(Received for publication, February 6, 1997, and in revised form, March 24, 1997)
From the Department of Pharmacology, § Division of
Cardiology, Department of Medicine, Medical University of South
Carolina, Charleston, South Carolina 29425 and Leiden Institute
of Chemistry, Leiden University, 2300 RA Leiden, The Netherlands
We report that two subtypes of
2-adrenergic receptors (
2A/D- and
2C-AR) are ectopically expressed with dramatically
different efficiencies and that this difference is due to a
288-nucleotide (nt) segment in the 3
-untranslated region (3
-UTR) of
the
2C-AR mRNA that impairs translational
processing. NIH-3T3 fibroblasts were transfected with receptor
constructs (coding region plus 552 nt,
2C-AR; coding
region plus 1140 nt,
2A/D-AR) and a vector conferring
G418 resistance. Transcription was driven by the murine sarcoma virus
promoter element, and the receptor gene segment was upstream of an SV40
polyadenylation cassette. Drug-resistant transfectants were evaluated
for expression of receptor mRNA and protein. 90% of the NIH-3T3
2C-AR transfectants expressed receptor mRNA, but
only 14% of the clonal cell lines expressed receptor protein. In
contrast, 90% of the NIH-3T3
2A/D-AR transfectants expressed receptor protein (200-5000 fmol/mg). Similar results were
obtained following transfection of DDT1MF-2 cells with the two receptor constructs. The role of the 3
-UTR of the
2C-AR in mRNA processing was determined by
generating new constructs in which the 3
-UTR was progressively
truncated from 552 to 470, 182, 143, or 74 nt 3
to the stop codon.
Truncation of the 3
-UTR resulted in the expression of receptor protein
in the G418-resistant transfectants (nt 74, 100%; nt 143, 80%; nt
182, 50%). The level of mRNA in the transfectants expressing the
receptor protein was not greater than that in nonexpressing clones, and
the differences in protein expression did not reflect altered mRNA
stability in the truncated construct. The
2C-AR mRNA
with the longer 3
-UTR underwent translational initiation as it was
found in the polysome fraction, indicating that the lack of receptor
protein was due to impaired translational elongation or termination.
These data suggest that translational efficiency is a key mechanism for
regulating
2C-AR expression and associated signaling
events.
The response of the cell to hormones/neurotransmitters is an integrated process that involves varying numbers of molecules. Several factors interact to engineer a specific cell response to a particular hormone. The cell-specific and developmentally regulated expression of entities involved in the signaling process is a key component in this process, allowing different cells to respond to the same hormone but with dramatically different results depending on the receptor subtype expressed and/or the cell phenotype. To maintain signaling specificity and diversity in higher organisms, the system has evolved such that the components of the signaling pathway are expressed as isoforms or closely related molecules subserving similar but distinct functions. The preceding thought is particularly evident for cell-signaling events initiated through heptahelical membrane receptors coupled to heterotrimeric guanine nucleotide-binding proteins. For example, the adrenergic signaling system includes two agonists (norepinephrine and epinephrine) that interact to varying degrees with nine different receptors. Signaling by this system is tightly regulated by mechanisms involving the expression and turnover of members of the adrenergic receptor family. Regulatory mechanisms influence receptor gene transcription, receptor mRNA stability, receptor trafficking, and posttranslational events such as receptor phosphorylation.
The 2 subfamily of adrenergic receptors consists of
three distinct proteins that differ in their ligand recognition
properties, tissue distribution, signaling efficiency, and regulation
(1, 2). Heterologous expression of the three
2-AR1 subtypes in various
cells indicates that the three subtypes also exhibit different
trafficking patterns within the cell (3, 4) and are selectively
phosphorylated by receptor kinases (5). The
2A/D-AR subtype is widely distributed
in both peripheral tissues and within the central nervous system,
whereas in the rat the
2B-AR is found primarily in the
kidney, liver, and neonatal lung. The rat
2C-AR is
primarily expressed in the central nervous system, and recently we
identified cis elements in the 5
upstream region of the rat
2C-AR important for cell type-specific transcription of
the receptor gene (6). In contrast to the
2A/D-AR, there is an apparent dissociation between
2C-AR protein
expression and receptor mRNA observed in discrete areas of the
central nervous system and the NG108-15 neuroblastoma × glioma
cell hybrid (7-10), suggesting that translation of the
2C-AR mRNA is a regulated event. To address this
possibility, we compared the relationship between mRNA and protein
expression following ectopic expression of the
2C-AR and
2A/D-AR in two cell lines. We report that the 3
-untranslated region of the
2C-AR impedes
translational processing of the receptor mRNA.
[3H]RX821002 (52 Ci/mmol) and [3H]rauwolscine (87 Ci/mmol) were purchased from Amersham Corp. [32P]dCTP (3000 Ci/mmol) and 35S-dATP (1320 Ci/mmol) were purchased from DuPont NEN. The multiprime DNA labeling system was obtained from Amersham Corp. Sequenase version 2.0 DNA sequencing kit was from U. S. Biochemical Corp. Restriction enzymes and DNA sizing markers were obtained from New England Biolabs Inc. (Beverly, MA). Tissue culture supplies were obtained from JRH Biosciences (Lenexa, KS). Rauwolscine was obtained from Atomergic Chemetals Corp. (Farmingdale, NY). RNA isolation kits were obtained from Stratagene (La Jolla, CA).
Cell Culture, Membrane Preparations, and Radioligand BindingNIH-3T3 fibroblasts were maintained in a monolayer culture in Dulbecco's modified Eagle's medium at 37 °C under 95% atmosphere and 5% CO2 supplemented with 10% bovine calf serum and containing penicillin (100 units/ml), streptomycin (100 µg/ml), and Fungizone (0.25 µg/ml). Cell membranes were prepared, and radioligand binding assays were performed as described previously (11).
Generation of Receptor Expression Constructs and Cell TransfectionThe 2C-AR (RG10) gene
(1929 nt) or
2A/D-AR (RG20) gene fragments
(2,493 nt) were inserted into the expression vector 3
to the MSV long
terminal repeat and upstream of the SV40 polyadenylation signal as
described previously (12). These gene segments began at the
translational start AUG within the context of a Kozak consensus sequence for translational initiation and contained varying lengths of
sequence 3
to the translational stop codon. To generate
2C-AR constructs with a truncated 3
-UTR, the 1,929-nt
gene segment was subcloned into the EcoRI-NotI
restriction sites of pSK. pSK.
2C-AR was linearized at
the 3
end of the gene fragment and digested with exonuclease III using
the Erase-A-Base system (Promega, Madison, WI) to generate clones
containing 74-, 143-, 182-, and 470-nt sequences 3
to the
translational termination codon. The
2C-AR constructs
were restricted with EcoRI-NotI, blunt-ended, and
modified with HindIII linkers for ligation into the
expression vector pMSV. We also generated constructs in a separate
vector that contained both the MSV long terminal repeat and the
neomycin drug resistance cassette. The
p
2A/D-AR/
2C-AR-3
-UTR
construct was generated by a three-component ligation using (I) pGEM7
containing the
2A/D-AR (nt 1-782 of the protein coding
region), (II) a fragment of
2A/D-AR (nt 782-1353 of the
protein coding region plus 64 nt 3
to the translational stop codon),
and (III) the
2C-AR 3
-UTR from nt 113 to 552 following
the translational stop codon. pGEM7.
2A/D-AR (receptor
gene inserted 5
at EcoRI and 3
at BamHI in the
polylinker) was digested with KpnI and HindIII to
remove the last 568 nt of the coding region and the 3
-UTR to generate
component I. To generate component II, pGEM7.
2A/D-AR was
digested with AccI (restriction sites at nt 644 and at nt 64 3
to the translational stop codon), and the 708-nt fragment was
purified, blunt-ended, and digested with KpnI, yielding
component II. To generate component III, we took advantage of an
RsaI restriction site at nt 113, 3
to the translational
stop codon. pGEM7.
2C-AR was restricted with
RsaI, and the 2237-nt fragment containing the 3
-UTR (nt
113-552) and a portion of the plasmid was purified and restricted with
HindIII, yielding component III. The three purified
components were then ligated to generate the
2A/D-AR/
2C-AR-3
-UTR construct in pGEM7, and its sequence was confirmed by restriction mapping and DNA sequencing. The
2A/D-AR/
2C-AR-3
-UTR
insert was then inserted into the HindIII cloning site of
the pMSV.neo vector as described above. Segments of the various
constructs were sequenced by the dideoxy chain termination method.
NIH-3T3 fibroblasts were transfected with a calcium phosphate
precipitate containing 16 µg of expression vector and 4 µg of a
plasmid encoding neomycin resistance or with 20 µg of plasmid when
the vector containing both the receptor construct and the neomycin
resistance cassette was used. Transfected cells were selected for their
resistance to the antibiotic G418 (0.5 mg/ml). Selection was begun 3 days after transfection and continued for 3-4 weeks. G418-resistant
clones were screened for expression of the receptor subtypes by RNA
blot analysis and by their ability to bind the
2-selective antagonists [3H]rauwolscine or
[3H]RX821002. The expected sizes of receptor mRNA
were calculated from the expression vector construct and served as
indicators of appropriate gene insertion. Selected transfectants were
characterized by determining the ligand recognition properties of the
receptor subtype proteins and their apparent molecular weight as
described previously (13, 14).
Total cellular RNA was isolated as described previously (15). For preparation of cytoplasmic RNA, NIH-3T3 cells were washed with ice-cold phosphate-buffered saline (137 mM NaCl, 2.6 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) and harvested by gentle scraping of the plate. Cells were pelleted and resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM MgCl, and 0.5% Nonidet P-40). The homogenate was centrifuged at 10,000 × g, and the supernatant was used to prepare cytoplasmic RNA. Cytoplasmic and total cellular RNA was isolated using the Stratagene RNA isolation kit according to the manufacturer's instructions. Isolated RNA was subjected to electrophoresis on 1% agarose, 3% formaldehyde gels followed by transfer to a nylon filter (Hybond-N) by pressure blotting. The filter was then baked for 2 h at 80 °C in a vacuum oven and prehybridized in phosphate buffer containing 0.5 M Na2HPO4, pH 7.2, 1% bovine serum albumin, 7% SDS, 1 mM EDTA at 65 °C for 1 h before the addition of probe as described previously (6, 15). Radiolabeled probes were generated by random priming using the receptor gene segment as a template. The stability of receptor mRNA was determined by harvesting cells at different times after the blockade of transcription with actinomycin D (5 µg/ml) (15). RNA blots were then hybridized with the appropriate probe as described above. mRNA degradation rate was calculated after densitometric scanning of the autoradiographs.
Analysis of Ribosomal Distribution of Receptor mRNAPolysomes were isolated as described previously (16, 17). Monolayer cultures of NIH-3T3 transfectants were washed with Hanks' balanced salt solution at 4 °C (5.4 mM KCl, 0.3 mM Na2HPO4, 0.4 mM KH2PO4, 4.2 mM NaHCO3, 1.3 mM CaCl2, 0.5 mM MgCl2, 0.6 mM MgSO4, 137 mM NaCl, 5.6 mM D-glucose, 0.02% phenol red) containing 0.01% cycloheximide and harvested by gentle scraping of the plate. Cells were pelleted and then resuspended in 1 ml of lysis buffer (16 mM Tris-HCl, pH 7.5, 250 mM KCl, 10 mM MgCl2, 0.5% Triton X-100, 2 mM dithiothreitol, 0.1 mg/ml cycloheximide, and 2 µl/ml RNasin. 150 µl of 10% Tween 80, 5% deoxycholate was added to the homogenate, the intact nuclei and mitochondria were removed by centrifugation, and the supernatant was loaded onto 15-50% linear sucrose gradients. The gradients were spun at 4 °C at 35,000 rpm in an SW 41 rotor for 2 h and then displaced upward through a modified 0.5-cm flow cell in an ISCO fractionator set to continuously monitor absorbance at 254 nm. Each fraction was extracted with phenol:chloroform, and the RNA was precipitated. The RNA pellet was dissolved in water and denatured by 50% formamide, 6% formaldehyde, 1 × SSC (150 mM NaCl, 15 mM NaC6H5N3O7) at 68 °C for 15 min, and ~5 µg of RNA was blotted directly onto a nylon membrane in a slot-blot apparatus. The blot was hybridized as described above. Following the removal of bound receptor subtype probe, the blot was hybridized with a nick-translated probe derived from rat 28 S RNA to provide controls for sample loading.
Secondary Structure of RNAThe secondary structure of the
2C-AR 3
-UTR was determined by the use of a genetic
algorithm, which is able to simulate RNA folding pathways. The
essential features of the algorithm involve mutations and crossovers in
the population of solutions, with subsequent processing of the fittest
solutions to generate new solutions as described previously (18, 19).
At every algorithm iteration, the population of structures was expanded
via the mutation/crossover process and subsequently diminished to that
of the original population by fitness criteria. A particular analysis
was considered completed when the free energy was not improved after a
chosen number of repetitions. The program MFOLD in the University of
Wisconsin GCG sequence analysis package was used for energy-minimum
calculations. The analysis was achieved using the APL programming
language in the program STAR.
A stable transfection system was used to evaluate the role
of the 3-UTR in regulating expression of the
2C-AR. A
fragment of the rat
2C-AR gene consisting of the 1374-nt
coding region and a 552-nt segment of the gene sequence 3
to the
translational stop codon was inserted into an expression vector
downstream of the pMSV promoter and upstream of an SV40 polyadenylation
cassette (11) (Fig. 1A). The
2C-AR gene construct was introduced into NIH-3T3
fibroblasts by calcium phosphate coprecipitation, and G418-resistant
clones were evaluated for receptor expression by radioligand binding
assays and RNA blot analysis. Only ~14% of the drug-resistant clones
expressed
2C-AR protein, whereas 90% of the clones
expressed receptor mRNA (Fig. 1, B and C).
Similar results were obtained in three different transfections in which a total of >100 individual clones were screened for receptor
expression, and results from a subset of such clones are shown in Fig.
1. In the few clones that expressed receptor protein, the level of
2C-AR mRNA was not greater than that in clones
lacking receptor protein (Fig. 1C). Similar results were
obtained when NIH-3T3 fibroblasts were transfected with a receptor
construct in which the drug resistance cassette was inserted into the
receptor expression vector.2 The
dissociation between
2C-AR protein and mRNA was also
observed following stable transfection of DDT1MF-2 cells
derived from hamster smooth muscle, indicating that the failure to
process the receptor mRNA is not restricted to a fibroblast cell
line (Fig. 1, B and C).
The dissociation between mRNA and expressed protein for the
2C-AR was not observed in similar experiments using
constructs encoding the
2A/D-AR subtype. A fragment of
the
2A/D-AR gene consisting of the 1350-nt coding region
and a 1140-nt segment of the gene sequence 3
to the translational stop
codon was inserted into the pMSV expression vector and introduced into
NIH-3T3 fibroblasts and DDT1MF-2 cells as described above
(Fig. 2A). Approximately 95% of the
2A/D-AR transfectants expressed receptor protein, suggesting that the
2C-AR and
2A/D-AR
mRNAs are processed with different efficiencies by the two cell
lines (Fig. 2B). The two receptor constructs contained
identical 5
upstream regions derived from the vector and exhibited
64% nucleotide sequence identity in the protein coding region. The two
receptor constructs encoded proteins that exhibited 55% overall
homology. A major difference between the two constructs was the gene
segment 3
to the translational stop codon, and subsequent studies
focused on the influence of this region on
2C-AR
expression.
Sequence analysis of the 3-UTR of the
2C-AR identified a polyadenylation signal AAUAAA at nt
469 (Fig. 3). The sequence of the genomic clone in this
region was identical to that of a rat
2C-AR cDNA
(20). The
2C-AR 3
-UTR sequence from nt 1 to 221 is rich
in GC nt (67%), whereas the GC content decreases to 40% from nt 222 to 481. To determine if the 3
-UTR of the
2C-AR influenced receptor expression, NIH-3T3 cells were transfected with
2C-AR gene constructs in which the 3
-UTR was
progressively truncated to 470, 182, 143, and 74 nt 3
to the
translational stop codon (Fig. 4A). In
contrast to the limited expression of the original receptor construct,
~50 (nt 182), 80 (nt 143), and 100% (nt 74) of the cells transfected
with
2C-AR constructs in which the 3
-UTR was truncated
expressed receptor protein (Fig. 4B). In terms of the number
of clones expressing receptor protein, the
p
2C-AR.3
-UTR-182 transfectants were intermediate
relative to the p
2C-AR.3
-UTR-143 and the
p
2C-AR.3
-UTR-470 transfectants. Photoaffinity labeling
of the expressed
2C-AR with the
2-AR photoprobe 125I-AzRAU and radioligand binding studies
indicated that the receptor exhibited the ligand recognition properties
and Mr expected of an
2C-AR (13,
14).2 The expression of receptor in the 3
-truncated
constructs but not in the p
2C-AR.3
-UTR-470 or the
p
2C-AR.3
-UTR-552 was independent of the relative levels
of receptor mRNA (Fig. 4C). Truncation of the 3
-UTR to
nt 470 removed the polyadenylation signal in the receptor gene
sequence, and p
2C-AR.3
-UTR-470 transfectants were
similar to p
2C-AR.3
-UTR-552 transfectants in that
~90% of the clones expressed receptor message but not receptor
protein (Fig. 4B). These data indicated that the presence of
a polyadenylation site in addition to that in the expression vector did
not account for the observed lack of mRNA processing and also
suggest that there is no long range interaction of this region with the
5
region of the transcript. These data indicated that a segment of the
2C-AR 3
-UTR from nt 183 to 470 (3
to the translational stop codon) regulated the processing of the receptor transcript.
Computer simulation of RNA folding in the 3-UTR generates a relatively
stable secondary structure (Fig. 5). The most stable structural elements of the
2C-AR 3
-UTR are the hairpins
from nt 13-90, 135-237, and the long branched hairpin between nt 260 and 450. The 3
-UTR also contains a motif (nt 469-476, UUUUUUAA) similar to sequences UUAUUUAU associated with message instability. Relative to the results of receptor expression in Fig. 4, the hairpin
from nt 13-90 is apparently not involved in the inhibition of
translational processing of the
2C-AR mRNA, as
receptor expression was observed with the
p
2C-AR.3
-UTR-143 construct. As the most efficient
processing of the receptor mRNA occurs with the
p
2C-AR.3
-UTR-74 and the
p
2C-AR.3
-UTR-143 constructs, the stable hairpin from nt
135-237 may contribute to the observed results. However, the p
2C-AR.3
-UTR-182 construct also expressed receptor
protein in ~50% of the transfectants, suggesting that the large
branched hairpin between nt 260-450 also played a role in the
translational processing of the receptor message.
Cellular Localization and Stability of
The processing of transcripts involves several steps
including capping, polyadenylation, splicing, transport out of the
nucleus, and movement through various populations of ribosomes in the
cytoplasm. The role of the 3-UTR in these events was addressed by
determining the distribution of full-length and truncated mRNA
species within the cell and the relative stability of the different
2C-AR transcripts. The apparently poor processing of the
full-length versus truncated mRNA may reflect a failure
of the full-length transcript to move out of the nucleus and associate
with a translationally active population of ribosomes in the cytoplasm.
This issue was addressed by comparing the relative amounts of
2C-AR mRNA in the cytosol in the transfectants that
expressed the receptor protein with those that did not. Analysis of
cytosolic versus total cellular
2C-AR
mRNA indicated that a portion of the mRNA species generated from the p
2C-AR.3
-UTR-74 and
p
2C-AR.3
-UTR-552 constructs were both found in the
cytosol (Fig. 6). Thus, the failure of the
p
2C-AR.3
-UTR-552 to be processed into receptor protein
was not due to the lack of potential mRNA access to the
translational machinery. Fig. 6 also indicates that the lack of
receptor protein in the p
2C-AR.3
-UTR-552 transfectants
was not due to lower amounts of receptor mRNA relative to those
observed in p
2C-AR.3
-UTR-74 transfectants.
Once in the cytosol, the p2C-AR.3
-UTR-552 mRNA
underwent translational initiation as indicated by the presence of the
mRNA in the polysome complex of translationally active ribosomes
(Fig. 7). These data suggest that the presence of the
3
-UTR segment in p
2C-AR.3
-UTR-552 impedes the movement
of the ribosome along the mRNA and that removal of the 3
-UTR
segment between nt 74 and 552 removes this constraint. The failure to
complete the translational processing of the mRNA was not
associated with any differences in the relative stabilities of the
full-length and truncated mRNAs (Fig. 8). The
stability of the
2C-AR mRNA species was determined following the transcription block with actinomycin D and compared with
that of
-actin as an internal control for RNA loading. Analysis of
the degradation rate of receptor mRNA in
p
2C-AR.3
-UTR-74 and p
2C-AR.3
-UTR-552
transfectants revealed a similar t1/2 (~6 h) for both
species, indicating that the 3
-UTR segment from nt 74 to 552 did not
influence mRNA stability (Fig. 8C). These data indicated
that the
2C-AR 3
-UTR was interfering with translational processing of the
2C-AR mRNA. To determine if the
3
-UTR of the
2C-AR could regulate translation of a
heterologous mRNA, we generated a construct in which the 3
-UTR of
the
2C-AR was substituted for the 3
-UTR of the
2A/D-AR (Fig. 9). The segment of the
2C-AR 3
-UTR appended to the
2A/D-AR
contained the portion that apparently impeded translation of the
2C-AR mRNA (Figs. 4 and 9A). NIH-3T3 cells were transfected with the
2A/D-AR/
2C-AR-3
-UTR construct, and
receptor expression was compared with that obtained in parallel transfections with the wild-type
2A/D-AR construct
indicated in Fig. 2. The
p
2A/D-AR/
2C-AR-3
-UTR and
p
2A/D-AR transfectants behaved similarly in terms of
receptor expression (Fig. 9). All of the six clonal cell lines examined
from each transfection expressed receptor protein as determined in
radioligand binding assays (Fig. 9). RNA blot analysis indicated that
similar amounts of
2A/D-AR/
2C-AR-3
-UTR and
2A/D-AR mRNA were expressed in the two series of
transfections.2
The processing of mRNA to the mature protein is subject to
regulation at several steps including translational initiation, elongation, and termination (21-23). These events are influenced by
several factors and often involve cis elements in the 5- and 3
-untranslated regions of the mRNA that are recognized by specific RNA-binding proteins and participate in mRNA masking, message stabilization, and/or movement of messages among different ribosome populations within the cell (24-33). The regulation of protein expression at a translational level has evolved to play significant roles in various aspects of cell-signaling events. One of the first
points of regulation in the translation process is translational initiation and the association of the mRNA with polysomes via the
43 S complex. This step is rate-limiting for the translation of most
mRNA molecules, primarily due to stoichiometric issues concerning
the factors required to form the translational initiation complex. One
of the best understood examples of translational regulation involves
the iron-responsive elements present in the 5
-UTR of ferritin mRNA
and their influence on translational initiation (30). Translational
initiation is also influenced by the 3
-UTR as indicated by the masking
of maternal mRNAs. The masking of maternal mRNAs in
Xenopus involves the binding of proteins to specific
sequences in the 3
-UTR of the mRNA, resulting in a translational block. At appropriate stages of development, the mRNA species are
unmasked with subsequent expression of the protein. These and other
observations related to posttranscriptional editing of the poly(A) tail
and its influence on translation indicate that there is a possibility
of physical interplay between the 5
- and 3
-untranslated regions of
mRNAs. Such an interaction of these two domains may be an important
component of translational regulation. The 3
-UTR is also an important
determinant of stability for several mRNA species (e.g.
-tubulin,
2-AR) and plays a key role in the
segregation of specific mRNAs within the cell (21, 31, 32, 34).
A second point of regulation occurs as the translational machinery
searches for the start codon positioned in the most favorable context
for initiation of protein synthesis. Once translation is initiated,
elongation proceeds at varying rates for different messages, eventually
terminating at the stop codon through the action of the release factor
and the subsequent dissociation of the peptide from the ribosome. The
elongation process is engineered through the action of elongation
factors, and it is fairly rapid, incorporating 4-6 amino acids per s.
The rate of the elongation process is also potentially subject to
regulation, although the mechanisms involved in such regulation are
poorly understood. A decrease in elongation rate (i.e.
translational stalling) may result in an increased amount of mRNA
associated with the polysome fraction in the cytosol, as it is not
efficiently processed through the translation process. In contrast,
mRNAs that are elongated at normal rates would spend less time in
the polysome complex. Thus, there are several points during translation
at which the processing of a particular mRNA can be specifically
regulated. Depending upon the type of translational regulation, a
situation could exist where there is detectable mRNA but the
corresponding protein is absent. Such is the situation for the
2C-AR mRNA.
The translational processing of 2C-AR mRNA appears
to be a regulated event, and this regulation involves a 278-nt segment in the 3
-UTR of the
2C-AR. The importance of this
segment in the processing of the
2C-AR mRNA is
indicated by the expression of the protein following removal of this
domain. As the protein coding region is identical in the truncated
construct, it is not possible to explain the observed data based on
differences in protein turnover. In terms of known mechanisms by which
the 3
-UTR can influence mRNA processing (i.e.
translational initiation and mRNA stabilization), the translation
of
2C-AR mRNA is of particular interest. Neither
message stability nor transport of the
2C-AR mRNA to
the cytosol is influenced by the 3
-UTR. In addition, the
p
2C-AR.3
-UTR-552 mRNA species was found in the
polysome complex, indicating that it undergoes translational
initiation. Thus the differences in the generation of the protein
product must be due to a decreased rate of elongation of the initiated products and/or failure to properly terminate protein synthesis in the
p
2C-AR.3
-UTR-552 transfectants. A similar mechanism is proposed to participate in the translational control of expression of
HSP70 in chicken reticulocytes (35) and of
-myosin in cardiac myocytes (36). The inability of the polysome complex to process the
p
2C-AR.3
-UTR-552 mRNA species is likely dependent
on the secondary structure generated by the 3
-UTR and/or RNA-binding proteins, both of which might impede elongation/termination. The inability of the
2C-AR 3
-UTR to influence mRNA
processing in a heterologous fashion suggests that there are additional
important interactions of the 3
-UTR with other domains of the
2C-AR mRNA.
Among the three 2-AR subtypes, the
2A/D-AR has the widest tissue distribution in the rat as
determined by both analysis of mRNA and radioligand binding
studies.
2A/D-AR mRNA is found in kidney, liver,
pancreas, adipocytes, vascular smooth muscle cells, and RIN-5AH
pancreatic beta cells. Each of these tissues also expresses the
receptor protein. In peripheral tissues and within the central nervous
system, the distribution of
2A/D-AR mRNA correlates
with receptor expression as determined by in situ hybridization, immunoblotting, and radioligand binding (37-39). In
contrast to the
2A/D-AR, the expression of the rat
2B-AR and
2C-AR is more restricted.
2C-AR mRNA and protein are primarily found in the
central nervous system, although low levels are detected by in
situ hybridization in the kidney (7, 38, 39). The distribution of
2C-AR and
2A/D-AR mRNA and
immunoreactivity in the central nervous system is discussed by Rosin
et al. (7) and Talley et al. (37). The
distribution of
2C-AR mRNA within the central
nervous system is not entirely consistent with the receptor
distribution defined by immunohistochemistry. In contrast to the close
relationship between mRNA and detectable protein for the
2A/ D-AR, there are selected sites within
the central nervous system (islands of Calleja, nucleus accumbens,
superior and inferior colliculus, caudate putamen) in which there is a dissociation between
2C-AR mRNA and detectable
protein as determined by either immunohistochemistry or radioligand
binding. A dissociation between
2C-AR mRNA and
detectable protein is also observed in the neuroblastoma × glioma
cell line NG108-15. Although transcripts encoding the
2B-AR and
2C-AR are identified in
NG108-15 cells (6-10), receptor purification and radioligand binding
studies indicate expression of the
2B-AR but not the
2C-AR protein (10, 40). The presence of
2C-AR mRNA, but the absence of receptor protein, is
exactly the situation observed in the present studies. Precise
interpretation of receptor mRNA versus protein
distribution in the rat central nervous system can be complicated by
potential neuronal transport of mRNA and/or proteins. However, the
dissociation between the
2C-AR mRNA and receptor
protein contrasts with the close relationship between mRNA and
protein for other membrane receptors in the central nervous system and
suggests that translation may be an important point of regulation for
the expression of the
2C-AR. The data presented in the
present manuscript are consistent with this possibility and suggest
that the 3
-UTR of the
2C-AR mRNA exerts a strong
influence on translational processing of the receptor message.
We thank Drs. Diane Rosin and Kevin R. Lynch (University of Virginia) for preprints of Refs. 7 and 37.