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. Author manuscript; available in PMC: 2007 Jun 22.
Published in final edited form as: Biochim Biophys Acta. 2006 Aug 22;1763(10):1076–1089. doi: 10.1016/j.bbamcr.2006.08.009

CRM 1-Mediated Degradation and Agonist-Induced Down-Regulation of β-Adrenergic Receptor mRNAs

Ying Bai 1, Huafei Lu 1, Curtis A Machida 1,2,*
PMCID: PMC1896136  NIHMSID: NIHMS14515  PMID: 16997396

SUMMARY

The β1-adrenergic receptor (β1-AR) mRNAs are post-transcriptionally regulated at the level of mRNA stability and undergo accelerated agonist-mediated degradation via interaction of its 3' untranslated region (UTR) with RNA binding proteins, including the HuR nuclear protein. In a previous report [Kirigiti et al. (2001). Mol. Pharmacol. 60:1308-1324), we examined the agonist-mediated down-regulation of the rat β1-AR mRNAs, endogenously expressed in the rat C6 cell line and ectopically expressed in transfectant hamster DDT1MF2 and rat L6 cells. In this report, we determined that isoproterenol treatment of neonatal rat cortical neurons, an important cell type expressing β1-ARs in the brain, results in significant decreases in β1-AR mRNA stability, while treatment with leptomycin B, an inhibitor of the nuclear export receptor CRM 1, results in significant increases in β1-AR mRNA stability and nuclear retention. UV-crosslinking/immunoprecipitation and glycerol gradient fractionation analyses indicate that the β1-AR 3' UTR recognize complexes composed of HuR and multiple proteins, including CRM 1. Cell-permeable peptides containing the leucine-rich nuclear export signal (NES) were used as inhibitors of CRM 1-mediated nuclear export. When DDT1MF2 transfectants were treated with isoproterenol and peptide inhibitors, only the co-addition of the NES inhibitor reversed the isoproterenol-induced reduction of β1-AR mRNA levels. Our results suggest that CRM 1-dependent NES-mediated mechanisms influence the degradation and agonist-mediated down-regulation of the β1-AR mRNAs.

Keywords: β1-adrenergic receptor, CRM 1, mRNA degradation and stability, agonist-induced down-regulation

INTRODUCTION

Post-transcriptional regulatory mechanisms participate in the stability and degradation of various cellular RNAs (1-4). Several G protein-coupled receptor (GPCR) mRNAs, including those encoding the β1- and β2-adrenergic receptors (ARs) (5-7), undergo agonist-induced decreases in transcript stability. The β2-AR mRNA contains an AU-rich 3' UTR that can recognize several RNA binding proteins, including 1) the β-adrenergic receptor mRNA-binding protein (βARB), a cytosolic protein that requires both AUUUA pentamers and U-rich domains for RNA recognition, 2) the Mr 37,000 RNA degradation factor AUF1, and 3) the T-cell-restricted intracellular-related protein (TIAR), a molecule involved in translational control (5-11). In the presence of β-AR agonist isoproterenol, βARB and AUF1 appear to be up-regulated, concomitant with the down-regulation of β2-AR mRNA. Specific AU-rich elements in the β2-AR 3' UTR, including a 20 bp core sequence, have been demonstrated to be essential for the agonist-mediated destabilization of β2-AR mRNA (12-14). Using chimeric receptor - beta globin recombinants, the human β1-AR mRNA 3' UTR was determined to target β-globin mRNA for accelerated decay, and was demonstrated to be necessary but not sufficient for agonist-mediated destabilization of transcripts (15). Using mass spectrometry, βARB has been identified as a complex protein mixture consisting of HuR and hnRNP A1 (16). HuR appears to be the predominant RNA binding component of βARB; however, the precise component composition of βARB appears to be dependent on the 3' UTR sequence of the target β-AR subtype mRNA (16). TIAR is believed to be a suppressor of β2-AR translation and provides further validation of the growing body of evidence linking ARE binding proteins to roles in post-transcriptional control mechanisms, including RNA degradation and translation (10, 11).

Cellular mRNAs are transported through the nuclear pore by interaction of complexes containing export receptor proteins and adapter molecules (17-21). RNA binding proteins that serve as potential adapters include HuR, hnRNP A1, hnRNP K, SRP20, 9G8, and ASF/SF2 (22-26). HuR selectively binds AREs and promotes the stabilization of many ARE-containing mRNAs, including those that encode c-fos, c-myc, tumor necrosis factor alpha, cyclooxygenase 2, myogenins, interleukins, vascular endothelial growth factor, and granulocyte-macrophage colony stimulating factor (27-29). HuR has been implicated in the regulation of a wide assortment of cellular responses, including cell division, immune cell activation and carcinogenesis (28). HuR, predominantly localized in the nucleus, can shuttle between the nuclear and cytoplasmic compartments; however, the major influence of HuR on mRNA stabilization and translation appears to be dependent on its cytoplasmic localization (28-30). HuR contains three RNA recognition motifs (RRMs). ARE recognition appears to be mediated by the first two RRMs, while the third RRM is believed to interact with the poly (A) tail of the targeted mRNA (27, 29). HuR undergoes nucleocytoplasmic shuttling using a novel shuttling sequence (HNS) located in the hinge region between the second and third RRMs (27, 29). The ability of HuR to undergo nucleocytoplasmic export has implicated that HuR may bind ARE-containing mRNAs in the nucleus for export to the cytoplasm. Four HuR ligands have been recently identified by affinity chromatography: SETα and SETβ, pp32, and acidic protein rich in leucine (APRIL) (31). All four HuR ligands have been found to form a multi-subunit heterocomplex with HuR; similar heterocomplexes containing SETα, SETβ, and pp32 possess histone acetyltransferase inhibitory activity and roles in chromatin remodeling and transcriptional regulation (32). Three of the HuR ligands (SETα, SETβ, and pp32) are inhibitors of protein phosphatase 2A (PP2A). Human-mouse heterokaryon fusion experiments and fluorescence-based nucleocytoplasmic transfer assays demonstrate that HuR, pp32, and APRIL undergo nucleocytoplasmic export (31). Using cell permeable peptide inhibitors, HuR nucleocytoplasmic export was determined to involve the association of HuR with pp32 and APRIL; both HuR ligands contain leucine-rich export signals (NES) that are recognized by the export receptor chromosome maintenance region 1 (CRM 1) (27, 29, 31, 33, 34). In addition, immunoprecipitation experiments support the contention of direct interaction of pp32 and APRIL with CRM 1. Inhibition of CRM 1 with leptomycin B results in the partial nuclear retention of pp32 and APRIL, and also results in the increased association of HuR with pp32 and APRIL and with nuclear poly (A) mRNA (33, 34). In addition, the ARE-containing c-fos mRNAs become partially retained in the nucleus following leptomycin B treatment (33, 34). In total, these findings implicate CRM 1 and HuR as a nuclear export receptor - adapter in the nucleocytoplasmic export of ARE-containing mRNAs, and that the mechanism of ARE-mediated mRNA degradation may be CRM 1-dependent and moderated by export of ARE-containing mRNAs into the cytoplasm.

Nuclear export pathways that are CRM 1-independent may also involve the HuR nucleocytoplasmic shuttling (HNS) sequence within HuR; this shuttling sequence is similar to the M9 shuttling sequence of hnRNP A1 (26, 29, 34). HnRNP A1 contains a 39 amino acid M9 domain that interacts with nuclear export receptor transportin 1 (Trn 1), a member of the importin β family; this export receptor – adapter pair has been implicated in the nuclear export of the dihydrofolate reductase mRNA using Xenopus oocytes as a model system (35, 36). The nuclear export receptor that recognizes HuR in this HNS-mediated pathway has not been unequivocally defined (28). However, the nuclear import receptors for HuR, facilitated by interaction with HNS, are transportin 1 and transportin 2 (Trn 1 and Trn 2, respectively) (28, 37, 38).

In this report, we examined and compared the agonist-mediated regulation of the rat β1-AR mRNAs in neonatal rat cortical neurons and in established cell lines endogenously or ectopically expressing β1-AR mRNAs. Nuclear transport and cytoplasmic localization of cellular mRNAs have been implicated as important determinants in mRNA stability. We have provided evidence that CRM 1-mediated mechanisms, including the use of leucine-rich export signals, are important components in post-transcriptional β1-AR mRNA degradation and agonist-mediated down-regulation.

MATERIALS AND METHODS

Cell lines, antibodies, chemicals, and statistical analyses

The rat C6 glioma (ATCCCCL 107) and hamster DDT1MF2 (ATCC CRL 1701) cell lines were cultured in Dulbecco's Modified Eagle medium (DMEM), supplemented with 10% heat-inactivated fetal calf serum (FCS), 1% penicillin - neomycin - streptomycin (PNS), and 1% L-glutamine. Low glucose (1.5 g/liter) and high glucose (4.5g/liter) DMEM were used to culture the C 6 and DDT1MF2 cell lines, respectively. DDT1MF2 cells transfected with the rat β1-AR expression recombinant are described in Kirigiti et al. (7). Antibodies recognizing HuR and CRM 1 were obtained from Dr. Henry F urneaux (Cornell University School of Medicine) and Dr. Gerard Grosveld (Department of Genetics, St. Jude Children's Research Hospital, Memphis, TN), respectively. Both antibodies were also obtained commercially from Santa Cruz Biotechnology (HuR antibodies: sc-5261; mouse monoclonal IgG1 and CRM 1 antibodies: sc-5595; rabbit polyclonal IgG). Leptomycin B was purified and provided by Dr. Minoru Yoshida (RIKEN, Japan) (39). Statistical analyses to ascertain significance of β1-AR mRNA half-lives were based on the two tailed unpaired t test derived from the Microsoft Excel software program.

Preparation of neonatal rat cortical neuron cultures

Primary cultures containing neonatal rat cortical neurons were prepared following methods described by Goforth et al. (40). Briefly, cortices were isolated from 1-2 day old Sprague-Dawley rats, minced in saline, trypsinized for 10 minutes at 37°C, and transferred to DMEM containing 4.5 g/l sucrose, 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine. The tissue was washed and dispersed by repeated triturations using Pasteur pipets and low-speed centrifugations, followed by filtration of the final suspension using an 88 μm nylon sieve. Cells were plated on poly-L-lysine coated dishes (Fisher Scientific) and incubated at 37°C in 5% CO2. Cells were then treated for 48 hours with ara-c (10 μm) to inhibit the growth of dividing cells, primarily glial cells, and allowing for the isolation of neurons. Culture medium were then replaced with DMEM supplemented with 30 mM glucose, 100 U/m penicillin, 100 μg/ml streptomycin, and 5% horse serum, prior to use in transfections and cell culture experiments.

RNA isolation, antisense probe preparation, and RNase protection assay procedures

Methods for RNA isolation, quantitation, and determination of sample integrity, and RNase protection assays (RPA) were conducted as described in Yang and Machida (41). The rat β1-AR mRNA has two functional polyadenylation signals at +2450 and +2732. While the downstream polyadenylation signal is highly preferred, the upstream polyadenylation signal is utilized in most tissues analyzed and lies close to the midpoint of the 3' UTR sequence. Thus, to circumvent issues concerning probe utilization, the probe pCS[−82, +273], which contains rat β1-AR sequence extending from position −82 to +273 (relative to the translational start site), recognizes all β1-AR transcripts and was used to measure total β1-AR mRNAs in RNase protection experiments. Antisense transcripts were generated with T7 RNA polymerase (20 Units, 37°C, 1 hour) using reaction components described in Yang and Machida (41). Total sample RNA (15 μg) was hybridized with antisense transcripts (β1-AR cRNAs and cyclophilin cRNA) and digested with RNase T1 (41). Ethanol-precipitated samples were suspended in formamide/dye solution and the products separated by electrophoresis on a 5% acrylamide, 7 M urea gel. RNase protection experiments were analyzed using the Biorad Molecular Imager phosphorimager system and radioactive signals were converted into digital quantitative values using the Quantity One software program. Intensity values for β1-AR protected fragments were normalized with corresponding intensity values for the cyclophilin-protected fragment obtained in the same lane. Normalization ratios (β1-AR-protected fragment intensity / cyclophilin-protected fragment intensity) for each lane were divided by the normalization ratio determined for the lane corresponding to time 0. This second normalization resulted in the assignment of the time 0 lane with a numerical value of 1.0.

UV-crosslinking and immunoprecipitation of RNA-protein complexes for SDS-polyacrylamide gel electrophoresis

Preparation of cell extracts, binding reactions, and UV-crosslinking of RNA-protein complexes were conducted as described in Kirigiti and Machida (42). Noncovalently bound ribonucleotides were degraded using RNase T1 (1 U) and RNase A (1 μl of 0.5 μg/μl) for 30 min at 37°C. The reaction mixtures were added to equal volumes of 2X loading buffer (100 mM Tris-HCl, pH 6.8, 200 mM dithiothreitol, 4% sodium dodecyl sulfate (SDS), 2% bromophenol blue, and 20% glycerol), boiled for 5 min, and subjected to electrophoresis in a 10% polyacrylamide gel (acrylamide:bis-acrylamide, 29:1; supplemented with SDS). Gels were transferred to nitrocellulose membranes prior to phosphorimager analysis. Following UV-crosslinking of RNA-protein complexes and RNase A/T1 digestion, 1-2 μl of specific antibody was added to the reaction mix and allowed to incubate overnight at 4° C. Phosphate buffered saline (PBS) was added to the mixture and 20-40 μl of protein A slurry (protein A-Sepharose 4B beads; obtained from Sigma, catalogue # P3391) was then added, and allowed to mix for 2 h at 4° C. The protein A beads were then washed three times by gentle centrifugation in PBS, and then resuspended in 6X loading buffer, prior to heating at 100° C for 2 min. RNA-protein complexes released from the protein A beads were then loaded and electrophoresed in SDS-polyacrylamide gels prior to phosphorimager analyses.

Electro-transfer to nitrocellulose membranes and immunoblotting procedures

Proteins were electro-transferred onto nitrocellulose membranes, and blots were blocked and prepared for immunostaining using procedures described in Kirigiti et al. (43). Primary antibodies include the mouse monoclonals to the HuR (1:5000 dilution) and rabbit polyclonal to CRM 1 (1:2000 dilution). The primary antibodies were then incubated for 2 - 5 h at room temperature with gentle agitation. The secondary antibodies were anti-mouse HRP or anti-rabbit HRP (1:10,000 dilutions). Membranes were then washed four times with 1X TTBS, and then developed using the Super-Signal Western pico chemiluminescence substrate (Pierce Chemicals).

Cell lysis, glycerol gradient centrifugation, and immunoblot procedures

Cell extracts using procedures known to preserve protein complexes are previously described (31, 44). Briefly, cells were extracted with 5 ml lysis buffer containing 25 mM HEPES-KOH, pH 8.0, 150 mM KCl, 2 mM EDTA, 1 mM DTT, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 μg/ml pepstatin A, 20 mM NaF, and 0.1% NP-40. Extracts were clarified by centrifugation at 40,000 rpm (SW 50.1 rotor, Beckman) for 40 minutes. The supernatant was loaded onto 4 ml 5-20% glycerol gradients and centrifuged at 40,000 rpm (SW 50.1 rotor, Beckman) for 12 hours. Gradient fractions were then subjected to polyacrylamide gel electrophoresis under nondenaturing conditions.

Cell-permeable peptides

Cell-permeable peptides contain the antenna-pedia internalization element (AP = RQIKIWFQNRRMKWKK) at its amino-terminus. Peptides were synthesized with COOH-extensions containing HNS (amino acids 205-237 of HuR; RRFGGPVHHQAQRFRFSPMGVDHMSGISGVNVP), NES (amino acids 73-84 of the human immunodeficiency virus protein rev; LQLPPLERLTLD); M9 (amino acids 268-305 of hnRNP A1; NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY), and a scrambled HNS or M9 sequence or a mutant NES with leucines 78 and 81 replaced by alanines, two sites known to be essential for NES function. Peptides were synthesized and purified by the Yale University Peptide Synthesis Facility.

RESULTS AND DISCUSSION

We have used an array of experimental methods to extend our understanding of the molecular determinants underlying the agonist-mediated degradation of the β1-AR mRNAs. Previous work implicates the existence of at least three regulatory mechanisms influencing β1-AR mRNA levels in C6 cells: 1) an initial up-regulation induced by transcriptional activation, 2) an intermediate down-regulation moderated by transcriptional repression, potentially by inducible cyclic AMP early repressor (ICER), and 3) a chronic down-regulation mediated by post-transcriptional degradation mechanisms, potentially influenced by HuR, a component of βARB and/or other RNA binding proteins (7). In our previous report, we provided evidence that the mammalian elav-like protein HuR and the heteronuclear protein hnRNP A1 interact with the β1-AR 3' UTR and that the induction of HuR by isoproterenol may play a role in the agonist-mediated modulation of β1-AR mRNA half-life (7). We had also presented evidence that availability of specific protein factors, including nuclear proteins and binding factors, may play a role in both the steady-state and agonist-mediated degradation of the β1-AR mRNAs, as observed with the differential β1-AR mRNA half-lives seen in C6 cells, L6 cells, and in the DDT1MF2 transfectants ectopically expressing β1-ARs (7). For this report, we wanted to extend our analysis to neonatal rat cortical neurons, an important central nervous system (CNS) cell type expressing β1-ARs, and provide identification of additional molecular determinants in the post-transcriptional degradation of the β1-AR mRNAs. We also wanted to substantiate the importance of CRM 1-mediated mechanisms of nuclear export in the agonist-mediated degradation of the β1-AR mRNAs.

Isoproterenol treatment of neonatal rat cortical neurons promotes decrease in β1-AR mRNA stability

Neonatal rat cortical neurons chronically treated with isoproterenol (24 hours) results in a significant decrease in β1-AR mRNA stability (Figure 1, Panels A-D). The β1-AR mRNA half-lives for both steady state conditions and during chronic isoproterenol treatment are 5.4 hours ± 0.3 hour and 3.0 hours ± 0.2 hour, respectively (Figure 1, Panels A-D). In a previous publication (7), we reported the β1-AR mRNA half-lives for the rat glioma C6 cell line and for hamster DDT1MF2 transfectants ectopically expressing β1-ARs under both steady state conditions and following chronic isoproterenol treatment (see also Table 1). In both cell lines, like in neonatal rat cortical neurons, the β1-AR mRNAs underwent accelerated degradation following chronic isoproterenol treatment (C6 cells: β1-AR mRNA half-lives are 63 minutes ± 1.7 min and 41 minutes ± 5.9 min for both steady state conditions and during chronic isoproterenol treatment, respectively; DDT1MF2 transfectants: β1-AR mRNA half-lives are 109 minutes ± 4.0 min and 83 minutes ± 5.1 min for both steady state conditions and during chronic isoproterenol treatment, respectively). These results confirm the importance of the availability of specific protein factors on β1-AR mRNA stability under both steady state conditions and during agonist-mediated degradation.

Figure 1.

Figure 1

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β1-AR mRNA half-life determinations in neonatal rat cortical neurons: Panels A, C, and E represent untreated controls, neurons undergoing chronic (24 hours) isoproterenol treatment, or neurons treated with leptomycin B (LMB). Panels A, C, and E depict representative RNase protection experiments. Neurons were pulsed (3 hours) with actinomycin D following isoproterenol or LMB treatment to inhibit de novo transcription. Panels B, D, and F: Normalization analyses of β1-AR mRNA levels. Gels were subjected to phosphorimager analyses. Normalization of β1-AR mRNA levels against corresponding cyclophilin mRNA levels was conducted. X- and Y-axis for both graphs are time following actinomycin D treatment and normalized levels of β1-AR mRNA, respectively. Data points on the y-axis were plotted on log scale to depict the first stage decline of β1-AR mRNA levels. Each data point was derived using 4 replicates. First-order decay equations were derived and used to determine β1-AR mRNA half-lives under agonist treatment, leptomycin B treatment, or control conditions.

Table 1.

β1-AR mRNA half life determinations under steady state conditions or under isoproterenol or leptomycin B induction

β1-AR mRNA half-life under treatment parameters
Steady state Isoproterenol Leptomycin
C6 cells 63 min ± 1.7 min (6)* (A) 41 min ± 5.9 min (6)* (B) 101 ± 3.9 min (4)*(C)
DDT1MF2 transfectants 109 min ± 4 min (6)*(D) 83 min ± 5.1 min (6)*(E) 8.6 hours ± 0.4 hour (4)*(F)
Rat Cortical neurons 5.4 hours ± 0.3 hr (4)*(G) 3.0 hours ± 0.2 hr (4)*(H) 16.4 hours ± 1.1 hr (4)*(I)
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*

Numbers placed within parentheses represent number of experiments used in determination of β1-AR mRNA half-life values. Half-life determinations were based on the equation: y = (intercept point on y-axis) × exp (mx) where y = percentage of β1-AR mRNA remaining; m = slope of the line; x = time (min). Statistical analyses were based on the two-tailed unpaired t test. Half-life determinations for A, B, D, and E were previously published (7). Statistical power of significance between A and B (p = 0.013) and between D and E (p = 0.01) were also previously published (7). New statistical analyses determined in this study: statistical power of significance between A and C (p = 0.00019); between E and F (p = 0.00071); between G and H (p = 0.0000025); between G and I (p = 2.29E-09).

Leptomycin B inhibition of CRM 1 nucleocytoplasmic export in neonatal rat cortical neurons results in significant increase in β1-AR mRNA stability

Leptomycin B (LMB) is an inhibitor of CRM 1 nucleocytoplasmic export and results in the nuclear retention of ARE-containing mRNAs, specifically c-fos mRNAs. Neonatal rat cortical neurons treated with leptomycin B display attenuated β1-AR mRNA half-lives of 16.4 hours ± 1.1 hour (Figure 1, Panels E and F).

Leptomycin B inhibition of CRM 1 nucleocytoplasmic export in DDT1MF2 transfectants results in significant increases in β1-AR mRNA levels and half-life

Using leptomycin B treatment, we examined both steady-state β1-AR mRNA levels and β1-AR mRNA stability in DDT1MF2 transfectants, which ectopically express rat β1-ARs. We observe a 10-fold increase in β1-AR mRNA levels in LMB-treated DDT1MF2 transfectants, occurring in either the presence or absence of isoproterenol (Figure 2, Panels A and B). Using actinomycin D, an inhibitor of de novo transcription, we have determined that the β1-AR mRNA half-life in LMB-treated DDT1MF2 transfectants is 8.6 ± 0.4 hours (n=4 experiments; Figure 2, Panels C an D; also Table 1), and that the half-life is not significantly different in the presence or absence of isoproterenol (8.2 hours for LMB + isoproterenol). This is compared to the β1-AR mRNA half-life of 109 ± 4 minutes and 83 ± 5.1 minutes observed in DDT1MF2 transfectants cultured in the absence or presence of isoproterenol, respectively (Figure 2; also Table 1). Leptomycin B inhibition of CRM 1 nucleocytoplasmic export in DDT1MF2 transfectants also results in the significant increase in β1-AR mRNA levels and transcript stability. In addition, the lack of a synergistic isoproterenol-mediated effect on β1-AR mRNA stability in leptomycin-treated cells indicates that the agonist-mediated down-regulation of β1-AR mRNAs may also be sensitive to LMB-inhibition of CRM 1 nucleocytoplasmic export. Alternatively, the isoproterenol-mediated effect on β1-AR mRNA stability in leptomycin-treated cells may potentially be too small or difficult to detect against the much larger LMB-mediated decrease in β1-AR mRNA levels.

Figure 2.

Figure 2

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Panels A and B: Rat β1-AR mRNA levels in DDT1MF2 transfectants undergoing independent or concurrent treatment with isoproterenol and leptomycin. Matched experiments using nontreated and isoproterenol-treated (without leptomycin) cells were also conducted in parallel with the leptomycin-treated cultures. Experiments measure steady-state β1-AR mRNA levels in nontreated control cultures, isoproterenol-treated cultures (10 μg/ml, 24 hours), leptomycin-treated cultures (10 ng/ml, 24 hours), and in cultures concurrently treated with isoproterenol (10 μg/ml, 24 hours) and leptomycin (10 ng/ml, 24 hours). Panel A depicts representative RNase protection experiment. Panel B depicts statistical analyses of n=4 experiments. Error bars represent one standard error. Note that the standard error bars for the histograms displaying the isoproterenol-treated and untreated control are indistinguishable from the height of the histograms. The observed decrease (54%) following isoproterenol treatment is statistically significant (p = 0.0015; see asterisk). Panels C-F: Rat β1-AR mRNA half-life analyses in DDT1MF2 transfectants undergoing independent or concurrent treatment with isoproterenol (10 μg/ml, 24 hours) and leptomycin (10 ng/ml, 24 hours) followed by actinomycin D treatment (3 hours) to inhibit de novo transcription. Panels C and D depict representative RNase protection experiments of isoproterenol-treated, leptomycin-treated (+ isoproterenol), or untreated cultures. Samples were removed at designated times following the initiation of actinomycin D treatment. Panels E and F contain normalization calculations of β1-AR mRNA levels against corresponding cyclophilin mRNA levels for isoproterenol-treated, leptomycin-treated (+ isoproterenol), and untreated cultures. Error bars represent one standard error. n=4 experiments for each treatment condition. Straight lines represent computer-generated first order decay for normalized levels of β1-AR mRNAs in isoproterenol-treated, leptomycin-treated, isoproterenol / leptomycin-treated, and untreated cultures. Decay rate for β1-AR mRNAs in leptomycin-treated cultures and cultures concurrently treated with leptomycin and isoproterenol are nearly identical.

We also treated C6 cells, which endogeneously express β1-AR mRNAs, with leptomycin B to ascertain effects on β1-AR mRNA stability. RNase protection experiments demonstrate that C6 cells treated with leptomycin B also resulted in enhanced β1-AR mRNA stability. β1-AR mRNA half-life determinations in leptomycin-treated C6 cells were 101 ± 3.9 minutes (n=4 experiments), compared to β1-AR mRNA half-lives of 59.2 ± 3.4 minutes in matched non-treated C6 cell controls (n=4 experiments; Table 1). This is compared to β1-AR mRNA half-lives of 63 ± 1.7 minutes and 41 ± 5.9 minutes in C6 cells cultured in the absence or presence of isoproterenol, respectively. This experiment provides additional validation that leptomycin increases the stability of β1-AR mRNAs, and that the decreased β1-AR mRNA stability may be moderated by cell type.

Leptomycin B inhibition of CRM 1 nucleocytoplasmic export in DDT1MF2 transfectants and in neonatal rat cortical neurons results in significant retention of β1-AR mRNAs in the nuclear fraction

Using matched parallel cultures, we have determined that leptomycin B treatment of DDT1MF2 tranfectants and neonatal rat cortical neurons result in an enhanced retention of β1-AR mRNAs in the nuclear fraction (10-fold increase for leptomycin-treated DDT1MF2 tranfectants compared to untreated controls; 3-fold increase for leptomycin-treated neonatal rat cortical neurons compared to untreated controls) (Figure 3, Panel D and Figure 4, Panel D). This experiment supports the contention that leptomycin B inhibits the nucleocytoplasmic export of β1-AR mRNAs in DDT1MF2 tranfectants ectopically expressing β1-ARs and in the neonatal rat cortical neurons. We believe that the leptomycin-induced nuclear retention of the β1-AR mRNAs was greatly enhanced in the DDT1MF2 transfectants, because of the much higher levels of CRM 1 in this cell line (see Figure 7 for comparison). This observation is consistent with the overall model implicating the role of CRM 1 in the nuclear export and subsequent down-regulation of the β1-AR mRNAs (see below). In addition, for the DDT1MF2 transfectants, the observed decrease (25%) in β1-AR mRNA levels following isoproterenol treatment, is statistically significant (p = 0.00004; Figure 3); note that the standard error bars for the histograms displaying the isoproterenol-treated and untreated controls, are nearly indistinguishable from the height of the histograms and accounts for the very high statistical significance. Also, as expected, these observations are very similar to the data displayed in Figure 2, Panels A and B; we determined that the difference between β1-AR mRNA levels in isoproterenol-treated and untreated controls in this experiment was also significant (p value = 0.0015). However, for the neonatal rat cortical neurons, the β1-AR mRNA levels in the isoproterenol-treated and untreated controls are not significantly different (Figure 4, Panels A and B; p = 0.551). We have previously compared β1-AR mRNA levels in C6 cells, L6 cells, and DDT1MF2 transfectants during steady state conditions and following chronic isoproterenol treatment. As described in our prior publication (7), steady-state β1-AR mRNA levels in the DDT1MF2 transfectants were approximately 3 fold higher than corresponding levels in C6 cells, and the isoproterenol-induced reduction in the latter cells was small. Our RNase protection experiments indicate that the steady-state β1-AR mRNA levels in the neonatal rat cortical neurons were approximately 15% lower than the β1-AR mRNA levels exhibited in C6 cells. It should be noted here that in cells exhibiting low steady state levels of β1-AR mRNAs, such as the neonatal rat cortical neurons, measurement of β1-AR mRNA stability, and not absolute levels of β1-AR mRNAs, would be a more sensitive determination of post-transcriptional β1-AR regulation. As noted in Figure 1, we have identified significant differences in the stability of the β1-AR mRNAs in the neonatal rat cortical neurons following isoproterenol treatment.

Figure 3.

Figure 3

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β1-AR mRNA levels in DDT1MF2 transfectants: Total RNA (Panels A and B) or nuclear RNA (Panel C and D). Panels A and C depict representative RNase protection experiments. For both experiments, DDT1MF2 transfectants were treated independently with isoproterenol (10 μg/ml, 24 hours) or leptomycin (10 ng/ml, 24 hours), or concurrently with both isoproterenol (10 μg/ml, 24 hours) and leptomycin (10 ng/ml, 24 hours), or untreated. Degraded RNA in RNase protection assays would be evident as smeared bands and/or greatly diminished signals. The cyclophilin band is highly resolved and of consistent intensity among all RNA preparations. These characteristics are indicative of the high integrity of the RNA preparation in both the total cell and nuclear fraction extractions. Panels B and D: Normalization analyses of β1-AR mRNA levels. Gels were subjected to phosphorimager analyses using the BioRad Molecular Imager. Normalization calculations of β1-AR mRNA levels against corresponding cyclophilin mRNA levels were conducted. Panels B and D depicts statistical analyses of n=4 experiments. Error bars represent one standard error. Note that the standard error bars for the histograms displaying the isoproterenol-treated and untreated controls are nearly indistinguishable from the height of the histograms. The observed decrease (25%) in total β1-AR mRNA levels following isoproterenol treatment is statistically significant (p = 0.00004; see single asterisk in Panel B). The observed decrease (19%) in nuclear β1-AR mRNA levels following isoproterenol treatment is also statistically significant (p = 0.00025; see double asterisk in Panel D).

Figure 4.

Figure 4

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β1-AR mRNA levels in neonatal rat cortical neurons: Total RNA (Panels A and B) or nuclear RNA (Panel C and D). Panels A and C depict representative RNase protection experiments. For both experiments, neonatal rat cortical neurons were treated independently with isoproterenol (10 μg/ml, 24 hours) or leptomycin (10 ng/ml, 24 hours), or concurrently with isoproterenol (10 μg/ml, 24 hours) and leptomycin (10 ng/ml, 24 hours), or untreated. Panels B and D: Normalization analyses of β1-AR mRNA levels. Gels were subjected to phosphorimager analyses using the BioRad Molecular Imager. Normalization calculations of β1-AR mRNA levels against corresponding cyclophilin mRNA levels were conducted. Panels B and D depicts statistical analyses of n=4 experiments. Error bars represent one standard error.

Figure 7.

Figure 7

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Glycerol gradient centrifugation and immunoblot analysis using HuR (Panel A) and CRM 1 (Panel B) antibodies. Cell extracts known to preserve protein complexes were prepared. The C6 cell line was chosen as the source of extract for this experiment because of its endogenous and natural expression of the β1-AR mRNAs and the moderately robust level of expression of HuR and CRM 1 that enables detection of protein complexes. We have chosen not to conduct the glycerol gradient centrifugation experiment with neonatal rat cortical neurons because of the low level of expression of CRM 1 and the low level of recovery of HuR – CRM 1 protein complexes from these sources. C6 cells were extracted with 5 ml lysis buffer containing 25 mM HEPES-KOH, pH 8.0, 150 mM KCl, 2 mM EDTA, 1 mM DTT, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 μg/ml pepstatin A, 20 mM NaF, and 0.1% NP-40. Extracts were clarified by centrifugation at 40,000 rpm (SW 50.1 rotor, Beckman) for 40 minutes. The supernatant was loaded onto 4 ml 5-20% glycerol gradients and centrifuged at 40,000 rpm (SW 50.1 rotor, Beckman) for 12 hours. Aliquots of the gradient fractions were subjected to polyacrylamide gel electrophoresis under nondenaturing conditions. Blots of the nondenaturing gels were sequentially probed with HuR (Panel A) and CRM 1 (Panel B) antibodies. The bottom panel was cropped to focus exclusively on the cross-immunoreactivity of the 110 kDa+ band with CRM 1 antibodies.

UV-crosslinking/immunoblot analysis verifies that the rat β1-AR 3' UTR interacts with the RNA binding protein HuR obtained from neonatal rat cortical neurons

Using extracts from neonatal rat cortical neurons, C6 cells, and DDT1MF2 transfectants ectopically expressing rat β1-ARs, we have demonstrated that all three extracts contain electrophoretically-equivalent proteins that recognize the rat β1-AR 3' UTR probe (Figure 5). As described in part in our previous publication (7) and by conducting additional immunoblot analysis, we have demonstrated that one of these molecules is the RNA binding protein HuR. HuR is contained in all three extracts, including the neonatal rat cortical neuron extract, and is recognized by the rat β1-AR 3' UTR probe. Interestingly, in the neonatal rat cortical neuron extract, additional molecules immunologically-related to HuR have been identified with our HuR antibody, and also appear to be recognized by the β1-AR 3' UTR probe. In the central nervous system, Hu genes encode several alternatively-spliced transcripts from distinct genes (HuB, HuC, and HuD) that produce immunologically-related proteins with specific developmental patterns of expression (45). We conjecture that the additional HuR-related molecules detected in rat cortical neurons with our HuR antibodies represent products derived from one of the Hu genes or from an alternatively spliced derivative. HuR interaction with the β1-AR 3' UTR probe has also been verified using UV-crosslinking / immunoprecipitation analysis, as displayed in our previous publication (7). Using a control β1-AR probe from positions −408 to +323 (comprising a portion of the promoter and 5' coding sequence), we demonstrated the absence of HuR immunoreactive proteins when used with extracts from C6 cells and DDT1MF2 transfectants in the UV-crosslinking assay (7). This experiment provides additional supporting evidence illustrating the importance of the β1-AR 3' UTR sequence in binding HuR and other molecules involved in the post-transcriptional control of the β1-AR mRNAs.

Figure 5.

Figure 5

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UV-crosslinking / immunoblot analyses of RNA-protein complexes. RNA-protein complexes containing the β1-AR probe (β1-AR sequence extending from +2084 to +2901) and extracts from C6 cells, DDT1MF2 transfectants, or neonatal rat cortical neurons (25 μg in all cases) were prepared and subjected to UV-crosslinking. Control protein extracts were also electrophoresed in parallel in polyacrylamide gels, and subsequently electrotransferred to nitrocellulose membranes for phosphorimager analyses. Blots were subsequently immunostained with HuR antibodies. Panel A shows phosphorimager analyses of UV-crosslinked proteins. Panel B shows HuR immunoblot detection of proteins in control cell extracts and UV-crosslinked protein complexes.

Interestingly, when immunoblot analysis was conducted using immunoprecipitated proteins that were previously UV-crosslinked to the β1-AR 3' UTR probe, we identified an additional HuR immunoreactive protein of 70 kDa that recognized the β1-AR 3' UTR probe (Figure 6). Note that the data displayed in Figures 5 and 6 originate from the experiment, and that the immunoblot experiment displayed in Figure 5 is the same experiment displayed in the left portion of Figure 6. The right portion of Figure 6, Panel A, contains an additional immunoblot experiment using a HuR antibody overlay on proteins UV-crosslinked to the β1-AR 3' UTR probe and immunoprecipitated with HuR antibodies prior to loading in SDS-PAGE. We conjecture that the detection of the 70 kDa protein using immunoblot analysis of immunoprecipitated UV-crosslinked proteins (and not using immunoblot analysis of UV-crosslinked proteins alone, as in Figure 5) is the result of the enhanced concentration of HuR-immunoreactive material following immunoprecipitation. Also, as shown in Figure 6, Panel B, with an over-exposure of the immunoblot analysis using immunoprecipitations with 4 μl of HuR antibody and UV-crosslinked proteins from neonatal rat cortical neurons (also as shown in Figure 6, Panel A, far right lanes), we readily detect the interaction of the additional Hu-related proteins with the β1-AR 3' UTR probe. The 70 kDa protein may represent an HuR dimer that becomes crosslinked during UV treatment with the β1-AR 3' UTR probe. The potential for HuR heterodimer formation is high, especially since HuR is known to interact with the HuR ligands, pp32 and APRIL, and is known to form the βARB complex composed of HuR and hnRNP A1.

Figure 6.

Figure 6

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Immunoblot analyses of RNA-protein complexes immunoprecipitated with HuR antibodies prior to electrophoresis. RNA-protein complexes containing the β1-AR probe (β1-AR sequence extending from +2084 to +2901) and extracts from C6 cells, DDT1MF2 transfectants, and neonatal rat cortical neurons (25 μg in all cases) were prepared and subjected to UV-crosslinking. UV-crosslinked RNA-protein complexes were subjected to immunoprecipitation using either 1 μl or 4 μl of HuR antibodies. Control protein extracts were also electrophoresed in parallel in polyacrylamide gels, and subsequently electrotransferred to nitrocellulose membranes. Blots were also subsequently immunostained with HuR antibodies. Panel A complete immunoblot analysis displaying control protein extracts, UV-crosslinked proteins, and immunoprecipitations using either 1 μl or 4 μl of HuR antibodies. Note that the experiment displayed in the left portion of Panel A is the same experiment displayed in the immunoblot panel in Figure 5, and comprise identical images. Panel B contains an overexposed image of the far right lanes in Panel A, and illustrates the interaction of the Hu-related proteins with the β1-AR probe.

Glycerol gradient centrifugation and sequential immunoblot analyses identify high molecular weight HuRimmunoreactive protein complexes in C6 cells

We have conducted extraction and fractionation procedures known to preserve RNA-protein complexes and have electrophoresed gradient fractions in nondenaturing gels. This extraction and glycerol gradient fractionation procedure was successfully utilized by Gallouzi et al. (46), in conjunction with immunoprecipitation, to verify interaction of HuR with the four HuR ligands, pp32, APRIL, SETα, and SETβ. Using the extraction and fractionation procedure with immunoblot analyses, we have determined that complex formation occurs between HuR and other molecules in the C6 cell line. Using HuR antibodies in the immunoblot analyses, we observe the 33-35 kDa HuR molecule in the upper portions of the glycerol gradient, along with HuR immunoreactive bands of 110+ kDa and 70 kDa, indicating multimer formation of HuR and/or interaction of HuR with other molecules (Figure 7, Top Panel). Our HuR antibody has defined immunoreactivity against the predominant HuR molecule with molecular weight of 33-35 kDa (7). We conjecture that the HuR immunoreactive band of 110+ kDa was visualized in this experiment, and not in the immunoblot analysis displayed in Figure 6, because of the preservation of native complexes isolated in the glycerol gradient fractionation and the use of nondenaturing gels. Interestingly, when the blot was reprobed using CRM 1 antibodies, the 110+ kDa band was also CRM 1-immunoreactive, indicating that this high molecular weight band may potentially be a protein complex composed of HuR and CRM 1 (Figure 7, Bottom Panel). As based on information from both Figure 6 and 7, we conjecture that the 70 kDa molecule could represent HuR dimers or HuR complexes containing other proteins, potentially HuR ligands pp32 or APRIL, both of which have defined molecular weight of 31-33 kDa. The composite molecular weight of complexes containing HuR and pp32 or APRIL would be consistent with the relative molecular weight of the 70 kDa molecule observed in the middle portion of the glycerol gradient.

UV-crosslinking/immunoblot analysis indicates that the β1-AR 3' UTR recognize HuR - CRM 1 protein complexes

The β1-AR 3' UTR recognizes molecules of 33-35 kDa, doublet proteins of 70-75 kDa, and a high molecular weight molecule of 110 kDa, that are a ll immunoprecipated using HuR antibodies (Figure 8, Left Panel). Proteins tagged with the β1-AR 3' UTR radioactive probe were not immunoprecipitated using normal rabbit serum (Figure 8, Left Panel). We have previously identified the 33-35 kDa molecule as HuR (7; also see Figure 5). CRM 1 immunoblot analysis of control extracts alone (Figure 8, Right Panel, left lane) identifies a predominant band of 110 kDa, consistent with the calculated molecular weight of 105 kDa for CRM 1, and a second minor component of 70 kDa. Both molecules appear to be electrophoretically equivalent to the 110 kDa and 70-75 kDa proteins that are immunoprecipitated using HuR antibodies and subsequently immunodetected by CRM 1 antibodies (Figure 8, Right Panel, right lane). We conjecture that at least one of the 70-75 kDa proteins identified in this UV crosslinking / immunoprecipitation analysis may be equivalent to the 70 kDa HuR complex identified in the glycerol gradient centrifugation experiment (see Figure 7). Interestingly, at least one of 70-75 kDA molecules retains immunoreactivity to CRM 1 antibodies; we hypothesize that the C6 cell line may have other CRM 1-related molecules that retain the ability to form complexes with HuR and with other HuR ligands. This experiment provides further evidence that the 110 kDa band is an HuR - CRM 1 protein complex and that this complex recognizes the β1-AR 3' UTR.

Figure 8.

Figure 8

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UV-crosslinking / immunoprecipitation analyses of RNA-protein complexes. RNA-protein complexes containing the β1-AR probe (β1-AR sequence extending from +2084 to +2901) and C6 extracts (25 μg) were prepared and subjected to UV-crosslinking and immunoprecipitation using HuR antibodies or normal rabbit serum (NRS) control. Immunoprecipitated proteins and control protein extracts were subjected to PAGE and subsequently electrotransferred to nitrocellulose membranes for phosphorimager analyses. Blots were also subsequently immunostained with CRM 1 antibodies. Panel A shows phosphorimager analyses of immunoprecipitated UV-crosslinked proteins. Panel B shows CRM 1 immunoblot detection of proteins in control cell extracts and UV-crosslinked protein complexes previously immunoprecipitated with HuR antibodies prior to electrophoresis.

Immunoblot analyses indicate that neonatal rat cortical neurons contain lower levels of CRM 1

Using CRM 1-specific antibodies and immunoblot analyses, we have determined that neonatal rat cortical neurons contain lower levels of CRM 1 compared to the DDT1MF2 cell line that ectopicallly expresses β1-ARs (Figure 9). The lower levels of CRM 1 and the reduced capacity to export ARE-containing mRNAs to the cytoplasm appears to correlate with the extended β1-AR mRNA half-life in neonatal rat cortical neurons compared to DDT1MF2 transfectants.

Figure 9.

Figure 9

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CRM 1 levels in neonatal rat cortical neurons and DDT1MF2 transfectants ectopically expressing β1-ARs. Cellular extracts (25 μg) were subjected to polyacrylamide gel electrophoresis, electrotransfer to nitrocellulose membranes, and immunoblot analyses using CRM 1 antibodies (provided by Dr. Gerard Grosveld). Equivalent loading of lanes were verified by Comassie blue staining of residual prote ins retained in the polyacrylamide gel following electrotransfer of the majority of the protein constituents.

Use of nuclear export receptor inhibitors provides evidence that CRM 1 is an important determinant in the agonist-mediated down-regulation of β1-AR mRNAs

To help determine that CRM 1 plays a distinct role in β1-AR mRNA down-regulation, we used cell-permeable peptides comprising the leucine-rich nuclear export signal (NES) contained in the HuR ligands pp32 and APRIL, and recognized by CRM 1. We also utilized peptides containing the HNS nuclear export sequence, to serve as in vivo inhibitors of HuR interaction with nuclear receptor importin β, and also scrambled or mutant forms of the HNS or NES peptides (scrambled HNS [S-HNS] or mutant NES [M-NES]) that have been demonstrated to not interact with their respective nuclear receptors. The nuclear receptor inhibitors appear to affect both the export and import pathways of the ARE-containing heat shock mRNAs. We used the DDT1MF2 transfectants for this experiment because of its resistance to the mild cytotoxic effects of the cell permeable inhibitory peptides documented for the heat shock system (34) and because of the more abundant quantities of CRM 1 present in this cell line compared to neonatal rat cortical neurons; we anticipated that CRM 1 inhibition in the DDT1MF2 transfectants would elicit a more pronounced and measurable effect on β1-AR mRNA down-regulation. Interestingly, when the DDT1MF2 transfectants are treated with the HNS inhibitor, we observe a 30% reduction in β1-AR mRNA levels; this reduction in β1-AR mRNA levels is reversed when using S-HNS (Figure 10, Panel D). When examining β1-AR mRNA levels in cells treated with the HNS peptide inhibitor, the best controls for comparison would be cells treated with the scrambled HNS (S-HNS) peptide. Unlike the untreated controls, the S-HNS treatment group would mimic all conditions in the HNS treatment group, and would control for ancillary effects due to cell entry of the peptides. The β1-AR mRNA levels between the HNS and S-HNS treatment groups are statistically significant (p = 0.072). There appears to be a no significant reduction in β1-AR mRNA levels following inhibition with NES peptides (Figure 10, Panel D) and treatment with M-NES does not induce e ffects on β1-AR mRNA levels that are different from corresponding levels in the untreated controls (p = 0.313 for comparison of data group using NES peptides versus M-NES peptides). Interestingly, the lack of measurable effects of the NES peptide on β1-AR mRNA levels in the DDT1MF2 transfectants appears to be inconsistent with the observed increase in β1-AR mRNA levels following leptomycin treatment (Figure 2). The mechanisms of action of leptomycin and NES peptide inhibition may not be identical. NES treatment specifically targets the interaction of CRM 1 with the NES signals contained in the HuR ligands. Alternatively, leptomycin binds to CRM 1 and allosterically displaces NES interaction from the CRM 1 nuclear export complex (47). Leptomycin also induces cell cycle arrest and apoptosis in certain cancer cells (48). Hence, while the CRM 1 target molecule for NES peptide inhibition and leptomycin inhibition may be the same, the resultant functional effects may potentially differ. When the DDT1MF2 transfectants are concurrently treated with isoproterenol and the peptide inhibitors, only the co-addition of the NES inhibitor significantly reversed the isoproterenol-induced reduction of β1-AR mRNA levels to levels observed for the untreated controls (Figure 10, Panel B) (p = 0.0004 for comparison of isoproterenol data group and isoproterenol plus NES peptide data group). This observation is consistent with the results obtained from the leptomycin inhibition experiments.

Figure 10.

Figure 10

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Use of nuclear export inhibitor peptides. DDT1MF2 transfectants expressing β1-ARs were treated with peptide inhibitors either individually (Panels C and D) or concurrently with isoproterenol (Panels A and B) for 24 hours. Panels A and C illustrate single representative RNase protection experiments for individual inhibitor (Panel C) or concurrent inhibitor + isoproterenol treatments (Panel A). β1-AR mRNA and cyclophilin mRNA levels were quantitated with the use of the BioRad Molecular Imager. All β1-AR mRNA measurements were normalized against cyclophilin mRNA levels; mRNA intensity ratios for the nontreated controls were assigned a value of 1.0. Panels D and B contain statistical analyses of RNase protection assay results for 3-4 culture dishes treated with either the individual inhibitors or concurrently with inhibitor plus isoproterenol, respectively. Error bars depict the range of one standard error unit. Histograms without apparent placement of error bars contain close sample values, with error bars nearly indistinguishable from the height of the histogram.

Interestingly, this use of the NES / CRM 1-mediated pathway for β1-AR mRNAs following isoproterenol induction is similar to the use of the CRM 1-mediated pathway observed for hsp70 mRNAs following heat shock (33). Our experiment provides evidence that HuR and its interaction with CRM 1 are important determinants in the agonist-mediated down-regulation of β1-AR mRNAs, and that the use of this export pathway may be enhanced compared to the export pathways used for agonist-independent β1-AR mRNA decay. Wang et al. (49) have also utilized similar inhibitory peptides to demonstrate that HuR-mediated mRNA stabilization of cytokine mRNAs is controlled at the level of HuR nuclear export and is a critical determinant of T cell activation.

CRM 1-mediated degradation of β1-AR mRNAs

We provide evidence that the nuclear export receptor CRM 1 moderates the steady state and agonist-mediated degradation of the β1-AR mRNAs in neonatal rat cortical neurons and in established cell lines. Leptomycin or peptide inhibition of CRM 1-mediated nuclear export result in significant increases of β1-AR mRNA levels and stability and enhanced retention in the nucleus (Figure 11). While β1-AR transcripts would have increased stability during the inhibition of CRM 1-mediated nuclear export, its retention in the nucleus may ultimately lead to decreased availability in the cytoplasm and resultant reduction in synthesis of β1-ARs. Our UV-crosslinking / immunoprecipitation analyses and glycerol gradient fractionation experiments provide evidence that HuR forms complexes with other molecules, including CRM 1, and that these complexes are recognized by the β1-AR 3' UTR. β1-AR mRNAs are regulated at the transcriptional, post-transcriptional, and translational levels, and RNA binding proteins that recognize AU-rich sequences in the 3' UTR can moderate nuclear export, mRNA stability, and translational efficiency (11, 30, 34). AUF-1 has been identified as an RNA degradation protein found in the cytoplasm that recognizes the human β1-AR 3' UTR. We conjecture that the β1-AR mRNAs are exported to the cytoplasm following agonist induction and that these mRNAs may be degraded in the cytoplasm by interaction with AUF 1 (Figure 11). mRNA stability and translation of ARE- containing mRNAs a re linked regulatory pathways. These pathways appear to be commonly dependent on the interaction of RNA binding proteins with AU-rich mRNAs that promote export to the cytoplasm, the predominant site of mRNA decay and suppression of translation.

Figure 11.

Figure 11

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CRM 1-mediated degradation of β1-AR mRNAs. Inset box denotes enlargement of the nuclear – cytoplasmic interface, and the nucleocytoplasmic export and agonist-mediated down-regulation of the β1-AR mRNAs.

ACKNOWLEDGEMENTS

We acknowledge C. Elisabeth Campbell and Madhavi Kondapalli for technical assistance during various stages of this project. We thank Drs. Steve Matsumoto and Agnieszka Balkowiec for help in the development of neonatal rat cortical neuron cultures in our laboratory, and for additional insights in the propagation of cortical neurons, respectively. We also thank Dr. Minoru Yoshida (RIKEN, Japan) for his generous gifts of leptomycin B, Drs. Henry Furneaux and Gerard Grosveld for the gifts of HuR and CRM 1 antibodies, respectively, Dr. Myriam Gorospe for advice concerning the nuclear fractionation procedures, and Dr. Imed Gallouzi for advice concerning the use of the cell permeable peptides and for his reading of the manuscript. Special thanks are extended to Philbert Kirigititi and Xiaorong Li for support and discussion during the early development of this project, and to Susan Bond for assistance in the development of Figure 11. We also thank Drs. Tom Shearer and Jack Clinton for their overall support of the program. We acknowledge the OHSU Molecular Microbiology and Immunology Molecular Biology Core Facility, the Oregon Cancer Institute Cell Culture Core Facility, and the Yale University Peptide Synthesis Facility for their assistance in automated DNA sequencing, cell culture support, and peptide synthesis respectively.

Footnotes

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This research was supported by NIH MH 63137, and by the National American Heart Association (AHA) grant 0250017N. CAM was also a former recipient of an AHA Established Investigator Award and was a recipient of the 2003 Dean's Research Grant Award from the OHSU School of Dentistry.

REFERENCES

  • 1.Beelman CA, Parker R. Degradation of mRNA in eukaryotes. Cell. 1995;81:179–183. doi: 10.1016/0092-8674(95)90326-7. [DOI] [PubMed] [Google Scholar]
  • 2.Mitchell P, Tollervey D. mRNA stability in eukaryotes. Curr. Opin. Genet. Dev. 2000;10:193–198. doi: 10.1016/s0959-437x(00)00063-0. [DOI] [PubMed] [Google Scholar]
  • 3.Ross J. mRNA stability in mammalian cells. Microbiol. Rev. 1995;59:423–450. doi: 10.1128/mr.59.3.423-450.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Shim J, Lim H, Yates JR, Karin M. Nuclear export of NF90 is required for interleukin-2 mRNA stabilization. Mol. Cell. 2002;10:1331–1344. doi: 10.1016/s1097-2765(02)00730-x. [DOI] [PubMed] [Google Scholar]
  • 5.Hadcock JR, Wang H-Y, Malbon CC. Agonist-induced destabilization of β-adrenergic receptor mRNAs. J. Biol. Chem. 1989;264:19928–19933. [PubMed] [Google Scholar]
  • 6.Port JD, Huang L-Y, Malbon CC. β-adrenergic agonists that down-regulate receptor mRNA up-regulate a Mr 35,000 Protein(s) that selectively binds to β-adrenergic receptor mRNAs. J. Biol. Chem. 1992;267:24103–24108. [PubMed] [Google Scholar]
  • 7.Kiriigiti P, Bai Y, Yang Y-F, Li X, Li B, Brewer G, Machida CA. Agonist-mediated down-regulation of rat β1-adrenergic receptor transcripts: role of potential post-transcriptional degradation factors. Mol Pharmacol. 2001;60:1308–1324. doi: 10.1124/mol.60.6.1308. [DOI] [PubMed] [Google Scholar]
  • 8.Huang L-Y, Tholanikunnel BG, Vakalopoulou E, Malbon CC. The Mr 35,000 β-adrenergic receptor mRNA-binding protein induced by agonists requires both an AUUUA pentamer and U-rich domains for RNA recognition. J. Biol. Chem. 1993;268:25769–25775. [PubMed] [Google Scholar]
  • 9.Pende A, Tremmel KD, DeMaria CT, Blaxall BC, Minobe, Sherman JA, Bisognano JD, Bristow MR, Brewer G, Port JD. Regulation of the mRNA-binding protein AUF 1 by activation of the β-adrenergic receptor signal transduction pathway. J. Biol. Chem. 1986;271:8493–8501. doi: 10.1074/jbc.271.14.8493. [DOI] [PubMed] [Google Scholar]
  • 10.Subramaniam K, Chen K, Joseph K, Raymond JR, Tholanikunnel BG. The 3' untranslated region of the β2-adrenergic receptor mRNA regulates receptor synthesis. J. Biol. Chem. 2004;279:27108–27116. doi: 10.1074/jbc.M401352200. [DOI] [PubMed] [Google Scholar]
  • 11.Kandasamy K, Joseph D, Subramaniam K, Raymond JR, Tholanikunnel BG. Translational control of β2-adrenergic receptor mRNA by T-cell-restricted intracellular antigen-related protein. J. Biol. Chem. 2005;280:1931–1943. doi: 10.1074/jbc.M405937200. [DOI] [PubMed] [Google Scholar]
  • 12.Danner S, Frank M, Lohse MJ. Agonist regulation of human β2-adrenergic receptor mRNA stability occurs via a specific AU-rich element. J. Biol. Chem. 1998;273:3223–3229. doi: 10.1074/jbc.273.6.3223. [DOI] [PubMed] [Google Scholar]
  • 13.Tholanikunnel BG, Malbon CC. A 20-nucleotide (A+U)-rich element of β2-adrenergic receptor (β2-AR) mRNA mediates binding to the β2-AR-binding protein and is obligate for agonist-induced destabilization of receptor mRNA. J. Biol. Chem. 1997;272:11471–11478. doi: 10.1074/jbc.272.17.11471. [DOI] [PubMed] [Google Scholar]
  • 14.Tholanikunnel BG, Raymond JR, Malbon CC. Analysis of the AU-rich elements in the 3' untranslated region of the β2-adrenergic receptor mRNA by mutagenesis and idenfification of the homologous AU-rich region from different species. Biochem. 1999;38:15564–15572. doi: 10.1021/bi9913348. [DOI] [PubMed] [Google Scholar]
  • 15.Mitchusson KD, Blaxall BC, Pende A, Port JD. Agonist-mediated destabilization of human beta 1-adrenergic receptor mRNA: Role of the 3' untranslated region. Biochem Biophys Res Comm. 1998;252:357–362. doi: 10.1006/bbrc.1998.9598. [DOI] [PubMed] [Google Scholar]
  • 16.Blaxall BC, Pellett AC, Wu SC, Pende A, Port JD. Purification and characterization of beta-adrenergic receptor mRNA-binding proteins. J. Biol. Chem. 2000;274:4290–4297. doi: 10.1074/jbc.275.6.4290. [DOI] [PubMed] [Google Scholar]
  • 17.Dahlberg JE, Lund E. Functions of GTPase Ran in RNA export from the nucleus. Curr. Opin. Cell Biol. 1998;10:400–408. doi: 10.1016/s0955-0674(98)80017-3. [DOI] [PubMed] [Google Scholar]
  • 18.Rodriquez MS, Dargemont C, Stutz F. Nuclear export of RNA. Biol. Cell. 2004;96:639–655. doi: 10.1016/j.biolcel.2004.04.014. [DOI] [PubMed] [Google Scholar]
  • 19.Antonin W, Mattaj IW. Nuclear pore complexes: round the bend? Nat. Cell Biol. 2005;7:10–12. doi: 10.1038/ncb0105-10. [DOI] [PubMed] [Google Scholar]
  • 20.Naklielny S, Dreyfuss G. Import and export of the nuclear pore protein receptor transportin by a mechanism independent of GTP hydrolysis. Curr. Biol. 1998;15:89–95. doi: 10.1016/s0960-9822(98)70039-9. [DOI] [PubMed] [Google Scholar]
  • 21.Cullen BR. Nuclear RNA export. J. Cell Sci. 2003;116:587–597. doi: 10.1242/jcs.00268. [DOI] [PubMed] [Google Scholar]
  • 22.Kutay U, Guttinger S. Leucine-rich nuclear-export signals: born to be weak. Trends Cell Biol. 2005;15:121–124. doi: 10.1016/j.tcb.2005.01.005. [DOI] [PubMed] [Google Scholar]
  • 23.Erkmann JA, Kutay U. Nuclear export of mRNA: from site of transcription to the cytoplasm. Exp. Cell Res. 2004;296:12–20. doi: 10.1016/j.yexcr.2004.03.015. [DOI] [PubMed] [Google Scholar]
  • 24.Huang Y, Gattoni R, Stevenin J, Steitz JA. SR splicing factors serve as adaptor proteins for TAP-dependent mRNA export. Mol Cell. 2003;11:837–843. doi: 10.1016/s1097-2765(03)00089-3. [DOI] [PubMed] [Google Scholar]
  • 25.Huang Y, Steitz J. Splicing factors SRp20 and 9G8 promote the nucleocytoplasmic export of mRNA. Mol. Cell. 2001;7:899–905. doi: 10.1016/s1097-2765(01)00233-7. [DOI] [PubMed] [Google Scholar]
  • 26.Cazalla D, Zhu J, Mache L, Huber E, Krainer AR, Caceres JF. Nuclear export and retention signals in the RS domain of SR proteins. Mol Cell. Biol. 2002:6871–6782. doi: 10.1128/MCB.22.19.6871-6882.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Brennan CM, Steitz JA. HuR and mRNA stability. Cell. Mol. Life Sci. 2001;58:266–277. doi: 10.1007/PL00000854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Wang W, Yang X, Kawai T, Lopez de Silanes I, Mazan-Mamczarz K, Chen P, Chook YM, Quensel C, Kohler M, Gorospe M. AMP-activated protein kinase-regulated phosphorylation and acetylation of importin α1. J. Biol. Chem. 2004;279:48376–48388. doi: 10.1074/jbc.M409014200. [DOI] [PubMed] [Google Scholar]
  • 29.Fan XC, Steitz JA. HNS, a nuclear-cytoplasmic shuttling sequence in HuR. Proc. Natl. Acad. Sci. USA. 1998;95:15293–15298. doi: 10.1073/pnas.95.26.15293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Kawai T, Lal A, Yang X, Galban S, Mazan-Mamczarz K, Gorospe M. Translation control of cytochrome c by RNA binding proteins TIA-1 and HuR. Mol Cell. Biol. 2006;26:3295–3307. doi: 10.1128/MCB.26.8.3295-3307.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Brennan CM, Gallouzi I-E, Steitz JA. Protein ligands to HuR modulate its interaction with target mRNAs invivo. J. Cell Biol. 2000;151:1–13. doi: 10.1083/jcb.151.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Santa-Coloma TA. Anp32e (Cpd1) and related protein phosphatase 2 inhibitors. Cerebellum. 2003;2:310–320. doi: 10.1080/14734220310017212. [DOI] [PubMed] [Google Scholar]
  • 33.Gallouzi I-E, Brennan CM, Steitz JA. Protein ligands mediate the CRM1-dependent export of HuR in response to heat shock. RNA. 2001;7:1348–1361. doi: 10.1017/s1355838201016089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Gallouzi I-E, Steitz JA. Delineation of mRNA export pathways by the use of cell-permeable peptides. Science. 2001;294:1895–1901. doi: 10.1126/science.1064693. [DOI] [PubMed] [Google Scholar]
  • 35.Izaurralde E, Jarmolowski A, Beisel C, Mattaj IW, Dreyfuss G, Fischer U. A role for the M9 transport signal of hnRNP A1 in mRNA nuclear export. J. Cell Biol. 1997;137:27–35. doi: 10.1083/jcb.137.1.27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Izaurralde E. Directing mRNA export. Nat. Struct. Mol. Biol. 2004;11:210–212. doi: 10.1038/nsmb0304-210. [DOI] [PubMed] [Google Scholar]
  • 37.Rebane A, Aab A, Steitz JA. Tranportins 1 and 2 are redundant nuclear import factors for hnRNP A1 and HuR. RNA. 2004;10:590–599. doi: 10.1261/rna.5224304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Guttinger S, Muhlhausser P, Koller-Eichhom P, Brennencke J, Kutay U. Tranportin 2 functions as an importin and mediates nuclear import of HuR. Proc. Natl. Acad. Sci. USA. 2004;101:2918–2923. doi: 10.1073/pnas.0400342101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Kudo N, Wolff B, Sekimoto T, Schreiner EP, Yoneda Y, Yanagida M, Horinouchi S, Yoshida S,M. Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1. Exp. Cell Res. 1998;241:540–547. doi: 10.1006/excr.1998.4136. [DOI] [PubMed] [Google Scholar]
  • 40.Goforth PB, Ellis EF, Satin LS. Enhancement of AMPA-mediated current after traumatic injury in cortical neurons. J. Neurosci. 1999;19:7367–7374. doi: 10.1523/JNEUROSCI.19-17-07367.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Yang Y-F, Machida CA. Ribonuclease protection assay for the detection of β1-adrenergc receptor RNA. In: Machida CA, editor. Adrenergic Receptor Protocols. Humana Press; Totowa, New Jersey: 2000. [DOI] [PubMed] [Google Scholar]
  • 42.Kirigiti P, Machida CA. Electrophoretic mobility shift assay for detection of DNA binding proteins recognizing beta-adrenergic receptor gene sequences. In: Machida CA, editor. Adrenergic Receptor Protocols. Humana Press; Totowa, New Jersey: 2000. [DOI] [PubMed] [Google Scholar]
  • 43.Kirigiti P, Yang Y-F, Li X, Li B, Midson CN, Machida CA. Rat β1-adrenergic receptor regulatory region containing consensus AP-2 elements recognizes novel transactivator proteins. Mol. Cell. Biol. Res. Comm. 2000;3:181–192. doi: 10.1006/mcbr.2000.0212. [DOI] [PubMed] [Google Scholar]
  • 44.Gu W, Shi XL, Roeder RG. Synergistic activation of transcription by CBP and p53. Nature. 1997;387:819–823. doi: 10.1038/42972. [DOI] [PubMed] [Google Scholar]
  • 45.Wakamatsu Y, Weston JA. Sequential expression and role of Hu RNA-binding proteins during neurogenesis. Development. 1997;124:3449–3460. doi: 10.1242/dev.124.17.3449. [DOI] [PubMed] [Google Scholar]
  • 46.Gallouzi I-E, Brennan CM, Stenberg MG, Swanson MS, Eversole A, Maizels N, Steitz JA. HuR binding to cytoplasmic mRNA is perturbed by heat shock. Proc. Natl. Acad. Sci. USA. 2000;97:3073–3078. doi: 10.1073/pnas.97.7.3073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Yashiroda Y, Yoshida M. Nucleocytoplasmic transport of proteins as a target for therapeutic drugs. Curr. Med. Chem. 2003;10:741–748. doi: 10.2174/0929867033457791. [DOI] [PubMed] [Google Scholar]
  • 48.Jang BC, Paik JH, Jeong HY, Oh HJ, Park JW, Kwon TK, Song DK, Park JG, Kim SP, Bae JH, Mun KC, Yoshida M, Suh SI. Leptomycin B-induced apoptosis is mediated through caspase activation and down-regulation of Mcl-1 and XIAP expression, but not through the generation of ROS in U937 leukemia cells. Biochem. Pharmacol. 2004;68:263–274. doi: 10.1016/j.bcp.2004.03.007. [DOI] [PubMed] [Google Scholar]
  • 49.Wang JG, Collinge M, Rangolam V, Ayalon O, Fan XC, Pardi R, Bender JR. LFA-1-dependent HuR nuclear export and cytokine mRNA stabilization in T cell activation. J. Immunol. 2006;176:2105–2113. doi: 10.4049/jimmunol.176.4.2105. [DOI] [PubMed] [Google Scholar]

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