(Received for publication, August 1, 1995; and in revised form, January 3, 1996)
From the
In both cell culture based model systems and in the failing
human heart, -adrenergic receptors (
-AR) undergo
agonist-mediated down-regulation. This decrease correlates closely with
down-regulation of its mRNA, an effect regulated in part by changes in
mRNA stability. Regulation of mRNA stability has been associated with
mRNA-binding proteins that recognize A + U-rich elements within
the 3`-untranslated regions of many mRNAs encoding proto-oncogene and
cytokine mRNAs. We demonstrate here that the mRNA-binding protein,
AUF1, is present in both human heart and in hamster DDT
-MF2
smooth muscle cells and that its abundance is regulated by
-AR
agonist stimulation. In human heart, AUF1 mRNA and protein was
significantly increased in individuals with myocardial failure, a
condition associated with increases in the
-adrenergic receptor
agonist norepinephrine. In the same hearts, there was a significant
decrease (
50%) in the abundance of
-AR mRNA and
protein. In DDT
-MF2 cells, where agonist-mediated
destabilization of
-AR mRNA was first described,
exposure to
-AR agonist resulted in a significant increase in AUF1
mRNA and protein (
100%). Conversely, agonist exposure
significantly decreased (
40%)
-adrenergic
receptor mRNA abundance. Last, we demonstrate that AUF1 can be
immunoprecipitated from polysome-derived proteins following UV
cross-linking to the 3`-untranslated region of the human
-AR mRNA and that purified, recombinant p37
protein also binds to
-AR 3`-untranslated region
mRNA.
The condition of heart failure is associated with heightened
activity of the adrenergic nervous system(1) , the severity of
failure correlating with increases in circulating and cardiac
concentrations of the catecholamine, norepinephrine(2) . As a
consequence of this increased ``adrenergic drive'' the
cardiac -AR(
)/G-protein/adenylyl cyclase pathway can
become markedly desensitized. One major component of the
desensitization is selective down-regulation of the dominant adrenergic
receptor subtype within the human myocardium, the
-AR (1, 3, 4, 5) . Recently, we (6) and others (7) have demonstrated that the observed
decrease in
-adrenergic receptors in failing human
heart is closely associated with a corresponding down-regulation of
AR mRNA. Therefore, it is of interest to better define
potential mechanisms responsible for down-regulation of
-AR mRNA.
Experiments performed using hamster DDT-MF2 smooth
muscle cells (8, 9) suggest that down-regulation of
the endogenously expressed
-AR mRNA does not appear to
be caused by a decrease in the rate of transcription; rather, it
appears that agonist exposure decreases the half-life of
-AR mRNA from approximately 12 to 5 h(8) .
This regulatory mechanism has been demonstrated previously to be
important for numerous mRNAs encoding proto-oncogenes, lymphokines, and
cytokines(10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) .
For these gene products regulation of mRNA stability has also been
associated with the interaction of the mRNA with a family of cytosolic
proteins (M
30,000-40,000) that often bind
to A + U-rich elements (AREs) commonly located within the
3`-untranslated region (3`-UTR) of the
mRNA(28, 29, 30, 31, 32, 33, 34) .
This interaction induces mRNA degradation by mechanisms only partially
understood. However, for some mRNAs including those containing
AREs(35, 36) , the degradation of mRNA may be
associated with the process of translation. The cytosolic A +
U-rich mRNA-binding proteins are in general considered to be distinct
from other mRNA-binding proteins such as the heterogenous nuclear
ribonucleoprotein particles(37, 38) , however, the
role of heterogenous nuclear ribonucleoprotein particles A1 and C
proteins as cytoplasmic factors regulating mRNA stability is currently
undergoing reassessment(39, 40) .
From previous
studies (41, 42, 43) using cytosolic extracts
produced from DDT-MF2 hamster smooth muscle cells, the
properties of a
-AR mRNA-binding protein (
-ARB), which binds
to hamster
-AR and human
-AR mRNAs,
has undergone preliminary characterization. Binding of
-ARB to
mRNA was determined to involve regions of the 3`-UTR of the hamster
-AR mRNA containing an ARE(41, 42) .
In addition, agonist stimulation of the
-AR pathway or protein
kinase A activation by a cAMP analogue resulted in significant
up-regulation (3-4-fold) of
-ARB protein as detected by UV
cross-linking. Conversely, treatment of DDT
-MF2 cells with
dexamethasone, which up-regulates
-AR mRNA,
down-regulated
-ARB by
50%. Therefore, agents that regulate
hamster
-AR mRNA stability and abundance appear to
affect reciprocally the abundance of
-ARB protein. Among the
family of G-protein-coupled receptors, the mRNAs of the hamster
-AR, the human
- and
-AR, and the thrombin receptor have all been
demonstrated to interact with
-ARB(41, 42, 43) . To date, the identity
of
-ARB has remained unresolved. However,
-ARB does share
characteristics in common with several described A + U-rich
mRNA-binding proteins(41) , including AUF1(31) .
The
cytoplasmic RNA-binding protein, AUF1 (A + U-rich element
RNA-binding/degradation factor), has recently been cloned and
characterized(31) . AUF1 binds to the 3`-UTRs of several highly
regulated mRNAs including c-myc, GM-CSF, and c-fos.
Furthermore, there is evidence of ``cause and effect''
between AUF1 and regulation of mRNA stability in that partially
purified AUF1 can selectively accelerate the degradation of c-myc mRNA in an in vitro mRNA decay system(34) . Based
on these findings, we endeavored to determine if AUF1 was expressed in
human heart and in DDT-MF2 cells, and if so, if AUF1
abundance was regulated by stimulation of the
-AR pathway. Here we
report that the mRNA encoding AUF1 protein is expressed in both human
heart and DDT
-MF2 cells. Furthermore, exposure of
DDT
-MF2 cells to
-AR agonist, or high adrenergic
drive, as manifest in the failing human heart, results in up-regulation
of the AUF1 gene product. In addition, we show that purified,
recombinant p37
protein binds to an ARE within the
3`-UTR of the human
-AR mRNA, and that cellular AUF1
can be immunoprecipitated from polysome-derived proteins following UV
cross-linking to the 3`-UTR of the human
-AR mRNA.
These data link for the first time a specific mRNA-binding protein
known to be associated with the regulation of mRNA stability, with the
mRNA of a G-protein-coupled receptor. In addition, they demonstrate
that the abundance of this mRNA-binding protein is up-regulated by
adrenergic stimulation, an effect known to destabilize
-AR mRNA.
For human heart tissues, approximately 100 mg of tissue frozen in
liquid N was placed in 200 µl of lysis buffer (20
mM Tris-HCl, 0.1% Triton X-100, 5 mM
-mercaptoethanol, aprotinin 10 µg/ml, and leupeptin 10
µg/ml). The tissue was homogenized using 25 strokes of a Teflon
pestle and a T-liner at 4 °C. The homogenate was subjected to 8
rounds of freeze/thaw and centrifuged at 16,250
g for
15 min. The supernatant was transferred to a fresh tube and the protein
concentration determined as above. To perform Western analysis, 100
µg of total cellular protein was subjected to SDS-PAGE as described
above and transferred to a polyvinylidine difluoride membrane
(Millipore, Marlborough MA) for 2.5 h at 40 V. Subsequent
immunodetection methods were as outlined above.
An Escherichia coli TOP10 (Invitrogen)
clone containing pTrcHisB/P37CR was induced to express plasmid-encoded
protein by culturing with 1 mM isopropyl--D-thiogalactopyranoside (U. S.
Biochemical). His
-AUF1 fusion polypeptide was purified
using the Xpress System (Invitrogen) under native conditions as
described by the manufacturer. Selected fractions were electrophoresed,
and the protein profile assessed by Coomassie staining. Fractions
4-11 were pooled, and human
-lactalbumin (Sigma) was added
to a final concentration of 100 µg/ml to aid in preserving the
activity of the recombinant AUF1 protein during storage at -80
°C. The concentration of purified recombinant AUF1 was determined
by comparison with known amounts of bovine serum albumin using
Coomassie-stained SDS-polyacrylamide gels and immunoblot analysis using
anti-AUF1 polyclonal antiserum.
Figure 1:
AUF1 proteins in human left ventricular
myocardium. A, a representative immunoblot of AUF1 proteins
from four nonfailing and four failing human hearts. Whole tissue
lysates of human ventricular myocardium were prepared and assayed for
AUF1 polypeptides using a polyclonal anti-AUF1 antibody(31) .
In each case, equivalent amounts of total cellular protein (100 µg)
were analyzed for each subject. The data from these hearts and an
additional eight hearts are summarized in B. B, the
relative abundance of AUF1 immunoreactive proteins was investigated in
nonfailing (n = 8) and failing (n = 8)
human heart. Data are presented as X ± S.E. The relative
abundance of p45 and p40 proteins are expressed in
arbitrary units (A.U.) Statistical analysis of nonfailing
compared to failing hearts was performed by use of a two-tailed,
unpaired t test.
Although the identity of p45
is currently unknown, it is obviously immunologically related to
p37 and to p40
. Furthermore, as
demonstrated below (Fig. 6), p45 recognized and bound to A
+ U-rich mRNAs thus appearing to be similar in this regard to AUF1
proteins. Lastly, like p40
, p45 abundance is
up-regulated in individuals with heart failure. The relationship of p45
to AUF1 or to other mRNA-binding proteins beyond these shared
characteristics remains to be determined.
Figure 6:
A,
immunoprecipitation and Western blot analysis of AUF1 polypeptides from
UV cross-linking reactions. KCl-extracted polysome-associated proteins
(RSW) from DDT-MF2 cells treated with isoproterenol (10
µM for 48 h) were pre-cleared with preimmune serum. RSW (2
10
cell equivalents) was UV cross-linked to 5
10
cpm of capped, uniformly labeled in vitro transcribed RNA corresponding to the human
-AR
3`-UTR. Following cross-linking, the reactions were treated with RNase
A + T1, diluted with NET-gel buffer(31) , and AUF1
proteins subjected to immunoprecipitation using polyclonal anti-AUF1
antiserum or nonimmune serum. Proteins were resolved by SDS-PAGE and
detected by autoradiography. B, polysome-associated proteins
(not subjected to KCl-extraction) were subjected to immunoprecipitation
as described in A. In addition, an input lane, labeled
``X-link,'' represents 10% of the cross-linking
reaction not subjected to immunoprecipitation. C,
polysome-associated proteins from isoproterenol-treated (10 µM for 24 h) DDT
-MF2 cells were UV cross-linked to
radiolabeled mRNA encoding the 3`-UTR of the human
-AR. The reactions were digested with RNase A +
T1, resuspended in Laemmli buffer, and subjected to SDS-PAGE. The gel
was transferred to a polyvinylidine difluoride membrane and proteins
detected as described under ``Materials and Methods'' (right panel). Proteins recognized by the anti-AUF1 antibody
are delineated by a bracket. The same membrane was also
analyzed by autoradiography (left panel). A band corresponding
to p38
is specifically labeled. Molecular weights
are as indicated.
As determined by
quantitative reverse transcription-PCR, -AR mRNA
abundance was significantly decreased (
40%) in failing as compared
to nonfailing, control hearts (Table 1). These results are
consistent with previous findings(6) .
-AR density and
subtype proportions also were determined in the same failing and
nonfailing hearts.
-AR density was also significantly
reduced (
61%) in failing compared to nonfailing hearts (Table 1). By contrast,
-AR density was not
different in failing compared to nonfailing hearts (23.2 ± 2.0 versus 27.4 ± 3.4 fmol/mg protein, respectively).
In
summary, these data indicate that (i) AUF1 mRNA and protein are
expressed in human ventricular myocardium; and (ii) in individuals with
heart failure, AUF1 mRNA and protein are significantly up-regulated
and, both -AR mRNA and protein are down-regulated.
From these data we conclude that up-regulation of AUF1 in human heart
may be involved in the regulation of
-AR mRNA
stability and thus may be at least partially responsible for the
decline in
-AR mRNA and subsequently protein abundance
in the failing heart.
Figure 2:
Immunoblot of AUF1 proteins in
DDT-MF2 cells. A, whole cell lysates of
DDT
-MF2 cells smooth muscle cells were prepared and assayed
for AUF1 polypeptides using a polyclonal anti-AUF1 antibody (31) . Three bands are in evidence,
p37
, p40
, and p45, a
polypeptide immunologically related to AUF1. B, post-nuclear
S130 cytosol and polysome fractions from DDT
-MF2 cells were
analyzed for the presence of AUF1
polypeptides.
As determined
by immunoblot analysis, p37 protein was increased to 230
± 50% of control (n = 5, pooled data) in cells
treated with 10 µM(-)-isoproterenol for 24 (n = 2) or 48 h (n = 3) compared to untreated
controls (Table 2). The relative abundance of p37
protein was increased to a similar extent by isoproterenol
treatment at both the 24- and 48-h time points. In the same
preparations, the relative amounts of p40
and p45 were
also determined. In cells treated with isoproterenol, p40
increased to 160 ± 30% of control (n = 5,
pooled data) and p45 increased to 180 ± 30% of control (n = 5, pooled data) (Table 2). At each time point, the
relative abundance of p40
and p45 were roughly similar,
both being of considerably greater abundance than p37
.
Also of note is the finding that the relative abundance values of
p37
and p40
are greater (in arbitrary
units) at 48 h compared to 24 h. This may indicate an increased
relative amount of AUF1 proteins as the cells continue to grow.
In
the same cells, the relative abundance of the endogenous
-AR mRNA was measured. Treatment of
DDT
-MF2 cells with 10
µM(-)-isoproterenol for 24 (n = 2)
or 48 h (n = 3) produced a decrease in
-AR mRNA to 63 ± 2% (p<0.05, n = 5) of control. The degree of down-regulation was highly
similar at 24 and 48 h (63% versus 62% of control). As with
AUF1, the protected signal for the hamster
-AR mRNA
was normalized to the invariant signal for 18 S rRNA (data not shown).
The degree of down-regulation of
-AR mRNA was in close
agreement with a previous investigation (8) and correlates well
with the degree of receptor down-regulation.
In summary, these
results demonstrate that in DDT-MF2 hamster smooth muscle
cells: (i) stimulation of the
-AR pathway produces an increase in
AUF1 mRNA and p37
protein. In addition, the abundance of
p40
and p45 proteins are also increased; (ii) there is a
reciprocal decrease in
-AR mRNA abundance; and (iii)
p37
protein is expressed and localized to a polysome
fraction. We conclude that agonist-mediated up-regulation of AUF1
protein in DDT
-MF2 cells may contribute to agonist-mediated
destabilization and down-regulation of the hamster
-AR
observed in these cells.
Figure 3:
Nucleotide sequence of the 3`-
untranslated region of the human -AR. The 3`-UTR of
the human
-AR was sequenced from the previously cloned
cDNA (49) using the dideoxy method. The nucleotide sequence
begins with the stop codon (UAG) at nucleotide 1432 and extends for an
additional 932 nucleotides. A uniquely long U-rich region as well as
several A + U-rich regions which are potential AREs are in bold and underlined including the putative mRNA
destabilizing sequence, UUAUUUAU. Four canonical poly(A) addition
sequences are shown in bold, underlined, and italics.
Figure 4:
UV cross-linking of RSW proteins to
multiple radiolabeled RNAs. Representative autoradiogram of RSW from
DDT-MF2 cells treated with(-)-isoproterenol (10
µM for 48 h) and UV cross-linked to capped, uniformly
radiolabeled in vitro transcribed RNAs. Equal amounts of RSW
(20 µl,
5
10
cell equivalents/µl) and
equimolar amounts of radiolabeled RNA were added to each reaction. Lane 1, non-UV cross-linked control; lane 2,
-AR 3`-UTR only; lane 3,
-AR coding region (CR) only; lane 4, c-myc 3`-UTR. A band at M
38,000,
previously designated as
-ARB(41, 42, 43) , binds to the
-AR 3`-UTR, and to the c-myc 3`-UTR, but not
to the
-AR CR.
Binding of all RSW
proteins to the -AR 3`-UTR is effectively competed by
a 10-fold molar excess of unlabeled
-AR 3`-UTR (Fig. 5). By contrast, only binding of M
38,000 protein(s) is competed by a 10-fold molar excess of the A
+ U-rich GM-CSF 3`-UTR RNA but not by a 50-fold molar excess of
GM-CSF. The
GM-CSF RNA contains only one of the five
pentameric AUUUA motifs present in the wild-type GM-CSF RNA (30) . By these criteria, and as demonstrated previously for
the hamster
-AR(41) , M
38,000 (
-ARB) has the properties of an A + U-rich
mRNA-binding protein.
Figure 5:
Competitive displacement of -ARB
protein binding to
-AR 3`-UTR RNA. Radiolabeled RNA
corresponding to the 3`-UTR of the human
-AR was UV
cross-linked to RSW proteins in the presence of increasing amounts (0-,
10-, and 50-fold molar excess) of unlabeled competitor RNAs encoding
the human
-AR 3`-UTR (lanes 1-3),
GM-CSF 3`-UTR (lanes 4-5) and
GM-CSF 3`-UTR (lanes 6 and 7).
-AR and GM-CSF but not
GM-CSF competed effectively for
-ARB
binding.
To test the hypothesis that the M 38,000 protein (
-ARB) may be an
AUF1-related protein, RSW proteins from isoproterenol (10 µM for 48 h) stimulated DDT
-MF2 cells were UV
cross-linked to radiolabeled
-AR 3`-UTR, as described
under ``Materials and Methods.'' The reaction was
immunoprecipitated using a polyclonal anti-AUF1 or with non-immune
serum. Anti-AUF1 serum selectively immunoprecipitated two proteins (Fig. 6A), a single major band of
M
45,000 and a band of weaker intensity immediately below the major
band. This finding is in exact concordance with that of Zhang et
al.(31) when immunoprecipitating AUF1 proteins UV
cross-linked to the c-myc ARE. No proteins were evident when
immunoprecipitating with non-immune serum.
In polysomes extracts
with KCl, as in the RSW preparations used above, proteins at
M
40,000 to 45,000 are not readily apparent in UV
cross-linking experiments. By contrast, polypeptides at M
40,000 to 45,000 are readily detected by UV
cross-linking to the 3`-UTR of the human
-AR when
using polysomes prior to KCl extraction. We therefore performed similar
UV cross-linking immunoprecipitation experiments using a polysome
preparation. Here, three proteins of
M
40,000
to 45,000 are immunoprecipitated corresponding to bands of similar
migration in the input lane (i.e. not subjected to
immunoprecipitation, labeled ``X-link''). By contrast, no
polypeptides co-migrating with the M
38,000
protein (
-ARB) were immunoprecipitated (Fig. 6B).
Therefore, all three proteins recognized by the anti-AUF1 antibody
associate with the 3`-UTR of the human
-AR mRNA and
can be immunoprecipitated after UV cross-linking.
An additional
experiment was performed to test whether or not -ARB and AUF1
proteins are the same. Polysome-derived proteins were UV cross-linked
to the radiolabeled mRNA corresponding to the
-AR
3`-UTR. Following separation by SDS-PAGE, the gel was transferred to a
polyvinylidine difluoride (Millipore) membrane and autoradiography and
immunoblotting were performed to ensure that signals could be
superimposed precisely. Here again, the proteins recognized by the
anti-AUF1 antibody do not co-migrate with the M
38,000 signal for
-ARB (Fig. 6C). By
immunologic and migratory criteria, the results presented above in Fig. 6, A-C, all argue strongly against
p38
-ARB being an AUF1 protein.
Lastly, to determine directly if
AUF1 could bind to the human -AR 3`-UTR, radiolabeled
RNA was incubated with purified, recombinant p37
,
subjected to UV irradiation, RNase A + T1 digestion, SDS-PAGE, and
autoradiography. Recombinant p37
protein binds to the
-AR 3`-UTR and to the c-fos ARE but fails to
bind to rabbit
-globin (R
) RNA (Fig. 7). Unlabeled
-AR 3`-UTR RNA effectively competes for AUF1 binding,
while a 100-fold molar excess of
-globin does not (data not
shown).
Figure 7:
UV cross-linking of purified, recombinant
p37 polypeptide to human
-AR 3`-UTR
RNA. Autoradiogram of recombinant p37
UV cross-linked to
radiolabeled in vitro transcribed human
-AR
3`-UTR RNA. Lane 1,
-AR RNA in the absence of
competitor RNA. Lanes 2 and 3,
-AR
RNA in the presence of 10-fold and 100-fold molar excess of unlabeled
-AR RNA. Lane 4, c-fos ARE only. Lane 5, rabbit
-globin RNA
only.
Together, the UV cross-linking and immunoprecipitation
experiments indicate that: (i) a number of polysome-derived proteins
between M
38,000 and 45,000 bind to the
3`-UTR of the human
-AR including a prominent
polypeptide at
M
38,000 as well as several
polypeptides between M
40,000 and 45,000 UV
cross-link to the 3`-UTR but not the coding region of the human
-AR mRNA (a protein of the same apparent molecular
weight binds to c-myc and GM-CSF mRNA; data not shown); (ii)
anti-AUF1 antibody immunoprecipitates proteins between
M
40,000 to 45,000 when cross-linked to the
human
-AR 3`-UTR, and (iii) purified recombinant
p37
binds to the 3`-UTR of the human
-AR mRNA. These results indicate that M
38,000 (
-ARB) is not an AUF1-related protein. The results
also demonstrate by multiple methods that AUF1 proteins bind to the
mRNA encoding the
-AR 3`-UTR.
Agonist-mediated down-regulation of G-protein-coupled
receptors is a well established regulatory paradigm. One of the ways in
which the amount of receptor protein may be down-regulated is by an
alteration in the steady-state abundance of its mRNA which, in turn, is
controlled by transcription rate, or by mRNA stability, or both. Since
the first report of agonist-mediated destabilization of an mRNA
encoding a G-protein-coupled receptor by Hadcock et
al.(8) , the mRNA abundance of a number of other
G-protein-coupled receptors have been reported to be regulated by
changes in mRNA half-life (Table 3). However, it should not be
assumed that for each receptor of a particular subtype that it is
regulated at the level of mRNA stability in all species or cell types.
For example, the -AR mRNA derived from rabbit aorta
smooth muscle cells is regulated by an agonist-mediated response
dependent on protein kinase C activity(54) . By contrast, in
DDT
-MF2 smooth muscle cells, the stability of hamster
-AR mRNA appears not to be regulated by agonist
exposure(55) . Furthermore, the hamster
-AR
mRNA from DDT
-MF2 cells is not A + U-rich and does not
interact with
-ARB(41) . It is unknown whether or not the
rabbit
-AR interacts with A + U-rich
mRNA-binding proteins. As another example, the half-life of the mRNA
encoding the m1-muscarinic receptor is decreased by its cognate
agonist, carbachol, an effect that necessitates an intact
3`-UTR(56) . While the 3`-UTR of the m1-muscarinic receptor
does not contain A + U-rich regions, there are sequence motifs
that may form hairpin loops that might act as binding sites for
RNA-binding proteins. For example, 5`-AUU-3`/5`-UAA-3` motifs appear to
be important for directing endonucleolytic cleavage in mRNAs containing
stem-loop structures (62) . These structures have been shown to
interact with mRNA- binding proteins of
M
34,000(63) .
Several other mRNAs important to
G-protein-coupled receptor signal transduction are also regulated at
the level of mRNA stability including protein kinase A1(64) ,
and the G-proteins G, and
G
(65) . Most recently, we have demonstrated
that the stability of the human
-AR mRNA, the subject
of the current investigation is regulated by
-agonist exposure in
a cell culture-based model system, an effect that is dependent entirely
on the presence of the 3`-UTR(61) .
AUF1 shares several
characteristics in common with the previously described -AR
mRNA-binding protein,
-ARB, which led us to hypothesize that they
might be the same or related proteins. First, both proteins have
similar electrophoretic mobilities. The apparent molecular weights of
AUF1 are 37 and 40 kDa, whereas
-ARB was reported to be
M
35,000 by UV cross-linking of
DDT
-MF2 S100 to the hamster
-AR mRNA (41) and
M
38,000, by UV
cross-linking of polysomal-derived proteins from DDT
-MF2
cells to the human
-AR 3`-UTR (this article). Second,
both proteins share the same cellular fractionation profile, i.e. both are minimally represented in cytosolic fractions and are
present at a much higher relative abundance in polysomes (Refs. 31 and
41; this article). Similarly, by immunoblotting, AUF1 is readily
detectable in whole cell extracts (Fig. 2A), in the
polysome fraction (Fig. 2B), and in RSW (data not
shown). Third, in response to
-AR stimulation, both
-ARB and
AUF1 are significantly up-regulated at both 24 and 48 h ((41) ;
this article). Fourth,
-ARB and AUF1 preferentially bind to the
AREs of multiple mRNAs.
-ARB binds to the mRNAs encoding the human
-AR and to the human and hamster
-ARs(41) . More specifically,
-ARB binds
the proximal U-rich region of the 3`-UTR of human
-AR
mRNA (data not shown). Similarly,
-ARB also binds to the AREs of
GM-CSF and the adenovirus AdIVa2(42) . More recently,
-ARB
has been demonstrated to bind to the thrombin receptor mRNA, another
G-protein-coupled receptor regulated at the level of
stability(43) . Like the hamster
-AR, the
half-life of the thrombin receptor mRNA is significantly decreased by
either agonist treatment or by stimulation of cells by cAMP
analogues(43) . By contrast,
-ARB does not bind to the
hamster
-AR mRNA or to the
-globin mRNA (41) and weakly or not at all to the rat
-,
-, or human
-AR mRNAs(43) .
-ARB, like purified recombinant p37
protein, binds
to the human
-AR mRNA, as well as to the AREs for
c-myc, c-fos, GM-CSF(31) , but not rabbit
-globin mRNA. Fifth, both
-ARB and p37
protein
increase with
-agonist exposure. However, the data from several
different experimental approaches presented herein indicate a lack of
concordance for the molecular weights of
-ARB and AUF1 proteins
and lead to the conclusion that
-ARB is not an AUF1 protein. These
experiments include immunoprecipitation of AUF1 proteins from polysomes
and immunoblotting of polysome-derived proteins to UV cross-linked
-AR mRNA. The biochemical and functional relatedness
of human AUF1 to hamster
-ARB will necessitate the future
purification and/or cloning of
-ARB.
The observation that AUF1
is up-regulated in the failing human heart and in agonist-treated
DDT-MF2 cells is intriguing and leads to several important
questions. The first and most obvious question: is AUF1 responsible for
the observed down-regulation of
-AR mRNA in the
failing human heart and/or the hamster
-AR in
DDT
-MF2 cells? The data presented here indicate that
-AR stimulation results in a reciprocal relationship: the
up-regulation of AUF1 gene product(s) and the down-regulation of
-AR and the destabilization of
-AR mRNA. This suggests that
an increase in AUF1 protein(s) may be associated with increased mRNA
turnover rates for the
-AR as well as for other mRNAs.
It is of
interest that AUF1 also binds to the human -AR mRNA
(data not shown). However, it is well established that the mRNA
-AR is not down-regulated in failing human
heart(1, 3, 4, 5, 6) . To
date, there is no ready explanation for this finding beyond
speculation. It is possible that human
-AR mRNA
undergoes agonist-mediated destabilization but that there is an
offsetting increase in transcription rate such that steady-state mRNA
abundance does not appear to change. It is also possible that there may
be differences in the affinity of AUF1 for the
-AR and
-AR mRNAs functionally affecting the role of AUF1.
Another possibility is that AUF1 and the target mRNA need to be
appropriately co-localized physically and temporally for mRNA turnover
to be accelerated. Lastly, multiple proteins co-immunoprecipitate with
AUF1, however, these proteins have not yet been
characterized(31) . By virtue of its protein structure, which
contains two consensus RNA recognition motifs(31) , AUF1
unquestionably binds to the RNA directly and thus must be central to
the actions of other proteins associated with this complex. However, it
is untested as to whether the same proteins always associate with AUF1
depending on which mRNA is bound. These points all address potential
mechanisms conferring specificity of action for AUF1, a topic for
future investigation.
In the failing human heart, changes in the expression of a number of other genes have also been documented including multiple components of several G-protein-coupled receptor signal transduction pathways, ion channels, and cytoskeletal/structural proteins. Also well documented is the observation that c-myc and other mRNAs encoding proto-oncogenes are transiently up-regulated by stimuli associated with myocardial failure, e.g. high adrenergic drive(66, 67, 68, 69) . By contrast to the transient nature of c-myc and other proto-oncogene expression associated with adrenergic stimuli, increased expression of AUF1 appears to be persistent. In many cases, the individual hearts assayed for AUF1 mRNA and protein abundance had been ``failing'' for months if not years. Therefore, unlike the expression of proto-oncogenes and other immediate-early genes, AUF1 expression does not appear to accommodate to the chronic stimulus causing its up-regulation. An issue of importance to address in the future will be to determine the precise mRNA targets of AUF1 in the human heart. The role of AUF1 or other mRNA-binding proteins in regulating the expression of these mRNAs in the context of heart failure or in myocardial hypertrophy remains to be explored.
We
conclude that AUF1 is present in both human heart and
DDT-MF2 cells and that it is up-regulated by
-agonist
exposure in cells and by the process of heart failure. Furthermore, we
have demonstrated that purified, recombinant AUF1 binds to the 3`-UTR
of the human
-AR mRNA and can be immunoprecipitated
from polysome-derived proteins UV cross-linked to human
-AR 3`-UTR mRNA. Future experiments will attempt to
address more precisely the role of AUF1 in the destabilization of
-AR mRNAs as well as exploring other potential target mRNAs for
AUF1 binding.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U29690[GenBank].