lactalbumin (Sigma) was added to a final concentration of 100 µg/ml to aid in preserving the activity of the recombinant AUF1 protein during storage at -80 °C. The concentration of purified recombinant AUF1 was determined by comparison with known amounts of bovine serum albumin using Coomassie-stained SDS-polyacrylamide gels and immunoblot analysis using anti-AUF1 polyclonal antiserum.

UV Cross-linking

UV cross-linking was performed as described previously(41) . Briefly, an aliquot of radiolabeled RNA (1-4 times 10^6 cpm) was added to a mixture containing 20 µl of RSW (5 times 10^6 cell equivalents/µl) from DDT(1)-MF2 cells, 5 µg of yeast tRNA, 4 mM dithiothreitol, 5 µg heparin, and 65 units of RNasin in a total volume of 50 µl. After incubation for 10 min at 22 °C, samples were placed in an ice slurry and exposed to short-wave (254 nm) UV radiation for 3 min in a Stratagene (model 1800) UV Stratalinker. The cross-linked RNA was digested with RNase A (0.5 mg/ml) and RNase T1 (10 units/ml) at 37 °C for 30 min. Samples were solubilized in 50 µl of Laemmli loading buffer for 10 min at 70 °C, and proteins were resolved by SDS-PAGE. Gels were stained with Coomassie Blue R-250 (Sigma) followed by destaining and drying, and subjected to autoradiography for 1-5 days.

Immunoprecipitation of AUF1

Polysomes and RSW from DDT(1)-MF2 cells and radiolabeled human beta(1)-AR 3`-UTR RNA were prepared as described above. Polysomes or RSW (2 times 10^6 cell equivalents) were mixed with 5 times 10^6 cpm of beta(1)-AR 3`-UTR RNA, UV cross-linked, and digested with RNase A and T1 as described above. Polysomes or RSW were precleared with preimmune serum and Pansorbin cells (Calbiochem, La Jolla, CA) and immunoprecipitated as described by Zhang et al.(31) . The pellet was resuspended in Laemmli buffer, boiled 5 min, and proteins resolved by SDS-PAGE (10%). Gels were dried, and radiolabeled proteins were visualized by autoradiography for 2 to 5 days.


RESULTS

Human Heart

In failing ventricular myocardium, beta(1)-AR mRNA and receptor protein are significantly down-regulated to a similar extent(6, 7) . Furthermore, as discussed below, sequencing of the cDNA for the 3`-UTR of the human beta(1)-AR has revealed that there is at least one potential ARE. Based on the precedent of agonist-mediated destabilization of the hamster beta(2)-AR mRNA (8) and the binding of beta-ARB to this mRNA(41) , we wished to determine if the gene encoding the mRNA-binding protein AUF1 was expressed in the human heart. Secondarily, we wished to determine if AUF1 gene expression was affected by heart failure. Left ventricular myocardium was obtained from two groups: (i) individuals with idiopathic dilated cardiomyopathy (n = 20) undergoing orthotopic cardiac transplantation, and (ii) organ donors whose hearts were unsuitable for cardiac transplantation (n = 14). To measure AUF1 and human beta(1)-AR mRNA, total cellular RNA was isolated from left ventricular myocardium. As determined by RPA, the mRNA encoding AUF1 was significantly up-regulated in failing heart (190% of control, p < 0.05) compared to nonfailing donor hearts (Table 1). In each case the signal for AUF1 mRNA was normalized to that of 18 S rRNA. Although 18 S rRNA is a significantly more abundant RNA that AUF1, the signal for 18 S was often less intense than that of AUF1 due to the intentionally low specific activity of the 18 S probe. Therefore, the signal strength ratio of AUF1 to that of 18 S RNA was often >1. Heart failure had no effect on 18 S rRNA expression (data not shown). In addition, immunoblot analysis of AUF1 proteins was performed on ventricular myocardium from nonfailing (n = 8) and failing (n = 8) human hearts. Fig. 1A is a representative immunoblot of eight of these hearts, four nonfailing and four failing. Compared to nonfailing hearts, the relative abundance of immunoreactive p45 and p40 protein were both significantly increased in failing hearts (Fig. 1B). p37 protein was distinctly present but variably detectable and at low abundance even when using as much as 100 µg of total cellular protein per lane. Therefore, quantitative analysis was not performed on the signal for p37. A more detailed analysis of AUF1 protein expression in response to beta-agonist stimulation was performed in a cell culture model system, described below.




Figure 1: AUF1 proteins in human left ventricular myocardium. A, a representative immunoblot of AUF1 proteins from four nonfailing and four failing human hearts. Whole tissue lysates of human ventricular myocardium were prepared and assayed for AUF1 polypeptides using a polyclonal anti-AUF1 antibody(31) . In each case, equivalent amounts of total cellular protein (100 µg) were analyzed for each subject. The data from these hearts and an additional eight hearts are summarized in B. B, the relative abundance of AUF1 immunoreactive proteins was investigated in nonfailing (n = 8) and failing (n = 8) human heart. Data are presented as X ± S.E. The relative abundance of p45 and p40 proteins are expressed in arbitrary units (A.U.) Statistical analysis of nonfailing compared to failing hearts was performed by use of a two-tailed, unpaired t test.



Although the identity of p45 is currently unknown, it is obviously immunologically related to p37 and to p40. Furthermore, as demonstrated below (Fig. 6), p45 recognized and bound to A + U-rich mRNAs thus appearing to be similar in this regard to AUF1 proteins. Lastly, like p40, p45 abundance is up-regulated in individuals with heart failure. The relationship of p45 to AUF1 or to other mRNA-binding proteins beyond these shared characteristics remains to be determined.


Figure 6: A, immunoprecipitation and Western blot analysis of AUF1 polypeptides from UV cross-linking reactions. KCl-extracted polysome-associated proteins (RSW) from DDT(1)-MF2 cells treated with isoproterenol (10 µM for 48 h) were pre-cleared with preimmune serum. RSW (2 times 10^6 cell equivalents) was UV cross-linked to 5 times 10^6 cpm of capped, uniformly labeled in vitro transcribed RNA corresponding to the human beta(1)-AR 3`-UTR. Following cross-linking, the reactions were treated with RNase A + T1, diluted with NET-gel buffer(31) , and AUF1 proteins subjected to immunoprecipitation using polyclonal anti-AUF1 antiserum or nonimmune serum. Proteins were resolved by SDS-PAGE and detected by autoradiography. B, polysome-associated proteins (not subjected to KCl-extraction) were subjected to immunoprecipitation as described in A. In addition, an input lane, labeled ``X-link,'' represents 10% of the cross-linking reaction not subjected to immunoprecipitation. C, polysome-associated proteins from isoproterenol-treated (10 µM for 24 h) DDT(1)-MF2 cells were UV cross-linked to radiolabeled mRNA encoding the 3`-UTR of the human beta(1)-AR. The reactions were digested with RNase A + T1, resuspended in Laemmli buffer, and subjected to SDS-PAGE. The gel was transferred to a polyvinylidine difluoride membrane and proteins detected as described under ``Materials and Methods'' (right panel). Proteins recognized by the anti-AUF1 antibody are delineated by a bracket. The same membrane was also analyzed by autoradiography (left panel). A band corresponding to p38 is specifically labeled. Molecular weights are as indicated.



As determined by quantitative reverse transcription-PCR, beta(1)-AR mRNA abundance was significantly decreased (40%) in failing as compared to nonfailing, control hearts (Table 1). These results are consistent with previous findings(6) . beta-AR density and subtype proportions also were determined in the same failing and nonfailing hearts. beta(1)-AR density was also significantly reduced (61%) in failing compared to nonfailing hearts (Table 1). By contrast, beta(2)-AR density was not different in failing compared to nonfailing hearts (23.2 ± 2.0 versus 27.4 ± 3.4 fmol/mg protein, respectively).

In summary, these data indicate that (i) AUF1 mRNA and protein are expressed in human ventricular myocardium; and (ii) in individuals with heart failure, AUF1 mRNA and protein are significantly up-regulated and, both beta(1)-AR mRNA and protein are down-regulated. From these data we conclude that up-regulation of AUF1 in human heart may be involved in the regulation of beta(1)-AR mRNA stability and thus may be at least partially responsible for the decline in beta(1)-AR mRNA and subsequently protein abundance in the failing heart.

DDT(1)-MF2 Cells

The use of a cell culture model system and the cloning of p37(31) has made it possible to explore in greater detail the role of beta-agonist stimulation in regulating the expression of the mRNA-binding protein, AUF1. DDT(1)-MF2 cells were chosen because: (i) agonist-mediated destabilization of the endogenous hamster beta(2)-AR mRNA was originally described in these cells(8) , and (ii) beta-ARB, which binds to the human beta(1)-AR and hamster beta(2)-AR mRNAs was also originally described in these cells(41) . When DDT(1)-MF2 cells were treated with 10 µm(-)-isoproterenol for 24 h (n = 2) or for 48 h (n = 3), steady-state AUF1 mRNA abundance, as determined by RPA, was modestly increased to 129 ± 6% (p<0.05, n = 5, pooled data) of untreated controls. AUF1 mRNA was increased to the same extent at both 24 and 48 h. In each case, mRNA abundance was normalized to signal for 18 S rRNA. Treatment with isoproterenol had no effect on 18 S rRNA expression (data not shown). Fig. 2A is a representative immunoblot of DDT(1)-MF2 whole cell lysates using polyclonal anti-AUF1 antibody. Three distinct bands are evident: p37, p40, and p45, an immunologically related polypeptide(31) . Fig. 2B demonstrates the presence of AUF1 polypeptides in the polysome fraction of DDT(1)-MF2 cells under both basal and isoproterenol stimulated conditions. As is evident, AUF1 proteins are preferentially localized to the polysome fraction. This finding is consistent with localization determined previously for AUF1 in K562 cells (31) and for beta-ARB in DDT(1)-MF2 cells(41) .


Figure 2: Immunoblot of AUF1 proteins in DDT(1)-MF2 cells. A, whole cell lysates of DDT(1)-MF2 cells smooth muscle cells were prepared and assayed for AUF1 polypeptides using a polyclonal anti-AUF1 antibody (31) . Three bands are in evidence, p37, p40, and p45, a polypeptide immunologically related to AUF1. B, post-nuclear S130 cytosol and polysome fractions from DDT(1)-MF2 cells were analyzed for the presence of AUF1 polypeptides.



As determined by immunoblot analysis, p37 protein was increased to 230 ± 50% of control (n = 5, pooled data) in cells treated with 10 µM(-)-isoproterenol for 24 (n = 2) or 48 h (n = 3) compared to untreated controls (Table 2). The relative abundance of p37 protein was increased to a similar extent by isoproterenol treatment at both the 24- and 48-h time points. In the same preparations, the relative amounts of p40 and p45 were also determined. In cells treated with isoproterenol, p40 increased to 160 ± 30% of control (n = 5, pooled data) and p45 increased to 180 ± 30% of control (n = 5, pooled data) (Table 2). At each time point, the relative abundance of p40 and p45 were roughly similar, both being of considerably greater abundance than p37. Also of note is the finding that the relative abundance values of p37 and p40 are greater (in arbitrary units) at 48 h compared to 24 h. This may indicate an increased relative amount of AUF1 proteins as the cells continue to grow.



In the same cells, the relative abundance of the endogenous beta(2)-AR mRNA was measured. Treatment of DDT(1)-MF2 cells with 10 µM(-)-isoproterenol for 24 (n = 2) or 48 h (n = 3) produced a decrease in beta(2)-AR mRNA to 63 ± 2% (p<0.05, n = 5) of control. The degree of down-regulation was highly similar at 24 and 48 h (63% versus 62% of control). As with AUF1, the protected signal for the hamster beta(2)-AR mRNA was normalized to the invariant signal for 18 S rRNA (data not shown). The degree of down-regulation of beta(2)-AR mRNA was in close agreement with a previous investigation (8) and correlates well with the degree of receptor down-regulation.

In summary, these results demonstrate that in DDT(1)-MF2 hamster smooth muscle cells: (i) stimulation of the beta-AR pathway produces an increase in AUF1 mRNA and p37 protein. In addition, the abundance of p40 and p45 proteins are also increased; (ii) there is a reciprocal decrease in beta(2)-AR mRNA abundance; and (iii) p37 protein is expressed and localized to a polysome fraction. We conclude that agonist-mediated up-regulation of AUF1 protein in DDT(1)-MF2 cells may contribute to agonist-mediated destabilization and down-regulation of the hamster beta(2)-AR observed in these cells.

Human beta(1)-AR 3`-UTR

Although previously cloned (49) , the nucleotide sequence of the 3`-UTR of cDNA for the human beta(1)-AR had not been determined. In order to determine if the beta(1)-AR 3`-UTR contained potential mRNA stability regulatory domains such as an ARE and to facilitate mapping of mRNA-binding proteins, we sequenced this portion of the cDNA (Fig. 3). The beta(1)-AR 3`-UTR contains a uniquely long poly(U) tract in its proximal region. This domain is similar to other AREs(20, 51) . Several other A + U-rich regions are denoted including a putative mRNA destabilizing sequence ``UUAUUUAU''(52, 53) . In addition, four potential poly(A) addition sites (AAUAAA or AUUAAA) are present. It is currently unknown which site or sites are used for poly(A) addition.


Figure 3: Nucleotide sequence of the 3`- untranslated region of the human beta(1)-AR. The 3`-UTR of the human beta(1)-AR was sequenced from the previously cloned cDNA (49) using the dideoxy method. The nucleotide sequence begins with the stop codon (UAG) at nucleotide 1432 and extends for an additional 932 nucleotides. A uniquely long U-rich region as well as several A + U-rich regions which are potential AREs are in bold and underlined including the putative mRNA destabilizing sequence, UUAUUUAU. Four canonical poly(A) addition sequences are shown in bold, underlined, and italics.



UV Cross-linking of Polyribosome-associated Proteins and Recombinant p37 Polypeptide to the beta(1)-AR mRNA

To determine which mRNA-binding proteins bind to the human beta(1)-AR mRNA, we performed UV cross-linking of radiolabeled RNA substrates to ribosome-associated proteins from isoproterenol (10 µM for 48 h) stimulated DDT(1)-MF2 cells. Proteins were solubilized from polyribosomes by extraction with 0.3 M KCl, termed a RSW. The rationale for using RSW rather than S100 cytosol or polysomes as a starting point was that this preparation has been shown to contain AUF1 in a partially purified form as well as being sufficient to reproduce decay of proto-oncogene mRNA in an in vitro mRNA decay system(18) . RNAs encoding the human beta(1)-AR coding region only, the beta(1)-AR 3`-UTR only, or the c-myc 3`-UTR, were in vitro transcribed and the radiolabeled RNAs incubated with RSW. Mixtures were UV irradiated, treated with RNase A + T1, and separated by SDS-PAGE. Prominent bands are apparent at M(r) of 65,000, 55,000, and 38,000. The M(r) 65,000 band binds only to the 3`-UTR of c-myc, whereas the M(r) 55,000 bands and the M(r) 38,000 band both bind to the 3`-UTRs of the beta(1)-AR and c-myc. By contrast, none of these bands bind to the coding region of the human beta(1)-AR mRNA (Fig. 4). In addition to the distinct band at M(r) 38,000, there is significant trailing of protein binding between M(r) 38,000 and 41,000 indicating that perhaps more than one protein in this size range binds to the 3`-UTR of the human beta(1)-AR mRNA. This is by contrast to the c-myc 3`-UTR where only the M(r) 38,000 band is in evidence. There are also bands of low intensity between M(r) 41,000 and 45,000 that bind to the beta(1)-AR and c-myc 3`-UTRs.


Figure 4: UV cross-linking of RSW proteins to multiple radiolabeled RNAs. Representative autoradiogram of RSW from DDT(1)-MF2 cells treated with(-)-isoproterenol (10 µM for 48 h) and UV cross-linked to capped, uniformly radiolabeled in vitro transcribed RNAs. Equal amounts of RSW (20 µl, 5 times 10^5 cell equivalents/µl) and equimolar amounts of radiolabeled RNA were added to each reaction. Lane 1, non-UV cross-linked control; lane 2, beta(1)-AR 3`-UTR only; lane 3, beta(1)-AR coding region (CR) only; lane 4, c-myc 3`-UTR. A band at M(r) 38,000, previously designated as beta-ARB(41, 42, 43) , binds to the beta(1)-AR 3`-UTR, and to the c-myc 3`-UTR, but not to the beta(1)-AR CR.



Binding of all RSW proteins to the beta(1)-AR 3`-UTR is effectively competed by a 10-fold molar excess of unlabeled beta(1)-AR 3`-UTR (Fig. 5). By contrast, only binding of M(r) 38,000 protein(s) is competed by a 10-fold molar excess of the A + U-rich GM-CSF 3`-UTR RNA but not by a 50-fold molar excess of DeltaGM-CSF. The DeltaGM-CSF RNA contains only one of the five pentameric AUUUA motifs present in the wild-type GM-CSF RNA (30) . By these criteria, and as demonstrated previously for the hamster beta(2)-AR(41) , M(r) 38,000 (beta-ARB) has the properties of an A + U-rich mRNA-binding protein.


Figure 5: Competitive displacement of beta-ARB protein binding to beta(1)-AR 3`-UTR RNA. Radiolabeled RNA corresponding to the 3`-UTR of the human beta(1)-AR was UV cross-linked to RSW proteins in the presence of increasing amounts (0-, 10-, and 50-fold molar excess) of unlabeled competitor RNAs encoding the human beta(1)-AR 3`-UTR (lanes 1-3), GM-CSF 3`-UTR (lanes 4-5) and DeltaGM-CSF 3`-UTR (lanes 6 and 7). beta(1)-AR and GM-CSF but not DeltaGM-CSF competed effectively for beta-ARB binding.



To test the hypothesis that the M(r) 38,000 protein (beta-ARB) may be an AUF1-related protein, RSW proteins from isoproterenol (10 µM for 48 h) stimulated DDT(1)-MF2 cells were UV cross-linked to radiolabeled beta(1)-AR 3`-UTR, as described under ``Materials and Methods.'' The reaction was immunoprecipitated using a polyclonal anti-AUF1 or with non-immune serum. Anti-AUF1 serum selectively immunoprecipitated two proteins (Fig. 6A), a single major band of M(r) 45,000 and a band of weaker intensity immediately below the major band. This finding is in exact concordance with that of Zhang et al.(31) when immunoprecipitating AUF1 proteins UV cross-linked to the c-myc ARE. No proteins were evident when immunoprecipitating with non-immune serum.

In polysomes extracts with KCl, as in the RSW preparations used above, proteins at M(r) 40,000 to 45,000 are not readily apparent in UV cross-linking experiments. By contrast, polypeptides at M(r) 40,000 to 45,000 are readily detected by UV cross-linking to the 3`-UTR of the human beta(1)-AR when using polysomes prior to KCl extraction. We therefore performed similar UV cross-linking immunoprecipitation experiments using a polysome preparation. Here, three proteins of M(r) 40,000 to 45,000 are immunoprecipitated corresponding to bands of similar migration in the input lane (i.e. not subjected to immunoprecipitation, labeled ``X-link''). By contrast, no polypeptides co-migrating with the M(r) 38,000 protein (beta-ARB) were immunoprecipitated (Fig. 6B). Therefore, all three proteins recognized by the anti-AUF1 antibody associate with the 3`-UTR of the human beta(1)-AR mRNA and can be immunoprecipitated after UV cross-linking.

An additional experiment was performed to test whether or not beta-ARB and AUF1 proteins are the same. Polysome-derived proteins were UV cross-linked to the radiolabeled mRNA corresponding to the beta(1)-AR 3`-UTR. Following separation by SDS-PAGE, the gel was transferred to a polyvinylidine difluoride (Millipore) membrane and autoradiography and immunoblotting were performed to ensure that signals could be superimposed precisely. Here again, the proteins recognized by the anti-AUF1 antibody do not co-migrate with the M(r) 38,000 signal for beta-ARB (Fig. 6C). By immunologic and migratory criteria, the results presented above in Fig. 6, A-C, all argue strongly against p38beta-ARB being an AUF1 protein.

Lastly, to determine directly if AUF1 could bind to the human beta(1)-AR 3`-UTR, radiolabeled RNA was incubated with purified, recombinant p37, subjected to UV irradiation, RNase A + T1 digestion, SDS-PAGE, and autoradiography. Recombinant p37 protein binds to the beta(1)-AR 3`-UTR and to the c-fos ARE but fails to bind to rabbit beta-globin (Rbeta) RNA (Fig. 7). Unlabeled beta(1)-AR 3`-UTR RNA effectively competes for AUF1 binding, while a 100-fold molar excess of beta-globin does not (data not shown).


Figure 7: UV cross-linking of purified, recombinant p37 polypeptide to human beta(1)-AR 3`-UTR RNA. Autoradiogram of recombinant p37 UV cross-linked to radiolabeled in vitro transcribed human beta(1)-AR 3`-UTR RNA. Lane 1, beta(1)-AR RNA in the absence of competitor RNA. Lanes 2 and 3, beta(1)-AR RNA in the presence of 10-fold and 100-fold molar excess of unlabeled beta(1)-AR RNA. Lane 4, c-fos ARE only. Lane 5, rabbit beta-globin RNA only.



Together, the UV cross-linking and immunoprecipitation experiments indicate that: (i) a number of polysome-derived proteins between M(r) 38,000 and 45,000 bind to the 3`-UTR of the human beta(1)-AR including a prominent polypeptide at M(r) 38,000 as well as several polypeptides between M(r) 40,000 and 45,000 UV cross-link to the 3`-UTR but not the coding region of the human beta(1)-AR mRNA (a protein of the same apparent molecular weight binds to c-myc and GM-CSF mRNA; data not shown); (ii) anti-AUF1 antibody immunoprecipitates proteins between M(r) 40,000 to 45,000 when cross-linked to the human beta(1)-AR 3`-UTR, and (iii) purified recombinant p37 binds to the 3`-UTR of the human beta(1)-AR mRNA. These results indicate that M(r) 38,000 (beta-ARB) is not an AUF1-related protein. The results also demonstrate by multiple methods that AUF1 proteins bind to the mRNA encoding the beta(1)-AR 3`-UTR.


DISCUSSION

Agonist-mediated down-regulation of G-protein-coupled receptors is a well established regulatory paradigm. One of the ways in which the amount of receptor protein may be down-regulated is by an alteration in the steady-state abundance of its mRNA which, in turn, is controlled by transcription rate, or by mRNA stability, or both. Since the first report of agonist-mediated destabilization of an mRNA encoding a G-protein-coupled receptor by Hadcock et al.(8) , the mRNA abundance of a number of other G-protein-coupled receptors have been reported to be regulated by changes in mRNA half-life (Table 3). However, it should not be assumed that for each receptor of a particular subtype that it is regulated at the level of mRNA stability in all species or cell types. For example, the alpha-AR mRNA derived from rabbit aorta smooth muscle cells is regulated by an agonist-mediated response dependent on protein kinase C activity(54) . By contrast, in DDT(1)-MF2 smooth muscle cells, the stability of hamster alpha-AR mRNA appears not to be regulated by agonist exposure(55) . Furthermore, the hamster alpha-AR mRNA from DDT(1)-MF2 cells is not A + U-rich and does not interact with beta-ARB(41) . It is unknown whether or not the rabbit alpha-AR interacts with A + U-rich mRNA-binding proteins. As another example, the half-life of the mRNA encoding the m1-muscarinic receptor is decreased by its cognate agonist, carbachol, an effect that necessitates an intact 3`-UTR(56) . While the 3`-UTR of the m1-muscarinic receptor does not contain A + U-rich regions, there are sequence motifs that may form hairpin loops that might act as binding sites for RNA-binding proteins. For example, 5`-AUU-3`/5`-UAA-3` motifs appear to be important for directing endonucleolytic cleavage in mRNAs containing stem-loop structures (62) . These structures have been shown to interact with mRNA- binding proteins of M(r) 34,000(63) .



Several other mRNAs important to G-protein-coupled receptor signal transduction are also regulated at the level of mRNA stability including protein kinase A1(64) , and the G-proteins Galpha, and Galpha(65) . Most recently, we have demonstrated that the stability of the human beta(1)-AR mRNA, the subject of the current investigation is regulated by beta-agonist exposure in a cell culture-based model system, an effect that is dependent entirely on the presence of the 3`-UTR(61) .

AUF1 shares several characteristics in common with the previously described beta-AR mRNA-binding protein, beta-ARB, which led us to hypothesize that they might be the same or related proteins. First, both proteins have similar electrophoretic mobilities. The apparent molecular weights of AUF1 are 37 and 40 kDa, whereas beta-ARB was reported to be M(r) 35,000 by UV cross-linking of DDT(1)-MF2 S100 to the hamster beta(2)-AR mRNA (41) and M(r) 38,000, by UV cross-linking of polysomal-derived proteins from DDT(1)-MF2 cells to the human beta(1)-AR 3`-UTR (this article). Second, both proteins share the same cellular fractionation profile, i.e. both are minimally represented in cytosolic fractions and are present at a much higher relative abundance in polysomes (Refs. 31 and 41; this article). Similarly, by immunoblotting, AUF1 is readily detectable in whole cell extracts (Fig. 2A), in the polysome fraction (Fig. 2B), and in RSW (data not shown). Third, in response to beta-AR stimulation, both beta-ARB and AUF1 are significantly up-regulated at both 24 and 48 h ((41) ; this article). Fourth, beta-ARB and AUF1 preferentially bind to the AREs of multiple mRNAs. beta-ARB binds to the mRNAs encoding the human beta(1)-AR and to the human and hamster beta(2)-ARs(41) . More specifically, beta-ARB binds the proximal U-rich region of the 3`-UTR of human beta(1)-AR mRNA (data not shown). Similarly, beta-ARB also binds to the AREs of GM-CSF and the adenovirus AdIVa2(42) . More recently, beta-ARB has been demonstrated to bind to the thrombin receptor mRNA, another G-protein-coupled receptor regulated at the level of stability(43) . Like the hamster beta(2)-AR, the half-life of the thrombin receptor mRNA is significantly decreased by either agonist treatment or by stimulation of cells by cAMP analogues(43) . By contrast, beta-ARB does not bind to the hamster alpha-AR mRNA or to the beta-globin mRNA (41) and weakly or not at all to the rat beta(1)-, beta(3)-, or human beta(3)-AR mRNAs(43) . beta-ARB, like purified recombinant p37 protein, binds to the human beta(1)-AR mRNA, as well as to the AREs for c-myc, c-fos, GM-CSF(31) , but not rabbit beta-globin mRNA. Fifth, both beta-ARB and p37 protein increase with beta-agonist exposure. However, the data from several different experimental approaches presented herein indicate a lack of concordance for the molecular weights of beta-ARB and AUF1 proteins and lead to the conclusion that beta-ARB is not an AUF1 protein. These experiments include immunoprecipitation of AUF1 proteins from polysomes and immunoblotting of polysome-derived proteins to UV cross-linked beta(1)-AR mRNA. The biochemical and functional relatedness of human AUF1 to hamster beta-ARB will necessitate the future purification and/or cloning of beta-ARB.

The observation that AUF1 is up-regulated in the failing human heart and in agonist-treated DDT(1)-MF2 cells is intriguing and leads to several important questions. The first and most obvious question: is AUF1 responsible for the observed down-regulation of beta(1)-AR mRNA in the failing human heart and/or the hamster beta(2)-AR in DDT(1)-MF2 cells? The data presented here indicate that beta-AR stimulation results in a reciprocal relationship: the up-regulation of AUF1 gene product(s) and the down-regulation of beta-AR and the destabilization of beta-AR mRNA. This suggests that an increase in AUF1 protein(s) may be associated with increased mRNA turnover rates for the beta-AR as well as for other mRNAs.

It is of interest that AUF1 also binds to the human beta(2)-AR mRNA (data not shown). However, it is well established that the mRNA beta(2)-AR is not down-regulated in failing human heart(1, 3, 4, 5, 6) . To date, there is no ready explanation for this finding beyond speculation. It is possible that human beta(2)-AR mRNA undergoes agonist-mediated destabilization but that there is an offsetting increase in transcription rate such that steady-state mRNA abundance does not appear to change. It is also possible that there may be differences in the affinity of AUF1 for the beta(1)-AR and beta(2)-AR mRNAs functionally affecting the role of AUF1. Another possibility is that AUF1 and the target mRNA need to be appropriately co-localized physically and temporally for mRNA turnover to be accelerated. Lastly, multiple proteins co-immunoprecipitate with AUF1, however, these proteins have not yet been characterized(31) . By virtue of its protein structure, which contains two consensus RNA recognition motifs(31) , AUF1 unquestionably binds to the RNA directly and thus must be central to the actions of other proteins associated with this complex. However, it is untested as to whether the same proteins always associate with AUF1 depending on which mRNA is bound. These points all address potential mechanisms conferring specificity of action for AUF1, a topic for future investigation.

In the failing human heart, changes in the expression of a number of other genes have also been documented including multiple components of several G-protein-coupled receptor signal transduction pathways, ion channels, and cytoskeletal/structural proteins. Also well documented is the observation that c-myc and other mRNAs encoding proto-oncogenes are transiently up-regulated by stimuli associated with myocardial failure, e.g. high adrenergic drive(66, 67, 68, 69) . By contrast to the transient nature of c-myc and other proto-oncogene expression associated with adrenergic stimuli, increased expression of AUF1 appears to be persistent. In many cases, the individual hearts assayed for AUF1 mRNA and protein abundance had been ``failing'' for months if not years. Therefore, unlike the expression of proto-oncogenes and other immediate-early genes, AUF1 expression does not appear to accommodate to the chronic stimulus causing its up-regulation. An issue of importance to address in the future will be to determine the precise mRNA targets of AUF1 in the human heart. The role of AUF1 or other mRNA-binding proteins in regulating the expression of these mRNAs in the context of heart failure or in myocardial hypertrophy remains to be explored.

We conclude that AUF1 is present in both human heart and DDT(1)-MF2 cells and that it is up-regulated by beta-agonist exposure in cells and by the process of heart failure. Furthermore, we have demonstrated that purified, recombinant AUF1 binds to the 3`-UTR of the human beta(1)-AR mRNA and can be immunoprecipitated from polysome-derived proteins UV cross-linked to human beta(1)-AR 3`-UTR mRNA. Future experiments will attempt to address more precisely the role of AUF1 in the destabilization of beta-AR mRNAs as well as exploring other potential target mRNAs for AUF1 binding.


FOOTNOTES

*
This work was supported in part by United States Public Health Services Grants HL51239 (to J. D. P.), and HL48013 (to M. R. B.), by Grant NP-884 from the American Cancer Society (to G. B.), and by Grant CBG-015-93 from the Colorado Affiliate of the American Heart Association (to J. D. P.). Additional support was obtained from the Temple Hoyne Buell Research Foundation at the University of Colorado HSC. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U29690[GenBank].

§
Supported by the Ospedale S. Martino, Genoa, Regione Liguria, Italy.

Supported by Training Grant T32-AI07401 from the National Institutes of Health.

**
Supported by Training Grant GM07635 from the National Institutes of Health.

§§
Supported by a Bugher Fellowship in Cardiology.

¶¶
To whom correspondence should be addressed: University of Colorado HSC, Div. of Cardiology, B139, 4200 East Ninth Ave., Denver, CO 80262. Fax: 303-270-3261.

(^1)
The abbreviations used are: beta-AR, beta-adrenergic receptor; ARE, A + U-rich element; GM-CSF, granulocyte/macrophage colony-stimulating factor; 3`-UTR, 3`-untranslated region; beta-ARB, beta-AR mRNA-binding protein; RPA, ribonuclease protection assay; RSW, ribosomal salt wash; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.


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