Human Molecular Genetics, 2003, Vol. 12, No. 3
DOI: 10.1093/hmg/ddg055

205–216

Synonymous mutations in the human dopamine
receptor D2 (DRD2) affect mRNA stability andsynthesis of the receptor
Jubao Duan1, Mark S. Wainwright2, Josep M. Comeron3, Naruya Saitou4, Alan R. Sanders1,
Joel Gelernter5 and Pablo V. Gejman1,*
1

Schizophrenia Genetics Research Program, Department of Psychiatry, The University of Chicago, Jules F. Knapp
Medical Research Center, Chicago, IL 60637, USA, 2Division of Pediatric Neurology, Northwestern University Medical
School, Children’s Memorial Hospital, Chicago, IL 60614, USA, 3Department of Biological Sciences, Center for
Comparative Genomics, University of Iowa, Iowa City, IA 52242, USA, 4Division of Population Genetics, National
Institute of Genetics, Mishima, Japan and 5Department of Psychiatry, Yale University School of Medicine,West Haven, CT 06516, USA

Received August 15, 2002; Revised and Accepted November 19, 2002

Although changes in nucleotide sequence affecting the composition and the structure of proteins are wellknown, functional changes resulting from nucleotide substitutions cannot always be inferred from simple
analysis of DNA sequence. Because a strong synonymous codon usage bias in the human DRD2 gene,suggesting selection on synonymous positions, was revealed by the relative independence of the G þ C
content of the third codon positions from the isochoric G þ C frequencies, we chose to investigate functional
effects of the six known naturally occurring synonymous changes (C132T, G423A, T765C, C939T, C957T, andG1101A) in the human DRD2. We report here that some synonymous mutations in the human DRD2 havefunctional effects and suggest a novel genetic mechanism. 957T, rather than being ‘silent’, altered thepredicted mRNA folding, led to a decrease in mRNA stability and translation, and dramatically changed
dopamine-induced up-regulation of DRD2 expression. 1101A did not show an effect by itself but annulled theabove effects of 957T in the compound clone 957T/1101A, demonstrating that combinations of synonymous
mutations can have functional consequences drastically different from those of each isolated mutation.
C957T was found to be in linkage disequilibrium in a European-American population with the -141C Ins/Del
and TaqI ‘A’ variants, which have been reported to be associated with schizophrenia and alcoholism,
respectively. These results call into question some assumptions made about synonymous variation inmolecular population genetics and gene-mapping studies of diseases with complex inheritance, and indicatethat synonymous variation can have effects of potential pathophysiological and pharmacogenetic
importance.

INTRODUCTION
In coding regions of genes, single nucleotide polymorphisms(SNPs) are categorized as either synonymous (also called‘silent’) for those that do not change amino acid sequence or
missense for those that do. In human genes, roughly 99.8%
of DNA sequence variations do not alter the primary sequencesof proteins (1), and synonymous SNPs show a higher frequency
than both missense SNPs and the SNPs in the non-coding
regions (1,2). Synonymous SNPs are the most polymorphic,
indicating that most such mutations may be functionallyneutral. However, this does not mean that every synonymoussite is non-functional or neutral. Studies in E.coli, yeast, andDrosophila support translation selection for major codons,
and there is a strong correlation between bias in synonymouscodon (SC) usage and the gene expression level (3). In thehuman genome, GC-ending codons are significantly more
frequent in constitutive than in alternative exons, suggesting
there may also be a translation selection for synonymous codon
usage (4). An effect of synonymous codon usage on gene
expression is supported by the detection of epistatic interactions between nucleotides that are important in maintaining*To whom correspondence should be addressed at: Schizophrenia Genetics Research Program, Department of Psychiatry, The University of Chicago,
Jules F. Knapp Medical Research Center, 924 East 57th Street, Room R-010, Chicago, IL 60637, USA; Tel: þ1 7738343060; Fax: þ1 7738342970.
Email: Pgejman@delphi.bsd.uchicago.edu

Human Molecular Genetics, Vol. 12, No. 3 # Oxford University Press 2003; all rights reserved

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pre-mRNA/mRNA secondary structures in the Drosophila
alcohol dehydrogenase genes (Adh and Adhr) (5–7). A
molecular evolutionary analysis of BRCA1 also provides
supportive evidence of selection for RNA or DNA structure
acting on synonymous codon usage (8). In addition, somesynonymous changes in humans have been shown tocause genetic disorders by exon skipping (9,10). Since it iscommonly believed that association studies with candidate
genes should be preferentially conducted with functional SNPs
and other functional mutations, the identification of nucleotide
substitutions associated with functional changes should haveimportant implications for the design and interpretation of
disease association studies.
G þ C content of the third positions of codons (GC3) has
been classically used to investigate whether natural selection
influences synonymous codon usage (3–5,11–13). However,
the isochoric structure of the human genome, in relation to the
distribution of G þ C content across the genome, might
confound the assessment of the influence of selection onsynonymous positions. This is because the base composition ofcoding sequences tends to reflect the variation in G þ C
content along chromosomes (11,14,15). Therefore, we investigated the departure between the GC3 of a gene and the G þ C
frequency observed in adjacent non-coding regions which
can be used as a measure of gene specific forces (e.g. selection)
on synonymous mutations beyond isochoric mutationaltendencies.
We studied those genes showing the strongest departure fromisochoric G þ C content as the best candidates for the detectionof functional changes resulting from synonymous mutations.
In particular, we focused on the study of human G proteincoupled receptor (GPCR) genes, many of which are implicatedin psychiatric disorders. Our analysis showed that among 35
closely related human GPCR genes (five dopamine receptors,
12 serotonin receptors, nine adrenergic receptors, five cholinergic receptors, and four histamine receptors), the GC3 in
DRD2 departed most clearly from its neighboring isochoric
G þ C content (Fig. 1). The results of our analysis suggested
that selection maintains the GC3 of DRD2 at a level well above
the G þ C content of the isochore in which the gene resides,
making DRD2 an ideal candidate to investigate the possiblefunctional consequences of synonymous SNPs.
Dopamine receptors are involved in motor control, neuroendocrine function, reward and reinforcement, and cognition,
and are targets in the pharmacologic therapy of schizophrenia,
Parkinson’s disease, Tourette’s syndrome, tardive dyskinesia,and Huntington’s disease (16). Furthermore, due to its central
role, DRD2 is among the most tested candidate genes in
psychiatric disorders. Multiple polymorphisms as well as
alternative splicing have been described for DRD2 (17–25).
DRD2 has two major isoforms (designated as long and short)
generated by alternative splicing of exon 6 (encoding 29 aminoacids) from the mRNA (17). Both isoforms display distinct
functions in vivo (24) as well as a differential ability to increase
the steady state receptor concentration induced by dopamine in
DRD2-transfected CHO-K1 cells (26). Four infrequent missense mutations (V96A, V154I, P310S and S311C) (19,21)
and a polymorphism (-141C Ins/Del) in the promoter
region (27) of DRD2 have been described. Some of themissense SNPs and -141C Ins/Del have been reported to be

Figure 1. Relationship between GC3 of 35 human GPCR genes and G þ C
content in adjacent non-coding (GC non-coding) sequences. Continuous anddashed lines indicate the linear regression and the 99.9% confidence intervals,
respectively. Note that the GC3 of DRD2 is significantly higher than its GC
non-coding (G ¼ 216.3, p < 1  106, G test for goodness of fit). GC3 wasestimated from the consensus human cDNA sequences for the 35 GPCRs,
and GC-noncoding was estimated using an average size of 3.5 kb.

associated with schizophrenia, although replication of thesefinding has yielded inconsistent results (22,23). Six known
DRD2 synonymous polymorphisms (C132T, G423A, T765C,
C939T, C957T, and G1101A) (17,18,20,21) had been presumed to be functionally silent and therefore have not been
investigated for a relationship with disease. In this study,
however, we have examined the functional properties of thesynonymous changes in DRD2, and have found evidence thattwo of the six synonymous SNPs have marked functional
consequences on DRD2 mRNA stability and dopamineregulated DRD2 expression. We also suggest a novel mechanism by which these two functional synonymous mutations in
DRD2 can affect its expression and regulation.

RESULTS
Codon-usage bias in DRD2
We first analyzed the departure between the GC3 of a gene and
the G þ C frequency observed in adjacent non-coding regions
as a measure of the isochoric G þ C content. We studied a totalof 35 human GPCR genes, including dopamine receptors (n ¼ 5),serotonin receptors (n ¼ 12), cholinergic receptors (n ¼ 5),
adrenergic receptors (n ¼ 9), and histamine receptors(n ¼ 4). As expected, there is a strong correlation(Spearman’s r ¼ 0.775, P < 1  106) between GC3 and GC
non-coding (isochoric G þ C content) (Fig. 1). However, GC3
in DRD2 departs strongly from the general trend, showing asignificant relative excess of GC3 (G ¼ 216.03, P < 1  106).
It could be argued that such a skewed GC3/GC non-coding
might manifest a selective pressure to maintain or diminish the
GC content of noncoding sequences that immediately flank agene and two genes with the same GC3 could show a markedly

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different GC3/GC non-coding relationship based upon thecharacteristics (and base compositions) of their promoters. To
address this possibility, and assuming that the isochoric
environment would affect equally all genes located in thesame genomic region, we have performed additional analysesof DRD2 and genes immediately located at its 50 and 30 end
based on the chromosome 11 reference genomic contig(NT_035088.1). The results of these analyses show that GC3
in DRD2 is higher than in genes flanking DRD2 while the GCnoncoding of these neighboring genes and DRD2 is notdifferent. Specifically, GC3 in DRD2 is greater (P < 1  106)than that in genes immediately flanking it on both sides, using
two (G ¼ 66.7), four (G ¼ 84.6) or six (G ¼ 142.4) flanking
genes. At the same time, the GC non-coding immediately 50(1 kb) to DRD2 does not show any reduction compared withthat in genes immediately flanking it using either two(G ¼ 1.55, P > 0.2), four (G ¼ 1.29, P > 0.25) or six(G ¼ 0.383, P > 0.5) flanking genes (both 50 and 30 relative toDRD2), and equivalent results are obtained using a larger 50
sequence (e.g. 3 kb) and also after removing from the analysis
the 300 bp immediately 50 to the start codon reflecting the mean
size of human 50 - UTRs (28). These analyses indicate that thehigh GC3 in DRD2 relative to the isochoric G þ C is restricted
to this gene and that there is no particular reduction of GC noncoding immediately flanking DRD2. Altogether, these results
strongly suggest that a selective effect on third codon positionsof DRD2 is present.
An independent line of evidence supporting the hypothesisthat bases at the third codon positions of DRD2 are underselection can been seen upon examination of data in Table 1.
Table 1 shows the SC usage for DRD2, 35 GPCRs, and 11
ribosomal proteins, and the average SC usage for the humangenome. We chose ribosomal proteins as a comparison groupsince they are highly expressed and thus potentially underselection for high codon usage bias. As can be seen from thelowest and highest 5% of codon usage for all the human genes
in Table 1, the variation of codon usage is rather high, and thisvariation is from multiple sources including the isochoreenvironment. We therefore have chosen to focus upon the
portion of data from Table 1 that allows separation of the forces
behind isochoric G þ C variation (which is by nature
independent of gene orientation, i.e. it is not ‘strand specific’)from other influences. The forces behind isochoric G þ Cvariation are expected to affect the G and C content
concurrently, but they could not explain a different behavior
for G- and C-ending synonymous codons. Hence, an
independent line of evidence from the previous analyses(Fig. 1) that the bases at the third codon positions of DRD2 areunder selection is provided by the study of G-ending and the
C-ending synonymous codons in 4-fold-degenerate (chosen tohave the opportunity to show, if present, independent variationof G- and C-ending codons) SC families (e.g. GUC and GUG
codons of the valine family). Note that if the isochoric G þ Cvariation was responsible for the increased GC3 in DRD2,
G- and C-ending synonymous codons in 4-fold-degenerate SC
families would be expected to be similarly influenced. Table 1
shows an independent behavior of G-ending and C-ending
synonymous codons in DRD2 from each other when comparedto the global human SC usage. This observation suggests that
selection is acting on synonymous sites not only to maintain a207high GC3 content but probably also on other levels. Because of
these preliminary findings, we next examined the functional
consequences of synonymous mutations naturally segregating
at DRD2 (17,18,20,21).
Decreased in vitro translation efficiency formRNA carrying 957T
We first investigated the in vitro translation of DRD2 mRNAs
carrying various synonymous mutations. We compared protein
synthesis at each time point by presenting the data aspercentage of the amount of protein synthesis by wild type
(WT) at 60 min. The DRD2 mRNA carrying the 957T mutation
showed a 50% decrease in the protein synthesis level as anindication of decreased translation compared with the others at
60 min (Fig. 2A). When expressed alone, the 1101A mutation
did not affect the translation activity. Interestingly, the mRNA
carrying the combined mutations 957T/1101A exhibited aprotein synthesis pattern similar to WT DRD2 mRNA,suggesting that the decreased translation associated with the
957T mutation was annulled in the presence of 1101A(Fig. 2A).
Mutant mRNA with the 957T mutation is less stable
Since the C957T mutation and another four of the studied
mutations (C132T, G423A, C939T, and G1101A) are allchanges from a preferred codon to a non-preferred one, weinferred that the decrease in protein synthesis associated with
957T is unlikely to be a general effect of codon preference.
Therefore, we next examined the possible effect of synonymous
SNPs on the mRNA stability. Stably transfected CHO (Chinese
hamster ovary)-K1 cells were treated with actinomycin D (ActD)
to inhibit transcription. DRD2 mRNA and the b-actin (ACTB)
control mRNA showed a similar decay in all clones except for
957T (Fig. 2C), a difference that is detectable with (Fig. 2B) or
without normalization by ACTB mRNA (data not shown). DRD2mRNA carrying the 957T mutation decayed significantly fasterthan WT mRNA, and the mRNA half-life was decreased fromapproximately 8 h (WT) to 4 h (957T) (Fig. 2B). The othersynonymous mutations (132T, 423A, 765C, 939T and 1101A)
did not show any effect on mRNA stability (Fig. 2B). While
1101A did not have an effect by itself, it again annulled the effectof 957T in the compound clone (Fig. 2B).
Effect of 957T on dopamine induced up-regulationof DRD2 expression
Work by others has shown that dopamine up-regulates mRNAof the DRD2 long isoform 2-fold in CHO cells transfected with
DRD2 cDNA through an unknown mechanism (26). Given thestrong selection for synonymous codon usage in DRD2 and thedemonstrated function of synonymous mutations 957T and
1101A, we hypothesized that 957T and possibly othersynonymous mutations might alter this dopamine-mediatedeffect, although most synonymous mutations are not expected
to be functional. We then examined the effect of synonymous
mutations on dopamine-mediated changes in DRD2 mRNA
expression. Transfected cells were treated with dopamine, andDRD2 mRNA levels in cultured cells were measured at various

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Table 1. SC usage in DRD2; all dopamine, serotonin, adrenergic, cholinergic, and histamine GPCRs; and 11 ribosomal proteins vs. the human genomea

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209

Figure 2. In vitro translation and stability of DRD2 mRNAs carrying various synonymous mutations. (A) Time course changes in the in vitro protein synthesis of
the receptor. Data are presented as proportions of the amount of WT at 60 min. Only the mutant 957T is significantly different from WT (P < 0.001, by two-way
ANOVA). Each data point represents the mean relative band intensity from two to three independent experiments. (B) Time course changes in the remaining
amount of DRD2 mRNA after ActD treatment. The data plotted are from at least three independent experiments. All the data were normalized by ACTB expression.
Only the 957T DRD2 mRNA showed a significant difference from WT (P ¼ 0.0014, by two-way ANOVA). (C) Representative autoradiograph of RPA. Cell lysate
used in RPA was from 5  105 cells at the indicated times (0, 4, 8 and 24 h) after treatment with ActD. The control showed the RNase digestion pattern of probes ina reaction without cell lysate.

time points. Dopamine up-regulated the cellular DRD2 mRNAlevel with the peak increase at 4 h [1.5–1.8-fold, P < 0.0001, by
two-way ANOVA (analysis of variance)] (Fig. 3A). However,
957T consistently showed a decreased response compared with
WT. The dopamine-induced up-regulation of DRD2 mRNA
could be blocked by ()-sulpiride (data not shown), indicatingthat the effect of dopamine here is specifically mediatedby DRD2. Clone 957T/1101A did not show a significantly
diminished response, once more suggesting a compensatory
effect of 1101A. These changes are likely to result in changesof receptor synthesis.
We next addressed whether the effects of 957T on dopamineinduced up-regulation of DRD2 mRNA could alter DRD2
expression in cell membrane preparations. Stably transfected
CHO cells expressing either WT or 957T DRD2 constructs
were used to quantitate [3H]methylspiperone-specific bindingsites determined by saturation binding studies. The density of
specific [3H]methylspiperone binding sites was similar between
the untreated DRD2 WT transfected cells (47.5  10.4 pmol/mgof membrane protein) and 957T (36.7  5.4 pmol/mg).
Treatment with dopamine resulted in a significant (P < 0.05
compared with baseline by two-tailed t-test) increase in[3H]methylspiperone-specific binding as determined by singlepoint measurements of radioligand binding after 6 h of exposure
to drug in both WT (107.9  16.8 pmol/mg) and 957T receptor
expressing cells (80.1  8.2 pmol/mg). This increase wassustained after 24 h of treatment with dopamine in both WT(118.0  6.6 pmol/mg) and 957T receptor expressing cells(90.5  12.9 pmol/mg). A significant difference of receptor
expression between WT and 957T clones appeared after 6 h(P ¼ 0.0076, t-test) and 24 h (P ¼ 0.0054, t-test) of treatment
with dopamine. These results indicated that 957T decreases the
dopamine-induced up-regulation of receptor expression at boththe mRNA level and the membrane protein level.
Dopamine differentially stabilized WT and
957T DRD2 mRNA
The effect of 957T on dopamine-induced up-regulation of DRD2mRNA in transfected CHO cells could be explained by an effectof on transcription of DRD2 mRNA or by a differential stabilityof DRD2 mRNA, i.e. a post-transcriptional event. We therefore
studied DRD2 mRNA turnover after blocking transcription bytreating cells with ActD. We found that dopamine treatment

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Figure 3. Dopamine induced up-regulation of DRD2 mRNA in transfected CHO cells and differential effects of dopamine on the stability of WT and 957T DRD2
mRNA. (A) Time course changes of DRD2 mRNA level upon dopamine treatment. Data are presented as a ratio calculated by dividing the DRD2 mRNA level at
the indicated time point by the mRNA level at 0 h of dopamine treatment. Only 957T cells had a significant difference from WT (P ¼ 0.0273, by two-way ANOVA).
(B) Time course changes of DRD2 mRNA level in the presence of ActD. Dopamine only significantly changed the mRNA stability of WT (P < 0.001, by ANOVA).
For both (A) and (B), data at each time point were from four independent experiments and were normalized by ACTB expression (assuming the same ACTB
decaying pattern in all the cell clones, and the acquired DRD2 data were adjusted accordingly). (C) Representative autoradiograph of an RPA in (B).
Dopamine was applied or not to stable-transfected CHO cells in the presence of ActD. Cell lysate used in the RPA was prepared at indicated time points(0, 4 and 8 h) after dopamine treatment. The control shows the RNase digestion pattern of probes in a reaction without cell lysate.

significantly increased DRD2 mRNA stability for WT but notfor 957T (Fig. 3B and C). In the absence of dopamine, 957T
again showed a faster mRNA decay rate than WT. These results
suggest that the increased amount of DRD2 mRNA induced bydopamine was probably due to the stabilization of DRD2 mRNA
and, interestingly here, dopamine stabilizes DRD2 mRNA
carrying the 957T mutation significantly less than WT.
Characterization of the folding of mRNA containing
synonymous SNPs
Previous study of the Drosophilia genes Adh and Adhr suggeststhat there maybe a relationship between mRNA secondary structure and gene expression (5,6,29). Therefore, we hypothesizedthat the observed effects of the synonymous mutations on theproperties of DRD2 mRNA may result from the changes inmRNA folding. Using MFOLD (30), we modeled the effectof the synonymous mutations on the DRD2 mRNA folding
structures of the coding region. Partial mRNA folding structures
of WT mRNA and mutant 957T, 1101A, 957T/1101A are shownin Fig. 4. Mutation 957T causes an obvious change in themRNA folding structures spanning the sequences 804-1212. The
mRNA carrying 957T/1101A displays a similar folding structure
to WT and mutant 1101A. The folding structures containing
other mutations (132T, 423A, 765C and 939T) are all similar tothat of WT (data available upon request). The larger effect of
957T on mRNA structure is consistent with experimental data
showing a specific effect of 957T on DRD2 mRNA stability andreceptor expression. Taken together, these findings suggest thatthe DRD2 mRNA secondary structure may account for theobserved differences of DRD2 mRNA stability and otherproperties resulted from synonymous substitutions.

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211

Figure 4. Examples of DRD2 mRNA folding structures predicted by MFOLD. DRD2 mRNA sequence carrying various synonymous SNPs was used for secondary
folding structure model building by the use of the computer program MFOLD (30). Only part (nt 804–1212 of the coding sequence) of each modeled structure forthe DRD2 mRNA carrying mutations 957T, 1101A, or 957T/1101A, and the WT are shown. Note an obvious change of folding patterns between the WT and the957T DRD2 mRNAs.

C957T is in LD with the -141C Ins/Del and
TaqI ‘A’ variants
To explore the possible population genetic implications of thefunctional synonymous mutations 957T and 1101A, we genotyped the C957T and G1101A variants in samples of 94
European-American (EA) and 51 African-American (AA)subjects who had been screened to exclude major psychiatricillness, as previously described (31). We observed allele
frequencies of 0.43 and 0.91 for C957 in these two populations(Table 2), respectively. We did not observe any instances of1101A. In a subset of the subjects (93 EA and 32 AA), we
evaluated linkage disequilibrium (LD) with two other DRD2
markers, -141C Ins/Del and the TaqI ‘A’system (dbSNP accession
number rs1800497), which have been used as markers in previous

Table 2. C957T genotype counts and allele frequency in EA and AA samples
Sample (numberof subjects)

Genotype counts
C/C

C/T

T/T

Allele frequency
CT

EA (94)
AA (51)

18
42

45
9

31
0

0.43
0.91

0.57
0.09

association studies of DRD2 and a variety of psychiatric disorders(23). Significant LD was detected in the EA sample between the
C957T marker and both the -141C Ins/Del marker (d ¼ 0.033,
d0 ¼ 0.724, G ¼ 7.58, P ¼ 0.0088) and the TaqI ‘A’ marker(d ¼ 0.071, d0 ¼ 0.832, G ¼ 16.20, P ¼ 0.0003). Significant LD
was not detected in the smaller AA sample.

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DISCUSSION
For mammalian genes, a translational effect of synonymoussites remains elusive. Some experiments show that altering
codon usage to G- and C-ending codons can enhance theexpression levels of genes in human cell lines (32,33). There isalso evidence for a correlation between tRNA abundance inhuman and rabbit reticulocytes and the codon usage of a- andb-globin mRNAs (34). The selection for translational accuracyof synonymous codon usage in Drosophila genes is supportedby the correlation between synonymous divergence and aminoacid conservation within mammalian genes (35,36). Further, atest for translational selection at silent sites in the humangenome revealed that GC-ending codons are more abundant inconstitutive than alternatively spliced exons (4). However, otherreports showed no relationship between expression patterns andsynonymous codon usage in human genes, and no relationshipbetween codon usage bias and synonymous divergence among
mammals (37,38).
In this study, we present experimental evidence that thenaturally occurring synonymous SNP C957T in DRD2 canmarkedly affect the in vitro expression of the gene, while theother five synonymous SNPs (C132T, G423A, T765C, C939T
and G1101A) were shown to be silent. We have showncompelling evidence that the synonymous mutation 957T in
DRD2 significantly affects mRNA stability. The striking effectof 957T on both DRD2 mRNA stability and receptor
expression is consistent with the changes of predicted mRNA
secondary structure (Fig. 4), suggesting that mRNA secondary
structure associated with the synonymous mutation 957T
may play an important role in the regulation of DRD2 gene
expression. Relevantly, it was shown that two distantsynonymous mutations in ITGB3 [integrin, beta 3 ( platelet
glycoprotein IIIa, antigen CD61)] were required to perturb premRNA folding and induce altered mRNA splicing (10). It has
been proposed that joint effects of mRNA secondary structure
and codon bias may interact to regulate the level of gene
expression (5). Selection may act on synonymous codons tomaintain the structural features of the mRNA that make it moreresistant to degradation (5,6,39). The high allele frequency of
957T in the population suggests that the functional C957T
dimorphism may not be very deleterious, otherwise strong
selection might preclude its observation in the population. Thisis consistent with a genetic model of complex disease in whichmildly deleterious mutations may have quite high allele
frequencies (40).
The mechanism underling the observed lower translational
efficiency and the decreased mRNA stability for 957T is not
obvious. The bulk of mRNA turnover is mediated either bymRNA decapping 50 -to-30 decay or exosome-mediated 30 -to-50
exonucleolytic decay (41). While it is possible that 957T
impairs translation initiation, typically this is produced bychanges in the 50 -UTR sequence, not the coding sequence.
According to a general model about mRNA translation
efficiency and mRNA decay (42), it is also conceivable (butdifficult to test) that 957T causes a global perturbation ofmRNP structure, precluding the normal bridging interactions
that basically involve 50 -cap structure, eukaryotic translation
initiation factor 4E (eIF4E) and eIF4G, poly(A)-binding protein(PABP), and the poly(A)-tail (42). Perturbation of this closed

loop conformation predisposes to decapping and the accelerated mRNA decay and also impairs translation efficiency bydecreasing recruitment of the 40S ribosome subunit (42,43). It
is also possible, although unlikely, that 957T might interferewith the base-pairing structure between 50 - and 30 -UTRs;
impaired long-range pairing between the UTRs could conceivably impair the translational efficiency and promotetranscript degradation by allowing accelerated decapping(44,45).
The regulation of mRNA decay is an important control pointof gene expression, and is mediated by a subset of sequence
elements, factors, and endoribonucleases (42). Most of themRNA stability elements are considered to be located in the
50 - and 30 -UTRs (untranslated regions) of genes. However,
we have shown here that a synonymous mutation in the coding
region of DRD2 could affect mRNA stability. Indeed, there is a
rapidly expanding literature describing determinants of mRNAstability in the coding region of multiple genes (5,46–50). The
mechanisms by which these coding region mRNA stability/
instability elements affect mRNA decay are complex. Thechange of mRNA stability induced by the synonymous 957T in
DRD2 suggests the existence of an mRNA stability/instability
determinant flanking or encompassing this locus that affects
mRNA conformation. This is supported by the fact that the
combined mutations, 957T/1101A, did not result in any
changes of mRNA stability. mRNA folding structures predicted
by MFOLD (Fig. 4) also support a change of mRNA secondary
structure. However, because of the limitation of structure
prediction of MFOLD, other RNA secondary structure
prediction methods (6) and more accurate assays (in vitro orin vivo) may be needed to perform additional analyses. Future
experimental efforts on the characterization of another synonymous change (not limited to the naturally occurring ones wehave investigated) in the vicinity of 957T that causes a similaralteration in the local mRNA folding would provide further
evidence to support our interpretation of the data. Furthermore,
to test our hypothesis more comprehensively, we are studying
whether naturally occurring synonymous mutations affect theperformance of transcripts of genes that do not show excess
GC3 [e.g. 5-hydroxytryptamine (serotonin) receptor 1B ¼
HTR1B] and also plan to test the effect on transcripts
harboring a variety of induced (but not naturally occurring)synonymous mutations in DRD2 with functional assays.
The DRD2 promoter variant (-141C Ins/Del) has previously
been reported to affect DRD2 mRNA expression and to be
associated with schizophrenia (27). The DRD2 TaqI ‘A’marker, a SNP located about 10 kb downstream of thetermination codon (51), has also been reported by someinvestigators to be associated with a range of psychiatricphenotypes, including drug dependence (52) and alcoholism(53), although this has not been replicated (54). The strong LD
observed between -141C Ins/Del and the functional synonymous SNP C957T could confound studies unless the functional
effects of 957T are considered, and it is possible these twomutations could have additive, multiplicative, or other interactions, though we have not tested this. Furthermore, no common
functional variant was previously known that could account forthe claimed associations observed with TaqI ‘A’. The strong
LD observed between C957T and TaqI ‘A’ could, finally,
provide a molecular basis for the traits attributed to TaqI ‘A’.

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Interestingly, the most closely related non-human primate
full coding DRD2 sequence (from one chimpanzee, Pantroglodytes; GenBank accession numbers AB080603-9 forexons 1–7, respectively) has the same nucleotides as the human
WT (i.e. human major alleles) at all of the human polymorphic
cSNP sites, including C957T, thus indicating that the C is likely
the most recent ancestral form. Several more distantly related
species have a T instead of a C at the same position in DRD2,
e.g. Cercopithecus aethiops (African green monkey; GenBank
accession number U18547) and Mus musculus (house mouse;
GenBank accession number X55674), indicating that the moredistant ancestral form might be a T, although the very frequent
occurrence of C to T mutations during evolution could accountfor this as well.
1101A did not have any effect by itself, but consistently
annulled the effects of 957T in the compound clone 957T/
1101A. This suggests another way in which reliance on isolated
SNPs, when mapping genes responsible for diseases withcomplex inheritance, may be insufficient from a functional
perspective. It may very well be that combinations ofmutations, rather than isolated mutations, are associated withdisease. Our demonstration of the interaction between two
DRD2 SNPs bolsters the case for studying SNP combinations
at particular candidate genes as opposed to single SNPs, and isconsistent with work indicating that interactions of multiple
SNPs within a haplotype affect biological traits of the humanbeta-2 adrenergic surface receptor (ADRB2) (55). This result isalso consistent with a proposed compensatory evolution model
involving mutations from two or more loci 56. In that model,
single mutations are assumed to be deleterious, but neutral inappropriate combinations. In the case of DRD2, 1101A does
not have a deleterious effect by itself, and compensates for theeffect of 957T in the compound clone 957T/1101A bychanging a potential mRNA secondary structure. Relevantly,
previous site-directed mutagenesis experiments found thatfunctional effects of synonymous changes in the Adh gene of
Drosophila melanogaster could be compensated by changes inthe 30 -UTR of Adh mRNA (6,7). The authors interpreted thisobservation as evidence for long-range interactions across the
Adh mRNA secondary structure.
In summary, we report for the first time that the synonymous
DRD2 mutation 957T has empirically measurable biologicalconsequences compared to the WT C957. These include
decreased mRNA translation, decreased mRNA stability, and aweakened response to dopamine-induced up-regulation of
DRD2. We suggest that 957T alters an mRNA secondary
structure that underlies these changes, possibly by altering abinding site for a protein factor, a hypothesis that remains to betested.

213

with mutagenic primers (sequences are available upon request)
to introduce the mutations (subsequently confirmed by DNA
sequencing).
GC3 and GC-noncoding analyses
Thirty-five GPCRs were selected, including five dopamine
receptors (DRD 1-5), 12 serotonin receptors (HTR 1A, 1B, 1D,
1E, 1F, 2A, 2B, 2C, 4, 5A, 6 and 7), nine adrenergic receptors(ADR A1A, A1B, A1D, A2A, A2B, A2C, B1, B2, and B3), fivecholinergic receptors (CHRM 1-5), and four histamine
receptors (HRH 1-4). GC3 was calculated from consensus
human cDNA sequences while the GC-noncoding (isochoric
G þ C content) was estimated from flanking (50 and 30 )noncoding sequences with an average size of 3.5 kb (Fig. 1).
The comparison between coding and noncoding sequences of
DRD2 and those of genes immediately flanking it was
conducted using the chromosome 11 reference genomic
contig NT_035088.1 (8.3 Mb), and only flanking genes
with mRNA evidence were used in the analyses. The following coding sequences were used as immediately flanking
DRD2:
centromeric–LOC143914–FLJ20535–LOC143915–
30 _DRD2_50 –LOC143916–TMPRSS5–ZW10–telomeric. In thisintra-isochoric analysis, three different sizes of noncoding 50
sequence were used: 1 kb 50 to the start codon, and 1 kb and3 kb 50 sequence after removing potential 50 UTR regions
assuming an average size of 300 bp (28).
Cell culture, cell transfection, and drug treatment
CHO-K1 cells (American Type Culture Collection) were
cultured in Ham’s F-12 medium supplemented with 10% fetal
bovine serum in a humidified atmosphere of 5% CO2 at 37 C.
CHO-K1 cells do not endogenously express DRD2, and are acommonly used model to study DRD2 gene expression andregulation (26,57,58). Stable transfections of plasmids into
CHO-K1 cells were performed using the LipoTAXI mammalian transfection kit (Stratagene). Transfected clones wereselected in medium supplemented with 800 mg/ml of G418, andresistant colonies (>300) were pooled by trypsinization forfurther study. Expression of the receptor on transfected cells
was studied by radioligand binding. The sequence of DRD2 instable transfectants was confirmed by RT-PCR followed bysequencing. For the mRNA stability assays, ActD (10 mg/ml)
was applied to the cell cultures grown overnight and cells were
harvested at selected intervals up to 24 h. For the dopamine
regulation experiments, the cell cultures grown overnight
were treated with dopamine (100 mM) in the presence of
0.2 mM sodium metabisulfite (to prevent oxidization of
dopamine in the culture medium), and cells were harvested
at selected intervals up to 24 h.

MATERIALS AND METHODS
In vitro translation
Mutagenesis and plasmid construction
A 2.5 kb cDNA fragment with 50 and 30 UTRs encoding thelong isoform of the human DRD2 (17) was subcloned into
plasmid cytomegalovirus (pCMV) Script (Stratagene). The
resultant pCMV-Script-DRD2 was used as a template formutagenesis by overlapping polymerase chain reaction (PCR)

mRNAs used for in vitro translation were generated by in vitrotranscription using plasmids linearized downstream of
the respective DRD2 inserts with HindIII as template. The
MEGAscript T3 kit (Ambion) was used according to the
manufacturer’s instructions. To control the quality and quantityof input mRNA, the in vitro transcribed mRNAs were checked

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Human Molecular Genetics, 2003, Vol. 12, No. 3

for integrity by electrophoresis on formaldehyde agrose gelsfollowed by quantification of concentration and purity byspectrophotometry using the GeneQuant pro RNA/DNA
Calculator (Amersham). The concentrations of transcripts weretypically 2.424  0.151 mg/ml. Subsequently, mRNA samples
were diluted to equal concentrations (1 mg/ml) and 1.5 ml of
diluted mRNA was added to the 25 ml translation reaction. In
vitro transcribed mRNA were translated in the Flexi1 rabbit
reticulocyte lysate system (Promega) in the presence of biotinlabeled TranscendTM tRNA (Promega) between 0 and 60 min at
30 C. Following electrophoresis and electro-blotting, theTranscendTM Non-Radioactive Translation Detection System(Promega) was used to detect protein synthesis.
RPA (RNase protection assay)
Detection and quantitation of DRD2 mRNA in transfected cells
was carried out using the Direct Protect Lysate RPA kit(Ambion) with ACTB as an internal control. Forty microlitersof cell lysate in Lysis/Denaturing solution from 5  105 cells
were directly used in each RPA reaction, and 5 ml 32P-labeled
DRD2 RNA probe and 5 ml 32P-labeled ACTB RNA probe wereadded to each reaction. The band intensities were quantitatedby Typhoon 8600 (Amersham Pharmacia). The RNA probes
used in RPA were synthesized using MAXIscript T7 (Ambion)and labeled by [a-32P]UTP (Amersham Pharmacia). For the
DRD2 RNA probe, a PCR-generated 310 bp DNA fragment of
DRD2 with a T7 promoter added by the No-cloning Promoter
Addition Kit (Ambion) was used as the template to generate theRNA probe. For the ACTB RNA probe, a plasmid
TRIPLEscriptTM–ACTB (Ambion) was used as template to
make an antisense mouse ACTB RNA 304 bp probe. The sizesof the expected protected fragments were about 310 and 250 bp
for DRD2 and ACTB, respectively. 32P-labeled RNA Century
Marker (Ambion) was used as a size marker.
Radioligand binding assays[3H]methylspiperone (New England Nuclear) was used to label
receptor binding sites (26,57). Disruption of the cells was
performed using a Sonic Dismembranator. Cell membranes
were collected by centrifugation at 34 000 g for 20 min at 4 C
and resuspended in binding buffer at a final protein concentration of 0.3 mg/ml. Membrane suspension (100 ml) was added totriplicate assay tubes containing [3H]methylspiperone (0.01–
1.2 nM) in a final volume of 1 ml. Non-specific binding was
defined using 1 mM (þ)-butaclamol. The assay tubes wereincubated for 1 h at 25 C. The assay was terminated by rapid
filtration through GF/C filters (Whatman) pretreated with 0.3%
polyethylenimine. The filters were rapidly washed three times
with 4 ml of 50 mM Tris–HCl, pH 7.4, at 4 C, and theradioactivity bound to the filters was quantitated by scintillation
counting. Protein concentrations were determined using thebicinchoninic acid reagent (Pierce). Radioligand binding data
was analyzed with the non-linear least-squares fitting program
GraphPad Prism version 3.0 (GraphPad Software, Inc.).
Genotyping, LD analysis, and sequencing
Genotyping was performed as previously described (31). We
designed a PCR restriction fragment length polymorphism

assay to detect the C957T and G1101A variants. Detailed
genotyping design is available upon request. The amplicon
includes both C957T and G1101A, so in dually heterozygous
subjects molecular haplotyping is possible via double digestion. LD was analyzed by the program 3LOCUS (59) andP-values were estimated with 10 000 Monte Carlo simulations.
Sequencing of the chimpanzee DRD2 was performed using
standard methods in the laboratory of N.S.

ACKNOWLEDGEMENTS
We thank the anonymous reviewers of this manuscript for theirinsightful and helpful comments. We thank Dr. Marcos
A. Antezana for useful discussions and for help with Table 1;
Dr David K. Grandy (Oregon Health Sciences University,
Portland, OR, USA) for the kind gift of the plasmid that
contains the whole length cDNA of DRD2; Dr Henry
R. Kranzler (University of Connecticut School of Medicine,
Farmington, CT, USA) for assistance in collecting anddiagnosing the control subjects; Dr Aya Takahashi (National
Institute of Genetics, Mishima, Japan) for sequencing of thecDNA of the chimpanzee DRD2; Dr Ikuo Hayasaka(Kumamoto Primates Park, Kumamoto, Japan) for providing
the chimpanzee genomic DNA; and Mr Eric B. Carpenter (The
University of Chicago, Chicago, IL, USA) and Ms Anne Marie
Lacobelle, M.S. (Yale University School of Medicine, West
Haven, CT, USA) for providing excellent technical assistance.
This project was supported by a Distinguished InvestigatorAward from the National Alliance for Research on
Schizophrenia and Depression (P.V.G.), the Women’s Boardof Children’s Memorial Hospital, Chicago, IL, USA (M.S.W.),
and NIAAA grant P50 AA12870.

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