The Regulation of D2 Dopamine Receptor Expression

NEWS & VIEWS

The regulation of D2 dopamine receptor expression

The question of whether or not there is a polymorphism in or near DRD2 which is associated

with receptor density remains unanswered. Both clinical and animal data demonstrate that D2 dopamine receptor density is highly variable among individuals; differences of more than

100% are not uncommon. Among inbred mouse strains and F2 intercrosses produced from these strains, it appears that most of the genetic variation in receptor expression is not associated with polymorphisms in or near Drd2.

Two reports in this issue of Molecular Psychiatry1,2 real. The schizophrenics actually showed a trend in the refocus our attention on an issue that has been contro- opposite direction, although these data may be con- versial for almost a decade, namely, ‘Is there a poly- founded by prior neuroleptic exposure. Overall, after morphism in or near DRD2 which is associated with reading these two reports, one is left in the rather the regulation of receptor density?’ Laruelle et al1 unsatisfying position of still being unsure as to whether examined the effects of both the Taq1 ‘A’ and ‘B’ poly- or not the A1 allele is associated with lower binding morphisms on D2 receptor-binding potential (Bmax/Kd) potential. using SPECT technology with the ligand 123I-IBZM in To understand the importance of this issue, it is 70 subjects; a previous postmortem study suggested necessary to put the studies in the proper historical that the A1 allele was associated with a mutation that perspective. Both studies trace their origin to the obser- decreases receptor expression.3 Laruelle et al1 found vation of Blum et al4 that there was an association that the A1 carriers (subjects homozygous or hetero- between the A1 allele and alcoholism. Some5–12 but not zygous for the allele) had the same binding potential all13–17 subsequent studies have confirmed this associ- as the non-carriers. Similarly, they found that the poly- ation for alcoholism and stimulant abuse. Population morphisms of the Taq1 ‘B’ system, which is in strong stratification is a potentially important confound in linkage disequilibrium with the ‘A’ system, had no studies of the A1 allele. Barr and Kidd18 examined 381 effect on binding potential. Pohjalainen et al2 exam- people from 16 different populations and found that ined the relationship between the Taq1 ‘A‘ system the frequency of the A1 allele varies widely from 0.09 polymorphisms and binding potential using PET and to 0.75. These significant differences make it impera- 11C-raclopride in 54 Finnish normal volunteers. A sig- tive that one should control for the ethnic origin of sub- nificant decrease (15–20%) in binding potential was jects when performing an association study with the found for the A1/A2 heterozygotes as compared to A1 allele; lack of concern for this issue appeared to be A2/A2 homozygotes; the effect was sustained after the a problem in some of the earlier studies. A recent study data were age or gender adjusted. Can the different by Chen et al19 illustrates the importance of this point. results between the two studies be explained? Both These authors examined the relationships among Taq1 groups used a substituted benzamide as the receptor and NcoI D2 receptor polymorphisms and alcoholism ligand, reported the data as binding potential and the in four aboriginal groups and the Han (Chinese) in Tai- A1 allele frequency was similar in the two study popu- wan. Haplotype analysis revealed a significant associ- lations. There was however, an important experimental ation of the A1N1 haplotype and alcoholism only in difference, namely the reduced coefficient of variation the Ami (one of the aboriginal populations) and the in the PET study. Thus, the Pohjalainen et al2 study Han. The authors conclude that the absence of the had significantly greater power to detect a small differ- association between the polymorphisms in DRD2 and ence. This point becomes important when one con- alcoholism in three of the aboriginal populations sug- siders that the study population used by Laruelle et al1 gests either a higher rate of phenocopies among abor- was composed of 47 normal controls and 23 schizo- iginal alcoholics or genetic heterogeneity in the suscep- phrenics. Among the normal control A1 carriers there tibility to alcoholism. was a trend to a lower binding potential but the sample The emphasis on the relationships between DRD2 size had insufficient power to detect the difference, if and alcohol/substance abuse builds from the proposed

role of the D2 dopamine system in reward mech- anisms20 and from the in vivo observations that chronic Correspondence: Dr RJ Hitzemann, Dept of Psychiatry, SUNY at Stony Brook, Stony Brook, NY 11794-8101, USA. E-mail: rhitzem- alcoholics, cocaine and heroin abusers have a lower annȰmail.psychiatry.sunysb.edu D2 receptor-binding potential which persists (for up to Received and accepted 28 January 1998 months) after drug withdrawal.21–25 The receptor data News & Views 199 come largely from a single group of investigators.22–24 potential are due either to environmental effects or It is important to note that in these studies the genetic effects on receptor density. The strongest evi- alcoholics and cocaine addicts were a highly selected dence that some of this receptor variance is related to group. For example, the alcoholics needed to use Ͼ3g genetic factors comes from animal studies. A useful of ethanol kg−1 daily but needed to be otherwise medi- starting point for reviewing this literature is the work cally stable, not abusing other substances and with a of Helmeste and Seeman38 and Ciarenello and family history positive for alcoholism. Approximately Boehme.39 These authors used membrane-binding one out of 20 alcoholics screened met these study cri- assays to examine striatal D2 receptor density among teria. The selection of the cocaine addicts was similarly inbred mouse strains; a difference among inbred strains stringent; importantly, from the genetic perspective, all is presumed to be of genetic origin. Five inbred strains of these subjects were also family history positive for were common to both studies; importantly the relative substance or alcohol abuse. For the cocaine addicts the order of receptor density for the three most commonly reduction of D2 receptor-binding potential was used strains in biomedical research was similar with detected in two separate studies, using two different the BALB/cJ Ͼ C57BL/6J (C57) Ͼ DBA/2J (DBA). receptor ligands.23,24 In none of the studies was it poss- Receptor density in the BALB/cJ strain was 60–80% ible to determine if the binding potential returned to higher than in the DBA strain. Subsequent studies40– normal. Thus, there are no data which support either 44 have conﬁrmed that there is a marked difference in a ‘state’ or ‘trait’ mechanism for the lower binding receptor density among inbred mouse strains. How- potential. ever, it is important to note that the relative difference

Interest in D2 receptor genetics also builds from the among strains depends on the region examined. For putative role of the D2 receptor system in a wide variety example, the C57 strain has a higher receptor density of neuropsychiatric disorders. For example, some data than the DBA strain in the striatum43,44 but a lower suggest that schizophrenics have a higher receptor den- receptor density in the substantia nigra and ventral teg- sity than controls and this difference is not the result mental area;44 the latter difference in part is related to of neuroleptic exposure.26 However, most27–29 but not a higher number of midbrain dopamine neurons in the all studies30 have been unable to detect a significant DBA strain.45 Differences in receptor density have also association between polymorphisms in DRD2 and been found among inbred rat strains in some46–48 but schizophrenia. Negative results have also been not all studies.49 Luedtke et al49 also examined the dif- obtained for affective disorders,31,32 obsessive compul- ferences among five inbred rat strains in the ratio of D2 sive disorder33 and Tourettes’ syndrome;34 in contrast, receptor mRNA for the long and short splice variants a positive association has been reported for (the long and short splice variants differ by a 29-amino schizoid/avoidant behaviors.35 These largely negative acid insert in the third cytoplasmic loop). No differ- results should not be unexpected; as will be shown ence in the long and short isoforms of the mRNA was below, most of the variance in receptor expression is noted among the strains. Similarly, we have found no probably not associated with polymorphisms in DRD2. difference in the ratio of the splice variants between There is remarkable variation among individuals in the C57 and DBA inbred mouse strains; further the the apparent availability of D2 receptors. The coef- amino acid sequences in both strains are identical ficients of variation in the studies of Laruelle et al1 and (unpublished observations). Pohjalainen et al2 range from 30 to 50%. Thus, those An important confound to detecting differences in individuals more than and less than one standard devi- D2 receptor density is the age at which the measure- ation from the mean will differ from each other by ments are made. For example, Lephohon-Greenwood 50 more than 85% in receptor-binding potential. Some of and Cinader examined D2 receptor binding among the difference among individuals may be associated females of five inbred strains (C57BL/6, SJL, A, DBA/1 with differences in synaptic dopamine concentrations. and C3H/He). At 7–15 weeks of age (the time of Both raclopride and IBZM have a ‘relatively’ low affin- measurement in most laboratory experiments), the Ͻ Ͻ = ity for the D2 receptor and thus, significant receptor order of receptor binding was C57 SJL A DBA/1 competition is expected between either of these ligands = C3H and the range of receptor density values was and endogenous dopamine. If this competition contrib- nearly 100%. Interestingly, these authors found that utes significantly to the variation in binding potential, there are marked age-related declines in receptor den- most of the variation in binding potential should come sity for all strains except the C57, such that by 31–45 2 from changes in KD and not Bmax. Pohjalainen et al weeks of age there was no difference among strains. No addressed this issue by administering raclopride at further difference among strains was seen up to 120 both low and high specific activity to each subject and weeks (even though there were age-related losses in 51 calculating individual KD values. No significant vari- receptor density). In a subsequent study, these ation in KD was observed. These data are consistent authors argued that in animals with a relatively low with the animal data which show that KD does not vary ‘youthful’ receptor density, aging had modest effects. among inbred strains of mice and rats.36 Further, nat- On the other hand, in animals with a high ‘youthful’ urally occurring variants in the human receptor eg the receptor density eg the MRL/Mp−2+ strain, aging causes Ser311 → Cys variant, do not appear to be associated a marked receptor loss. These authors termed the with differences in receptor-binding affinity.37 Thus, it phenomenon of receptor loss being greatest in the appears that the individual differences in binding strains with the highest receptor density ‘economic News & Views 200 correction’. The obvious question arises as to whether (including catalepsy) show a unimodal distribution or not such economic correction occurs in humans. We which is consistent with the involvement of multiple must consider the possibility that disorders with a genes. The chromosomal location of these genes is esti-

young adult onset eg schizophrenia, may be associated mated by QTL analysis. The strain means (ED50 values with significant abnormalities in receptor density; for haloperidol-induced catalepsy) were correlated however, at the time of ascertainment (usually years with the strain distribution patterns of 1300 marker later) economic correction will have muted differences loci, which are polymorphic between the progenitor between patients and normal controls. strains and whose chromosomal location is known. In addition to the work on receptor density, there is The significance of this correlation is the same as the a large body of data which documents marked differ- regression of phenotype on gene dosage for a particular ences among inbred strains in their behavioral locus.59 Six provisional or candidate QTLs were ident- Ͻ responses to the administration of D2 receptor agonists, ified in this step at P 0.01. The second step of the antagonists and related compounds (reviewed in52). analysis focused on determining which of these QTLs Studies from our laboratory have focused on the cata- were real and which resulted from chance correlation

lepsy response induced by D2 receptor antagonists such because of multiple comparisons. To accomplish this as haloperidol. Among 45 inbred mouse strains the goal, 150 B6D2 F2 intercross animals were phenotyped range of ED50 values for haloperidol-induced catalepsy for the catalepsy response and then genotyped using is 50-fold; lowest in the I/J strain (0.2 mg kg−1) and microsatellite markers found in the candidate QTL highest in the LP/J strain (10 mg/ kg−1).44,52,53 This dif- intervals. Two of the QTLs were confirmed at P Ͻ 0.01 ference in response is not the result of differences in or better. One of the confirmed QTLs was near the b or drug uptake and/or metabolism.44 Further, drugs with tyrosinase-related protein (Tyrp1) locus on chromo- a more selective pharmacological profile than haloperi- some 4; the homologous locus in man is found at 9p23.

dol eg raclopride, a speciﬁc D2/D3 receptor antagonist, The other conﬁrmed QTL appeared to be either near or show the same range of response differences among the part of the Drd2 locus on chromosome 9. One hundred

inbred strains. Interestingly, there is essentially no dif- of the intercross animals were also phenotyped for D2 ference among inbred strains in catalepsy induced by receptor density. Focusing on the data from the core 44 the D1 antagonist, SCH 23390. In addition to the work of the nucleus accumbens, the range of difference in on inbred strains, we have also selectively bred mice receptor binding was more than 200%; further, it was for increased and decreased responsiveness to haloper- observed that there was a signiﬁcant relationship idol-induced catalepsy.54,55 Beginning with a hetero- between genotype for D9Mit21 which is closely linked geneous stock formed by crossing eight inbred strains, to Drd2, and receptor binding. Binding in the hetero- selective breeding leads to a rapid divergence of the zygote and the D2D2 homozygote was signiﬁcantly responsive and non-responsive lines such that by the higher (approximately 20%) than in the B6B6 homozy-

ﬁfth selected generation, the lines differ in their ED50 gote. Analogous experiments were conducted for values by approximately 10-fold. These studies on the microsatellite markers near the b locus; no association inbred strains and selected lines are particularly perti- was detected between receptor phenotype and geno- nent to the topic of this report since we have repeatedly type. Overall, these data appeared to provide murine made the paradoxical observation that the non-respon- support for the hypothesis that a polymorphism in the

sive animals have the highest D2 receptor den- D2 receptor gene affects receptor density (the Taq 1 sity.54,56,57 This point has also been conﬁrmed in the hypothesis). However, the data also strongly argued phenotypic extremes (for the catalepsy response) of that most of the genetic variance in receptor density is

two unique F2 intercross populations—one formed associated with other genes. from the C57 and DBA strains and another formed from Recent work in the laboratory now suggests that the the BALB/c and LP strains.53,58 Thus, we conclude that QTL (for catalepsy) on chromosome 9 is probably some of the genes which regulate the catalepsy somewhat distal to Drd2. It is important to note that

response also regulate D2 receptor expression. the QTLs described above have 95% confidence inter- What are these genes? Our first attempt to find ‘cata- vals of 20–30 cM. To both reduce the confidence inter- lepsy related’ genes was a two-step quantitative trait vals and to search for additional QTLs with effect sizes loci (QTL) analysis.53 In step one, provisional QTLs smaller than those that could be detected in the BXD

were identified using the BXD recombinant inbred (RI) analysis, we have phenotyped 1500 B6D2 F2 intercross strains. This RI series which contains 26 strains was progeny and begun a genome-wide scan focusing on formed by Taylor and colleagues over 20 years ago the phenotypic extremes. The QTL near the b locus is beginning with the C57BL/6 and DBA/2 inbred strains again confirmed but only for the female progeny; gen- and was originally intended to find single gene effects. der-specific QTLs may prove to be an important con- The influence of a single major gene was inferred found to mapping studies. The QTL on chromosome 9 whenever a bimodal distribution was observed among was first confirmed simply on the basis of dilute coat the strain means, with one of the progenitor strains in color—160% higher in the non-responsive extreme. each mode. The expected ratio of the two modes is 1:1 Microsatellite-based genotypic analysis has revealed because all animals are homozygous. However, for that the chromosome 9 QTL is indeed centered on the behavioral and neurochemical phenotypes, a bimodal d locus, which is approximately 15 cM distal to the distribution is rarely observed; rather most phenotypes Drd2 locus. The estimated LOD score for this QTL is News & Views 201 7. A likely candidate gene near the d locus is Htr1b. between the increase in receptor density which occurs

High densities of 5-HT1B receptors are found on nerve after chronic haloperidol administration and mRNA terminals in the basal ganglia output nuclei eg the sub- levels.65,66 Mismatches have also been found for the thalamic nucleus and substantia nigra zona reticul- age-related loss of receptor density.67 There is a reason- ata,60 where their function is to regulate transmitter, ably good association between mRNA levels and the largely GABA release. The question of whether or not striatal receptor gradients;57 however, these gradients there is a polymorphism between the C57 and DBA appear to be genetically invariant.44 Among the selec- strains in Htr1b and whether or not this polymorphism ted lines we concluded that the receptor density differ- affects catalepsy and/or D2 receptor density remains to ences were associated with post-translational effects on be determined. Further, these data do not exclude the receptor production; receptor degradation appeared possibility that a functionally important polymorphism unchanged. The important point here is that receptor exists at Drd2. The data only suggest that if two QTLs density can be affected at numerous points in the ’re- are present, the QTL near or in Htr1b has a larger effect ceptor life cycle’ and no single main point of regulation on the phenotypes of interest. The strategy we are has emerged. using to untangle this issue is to develop congenic The general argument which underlies the research strains in which segments of DNA from the DBA strain of Laruelle et al1 and Pohjalainen et al2 as well as the either containing Drd2 and/or Htr1b are introgressed research conducted in our laboratory is that there are into the recipient C57 strain. This strategy will allow genes which regulate the function of the basal ganglia us to examine the effects of the naturally occurring and in turn regulate a wide variety of behaviors. The polymorphisms. Once a functional effect is estab- complexity of this statement cannot be underesti- lished, interval-speciﬁc congenics can be formed to mated. In addition to the general problem of detecting precisely locate the polymorphism.61 Importantly, the multiple genes each with small effects on the pheno- QTL near or a part of Htr1b has been conﬁrmed in a types of interest, one must deal with the enormous completely independent F2 cross formed from the plasticity of the basal ganglia. The observation that BALB/cJ and LP/J inbred strains.58 Of course these data most of the dopamine neurons can be lost before signs do not demonstrate that the same gene or genes are of Parkinsonism appear are well known to most inves- involved in both crosses. However, these data do sug- tigators. It is also remarkable that ‘knockout’ mice have gest that this region of mouse chromosome 9 is gener- been formed for all the dopamine receptors, including ally important to the regulation of the catalepsy D2, and for the dopamine transporter. While these mice response (and probably to the regulation of D2 receptor are clearly not ‘normal’, the fact that they can survive is density). Further, these data suggest that it would be a testament to the fact that the basal ganglia can make reasonable to examine in human samples, the relation- enormous accommodations to insure the development ships between polymorphisms in HTR1B and D2 recep- of necessary functions. This point is underscored by tor density (or binding potential). Interestingly, HTR1B the wide natural variation in D2 receptor density is found at 6q13, in a region of chromosome 6 which among normal controls and the apparent lack of asso- has also been implicated in schizophrenia.62 ciated pathology.

Overall, we have concluded that murine D2 receptor density appears to be a complex phenotype, under the RJ Hitzemann, PhD control of multiple genes, each of which is likely to Department of Psychiatry, SUNY at Stony Brook have only a moderate effect size. Further, although Stony Brook, NY 11794-8101, USA there may be a polymorphism in Drd2 that is associated with the regulation of receptor density, other genes References control most of the variance. The search for these genes is hindered by a lack of understanding of the molecular 1 Laruelle M, Gelernter J, Innis RB. D2 receptors binding potential is mechanisms responsible for the variance in receptor not affected by Taq1 polymorphism at the D2 receptor gene. Mol Psychiatry 1998; 3: 261–265. expression. It would be natural to point to differences 2 Pohjalainen T, Rinne JO, Nagreu K, Lehikoinen P, Anttila K, Syval- in the rate of transcription as the source of the variance ahti EKG et al. The A1 allele of the human D2 dopamine receptor in receptor expression. Although it is clear that the gene predicts low D2 receptor availability in healthy volunteers. receptor can be up and down regulated through phar- Mol Psychiatry 1998; 3: 256–260. 3 Noble EP, Blum K, Ritchie T, Montgomery A, Sheridan PJ. Allelic macological manipulation, little is known about the association of the D2 dopamine receptor gene with receptor-bind- actual molecular mechanisms. The promoter for DRD2 ing characteristics in alcoholism. Arch Gen Psychiatry 1991; 48: has not been extensively characterized.63,64 Further- 648–654. more, mismatches between mRNA levels and receptor 4 Blum K, Noble EP, Sheridan PJ, Montomery A, Richie T, Jagadees- density are common for the D receptor (as they are waran P et al. Allelic association of human dopamine D2 receptor 2 gene in alcoholism. JAMA 1990; 263: 2055–2059. for many other receptors). For example, we have been 5 Parisan A, Todd RD, Devor EJ, O’Malley KL, Suarez BK, Reich T unable to show that there is a relationship between et al. Alcoholism and alleles of the human D2 dopamine receptor mRNA levels (as determined by quantitative nuclease locus. Studies of association and linkage. Arch Gen Psychiatry protection assay) and the differences in receptor den- 1991; 48: 655–663. 6 Comings DE, Comings BG, Muhleman D, Dietz G, Shahbahrami B, sity introduced by selective breeding for the catalepsy Tast D et al. The dopamine D2 receptor locus as a modifying gene 56,57 response (see above). Similarly, several investi- in neuropsychiatric disorders. JAMA 1991; 266: 1793–1800. gators have been unable to show a relationship 7 Blum K, Noble EP, Sheridan PJ, Montgomery A, Ritchie T, Ozkara- News & Views 202 goz T et al. Genetic predisposition in alcoholism: association of S et al. No major role for the dopamine D2 receptor SerFCys311

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