INTRODUCTION

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.

EXPERIMENTAL PROCEDURES

[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).

NIH-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).

The _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.

Polysomes 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.

The 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.

RESULTS

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).

Expression construct and transfection efficiency for the _2C-AR in NIH-3T3 fibroblasts and DDT₁MF-2 cells.

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.

Expression construct and transfection efficiency for the _2A/D-AR in NIH-3T3 fibroblasts and DDT₁MF-2 cells.

_2C-Adrenergic Receptor Expression Using Truncated Constructs

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 M_r 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.

Nucleotide sequence of the _2C-AR gene 3 to the protein coding region.

Expression of _2C-AR in NIH-3T3 fibroblasts following truncation of the 3-UTR.

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.

Predicted structure of the 3-UTR of the _2C-AR mRNA.

Cellular Localization and Stability of _2C-AR Transcripts

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.

Cellular distribution of _2C-AR mRNA in _2C-AR.3-UTR-74, _2C-AR.3-UTR-470, and _2C-AR.3-UTR-552 NIH-3T3 transfectants.

Once in the cytosol, the p_2C-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

Distribution of _2C-AR.3-UTR-552 mRNA in polysome preparations.

Stability of _2C-AR mRNA in _2C-AR.3-UTR-74 and _2C-AR.3-UTR-552 NIH-3T3 transfectants.

Influence of the _2C-AR 3-UTR on the transfection efficiency of the _2A/D-AR.

DISCUSSION

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.