©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The M 35,000 -Adrenergic Receptor mRNA-binding Protein Binds Transcripts of G-protein-linked Receptors Which Undergo Agonist-induced Destabilization (*)

Baby G. Tholanikunnel (1)(§), James G. Granneman (2), Craig C. Malbon (1)

From the (1) Department of Pharmacology, Diabetes and Metabolic Diseases Research Program, University Medical Center, State University of New York, Stony Brook, New York 11794-8651 and the (2) Department of Psychiatry, Wayne State University, Detroit, Michigan 48201

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The M 35,000 -adrenergic receptor mRNA-binding protein, termed -ARB protein, is induced by -adrenergic agonists and binds to -receptor mRNAs that display agonist-induced destabilization. Recently a cognate sequence in the mRNA was identified that provides for recognition by -ARB protein. In the present work we test the ability of the -ARB to discriminate among G-protein-linked receptor mRNAs that either do or do not display agonist-induced destabilization and test the predictive value of the presence of the cognate sequence to identify receptors displaying post-transcriptional regulation. Transcripts of -, but not rat -, rat -, or human -adrenergic receptors bind -ARB protein, linking agonist-induced destabilization of mRNA to transcripts with the cognate sequence. Scanning GeneBank for G-protein-linked receptor transcripts with the cognate sequence revealed several candidates, including the thrombin receptor. We demonstrate that the thrombin receptor mRNA is recognized by -ARB protein and like the -receptor is regulated post-transcriptionally by agonist and cAMP. Thus, the domain of regulation by -ARB protein includes transcripts of G-protein-linked receptors other than -adrenergic receptors.


INTRODUCTION

Agonist-induced down-regulation of G-protein-linked receptors, like the -adrenergic receptor, provides an explanation for long-term adaptation to chronic stimuli characteristically observed for members of this receptor family (Collins et al., 1988, 1991; Hadcock and Malbon, 1988, 1991, 1993). For -adrenergic receptors, steady-state levels of the receptor and its mRNA decline following a challenge with agonist (Hadcock and Malbon, 1988; Collins et al., 1991). The basis for the decline in receptor mRNA induced by agonist is not transcriptional suppression, but rather post-transcriptional destabilization of receptor mRNAs (Hadcock et al., 1989b). Recently, we identified a M 35,000 protein with properties consistent with those expected for an RNA-binding protein selective for mRNAs of receptors which display agonist-induced down-regulation of their messages and protein expression. We termed this M 35,000 -adrenergic receptor mRNA-binding protein, -ARB protein (Port et al., 1992).

The steady-state levels of highly regulated mRNAs (e.g. mRNAs of granulocyte/macrophage colony-stimulating-factor, tumor necrosis factor-, and the oncogenes c-myc and c-fos) are markedly influenced by the rate of degradation (Shaw and Kamen, 1986; Brawerman, 1987, 1989; Raghow, 1987; Ross, 1988; Cleveland, 1988; Hargrove and Schmidt, 1989; Peltz et al., 1991). Regulation of mRNA stability and turnover is multifaceted, reflecting not only various cytosolic and nuclear-associated factors, and polyadenylation, but also cognate sequences of the 3`-untranslated regions (UTR)() of mRNA, such as the AUUUA pentamer (Brewer and Ross, 1988, 1989; Wreschner and Rechavi, 1988; Stolle and Benz, 1988; Pei and Calame, 1988; Schuler and Cole, 1988; Bernstein et al., 1989; Malter, 1989; Shyu et al., 1991; Brewer, 1991; Vakalopoulou et al., 1991; Wisdom and Lee, 1991; Chen et al.,1994; Chen and Shyu, 1994).

Several classes of RNA-binding proteins have been implicated in regulating mRNA stability and turnover. The heterogeneous, nuclear ribonucleoprotein particles participate in several steps of mRNA maturation including packaging, translocation, and splicing of heterogenous nuclear RNA (Dreyfuss, 1986; Swanson and Dreyfuss, 1988; Wilusz and Shenk, 1990). Splicing and further processing of pre-mRNAs possessing introns, a 5`-cap and 3`-poly(A) tract involves the small nuclear RNA-binding proteins (Steitz et al., 1983; Konarska and Sharp, 1987). Cytosolic mRNA-binding proteins include the M 72,000 poly(A)-binding protein, which binds to long stretches (25 nucleotides/protein) of poly(A) and stabilizes the RNA to 3` 5` nuclease activity (Bernstein et al., 1989). A subset of smaller (M in the range of 30,000-40,000) cytosolic mRNA-binding proteins have been identified that display recognition of AU-rich domains in the 3`-UTR have been identified (Malter, 1989; Brewer, 1991; Vakalopoulou et al., 1991; Bohjanen et al., 1991; Port et al., 1992; Huang et al.,1993). The precise role that these smaller RNA-binding proteins play in regulating mRNA stability and turnover has not been elucidated.

Recently, we identified a M 35,000 cytosolic RNA-binding protein (-ARB) that binds selectively -adrenergic receptor mRNA; does not bind to either -adrenergic receptor mRNA which does not undergo agonist-induced down-regulation, or to -globin mRNA; displays binding of -adrenergic receptor mRNA that is selectively competed by poly(U) RNA, but not poly(A), -(C), or -(G) RNA; and varies inversely with the level of receptor mRNA, being induced by agonists that down-regulate receptor mRNA (Port et al., 1992). We also identified the cognate sequences of -adrenergic receptor mRNA important for binding to -ARB protein via competition studies with 3`-UTRs of highly regulated mRNAs and RNA variants with specific mutations in the cognate domains, as well as via radiolabeling of these 3`-UTRs followed by UV cross-linking to cytosolic preparations containing -ARB protein (Haung et al.,1993).

In the present report we explored the role of the cognate sequence identified for binding of -ARB protein by using radiolabeled mRNAs of -adrenergic subtypes and also by competition studies using unlabeled mRNAs of subtypes. In addition, we tested the predictive value of the presence of the cognate sequence to identify receptors displaying post-transcriptional regulation.


EXPERIMENTAL PROCEDURES

Cell Culture

DDT-MF2 vas deferens smooth muscle cells were cultured in Dulbecco's modified Eagle's medium supplemented with heat-inactivated 5% fetal bovine serum (HyClone), penicillin (60 µg/ml), and streptomycin (100 µg/ml) as described by Scarpace et al.(1985). Cells were treated with either drugs prepared in a vehicle or with the vehicle alone, as described in each individual protocol. HEL cells were maintained in suspension culture in RPMI with 10% calf serum.

Preparation of Cytosolic (S100) Extracts

Following drug treatment, cells were washed twice with phosphate-buffered saline, and removed from the plate with 1.0 mM EDTA in phosphate-buffered saline. Approximately 5 10 cells were collected gently by low-speed (1,000 g) centrifugation, resuspended in phosphate-buffered saline, transferred to a sterile polypropylene ultracentrifuge tube, and collected again gently by centrifugation. The phosphate-buffered saline was aspirated from the cell pellet, and 5-µl aliquots each of the protease inhibitors (10 mg/ml) aprotinin and leupeptin were added to the cell pellet. The cells were then subjected to ultracentrifugation (100,000 g) for 90 min at 4 °C. The resulting supernatant fraction was transferred to Eppendorf tubes and maintained in an ice-bath for immediate use. This cytosolic fraction is referred to as the ``S100 fraction'' throughout the paper. Protein concentration was determined by method of Lowry et al.(1951).

In Vitro Transcription

The cDNAs for the hamster -adrenergic receptor (Dixon et al., 1986) were harbored pSP70 plasmid vector. The rat and cDNAs and the 3`-UTR region of human -adrenergic receptor were cloned in to pGEM 7z. The cDNA for thrombin receptor was harbored in pBluescript (Vu et al., 1991). Thrombin receptor coding region corresponding to nucleotide 222-1502 and the 3`-untranslated region of thrombin receptor corresponding to nucleotide 1504-3110 were cloned into pGEM (Bahou et al.,1991). Each plasmid (10 µg) was linearized by restriction using an enzyme to cut the plasmid immediately 3` to the receptor or globin cDNA insert. In vitro transcription was performed using SP6 or T7 DNA-directed RNA polymerase to produce full-length, 5`-capped, uniformly labeled poly(A) mRNAs based on the technique of Melton et al.(1984). Briefly, mRNAs were transcribed in the presence of RNasin (Promega), radiolabeled [-P]UTP (800 Ci/mmol, DuPont NEN), with nucleotides and buffer conditions as detailed by Promega. Co-transcriptional capping was performed by using the cap analogue m(5`)Gppp(5`)G (New England Biolabs) at a concentration that was 10-fold in excess to the concentration of GTP. After the mRNA was transcribed, RNase-free DNase was added to the mixture to remove template DNA. The labeled transcript was extracted with phenol, then with chloroform, and precipitated finally with 2.5 volumes of ice-cold ethanol and 0.1 volume of 3 M sodium acetate. The labeled transcript was then reconstituted in RNase-free water, maintained at -80 °C, and used within 24 h of synthesis. Size and integrity of the transcripts were verified immediately prior to use by agarose-formaldehyde gel electrophoresis.

UV Cross-linking and Label Transfer

An aliquot of radiolabeled mRNA (1-4 10 cpm), 5 µg of yeast tRNA, and competing unlabeled RNA transcripts (at the molar excess over probe indicated) were each added to a mixture containing the S100 cytosolic fraction (30-100 µg of total protein), 4 mM dithiothreitol, 5 µg of heparin, and 65 units of RNasin in a total volume of 50 µl. Aliquots of the mixture of S100 cytosolic fraction and radiolabeled mRNA were distributed in wells of a 24-well microliter plate and allowed to incubate for 10 min at 22 °C. Samples were exposed to short-wave (254 nm) UV irradiation at a distance of 7 cm for 30 min, mRNA not cross-linked to protein was digested with RNase A (0.5 mg/ml) and RNase T1 (10 units/ml) at 37 °C for 30 min.

SDS-Polyacrylamide Gel Electrophoresis of Cross-linked Proteins

Samples were solubilized in 50 µl (1:1) of Laemmli (1970) loading buffer for 10 min at 67 °C. The samples were then loaded onto a 10% SDS-polyacrylamide gel (5% stack) and subjected to gel electrophoresis for 110 mA h. Gel proteins were then stained with Coomassie Blue R, and the gels destained, dried, and subjected to autoradiography for 3-7 days. The relative intensity of radiolabeled species on the gel autoradiograms was quantified by direct analysis of radioactivity using a Beta-scope 603 PhosphorImager.

Northern Blot Analysis

Total cellular RNA was isolated by a single-step guanidine isothiocyanate/phenol-chloroform extraction method (Chomczynski and Sacchi, 1987) from untreated control, thrombin- (8 nm) and cAMP (50 µm)-treated cells. Duplicate aliquots (40 µg) of total RNA were denatured with formamide, fractionated through 1.2% agarose, 6% formaldehyde gel electrophoresis, and transferred to nitrocellulose filter by capillary action. The membrane blot was baked at 80 °C for 2 h and prehybridized with 50% formamide, 0.02% polyvinylpyrrolidone-Ficoll, 0.02% bovine serum albumin, 100 mM NaPO, pH 7.0, 0.75 M NaCl in 0.075 M sodium citrate, pH 7.0, 1.0% SDS and denatured salmon sperm DNA (100 µg/ml) at 42 °C for 12 h. The blot was hybridized at high stringency with random primer-generated P-labeled thrombin receptor probe corresponding to the coding region for 12-15 h at 42 °C with constant agitation. The blot was washed twice with 2 SSC (2 SSC = 0.3 M NaCl, 0.03 M sodium citrate) containing 0.5% SDS for 5 min each at room temperature, followed by two more washes with 0.1 SSC containing 0.5% SDS for 30 min at 60 °C. The blot was scanned by Betascope 603 PhosphorImager to determine the steady-state level of mRNA in control and treated cells. The size of the mRNA species (kilobases) was established with RNA standards obtained from Life Technologies, Inc., and also by subjecting in vitro transcribed full-length thrombin receptor mRNA to RNA blot analysis along with samples.

Determination of mRNA Half-life

HEL cells were pretreated with thrombin, cAMP, or vehicle. Six hours after thrombin treatment or 12 h after cAMP treatment, actinomycin D (5 µg/ml) was added to arrest transcription. At the indicated times, total RNA was extracted from individual dishes and RNase protection assay was performed using antisense radiolabeled riboprobes corresponding to the coding region of thrombin receptor mRNA. Hybridizations were performed at 50 °C for 18 h by incubating labeled RNA probes (1-4 10 cpm) with total RNA (40 g) from HEL cells in 20 µl of hybridization buffer (80% deionized formamide, 40 mM PIPES, pH 6.4, 0.4 M NaCl, and 1 mM EDTA). Upon completion of hybridization, 200 µl of ribonuclease digestion buffer (10 mM Tris-HCl, pH 7.5, 300 mM NaCl, and 5 mM EDTA) containing RNase T1 (500 units/ml) was added to each assay tube followed by incubation at 30 °C for 45 min. RNase digestion was stopped by addition of 225 µl of 4 M guanidinium thiocyanate solution (4 M guanidium thiocyanate, 0.5% sodium N-lauroylsarcosine, 25 mM sodium citrate, pH 7.0, and 0.1 M -mercaptoethanol). The RNase-resistant hybrids were ethanol-precipitated in the presence of 5 µl of 5 µg/µl carrier RNA (yeast tRNA) and loaded on a 5% acrylamide, 7 M urea gel.


RESULTS

The rodent -AR mRNA is strongly down-regulated by agonist or cAMP analogues, whereas the human -AR mRNA is not affected by agonist and is up-regulated by cAMP (Granneman and Lahners, 1994). The rat -AR mRNA appears to be down-regulated by agonist in some cells (Hough and Chuang, 1990; Bahouth, 1992) but not others (Granneman and Lahners, 1992). According to our hypothesis, if the rodent -AR displays agonist-induced destabilization of mRNA then the mRNA should bind -ARB protein via the 3`-UTR. We tested the hypothesis, exploring the -AR mRNA against -AR and -AR transcripts for recognition of -ARB using label transfer. In vitro transcribed, capped, uniformly labeled mRNAs and cytosolic S100 fractions were incubated and subjected to UV-catalyzed cross-linking to identify RNA-binding proteins (Wilusz and Shenk, 1988), as described earlier (Port et al.,1992; Huang et al.,1993). The binding of transcripts to -ARB protein was assessed by use of radiolabeled mRNA of either rat -, hamster -, rat -, and human -ARs in direct label transfer, as well as by evaluating the ability of unlabeled RNA to compete for binding of P-labeled -AR transcripts to -ARB protein. The cross-linked, radiolabeled RNA-binding proteins were made visible by autoradiography (Fig. 1). Direct label transfer studies identified several classes of RNA-binding proteins using labeled transcripts for -, -, and -AR. -ARB protein (M 35,000) was prominently labeled by the -AR mRNA. Several other slower migrating species of 40,000, 55,000, 70,000 (doublet), and 90,000 M were also identified. Using radiolabeled -AR transcript, the amount of label transfer to -ARB protein was substantially less than that obtained with the -AR mRNA. For the -AR mRNAs (both human and rat species), binding to -ARB protein was virtually absent, in spite of the fact that the rodent -AR mRNA is known to display agonist-induced down-regulation. Quantification of several label transfer experiments by phosphorimaging reveals a rank order of label transfer to -ARB protein, -AR -AR > -AR. The inability of unlabeled -AR and -AR mRNA to compete with [P]-AR mRNA for label transfer to -ARB (Fig. 2) provides further evidence that these mRNAs bind -ARB poorly (-AR), if at all (-AR). These data suggest that the agonist-induced decline in -AR mRNA, unlike that of the -AR mRNA, does not involve binding to -ARB and perhaps, post-transcriptional regulation.


Figure 1: UV cross-linking of the M 35,000 -ARB protein(s) to mRNA of -adrenergic subtypes. Representative autoradiogram of UV cross-linking between S100 cytosolic fractions from DDT-MF2 cells and full-length, capped, and uniformly labeled in vitro transcribed mRNAs corresponding to rat (lane 1), hamster (lane 2), rat (lane 3), and human (lane 4). Equal amounts of S100 cytosolic protein and equimolar concentration for each radiolabeled mRNA were added into the appropriate lane. A distinct band appears at approximately M 35,000 in lane 2 corresponding to the -adrenergic receptor mRNA. The right-hand panel shows the actual P (cpm) transferred from -adrenergic receptor mRNA subtypes to the M 35,000 -ARB proteins as determined by analysis of radioactivity by Betascope 603 PhosphorImager. The values are mean ± S.D. of at least three separate experiments.




Figure 2: Analysis of -ARB protein binding of receptor mRNAs: recognition of hamster -, but not rat - or -adrenergic receptor mRNAs. Representative autoradiogram of UV cross-linking between S100 cytosolic fractions prepared from DDT-MF2 cells and full-length, capped, and uniformly labeled in vitro transcribed mRNA encoding the hamster -adrenergic receptor. P-Labeled -adrenergic receptor mRNA was subjected to competition by adding increasing amounts (10- and 50-fold molar excess) of unlabeled RNAs from rat (lanes 1-3), hamster (lanes 4-6), or rat (lanes 7-9) adrenergic receptor. Unlabeled RNAs and P-radiolabeled RNA were added simultaneously to the mixture containing the S100 cytosolic extracts. The mixture was incubated for 10 min prior to UV cross-linking.



The predictive value of the presence of an AUUUA motif in the 3`-UTR of an mRNA for agonist-induced post-transcriptional regulation was explored in a subset of G-protein-linked receptors. The GeneBank subset was scanned for mRNAs harboring an AUUUA motif. In addition to the -AR, several G-protein-linked receptors were identified which possess from 1 to 5 AUUUA pentamers in the 3`-UTR of their mRNAs (). The thrombin receptor mRNA harbors 6 AUUUA pentamers, the most 5`-pentamer residing in the 3` end of the open reading frame (ORF) and the five remaining confined to the 3`-UTR. The organization of the AUUUA pentamers in the thrombin receptor is similar to that of c-fos and c-myc mRNAs, post-transcriptionally regulated immediate early gene products (Jones and Cole, 1987; Shyu et al., 1991).

Uniformly labeled, full-length, capped and polyadenylated mRNAs for the -AR and thrombin receptor, respectively, were prepared as probes with which to study the RNA-binding proteins to which each binds (Fig. 3, lanes 1 and 2). The 35,000-M -ARB protein was a prominent target for label transfer from both the -AR and the thrombin receptor mRNAs. In contrast to label transfer with -AR mRNA, the pattern of label transferred from the thrombin receptor mRNA was more complex, displaying binding to several other classes of RNA-binding proteins with M 18,000, 43,000, and 55,000 and some slower migrating species that bind the -AR mRNA poorly. When the 3`-UTR of the thrombin receptor mRNA (harboring 5 AUUUA pentamers) was employed in the label transfer, the labeling to proteins clustered at M 18,000 and 55,000 was abolished while the binding to -ARB protein remained (Fig. 3, lane 4). The ORF of the thrombin receptor mRNA, which possess a single AUUUA pentamer, provided a pattern of label transfer similar to that of the entire mRNA, recognizing -ARB and other RNA-binding proteins (Fig. 3, lane 3).


Figure 3: -Adrenergic receptor mRNA-binding protein recognizes AU-rich domains of the thrombin receptor mRNA. Autoradiogram of UV cross-linking between S100 cytosolic fractions prepared from DDT-MF2 cells and uniformly labeled in vitro transcribed mRNA corresponding to full-length hamster -adrenergic receptor (lane 1), full-length (lane 2), open reading frame (lane 3), and 3`-UTR (lane 4) regions of thrombin receptor RNAs.



Binding of the thrombin receptor mRNA to -ARB protein was investigated further through studies in which the ability of the unlabeled thrombin receptor mRNA to compete with the labeled -AR mRNA for binding to -ARB protein was assessed (Fig. 4). Increasing the amount of unlabeled thrombin receptor mRNA from 1 to 10-fold molar excess over P-labeled -AR mRNA decreased the amount of binding to -ARB protein (Fig. 4, lanes 2-4). At a 10-fold molar excess of thrombin receptor mRNA, -AR mRNA binding to -ARB protein was essentially abolished.


Figure 4: The M 35,000 -adrenergic receptor mRNA-binding protein specifically binds both -adrenergic and thrombin receptor mRNAs. Autoradiogram of UV cross-linking between S100 cytosolic fractions prepared from DDT-MF2 cells and full-length, capped, and uniformly labeled in vitro transcribed mRNA corresponding to full-length hamster -adrenergic receptor, in the presence of increasing amounts of (1-, 5-, and 10-fold molar excess) unlabeled thrombin receptor mRNA. Similarly, label transfer experiments were performed using P-labeled RNA from full-length thrombin receptor and S100 cytosolic preparations from DDT-MF2 cells in the presence of increasing amounts (5- and 10-fold molar excess) of unlabeled -AR mRNA. The bottom panel shows the actual [P] (cpm) transferred from the respective labeled receptor mRNA to the M 35,000 -ARB proteins. The values are mean ± S.D. of at least three separate experiments.



The ability of the unlabeled -AR mRNA to compete with P-labeled full-length mRNA of the thrombin receptor in label transfer experiments was explored next. Label transfer with P-labeled thrombin receptor mRNA was performed in the absence (lane 5) or presence of 5-fold (lane 6) or 10-fold (lane 7) molar excess of -AR mRNA (Fig. 4). The unlabeled -AR mRNA competed effectively with the thrombin receptor mRNA for binding to -ARB protein. At 10-fold molar excess over labeled probe, -AR mRNA abolished binding of the thrombin receptor mRNA to -ARB (Fig. 4, lane 7). The ability of the -AR mRNA to block label transfer from thrombin receptor mRNA to -ARB contrasts with the inability of the -AR mRNA to alter label transfer from the thrombin receptor to RNA-binding proteins other than -ARB (Fig. 4, lanes 5-7).

The ability of the ORF and 3`-UTR of the thrombin receptor mRNA to compete with the -AR full-length mRNA was investigated next. Both unlabeled ORF and 3`-UTR of the thrombin receptor possess AUUUA pentamers and were able to compete with P-labeled -AR mRNA for binding to -ARB (Fig. 5). When comparing the relative ability of unlabeled RNAs to compete with P-labeled -AR mRNA for binding to -ARB, the rank order is as follows: full-length thrombin receptor mRNA (Fig. 4, lanes 2-4), thrombin receptor 3`-UTR (Fig. 5, lanes 2-4) > thrombin receptor ORF RNA (Fig. 5, lanes 6-8).


Figure 5: The thrombin receptor 3`-UTR and ORF compete for binding to M 35,000 -ARB protein with labeled -adrenergic receptor mRNA. Autoradiogram of UV cross-linking between S100 cytosolic fractions prepared from DDT-MF2 cells and full-length, capped, and uniformly labeled in vitro transcribed mRNA corresponding to full-length hamster -adrenergic receptor, in the presence of increasing amounts of (1-, 5-, and 10-fold molar excess) unlabeled thrombin receptor mRNA corresponding to 3`-UTR (lanes 1-4) and open reading frame (lanes 5-8) regions.



We explored if -ARB protein is present in human megakaryoblastic HEL cells, which express significant levels of thrombin receptors. HEL cells were treated with a non-hydrolyzable analogue of cAMP (50 µM, 8-(4-chlorophenylthio)-cAMP (CPT-cAMP)) for 12 h to induce -ARB protein (Port et al.,1992) and cytosolic S100 fractions prepared for label transfer studies (Fig. 6). Label transfer from uniformly labeled, full-length thrombin receptor mRNA revealed -ARB protein (M 35,000). Competition studies with unlabeled, full-length thrombin receptor mRNA, the thrombin receptor ORF, and the thrombin receptor 3`-UTR demonstrate that -ARB protein is present and interacts with the thrombin receptor mRNA of HEL cells, as well as of DDT-MF2 smooth muscle cells.


Figure 6: HEL cells express M 35,000 -ARB protein that recognizes thrombin receptor mRNA. HEL cells were treated with CPT-cAMP (50 µm) for 12 h to induce -ARB (Port et al., 1993). Autoradiogram of UV cross-linking between S100 cytosolic fractions prepared from cAMP-treated HEL cells and full-length, capped, and uniformly labeled in vitro transcribed thrombin receptor mRNA, in the presence of 1- and 10-fold molar excess unlabeled thrombin receptor mRNA. Competion studies were performed with full-length thrombin receptor mRNA (lanes 1-3), ORF (lanes 4-6), and 3`-UTR (lanes 7-9) regions.



The results from the label transfer studies reveal the thrombin receptor mRNA identified by the presence of AUUUA motifs to recognize the M 35,000 -ARB protein. The hypothesis that the presence of the AUUUA motif was predictive of a regulatable mRNA was tested directly using the thrombin receptor. HEL cells were selected as the model system in which to address the prediction that the presence of the AUUUA motif identifies the thrombin receptor as a target for post-transcriptional regulation. Cells were challenged with either cAMP (50 µM, which has been shown to induce down-regulation and mRNA instability for the AR) or agonist (thrombin, 8 nM) for 6-24 h in culture. Steady-state levels of the thrombin receptor mRNA were defined by Northern analysis (Fig. 7). When cells were challenged with cAMP there was a frank decline in thrombin receptor mRNA from 12 to 24 h. When challenged with thrombin, in contrast, receptor mRNA levels declined sharply from 6 to 12 h (>80%) and re-bounded significantly by 24 h, despite the continued challenge by agonist. Steady-state levels of -AR mRNA also decline precipitously in response to agonist stimulation, but do not show the rebound observed in each of three trials with the thrombin receptor mRNA. A modest increase in thrombin receptor mRNA was observed at 6 h in cells challenged with either cAMP or thrombin.


Figure 7: Thrombin and cAMP regulate thrombin receptor mRNA levels: Northern blot analysis. The time course of the effect of cAMP and thrombin on the steady-state level of thrombin receptor mRNA. Total RNA extracted from cells were fractionated on 1.2% formaldehyde gel and transferred to nitrocellulose and hybridization was performed with coding region thrombin receptor probe as described under ``Experimental Procedures.''



The half-life (t) of the thrombin receptor mRNA was determined in order to investigate if agonist induces the decline in receptor mRNA levels via message destabilization. HEL cells were challenged with thrombin (8 nM for 6 h) or CPT-cAMP (50 µM for 12 h) and then treated with actinomycin D to arrest transcription (Fig. 8). Thrombin receptor mRNA levels were quantified by RNase protection assay at various times after transcriptional arrest to establish the half-life of the mRNA. The t for thrombin receptor mRNA in the untreated cells was approximately 10 h. In cells challenged with thrombin, receptor mRNA t declined sharply from 10 to 4 h, similar to the decline in -AR mRNA t observed in cells challenged with -adrenergic agonist (Hadcock et al., 1989a, 1989b). cAMP also promoted a post-transcriptional decline in thrombin receptor mRNA, just as observed for the -AR message (Hadcock et al., 1989b). The t for thrombin receptor mRNA declined from 10 to 6 h following challenge with cAMP. These studies of receptor mRNA stability highlight the similarities in the biology of -AR and thrombin receptors, first implicated by the presence of the AUUUA motif in the mRNA of the latter.


Figure 8: Thrombin and cAMP induce destabilization of thrombin receptor mRNA. A, representative autoradiogram obtained from RNase protection analysis of thrombin receptor mRNA. Thrombin-induced decay of thrombin receptor mRNA was determined as described under ``Experimental Procedures.'' B, estimation of half-life of thrombin receptor mRNA. The RNase-resistant species were quantified by analysis of radioactivity by Betascope 603 PhosphorImager.




DISCUSSION

Agonist-induced down-regulation of receptor expression is commonly observed for members of the G-protein-linked receptor superfamily. Clues to mechanisms underlying this mode of receptor down-regulation were provided when it was observed that -adrenergic agonists induce both a decline in -AR protein and mRNA levels in cells challenged chronically (Hadcock and Malbon, 1988). The basis for the agonist-induced decline in -AR mRNA levels was shown subsequently to reflect a destabilization of pre-existing receptor message (Hadcock et al., 1989b). These studies provided the first description of post-transcriptional regulation of G-protein-linked receptors.

To identify candidate proteins involved in the agonist-induced destablization, we employed uniformly labeled, full-length, capped and polyadenylated -AR mRNA and label transfer following UV-catalyzed cross-linking to putative RNA-binding proteins (Port et al., 1992). A prominently labeled M 35,000 cellular protein was identified displaying specificity for binding of -AR mRNA, but not the -AR mRNA which is not post-transcriptionally regulatable. Expression of this RNA-binding protein increased in the cytosol from cells treated with agonist, varying inversely with -AR mRNA levels. The binding of -AR mRNA to this protein was sensitive to competition by poly(U) RNA (Port et al., 1992). This protein was termed ``-ARB protein,'' reflecting its nature, i.e. a -adrenergic receptor RNA-binding protein.

The identification of an AUUUA pentamer in the -AR mRNA 3`-UTR provided a second, major lead for further investigation. Using both labeled as well as unlabeled RNA of 3`-UTRs of genes whose messages display highly regulated degradation (e.g. mRNAs of granulocyte/macrophage colony-stimulating-factor, tumor necrosis factor-, and the oncogenes c-myc and c-fos), we demonstrated that recognition by -ARB protein requires not only an AUUUA pentamer, but also flanking U-rich domain(s) in the target mRNAs (Haung et al.,1993). Extensive mutagenesis studies revealed that the integrity of the AUUUA pentamer was absolute and that interruption in the poly(U) stretches either 3` or 5` to the pentamer were not tolerated. We hypothesized that the cognate sequence containing a well known destabilizing domain defined in the context of the -AR mRNA reflected its post-translational regulation by agonist and cAMP.

Our ability to generate uniformly labeled, full-length, capped and polyadenylated mRNAs for the three -AR subtypes (rat , -, and human ) afforded us the opportunity to test the hypothesis by direct label transfer. Comparison of the ability of the labeled mRNAs to bind -ARB protein revealed a rank order from hamster -AR (greatest) >> rat -AR > human -AR > rat -AR (least). Inspection of GeneBank sequence information of rat -AR and human -AR mRNA showed the following. The rat -AR does not have any AUUUA pentamers, but does have a poly(U) region in its 3`-UTR region (Granneman et al., 1991). The human -AR mRNA lacks both the poly(U) tract and the AUUUA pentamer (Lelias et al.,1993). These data agree well with the recent report that agonists induce a transcriptional repression of the -AR gene, without alterations in the t of the receptor mRNA (Granneman and Lahners, 1995).

The importance of flanking poly(U) regions about an AUUUA pentamer is highlighted by the fact that rat -AR mRNA possesses an AUUUA pentamer in its 3`-UTR and has a higher U content than the -AR mRNA. Although rich in U content, the rat -AR mRNA lacks the poly(U) tracks necessary for recognition by -ARB protein. There exists little doubt that the pentameric motif AUUUA plays an critical role in the selective degradation of immediate early gene mRNAs (Shaw and Kamen, 1986; Chen and Shyu, 1994; Chen et al., 1994). Comparative analysis of U richness in the AUUUA flanking regions of immediate early genes led to the conclusion that the pentamer exist in a uridine-rich (40-50%) context (Alberta et al., 1994). Analysis of -adrenergic subtypes suggests that poly(U) regions are more important than over-representation of U in the message (). Lacking the poly(U) regions, the rat -AR mRNA does not bind -ARB protein although harboring an AUUUA pentamer and a U content in the flanking sequences higher than that of the -AR mRNA.

The most exciting outcome of the present work was evaluating the predictive value of the presence of the cognate sequence for -ARB protein in an mRNA for susceptibility to post-transcriptional regulation. Scanning a subset of G-protein-linked receptor genes in the GeneBank for the presence of the cognate sequence revealed several possible test candidates. The availability of molecular probes and limitations in our knowledge of its biology, fostered our selection of the thrombin receptor for this test. The thrombin receptor transcript harbors AUUUA pentamers and flanking poly(U) regions that meet the strict requirements for -ARB recognition. Direct analysis of the ability of thrombin receptor mRNA to be recognized by RNA-binding proteins identified the M 35,000 -ARB protein. Treating HEL cells with thrombin or cAMP resulted in a decline in the steady-state thrombin receptor mRNA levels, with similarities to agonist-induced decline in -AR mRNA observed following stimulation of cells with -adrenergic agonists. Moreover, the decline in thrombin receptor mRNA was shown to reflect post-transcriptional regulation, i.e. the t of the message declined, reflecting altered stability. Interestingly, the decline in thrombin receptor mRNA observed at 12 h was largely lost by 24 h (Fig. 7). Thrombin has been shown to induce a transient homologous desensitization and loss of thrombin receptors that rebounds after 24 h (Brass et al.,1991; Brass, 1992), perhaps reflecting a need of the cell to replenish the complement of receptor persistently activated by proteolytic cleavage. The decline induced by cAMP, in contrast to that observed in response to thrombin, was not restored by 24 h, but remained reduced like that observed for -AR mRNA under similar circumstances. Treating cells with -adrenergic agonists or with CPT-cAMP induces a down-regulation of -AR mRNA, reflecting destabilization of existing message (Hadcock et al.,1989b; Port et al.,1992; Huang et al.,1993). In the present work we demonstrate that either elevating cAMP or activating a G-coupled receptor (data not shown) down-regulates the thrombin receptor mRNA in a similar fashion, reflecting cross-regulation between two distinct, G-protein-linked pathways.

The present study expands our understanding of the cognate sequence of mRNA recognized by the M 35,000 -ARB protein. mRNAs of G-protein-linked receptors lacking the AUUUA destabilization pentamer are not recognized by -ARB protein. Likewise, mRNAs harboring an AUUUA pentamer and no flanking poly(U) tracks are not recognized by -ARB protein. Most revealing is the rat -AR mRNA that harbors an AUUUA pentamer and is relatively rich in U content of flanking regions, but lacking poly(U) tracks. The rat -AR mRNA fails to bind -ARB protein. Using the expanded knowledge of the AUUUA motif and a subset of G-protein-linked receptors in GeneBank, several candidate receptor mRNAs were identified and one, the thrombin receptor was tried. Although limited to a study of this one candidate, our results in vitro (label transfer) and in vivo (cell culture) demonstrate a predictive value of the presence of the cognate sequence for -ARB in receptor biology, more specifically post-transcriptional regulation. We show the thrombin receptor to be subject to agonist-induced down-regulation of mRNA via message destabilization. Although these studies do not establish -ARB protein as the mediator for the post-transcriptional regulation of the thrombin receptor, they do provide a compelling linkage. Based on these observations it may be speculated that members of the G-protein-linked receptor superfamily with the cognate sequence can be expected to include agonist-induced (and perhaps cAMP-induced) destabilization of receptor mRNA as a mechanism for receptor down-regulation.

  
Table: Several G-protein-coupled receptors which possess one or more AUUUA pentamer in their 3`-UTR region


  
Table: Uridine richness in the 3`-UTR region flanking the AUUUA pentamer of -adrenergic receptor mRNA subtypes

The rat -AR displays a poly(U) region in its 3`-UTR, but no AUUUA pentamer and the human -AR mRNA lacks both the poly(U) tract and the AUUUA pentamer.



FOOTNOTES

*
This work was supported in part by United States Public Health Services Grant DK25410 from the National Institutes of Health. 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.

§
To whom correspondence should be addressed: Dept. of Pharmacology, DMDRP, HSC, SUNY, Stony Brook, NY 11794-8651. Tel.: 516-444-7873; Fax: 516-444-7696.

The abbreviations used are: UTR, untranslated region; -AR, -adrenergic receptor; -ARB protein, -AR mRNA-binding protein; ORF, open reading frame; PIPES, 1,4-piperazinediethanesulfonic acid; CPT-cAMP, 8-(4-chlorophenylthio)-cAMP.


ACKNOWLEDGEMENTS

We thank Drs. S. R. Coughlin, Department of Medicine, Cancer Research Institute, University of California at San Francisco, and W. F. Bahou, Department of Medicine, State University of New York, Stony Brook, NY, for the thrombin receptor cDNAs.


REFERENCES
  1. Alberta, J. A., Rundell, K., and Stiles, C. D.(1994) J. Biol. Chem. 269, 4532-4538 [Abstract/Free Full Text]
  2. Bahou, W. F., Coller, B. S., Potter, C. L., Norton, K. J., Kutok, J. L., and Goligorsky, M. S.(1991) J. Clin. Investi. 91, 1405-1413
  3. Bahouth, S. W.(1992) Mol. Pharmacol. 42, 971-981 [Abstract]
  4. Bernstein, P., Peltz, S. W., and Ross, J.(1989) Mol. Cell. Biol. 9, 659-670 [Medline] [Order article via Infotrieve]
  5. Bohjanen, P. R., Petryniak, B., June, C. H., Thompson, C. B., and Lindsten, T.(1991) Mol. Cell. Biol. 11, 3288-3295 [Medline] [Order article via Infotrieve]
  6. Brawerman, G.(1987) Cell 48, 5-6 [Medline] [Order article via Infotrieve]
  7. Brawerman, G.(1989) Cell 57, 9-10 [Medline] [Order article via Infotrieve]
  8. Brass, L. F.(1992) J. Biol. Chem. 267, 6044-6050 [Abstract/Free Full Text]
  9. Brass, L. F., Manning, D. R., Williams, A. G., Woolkalis, M. J., and Poncz, M.(1991) J. Biol. Chem. 266, 958-965 [Abstract/Free Full Text]
  10. Brewer, G.(1991) Mol. Cell. Biol. 11, 2460-2466 [Medline] [Order article via Infotrieve]
  11. Brewer, G., and Ross, J.(1988) Mol. Cell. Biol. 8, 1697-1708 [Medline] [Order article via Infotrieve]
  12. Brewer, G., and Ross, J.(1989) Mol. Cell. Biol. 9, 1996-2006 [Medline] [Order article via Infotrieve]
  13. Chen, C. A., and Shyu, A.(1994) Mol. Cell. Biol. 14, 8471-8482 [Abstract]
  14. Chen, C. A., Chen, T., and Shyu, A.(1994) Mol. Cell. Biol. 14, 416-426 [Abstract]
  15. Chomczynski, P., and Sacchi, N.(1987) Anal. Biochem. 162, 152-159
  16. Cleveland, D. W.(1988) Trends Biochem. Sci. 13, 339-343 [CrossRef][Medline] [Order article via Infotrieve]
  17. Collins, S., Caron, M. G., and Lefkowitz, R. J.(1988) J. Biol. Chem. 263, 9067-9070 [Abstract/Free Full Text]
  18. Collins, S., Caron, M., and Lefkowitz, R. J.(1991) Annu. Rev. Physiol. 53, 497-508 [CrossRef][Medline] [Order article via Infotrieve]
  19. Dixon, R. A. F., Kobilka, B. K., Strader, D. J., Benovic, J. L., Dohlman, H. G., Frielle, T., Bolanowski, M. A., Bennett, C. D., Rands, E., Diehl, R. E., Mumford, R. A., Slater, E. E., Sigal, I. S., Caron, M. G., Lefkowitz, R. J., and Strader, C. D.(1986) Nature 321, 75-79 [Medline] [Order article via Infotrieve]
  20. Dreyfuss, G.(1986) Annu. Rev. Cell Biol. 2, 459-498 [CrossRef]
  21. Granneman, J. G., and Lahners, K.(1992) Endocrinology 130, 109-114 [Abstract]
  22. Granneman, J. G., and Lahners, K.(1994) Endocrinology 135, 1025-1031 [Abstract]
  23. Granneman, J. G., and Lahners, K.(1995) Am. J. Physiol., in press
  24. Granneman, J. G., Lahners, K., and Chaudhery, A.(1991) Mol. Pharmacol. 40, 895-899 [Abstract]
  25. Hadcock, J. R., and Malbon, C. C.(1991) Trends Neurosci. 14, 242-247 [CrossRef][Medline] [Order article via Infotrieve]
  26. Hadcock, J. R., and Malbon, C. C.(1993) J. Neurochem. 60, 1-9 [Medline] [Order article via Infotrieve]
  27. Hadcock, J. R., and Malbon, C. C.(1988) Proc. Natl. Acad. Sci U. S. A. 85, 5021-5025 [Abstract]
  28. Hadcock, J. R., Ros, M., and Malbon, C. C. (1989a) J. Biol. Chem. 264, 13956-13961 [Abstract/Free Full Text]
  29. Hadcock, J. R., Wang, H., and Malbon, C. C. (1989b) J. Biol. Chem. 264, 19928-19933 [Abstract/Free Full Text]
  30. Hargrove, J. L., and Schmidt, F. H.(1989) FASEB J. 3, 2360-2370 [Abstract/Free Full Text]
  31. Hough, C., and Chuang, D.-M.(1990) Biochem. Biophys. Res. Commun. 170, 46-52 [Medline] [Order article via Infotrieve]
  32. Huang, L.-Y., Tholanikunnel, B. G., Vakalopoulou, E., and Malbon, C. C. (1993) J. Biol. Chem. 268, 25769-25775 [Abstract/Free Full Text]
  33. Jones, T. R., and Cole, M. D.(1987) Mol. Cell. Biol. 7, 4513-4521 [Medline] [Order article via Infotrieve]
  34. Konarska, M. M., and Sharp, P. A.(1987) Cell 49, 763-774 [Medline] [Order article via Infotrieve]
  35. Laemmli, U. K.(1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  36. Lelias, J. M., Kaghad, M., Rodriguez, M., Chalon, P., Bonnin, J., Dupre, I., Delpech, B., Bensaid, M., LeFur, G., Ferrara, P., and Caput, D.(1993) FEBS Lett. 324, 127-130 [CrossRef][Medline] [Order article via Infotrieve]
  37. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.(1951) J. Biol. Chem. 193, 265-275 [Free Full Text]
  38. Malter, J. S.(1989) Science 246, 664-666 [Medline] [Order article via Infotrieve]
  39. Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K., and Green, M. R.(1984) Nucleic Acids Res. 12, 7035-7056 [Abstract]
  40. Pei, R., and Calame, K.(1988) Mol. Cell. Biol. 8, 2860-2868 [Medline] [Order article via Infotrieve]
  41. Peltz, S. W., Brewer, G., Bernstein, P., Hart, P. A., and Ross, J. (1991) Crit. Rev. Eukaryotic Gene Exp. 1, 1-78
  42. Port, J. D., Huang, L.-Y., and Malbon, C. C.(1992) J. Biol. Chem. 267, 24103-24108 [Abstract/Free Full Text]
  43. Raghow, R.(1987) Trends Biochem. Sci. 12, 358-360
  44. Ross, J.(1988) Mol. Biol. Med. 5, 1-14 [Medline] [Order article via Infotrieve]
  45. Scarpace, P. J., Baresi, L. A., Sanford, D. A., and Abrass, I.(1985) Mol. Pharmacol. 28, 495-501 [Abstract]
  46. Schuler, G. D., and Cole, M. D.(1988) Cell 55, 1115-1122 [Medline] [Order article via Infotrieve]
  47. Shaw, G., and Kamen, R.(1986) Cell 46, 659-667 [Medline] [Order article via Infotrieve]
  48. Shyu, A.-B., Belasco, J. G., and Greenberg, M. E.(1991) Genes & Dev. 5, 221-231
  49. Steitz, J. A., Wolin, S. L., Rinke, J., Pettersson, I., Mount, S. M., Lerner, E. A., Hinterberger, M. E., and Gottlieb, E.(1983) Cold Spring Harbor Symp. Quant. Biol. 47, 893-900 [Medline] [Order article via Infotrieve]
  50. Stolle, C. A., and Benz, E. J., Jr.(1988) Gene (Amst.) 62, 65-74 [Medline] [Order article via Infotrieve]
  51. Swanson, M. S., and Dreyfuss, G.(1988) Mol. Cell. Biol. 8, 2237-2241 [Medline] [Order article via Infotrieve]
  52. Vakalopoulou, E., Schaack, J., and Shenk, T.(1991) Mol. Cell. Biol. 11, 3355-3363
  53. Vu, T.-K. H., Hung, D. T., Wheaton, V. I., and Coughlin, S. R.(1991) Cell 64, 1057-1068 [Medline] [Order article via Infotrieve]
  54. Wilusz, J., and Shenk, T.(1988) Cell 52, 221-228 [Medline] [Order article via Infotrieve]
  55. Wilusz, J., and Shenk, T.(1990) Mol. Cell. Biol. 10, 6397-6407 [Medline] [Order article via Infotrieve]
  56. Wisdom, R., and Lee, W.(1991) Genes & Dev. 5, 232-243
  57. Wreschner, D. H., and Rechavi, G.(1988) Eur. J. Biochem. 172, 333-340 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.