From the
The M
Agonist-induced down-regulation of G-protein-linked receptors,
like the
The steady-state levels of highly regulated mRNAs (e.g. mRNAs of granulocyte/macrophage colony-stimulating-factor, tumor
necrosis factor-
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)
Recently, we identified a M
In the present report we
explored the role of the cognate sequence identified for binding of
The rodent
Uniformly labeled,
full-length, capped and polyadenylated mRNAs for the
The ability of the ORF and 3`-UTR of the
thrombin receptor mRNA to compete with the
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
To
identify candidate proteins involved in the agonist-induced
destablization, we employed uniformly labeled, full-length, capped and
polyadenylated
The identification of an AUUUA pentamer in the
Our ability to generate uniformly
labeled, full-length, capped and polyadenylated mRNAs for the three
The importance of flanking poly(U) regions about an
AUUUA pentamer is highlighted by the fact that rat
The most exciting outcome of the present work was evaluating
the predictive value of the presence of the cognate sequence for
The present study expands our
understanding of the cognate sequence of mRNA recognized by the
M
The
rat
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
-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).
, 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).
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.
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).
-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.
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.
-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).
-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).
-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.
-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.
-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.
-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.
-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).
-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.
-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.
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
-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.
-AR,
-adrenergic receptor;
-ARB protein,
-AR mRNA-binding
protein; ORF, open reading frame; PIPES, 1,4-piperazinediethanesulfonic
acid; CPT-cAMP, 8-(4-chlorophenylthio)-cAMP.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.