(Received for publication, December 12, 1996, and in revised form, February 10, 1997)
From the Department of Pharmacology, Diabetes and Metabolic Diseases Research Program, School of Medicine, Health Sciences Center, SUNY/Stony Brook, Stony Brook, New York 11794-8651
The Mr 35,000 -adrenergic receptor mRNA-binding protein, termed
ARB
protein, is induced by
-adrenergic agonists and binds to
2-receptor mRNAs that display agonist-induced
destabilization. A cognate sequence in the mRNA was identified
previously that provides for
ARB protein binding in
vitro. In the present work, the sequence established in
vitro for binding of
ARB protein to hamster
2-adrenergic receptor mRNA was probed in
vivo by site-directed mutagenesis of the 3
-untranslated region
and expression in Chinese hamster ovary cells. A 20-nucleotide, (A + U)-rich region in the 3
-untranslated region consisting of an AUUUUA
hexamer flanked by defined U-rich regions constitutes the binding
domain for
ARB protein. U to G substitution in the hexamer region
attenuates the binding of
ARB protein, whereas U to G substitution
of hexamer and flanking U-rich domains abolishes binding of
ARB
protein and stabilizes
2-adrenergic receptor mRNA
levels in transfectant clones challenged with either isoproterenol or
cyclic AMP. These results demonstrate that binding of
ARB protein to
the 20-nucleotide, (A + U)-rich domain mediates the agonist and cyclic
AMP-induced mRNA decay of G protein-linked receptors, such as the
2-adrenergic receptor.
Agonist-induced down-regulation of G protein-linked receptors,
such as the 2-adrenergic receptor, provides an
explanation for long term adaptation to chronic stimuli
characteristically observed for members of this receptor family (1-5).
For
2-adrenergic receptors, steady-state levels of the
receptor and its mRNA decline following a challenge with agonist
(2, 3). The basis for the decline in receptor mRNA induced by
agonist is not transcriptional suppression, but rather
post-transcriptional destabilization of receptor mRNAs (6).
Recently, we identified a Mr 35,000 protein with
properties consistent with those expected for an RNA-binding protein
selective for mRNAs of receptors that display agonist-induced down-regulation of their messages and protein expression. This Mr 35,000
-adrenergic receptor
mRNA-binding protein, termed
ARB1 protein (7),
demonstrates several RNA binding properties (8) similar
to (A + U)-rich element (ARE)-binding proteins reported by others
(9-12).
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
(13-20). 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
tandem repeats of AUUUA pentamers (9-12, 21-23) and nonamers, such as
UUAUUUA(U/A)(U/A) (24) and UUAUUUAUU (25).
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 (26-28). Splicing and further processing of
pre-mRNAs possessing introns, a 5-cap and 3
-poly(A)+
tract involves the small nuclear RNA-binding proteins (29, 30).
Cytosolic mRNA-binding proteins include the
Mr 72,000 poly(A)-binding protein, which binds
to long stretches (~25 nucleotides/protein) of poly(A)+
and stabilizes the RNA to 3
- to 5
-nuclease activity (31). A subset of
smaller (ranging from Mr 30,000 to 40,000),
cytosolic mRNA-binding proteins have been identified that display
recognition of AU-rich domains in the 3
-UTR (7, 9-12, 32, 33).
Although several of these proteins have been purified (32, 33), the precise role that these smaller RNA-binding proteins play in regulating mRNA stability and turnover has not been elucidated.
The Mr 35,000 cytosolic ARB RNA-binding
protein displays the following properties: binds selectively to
1- and
2-adrenergic and thrombin receptor
mRNAs, examples of G protein-linked receptors with mRNAs
displaying AU-rich domains; fails to bind both rat and human
3 mRNA (34); and is induced by agonist treatment, its levels varying inversely with the level of receptor mRNA (7). The cognate sequences of
2-adrenergic receptor mRNA
important for binding to
ARB protein has been established in
vitro 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-catalyzed cross-linking to cytosolic preparations containing
ARB
protein (8). The predictive value of the presence of the cognate
sequence to identify receptors displaying post-transcriptional
regulation was tested using thrombin receptor as a model system. The
thrombin receptor, whose mRNA harbors several AU-rich sequences in
its 3
-UTR region, was found to display agonist-induced destabilization and its mRNA to bind
ARB protein (34). In the present work the
sequences established for binding of
ARB protein to hamster
2-adrenergic receptor mRNA in vitro were
tested in vivo following site-directed mutagenesis in tandem
with stable expression of receptor mRNA with mutated 3
-UTRs in
Chinese hamster ovary cells. Although both pentamer and hexamer core
AREs bind the
ARB protein (34), a 20-nucleotide, (A + U)-rich
sequence consisting of an AUUUUA hexamer flanked by U-rich regions is
shown to be obligate for binding of
ARB protein and regulation of
2-adrenergic receptor mRNA stability in
vivo.
DDT1MF2 vas deferens smooth muscle cells were cultured in Dulbecco's modified Eagle's medium supplemented with 5%, heat-inactivated fetal bovine serum (HyClone), penicillin (60 µg/ml), and streptomycin (100 µg/ml) as described by Scarpace et al. (35). CHO cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (HyClone). Cells were treated with either drugs prepared in a vehicle or with the vehicle alone, as described in each individual protocol.
Preparation of Cytosolic (S100) ExtractsFollowing 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 × 107 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 this work. Protein concentration was determined by method of Lowry et al. (36).
Mutagenesis and Plasmid ConstructionMutagenesis of
2-adrenergic receptor cDNA in pSP70 was performed by
overlap extension polymerase chain reaction. Briefly, mutagenic primers
were constructed containing complementary sequences to
2AR cDNA immediately 5
or 3
to the flanking 3
-UTR
AUUUA pentamer (nucleotide 1520-1524) designated mutant 1, or to
AUUUUA hexamer nucleotide (1598-1603) designated mutant 2 (Fig. 1).
The polymerase chain reaction was performed on
2AR
cDNA template using SP6 or T7 promoter primer and one of the
mutagenic primers. Products were separated by agarose gel
electrophoresis, made visible by staining with ethydium bromide and UV
irradiation, excised, and purified with a GeneClean-11 kit, as
described by the manufacturer (Bio 101 Inc., La Jolla, CA). Amplified
fragments (5-10 ng) from the forward and reverse polymerase chain
reaction were mixed and subjected to a second round of amplification by
polymerase chain reaction, and the products were separated and
identified as above. The fragment corresponding to full-length, mutant
2AR cDNA was excised and gel-purified. The fragment
was subjected to digestion with EcoRI, and the
EcoRI-digested fragment was then cloned into EcoRI sites of both pSP70 and pCMV5. After identification of
the appropriate recombinants, orientation was determined by restriction digestion mapping. The mutated cDNAs were sequenced by dideoxy method to verify the sequence for the appropriate base substitution. Plasmid vector pSP70, into which wild-type and various mutants of
2AR cDNA were inserted, was used for in
vitro transcription after linearization with a restriction enzyme
that cleaves the plasmid immediately 3
to the receptor cDNA
insert.
Expression vector pCMV5 was used for stable transfection in CHO cells.
Mutant 2 was used as the template for engineering both mutants 3 and 4 (Fig. 1). Plasmids containing the 20-nucleotide AUUUUA hexamer flanked
by poly(U) regions as well as those containing 20-nucleotide pentamer
flanked by poly(U) regions were constructed by use of complimentary
synthetic oligonucleotides flanked by restriction sequences for
HindIII at the 5-end and ClaI at the 3
-end.
Complimentary oligonucleotide were annealed and cloned into pSP70. The
resultant plasmids were linearized immediately 3
to the AU-rich region
and employed as templates for in vitro transcription.
The wild-type (37) and mutant
cDNAs for the hamster 2-adrenergic receptor were
inserted in pSP70 plasmid vector, which then was linearlized.
Transcription was performed in vitro using SP6 DNA-directed
RNA polymerase to produce full-length, 5
-capped, uniformly labeled,
poly(A)+ mRNAs, based on the technique of Melton
et al. (38). Briefly, mRNAs were transcribed in the
presence of RNasin (Promega), radiolabeled
-[32P]UTP
(800 Ci/mmol, DuPont NEN), nucleotide, and buffer conditions as
detailed by Promega. Co-transcriptional capping was performed by using
the cap analogue m7(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
Na-acetate. The labeled transcript was 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.
An aliquot of radiolabeled mRNA (1-4 × 106 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 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 aspirated, placed in an ice slurry, and then exposed to short wave (254 nm) UV irradiation at a distance of 7 cm for 30 min. The 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 RNA Protein AdductsSamples were solubilized in 50 µl (1:1) of Laemmli
loading buffer (39) for 10 min at 67 °C. The samples were then
loaded onto a SDS-polyacrylamide gel (10% acrylamide separating gel
with 5% acrylamide stack) and subjected to gel electrophoresis for 110 mA/h. Resolved proteins were 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 was
quantified by direct analysis of radioactivity using a
-phosphorimager.
CHO wild-type cells were
co-transfected with vectors harboring mutant or wild-type receptor
cDNAs, or empty vector plasmids, each in combination with plasmid
pCW1 containing the neomycin resistance gene, using Lipofectin (Life
Technologies, Inc). Stable transfectant clones were selected by
neomycin resistance in Dulbecco's modified Eagle's medium containing
10% fetal bovine serum and G418 (10 µg/ml). Expression of
2-adrenergic receptor was determined for CHO clones
stably expressing receptors with wild-type and mutant 3
-UTRs, by ICYP
binding (6).
CHO
cells were pretreated with isoproterenol (10 µM),
CTP-cyclic AMP (10 µM), or vehicle. After 24 h of
treatment with isoproterenol or after 12 h of treatment with
CPT-cyclic AMP, actinomycin D (5 µg/ml) was added to arrest
transcription at specific times. Total RNA was extracted from
individual culture dishes at the indicated time, and the amount of
receptor mRNA was established by use of an RNase protection assay
performed as described previously (34). Two different radiolabeled,
antisense riboprobes corresponding to 600 (740-1338) and 285 (1201-1486) nucleotides from the coding region of 2AR
mRNA were employed for the RNase protection assay.
Previous studies using
2-adrenergic receptor mRNA and 3
-UTRs of highly
regulated mRNAs and RNA variants with specific mutations in the
AU-rich region demonstrated the importance of AUUUA pentamers flanked
by poly(U) regions in the binding of
ARB protein (8). 3
-UTR of
2AR mRNA contains one AUUUA pentamer, which is not flanked by poly(U) regions, and another AUUUUA hexamer, which is
flanked immediately on either side by poly(U) regions. These regions
were mutated with U to G substitutions (Fig. 1)
and then tested for the ability of the mRNA transcribed from
these mutants to bind
ARB protein in vitro. The mutations
were focused on the AUUUA pentamer (1524-1528) and AUUUUA hexamer and
the flanking poly(U) regions (1592-1611). U to G substitutions, in the
most 5
-localized AUUUA pentamer of the 3
-UTR, yield mutant 1. U to G
substitutions in the AUUUUA hexamer yield mutant 2. Transcribed in vitro, capped, uniformly labeled mRNAs and cytosolic
S100 fractions were incubated and subjected to UV-catalyzed
cross-linking to identify the RNA-binding proteins (28), as described
earlier (7, 8).
The binding of transcripts to ARB protein was assessed by direct
label transfer using radiolabeled mRNA of hamster wild-type and
various mutant
2ARs as well as by evaluating the ability of unlabeled wild-type and mutant mRNAs to compete with
32P-labeled
2AR transcripts for
ARB
protein binding. The cross-linked, radiolabeled RNA-binding protein
adducts were subjected to SDS-polyacrylamide gel electrophoresis and
made visible by autoradiography (Fig. 2). The direct
label transfer studies identify several classes of RNA-binding proteins
capable of binding labeled transcript of wild-type (8) and of the
mutant
2ARs.
ARB protein (Mr = 35,000) was labeled prominently by the radiolabeled transcripts of
wild-type
2AR as well as those of the mutant with the U
to G substitution in the most 5
-AUUUA pentamer (mutant 1). Several other, slower migrating proteins, less prominently labeled, were also
visible. Using radiolabeled
2AR transcripts from the
mutant in which U to G substitutions occur in the AUUUUA hexamer at
1592 (mutant 2) substantially reduced the amount of label transfer to
ARB protein compared with that obtained with the wild-type and
mutant 1 transcripts. U to G substitutions to the 5
-flanking poly(U)
region of the AUGGUA hexamer as well as U to G substitutions to both
the 5
- and 3
-flanking poly(U) regions of the mutated hexamer
abolished binding of the transcripts to
ARB protein. Quantification
by
-phosphorimaging of the data from replicate label transfer
experiments reveals that a more than 90% label transfer to
ARB
protein is abolished by these U to G substitutions (Fig. 2, right
panel). The results from the direct label transfer studies using
labeled transcripts of each mutant were tested further via competition
studies in which the unlabeled transcript of mutants 2 and 3 were
employed to compete with the binding of the radiolabeled wild-type
2AR mRNA to
ARB protein (Fig. 3).
Unlike the wild-type transcripts, the unlabeled transcripts for mutants
2 and 3 failed to compete with 32P-labeled
2AR mRNA for label transfer to
ARB protein (Fig.
3).
Since mutation in
the 20-nucleotide region abolished binding of ARB protein, it was of
interest to test whether this 20-nucleotide AU-rich region is the major
element involved in the binding of
ARB protein. The AUUUA pentamer
is a highly conserved sequence that is often repeated in tandem in the
3
-untranslated region of RNAs encoding short-lived cytokines and
proto-oncogenes, and their presence appears to alter stability of some
mRNAs (13). In contrast, the element identified in hamster
2AR mRNA is an AUUUUA hexamer flanked by U-rich
regions uniquely different from the known cis-acting
elements identified in several, highly regulated mRNAs. To compare
the ability of hexamer and pentamer AREs to binding to
ARB protein,
we engineered plasmids expressing the 20-nucleotide ARE found in the
wild-type
2AR mRNA (see Fig. 1) and a second plasmid
expressing the same 20-nucleotide ARE in which the AUUUUA hexamer is
replaced by a AUUUA pentamer. Uniformly radiolabeled, capped, RNA
corresponding to each of these probes was prepared together under
identical conditions, and binding to
ARB protein was tested by
direct label transfer (Fig. 4). The radiolabeled,
20-nucleotide hexamer ARE (*HEXAMER) displays prominent
binding to the Mr 35,000
ARB protein. In
sharp contrast, the uniformly labeled 20-nucleotide ARE in which the
hexamer is replaced by a pentamer (*PENTAMER) binds
ARB
protein less well, displaying less than 50% of the binding observed
with hexamer ARE. Binding of hexamer versus pentamer was
investigated further through competition studies in which the ability
of the unlabeled, 20-nucleotide hexamer ARE competed with radiolabeled
hexamer and pentamer AREs for binding to
ARB protein (Fig. 4). The
unlabeled, 20-nucleotide hexamer ARE competes effectively with the
binding of the labeled hexamer ARE as well as the pentamer ARE, as
displayed in the autoradiogram (Fig. 4, top). Quantification
of data from replicate, independent label transfer studies in which
both labeled AREs were prepared and used simultaneously reveal the
hexamer-containing ARE as the preferred binding site for
ARB (Fig.
4, bottom).
To more fully explore the binding character of ARB protein with the
hexamer- as compared with pentamer-containing AREs, we examined the
ability of the unlabeled pentamer-containing ARE to compete with
labeled pentamer- and hexamer-containing AREs (Fig. 5).
The autoradiogram reveals the more prominent binding of the
hexamer-containing versus pentamer-containing AREs (Fig. 5,
top). Unlabeled pentamer ARE competed for binding to
ARB
protein with labeled hexamer and pentamer AREs, confirming earlier
studies in which the binding of the pentamer-containing ARE was used to elucidate the properties of RNA binding to
ARB protein (8, 34).
Analysis of the combined results from the competition studies suggests
that the hexamer-containing ARE displays greater binding to
ARB
protein, competing more robustly than the pentamer in these studies
(Figs. 4 and 5).
Wild-type and Mutant
Agonist-mediated changes in receptor mRNA
levels can be mimicked in culture using DDT1-MF2 smooth
muscle cells (6) as well as cell lines stably transfected and
expressing G protein-linked receptors (40, 41). Wild-type CHO cells
express very few endogenous 2AR and provide an ideal
cell-type for the study of agonist-induced regulation of
2AR with essentially no background signal, once stably
transfected with expression vectors (42, 43). To address not only the
binding character of
ARB protein, but more importantly the influence
that mutations of the hexamer-containing ARE would have on the
steady-state level of
2AR mRNA harboring these
mutated 3
-untranslated regions in vivo, we adopted the CHO
cells and transfected the cells with vectors expressing wild-type
2AR mRNA as well as mutants 1, 2, 3, and 4.
CHO clones stably transfected and expressing 2AR
mRNA with wild-type and mutated 3
-untranslated regions of interest
were created. S100 fractions prepared from CHO cells display
ARB
protein binding of uniformly labeled, capped, and polyadenylated
2AR mRNA, as evidenced in autoradiograms of the
Mr 35,000 protein following direct label
transfer studies (Fig. 6). The results of the direct
label transfer studies performed with S100 fractions from the CHO cells
agree well with those performed with S100 fractions from
DDT1-MF2 smooth muscle cells (Fig. 2). Due to the
significant expression of endogenous
2AR in the
DDT1-MF2 cells, the CHO cells with virtually no endogenous
expression of
2AR presented the only real candidate for
these studies. In agreement with the results obtained with S100 from
the smooth muscle cells (Fig. 2), mutation of the hexamer ARE
attenuated its ability to bind to
ARB protein (Fig. 6, lane
3, M3). Further mutation of the hexamer ARE with 5
(mutant 3)- and both 5
- and 3
(mutant 4)-flanking sequences abolishes
the ability of these mRNAs to bind
ARB protein (Fig. 6,
lanes 4 and 5).
As a prelude to a determination of the effects of the mutated
3-untranslated regions on the steady-state levels of
2AR mRNA, the expression of
2ARs and
their capacity for down-regulation was examined. Expression of
2AR was determined by radioligand binding studies with
the high affinity,
-adrenergic antagonist ligand ICYP. Stable
transfectant CHO clones were found to express similar levels of
2AR (2.0-3.0 fmol/105 cells). The
transfected clones expressing the wild-type receptors displayed
agonist-induced down-regulation of receptor expression. Treatment with
agonist (10 µM isoproterenol) for 24 h resulted in a
40-50% decline in receptor number, as measured by ICYP binding (not
shown).
Agonist-induced destabilization of the 2AR mRNA was
explored in the CHO transfectant clones expressing
2AR
mRNA with the wild-type 3
-untranslated region. The stability of
the
2AR transcript was assessed in cells challenged with
-adrenergic agonist as well as with the second messenger cyclic AMP.
Clones stably transfected with vector expressing wild-type
2AR were challenged with either isoproterenol (10 µM, 24 h) or CPT-cyclic AMP (50 µM,
12 h), and actinomycin D was added to arrest transcription for the
periods indicated.
-Adrenergic receptor mRNA levels were
quantified by use of an RNase protection assay (Fig.
7A). In cells not stimulated with either
agonist or cyclic AMP (i.e. control) the half-life for the
2AR mRNA is greater than 10 h. The half-life
for
2AR mRNA in DDT1-MF2 smooth muscle
cells was established to be 12-15 h (6). Treating cells with the
-adrenergic agonist isoproterenol or the poorly hydrolyzed,
water-soluble analogue of cyclic AMP, CPT-cyclic AMP altered
dramatically the stability of the
2AR transcript (Fig.
7A). In the clone expressing the wild-type
2AR mRNA, the half-life is reduced to ~ 7 h by challenge with agonist or with CPT-cyclic AMP (Fig.
7A, lower panel). The half-life for the
2AR mRNA following agonist-induced down-regulation
in CHO cells is similar to that obtained in the DDT1-MF2
smooth muscle cells in culture (5).
Identification of cis-Acting Elements Responsible for
The mutations in the
pentamer and hexamer ARE that altered the ability of the transcripts to
bind to ARB protein were investigated to ascertain what influence,
if any, these mutations would have upon mRNA stability and
agonist-induced destabilization of receptor mRNA. As shown in Fig.
6, disruption of the 3
-untranslated region pentamer with U to G
substitutions (Fig. 1) had no significant influence in the stability of
the
2AR mRNA. In CHO cells expressing
2AR with either wild-type 3
-UTR or a 3
-UTR with U to G
substitutions in the AUUUA pentamer,
2ARs displayed
agonist-induced down-regulation, mutant 1 clones displaying a decline
from 2.1 to 1.09 fmol/105 cells in response to agonist. The
half-life of the receptor mRNA was found to be very similar for
2AR mRNA with either wild-type 3
-untranslated
region or those with the most 5
-AUUUA pentamer disrupted to AUGUA
(Fig. 7B). These data clearly indicate that the integrity of
the pentamer itself is not a requisite for the destabilization of the
2AR mRNA. Furthermore, the results agree well with
the demonstration by others that the presence of an isolated AUUUA
sequence itself does not ensure destabilization of the mRNA
(44).
U to G substitutions in the hexamer of the ARE sharply reduced the
ability of the 2AR mRNA with mutated 3
-untranslated
region to bind
ARB protein. Further U to G substitutions in the
poly(U) regions of the flanking 5
and 3
sequences of the mutated
hexamer led to complete loss of the ability of the mutated
2AR mRNAs to bind
ARB protein. Mutants 2-4 were
transfected into CHO cells and the clones tested for agonist-induced
down-regulation of
2AR mRNA and of
2AR, in response to agonist and CPT-cyclic AMP
treatment. U to G substitutions disrupting the AUUUUA hexamer (mutant
2) not only attenuated the binding of the transcript to
ARB protein, but also attenuated sharply the destabilization of the
2AR mRNA in response to either agonist or treatment
with CPT-cyclic AMP (Fig. 8A). When mutation
of the AUUUUA hexamer was accompanied by U to G substitutions in the
5
-flanking U-rich region (mutant 3), both binding to
ARB protein
and agonist-induced destabilization were abolished (Fig.
8B). The disruption of the 5
- and 3
-flanking regions of
the mutated hexamer AUGGUA displayed the same loss of destabilization
of the
2AR mRNA in response to either agonist challenge or treatment with CPT-cyclic AMP (Fig. 8C).
Agonist-induced down-regulation of
2AR was sharply
reduced by mutation 3 in the 3
-UTR, declining only 15%. In clones
expressing
2AR with mutation of the hexamer alone,
agonist-induced down-regulation of
2AR was variable in
extent ranging from 10 to 30%. Taken together, these results
demonstrate that the integrity of the hexamer ARE is obligate for
agonist-induced destabilization of the
2AR mRNA (Fig. 8, A-C, lower panels). Disruption of the
hexamer, but not the pentamer, attenuated, whereas further mutation of
the U-rich regions flanking 5
and 3
to the hexamer nearly abolished
binding of the transcript to
ARB protein and agonist-induced
destabilzation of
2AR mRNA. To confirm that the
600-nucleotide probe hybridizes with the target RNA to near completion
(more than 90%) we established a standard curve using different
quantities of in vitro transcribed
2AR
mRNA and a 285-nucleotide riboprobe as well as the 600-nucleotide riboprobe used throughout the earlier work (Fig.
9A and B). Receptor mRNA was
quantified equally well with either the 285- or the 600-nucleotide riboprobe. Agonist-induced destabilization data and the mRNA
half-life, as determined using the 285-nucleotide riboprobe in RNA
extracted from CHO cells expressing
2AR with wild-type
3
-UTR, agree well with data obtained with the 600-nucleotide probe
(Fig. 9C).
Agonist-mediated down-regulation of receptor expression is
commonly observed for members of the G protein-linked receptor (GPLR)
superfamily. Clues to mechanisms underlying this mode of receptor
down-regulation were provided when it was first observed that
-adrenergic agonists induce both a decline in
2ARs
and
2AR mRNA levels in response to chronic
stimulation (3). The basis for the agonist-induced decline in
2AR mRNA levels was shown subsequently to reflect a
destabilization of preexisting receptor mRNA (6). Since this first
report of agonist-mediated destabilization of an mRNA encoding a
GPLR by Hadcock et al. (6), other members of this
superfamily of receptors have been reported to be regulated at the
level of mRNA stability, including rat m1-muscarinic (41),
AT1-angiotensin II (45), rat 5-HT 2A-serotonin (46), and human thrombin
(34) receptors. A search for well known destabilizing elements,
i.e. AUUUA pentamer flanked by poly(U) regions, identified
these and other GPLRs (34).
To identify candidate proteins involved in the agonist-induced
destabilization, uniformly labeled, full-length, capped and polyadenylated 2AR mRNA in tandem with UV-catalyzed
direct label transfer to cross-link putative RNA-binding proteins (8).
Prominently labeled is the Mr 35,000
ARB
protein, displaying specificity for binding to mRNA of
2AR and thrombin receptors which display agonist-induced
destabilization of mRNA, while not recognizing mRNA of
-adrenergic subtypes (rat and human
3) (34) which shows transcriptional repression without alterations in the
t1/2 of the receptor mRNA (47).
In the present study the sequence of hamster 2AR
mRNA necessary for binding to
ARB protein was revealed to be a
20-nucleotide ARE consisting of an AUUUUA hexamer flanked on 5
and 3
reaches by U-rich regions. Several eukaryotic, mRNA-stability
determinants rich in AREs have been identified in the 3
-untranslated
regions of transiently expressed mRNAs. Extensive mutational analysis and preparation of chimeric RNAs using AU-rich sequences of various proto-oncogenes and cytokine mRNAs as well as stable mRNA such as that for
-globin have permitted identification of AUUUA pentamers that are present both in tandem repeats (13) and nonamers such as
UUAUUUA(U/A)(U/A) (24) and UUAUUUAUU (25), as potent destabilizing elements in these RNAs.
Regulatory, trans-acting factors identified to date, include
several cytoplasmic mRNA-binding proteins, which specifically interact with the ARE (7, 9, 11, 12, 32, 48, 49). Based on their
patterns of induction as well as their subcellular localization, these
RNA-binding proteins have been shown to act as stabilizers (9, 33),
destabilizers (7, 11, 12, 32), and nucleocytoplasmic transporters (50).
Agonist stimulation of 2ARs provokes a significant
up-regulation of
ARB protein, as established by UV-catalyzed
cross-linking (7). Treatment of DDT1-MF2 smooth muscle
cells with the glucocorticoid dexamethasone up-regulates
2AR expression and
2AR mRNA levels,
and simultaneously down-regulates the level of
ARB protein (7).
Based on the pattern of induction and the sign of the change in
2AR mRNA levels, the Mr
35,000
ARB protein appears to be a destabilizer of mRNA.
Bohjanen et al. (51) identified three different kinds of
RNA-binding proteins, termed AU-A, AU-B, and AU-C in human lymphocytes. The Mr 34,000 AU-A protein is constitutively
expressed, interact with AUUUA multimers, and other U-rich sequences,
including poly(U) sequences. AU-B and AU-C are 30,000 and
Mr 43,000 cytoplasmic proteins requiring at
least three tandem repeats of AUUUA pentamers for efficient binding and
do not bind to AUUUUA-containing AREs. ARB protein, in sharp
contrast, binds efficiently hexamer AREs. The binding by
ARB protein
is reduced by 50% when the hexamer ARE of the
2AR
mRNA is replaced by a pentamer.
ARB protein also does not bind
to poly(U) sequences (34). An additional AU-binding factor, AUF1, is a
Mr 37,000 protein purified and cloned from the
cytoplasmic extract of human leukemia cells (32). Although AUF1 has
been shown to bind to
2AR mRNA via the 3
-UTR, its
nonidentity with
ARB protein was established by immunoprecipitation
and immunoblotting analyses of AUF1 polypeptides of UV cross-linking
products (52). DeMaria and Brewer (53) demonstrated that AUF1-ARE
binding affinity is directly related to the potency with which an ARE
destabilizes a heterologous mRNA. The more prominent binding of the
hexamer-containing ARE as compared with the pentamer-containing ARE to
ARB protein suggests the
ARB protein binds preferentially to the
hamster
2AR mRNA hexamer-containing ARE than to
other mRNAs with ARE harboring AUUUA pentamers, such as those found
in regulated mRNAs.
Although AU-rich sequences have been identified within the
3-untranslated regions of many GPLR mRNAs (34, 52), the current report is the first to identify a particular cis-acting
element obligate for binding to a specific RNA-binding protein, in this case
ARB protein. Although having some similarities with other known
AREs, the ARE identified in the present work is unique in several
important ways. First, the core region is an AUUUUA hexamer and not the
more commonly found pentamer. Second,
ARB protein binding is
significantly greater for the hexamer rather than pentamer-containing ARE. Many ARE-binding proteins display just the opposite,
i.e. much less binding affinity for hexamer- than
pentamer-containing AREs as the core region (51). Finally, we
demonstrate that the hexamer-ARE is obligate for agonist-induced
destabilization of the
2AR mRNA in vivo.
Although preferring the hexamer- to pentamer-containing ARE of the
2AR mRNA,
ARB protein fails to bind, and
2AR mRNA fails to undergo destabilization if the
integrity of the hexamer-containing ARE is lost.