research report new dopamine receptor, d2 , with unique tg...

Ž .Molecular Brain Research 76 2000 132–141www.elsevier.comrlocaterbres

Research report

New dopamine receptor, D2 , with unique TG splice site, in humanLonger

brain 1

Philip Seeman a,b,), Diane Nam a, Carla Ulpian a, Ivy S.C. Liu a,b, Teresa Tallerico a,b

a Department of Pharmacology, Medical Science Building, 8 Taddle Creek Road, UniÕersity of Toronto, Toronto, Ontario, Canada M5S 1A8b Department of Psychiatry, Medical Science Building, 8 Taddle Creek Road, UniÕersity of Toronto, Toronto, Ontario, Canada M5S 1A8

Accepted 7 December 1999

Abstract

Brain dopamine receptor agonists alleviate the signs of Parkinson’s disease, while dopamine receptor antagonists alleviate hallucina-Ž .tions and delusions in psychosis. The dopamine type 2 receptor or D2 is blocked by antipsychotic drugs, including even the ‘‘atypical’’

drugs such as clozapine or remoxipride, in direct relation to their clinical potencies. Compared to the long form of the D2 receptorŽ . Ž .D2 , the short form D2 may be three times more sensitive to benzamide antipsychotic drugs. Hence, it is essential to identifyLong Short

additional variants of dopamine receptors for which more selective antipsychotic drugs can be found. Although no family linkage hasbeen found between the D2 receptor and schizophrenia, there can be brain region abnormalities in the RNA transcript expression ofdopamine receptors. Therefore, in order to identify variant dopamine D2 receptors, we searched for mutations in the RNA transcripts forthe dopamine D2 receptor in the striatum of post-mortem brains from individuals who died with psychosis, including schizophrenia. Anew splice variant of the D2 receptor, D2 , with a unique TG splice site, was found in one control brain and in two psychotic brains.Longer

q 2000 Elsevier Science B.V. All rights reserved.

Keywords: Dopamine receptor; D2 splice variant; D2 mRNA; D2 ; PsychosisLonger

1. Introduction

Dopamine receptor antagonists are effective antipsy-chotic drugs, alleviating hallucinations and delusions in

w xneuropsychiatric disorders 40 . Improved therapy may beobtained by developing drugs that selectively target one of

w xthe several types of dopamine receptors 40 . For example,Ž .the dopamine type 2 receptor or D2 is blocked by

antipsychotic drugs, including even the ‘‘atypical’’ drugssuch as clozapine or quetiapine, in direct relation to their

w xclinical potency 39,41 . Compared to the long form of theŽ . Ž .D2 receptor D2 , the short form D2 may beLong Short

w xmore sensitive to benzamide antipsychotic drugs 3,20,22 .Moreover, three other genomic variants of the D2 receptorw x10,16 , each with a single amino acid substitution, exhibit

) Corresponding author. Departments of Pharmacology and Psychiatry,Medical Science Building, Room 4344, 8 Taddle Creek Road, Toronto,Canada M5S 1A8. Fax: q1-416-971-2445; e-mail:[email protected]

1 The sequence reported in this paper has been deposited in thew xGenBank database accession no. AF176812 .

marked differences in their ability to inhibit cyclic AMPw xsynthesis 6 . Hence, it is essential to identify additional

variants of dopamine receptors for which more selectiveantipsychotic drugs can be found.

Although the dopamine D1 receptor is not the primaryw xtarget of antipsychotic drugs 39 , it indirectly affects the

existence of the high-affinity state of the D2 receptor, anw xinfluence which is reduced in psychosis 42 . Although no

association has been found between markers for either ofwthese two receptors and schizophrenia 4,5,19,24,28,

x29,33,34,43 there can be brain region abnormalities in theRNA transcript expression of dopamine receptors, such asthat for D3 and D4 dopamine receptors in schizophrenia

w x w xfrontal cortex 23 . Moreover, mutations 47,48 and edit-w xing 2,14,37,48 of RNA can occur independent of a

normal genomic DNA. Therefore, in order to identifyvariant dopamine D2 receptors, we searched for mutationsin the RNA transcripts for the dopamine D2 receptor in thestriatum of post-mortem brains from individuals who diedwith psychosis, including schizophrenia. A new splicevariant of the D2 receptor, D2 , with a unique TGLonger

splice site, was found in control and psychotic brains.

0169-328Xr00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.Ž .PII: S0169-328X 99 00343-5

( )P. Seeman et al.rMolecular Brain Research 76 2000 132–141 133

2. Materials and methods

2.1. Human tissues

A summary of the case histories of the post-mortemhuman brains is given in Table 1. The frozen striataŽ .caudate nucleus and putamen and the frozen frontalcerebral cortices were used to search for variants in theRNA transcripts coding for the dopamine D2 receptor.

2.2. RNA and cDNA from post-mortem human tissues

The tissues were homogenized in TRIZOLw ReagentŽ .Life Technologies, Burlington, Ontario, Gibco and chlo-

Ž w .roform 0.2 ml per 1 ml of TRIZOL . The RNA in theupper phase was precipitated from the aqueous phase byadding 0.5 ml of isopropanol per 1 ml of TRIZOLw

Reagent. The samples were incubated at 15–308C for 10min and then centrifuged at 12,000=g for 10 min at 48C.After removing the supernate, the RNA pellet was washed

Ž wonce with 75% ethanol 1 ml per ml original TRIZOL.Reagent . The mixed sample was centrifuged at 7500=g

for 5 min at 48C. The final RNA pellet was brieflyair-dried, dissolved in 0.5% sodium dodecyl sulfate, andincubated for 10 min at 55–608C. Although it has gener-ally been assumed that mRNA degradation renders post-mortem human brain research unreliable, many studieshave shown that mRNA transcripts are relatively stable in

w xhuman post-mortem tissues 11,17,30,32 . cDNA was thenprepared for each tissue, using 2 mg of total RNA, andusing the procedure described in the Superscripte Pream-

Žplification System for First Strand cDNA synthesis Cata-.logue 18089-011, Gibco BRL products, Life Technologies .

The cDNA was amplified by the polymerase chain reac-Ž . Žtion PCR procedure, using primers N25 and N1495 see

.following sequences for the D2 receptor, using a Ge-ŽneAmp PCR System 9600 Perkin-Elmer Cetus, Foster

. w ŽCity, CA thermocycler, and using Advantage -HF High

. ŽFidelity DNA polymerase Kit PT3139-1, Catalogue.aK1909-1, CLON-TECH Laboratories, Palo Alto, CA .

Ž X.The sequence of primer N25 was 5 ACC GCC CTGŽ X .ATG GAT CCA CTG AAT CTG TCC 3 , and the se-

Ž X.quence of primer N1495 was 5 TGA GGA GCA TGGŽ X.AGC CAA GCG AAC ACT GC 3 . The manufacturer’s

procedure for Advantagew-HF DNA polymerase was fol-lowed. Thus, to amplify the cDNA for the D2 receptor,each PCR tube received 0.8 mg of cDNA, 2.5 ml of

Ž .High-Fidelity buffer supplied by the manufacturer , 2.5 mlŽ . Žof 10 mM dNTPs dATP, dTTP, dCTP, dGTP , 0.5 ml 0.5

. Ž .mg of primer N25, 0.5 ml 0.5 mg of primer N1495, 0.5Ž w .ml of DNA polymerase Advantage -HF , and 17.5 ml

water. A total of 35 thermocycles was done, each consist-ing of 15 s for denaturation at 948C, 4 min for annealingand extension at 688C, followed by a final period of 7 minfor extension at 688C. The PCR products were elec-

Ž .trophoresed on agarose gels 1.5% to confirm the MW ofŽ .the expected bands 1.4 kb for D2 . An aliquot of the PCR

w Žproducts was subcloned into the vector pCR 2.1 Invitro-. wgen, San Diego, CA , using the Original TA Cloning Kit

Ž .Invitrogen, Catalog nos. K2000-01 and K2000-40 .Colonies were picked for DNA minipreparations and se-quencing.

2.3. PCR amplification of cDNA for D2Lon g er

Each PCR tube received 9.5 ml of water, 5 ml of 5XŽGC cDNA PCR Reaction buffer Kit number PT3091-1,

.CLON-TECH Laboratories , 0.5 ml of 10 mM dNTPs, 0.5Žml of 10 mM primer 863 General Synthesis and Diagnos-

. Žtics, Toronto , 0.5 ml of 10 mM primer 253 see following. wfor primer sequences , 0.5 ml of Advantage -GC cDNA

Ž . Žpolymerase Mix 50X Kit number PT3091-1, CLON-. ŽTECH Laboratories , 7.5 ml of GC-Melte CLON-TECH

.Laboratories , 1 ml of cDNA from human tissue or fromthe cloned DNA of D2 or D2 , making a totalLong Longer

Ž X .volume of 25 ml. The sequence of primer 253 was 5 GTC

Table 1Clinical summaries for post-mortem brain tissuesAbbreviations: L indicates that the brain tissue came from the National Neurological Research Specimen Bank in Los Angeles; T indicates that the braintissue came from the Canadian Brain Tissue Bank in Toronto; S indicates that the brain tissue came from the Cambridge Brain Bank in Cambridge,England. Halo. is haloperidol; CPZ is chlorpromazine.

Brain Age, sex P.M. delay Death Diagnosis; symptoms Duration antipsychotic Rx of psychosis

Control tissuesL 964 29, m 24 h Cycle accident Control None NoneT 1292 41, m 4 h Car accident Control None None

Psychosis tissuesL 2161 53, m 10 h Cardiac infarct Schizophrenia; voices 10 yr HaloperidolT 638 73, m 7 h Coma Alzheimer; delusional )7 yr NoneS 117 64, f 32 h Pneumonia Schizophrenia; delusions 34 yr Halo., CPZS 130 65, m 6 h Cardiac infarct Schizophrenia; incoherent 25 yr No Rx last 6 months; Halo. before 6 monthsS 132 83, m 48 h Cardiac? Schizophrenia; delusions 57 yr No Rx for last 3 y; Perphenazine for 7 y

( )P. Seeman et al.rMolecular Brain Research 76 2000 132–141134

Table 2D2 cDNA variants

D2 transcripts Transcript variantsexamined D2 D2 D2Longer Short Long

Schizophrenia 117 14 14Schizophrenia 130 100 3 29 68Schizophrenia 132 5 5Schizophrenia 2161 3 3Alzheimer 638 58 1 7 50Control 964 1 1Control 1292 38 1 4 33

Ž . Ž . Ž .Total 219 5 2.3% 40 18% 174 79%

Ž X .AGC CTC GCA GTG GCC GAC CTC CTC G 3 , andŽ X.the sequence of primer 863 was 5 TGG GCT CGC CGG

Ž X.GCA GCC TC CTG CAC 3 , where the latter six boldedletters indicate the bases complementary to the six addi-

Žtional bases found in the dopamine D2 receptor seeLonger.Section 3 . The mixture was preincubated at 948C for 5

Ž .min ‘hot start’ , followed by 40 thermocycles, each con-sisting of 30 s for denaturation at 948C, 60 s for annealingat 758C, and followed by a final period of 5 min forextension at 758C. Because of the low levels of the D2 Longer

cDNA in the extracts from the human frontal cerebralcortex, an aliquot of 1 ml of the latter PCR products forthese tissues was subjected to a further 25 thermocycles.The predicted size of the PCR product was 610 bp.

2.4. Measurement of inhibition constants for drugs

w3 xThe competition between a drug and a H ligand forbinding at the dopamine D2 receptors was done as follows.

Ž .Each incubation tube 12=75 mm glass received, in theŽfollowing order, 0.5 ml buffer 50 mM Tris–HCl, pH 7.4

at 208C, 1 mM EDTA, 5 mM KCl, 1.5 mM CaCl , 4 mM2. ŽMgCl and 120 mM NaCl with or without an excess of2

. w3 x ŽS-sulpiride; see below , 0.5 ml H ligand final concentra-w3 x wtion of approximately 200 pM for H spiperone 60–100

Cirmmol; 2.2–3.7 TBqrmmol; Amersham Life Sciences,x w3 xOakville, Ontario, Canada and 2 nM for H raclopride

w60–70 Cirmmol; New England Nuclear, E.I. du Pont dex.Nemours, Boston, MA , and 0.5 ml of a suspension of the

disrupted COS or 293 cells. The tissue culture cells werecollected and suspended at approximately 200 mgproteinrml. The cells were not washed or centrifuged. The

Žcell suspension was homogenized for 5 s Polytron, setting.5; Brinkmann Instruments, Westbury, NY immediately

before being used.The tubes containing a total volume of 1.5 ml were

Ž .incubated for 2 h at room temperature 208C , after whichthe incubates were filtered, using a 12-well cell harvesterŽ .Titertek, Skatron, Lier, Norway and buffer-presoaked

Ž .glass fiber filter mats No. 7034, Skatron, Sterling, VA .After filtering the incubate, the filter mat was rinsed with

Ž .buffer for 15 s 7.5 ml buffer . The filters were pushed outŽand placed in scintillation minivials Packard Instruments,

.Chicago, IL . The minivials received 4 ml each of scintil-Ž .lant Ready Solve, Beckman, CA , and were monitored 6 h

later for tritium in a Packard 4660 scintillation spectrome-ter at 55% efficiency. Nonspecific binding for dopamineD2 receptors was defined as that which occurred in thepresence of 10 mM S-sulpiride.

Ž . w3 xThe K values dissociation constants of H spiperoneDw3 xand H raclopride for the dopamine D2 receptors were

measured separately. This was done using final concentra-w3 xtions of 10 to 2000 pM for H spiperone, and 0.2 to 12

w3 xnM for H raclopride, and using the same procedureoutlined above for the competition-type experiments. Thedissociation constants were derived by Scatchard analysis.

The inhibition constant, K , for each drug was theniŽderived by the traditional relation, K s C r 1 qi 50

w U x.C rK , where C is the concentration of drug whichD 50w3 x Uinhibits the binding of the H ligand by 50%, where C is

w3 xthe final concentration of the H ligand, and where thew3 xK is the dissociation constant of the H ligand directlyD

determined as outlined above.

3. Results

The objective of this study was to identify variantdopamine D2 receptors. Because mutations and editing ofRNA can occur independent of a normal genomic DNAw x2,14,37,47,48 , we searched for mutations in the RNAtranscripts for the dopamine D2 receptor in the striata ofpost-mortem brains. Because the dopamine D2 receptor isthe main target for antipsychotic drugs, we hypothesizedthat there may be mutations in the D2 receptor whichconceivably might be found in psychotic brain tissue from

Fig. 1. Exon sequence for D2 , as found in the striata fromLongerŽschizophrenia brain 130, Alzheimer brain 638 who had suffered from

.delusions , and control brain 1292. D2 has the same sequence asLonger

D2 , except that exon 6 has two extra codons, GTG and CAG.Long

()

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–141

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Ž . Ž .Fig. 2. Exon and intron sequence for the dopamine D2 receptor. The six bases which are normally included in intron 5 of D2 gtg and cag are part of exon 6 in D2 GTG and CAG .Longer Long Longer


Table 3Ž . aInhibition constants K of drugsi

Drug K i

D2 COS cells D2 COS cellsLong Longer

AgonistsDopamine 2.2"0.3 mM 2.4"0.4 mM

bDopamine 3.9"0.4 mM 5.8"0.8 mMbDopamine qG.N. 10"4 mM 27"8 mM

Serotonin 11"3 mM 11"3 mMNoradrenalin 15"5 mM 18"5 mM

AntagonistsClozapine 45"12 nM 66"15 nMHaloperidol 0.21"0.1 nM 0.22"0.1 nMLoxapine 1.5"0.5 nM 1.8"0.4 nMOlanzapine 7"2 nM 9.2"0.9 nMQuetiapine 220"80 nM 190"60 nMRaclopride 2.2"0.4 nM 1.7"0.4 nM3w xH Raclopride 1.9"0.5 nM 1.8"0.6 nM

Remoxipride 79"12 nM 69"15 nMRisperidone 0.53"0.2 nM 0.7"0.3 nMSertindole 0.39"0.1 nM 1"0.4 nM3w xH Spiperone 0.076"0.01 nM 0.12"0.05 nM

Thioridazine 0.78"0.1 nM 0.52"0.2 nMZiprasidone 2.6"0.7 nM 3.3"0.4 nM

a w3 x Ž . Ž .Using 2 nM H raclopride 70 Cirmmol ns3 ; average"S.E.M.b w3 x Ž . Ž .Using 200 pM H spiperone 101 Cirmmol and 293 cells ns5 .

Ž .G.N.: In the presence of 200 mM guanilylimidodiphosphate ns5 .

individuals who have died with psychosis, despite the factthat no abnormal sequences have been found in the ge-nomic DNA of dopamine D2 receptors in such individuals.Hence, we examined brain tissues from individuals who

Ždied with psychosis four individuals who had schizophre-.nia, and one who had Alzheimer’s disease with delusions

and from two individuals who died suddenly of accidentsŽ . Ž .car accident and motorcycle accident Table 1 .

After preparing the RNA and cDNA from each tissueŽ .see Section 2 , the full-length cDNA for the dopamine D2receptor was amplified by the PCR, using primers N25 and

Ž .N1495 see Section 2 .A total of 219 transcripts of the RNA for the dopamine

D2 receptor were examined in tissues from seven brains,as summarized in Table 2. The full-length cDNA se-quences were examined for almost all of these 219 tran-scripts. The findings in Table 2 show that 79% of thetranscripts were D2 , while 18% of the transcripts wereLong

D2 .Short

During the course of sequencing the 219 cDNA tran-scripts, we were surprised to discover five sequences for

Žthe D2 receptor which contained six extra bases GTG.CAG located immediately before the start of exon 6, as

shown in Figs. 1 and 2. This variant of the D2 receptor,which may be named D2 , is essentially a new spliceLonger

Fig. 3. Selective amplification of the cDNA for D2 from RNA extracted from post-mortem human brain striata and frontal cortices. Although theLonger

frontal cortex cDNA required a total of 65 thermocycles of PCR to amplify D2 , the cDNA for D2 did not amplify under these PCR conditions.Longer Long


variant of the D2 receptor sequence, because the sequenceŽof GTG CAG is normally found in the intron i.e., intron

.5 preceding the normal start of exon 6.An additional surprise was to discover that this new

splice variant of the D2 receptor, D2 , had a uniqueLonger

TG splice site, as shown in Fig. 2. The unusual nature ofthis TG splice site is considered in Section 4.

The transcript variant, D2 , was found in threeLongerŽ wstriata schizophrenia S-130, Alzheimer T-638 who was

x .delusional , and control brain T-1292 . This transcript vari-ant was identical to that for D2 , except that the spliceLong

site between intron 5 and exon 6 occurred six bases beforethe start of exon 6 in D2 , as shown in Figs. 1 and 2.Long

Because the new dopamine D2 receptor which would besynthesized by this variant mRNA would contain an extra

Ž .two amino acids compared to D2 , the new variantLong

was named D2 .Longer

Because it was possible that these extra two aminoŽ .acids valine and glutamine could alter the binding of

dopamine agonists and antagonists to the dopamineŽ .D2 receptor in comparison to D2 or D2 , itLonger Short Long

was necessary to examine this directly. Therefore, thecDNA for the dopamine D2 receptor was transfectedLonger

into, and expressed in, both COS and 293 cells, and theaffinities of the expressed dopamine D2 receptor forLonger

various drugs were obtained. Both the COS cells and the293 cells expressed approximately 1.5 to 2 pgrmg ofprotein.

The data in Table 3 indicate that the inhibition constantsof the dopamine D2 receptor for various agonists andLonger

antagonists were similar, but not always identical, to thosefor the dopamine D2 receptor. Parenthetically, it mayLong

be noted that the inhibition constant for dopamine isw3 xslightly higher when competing against H spiperone than

w3 xwhen competing against H raclopride. This apparent de-w3 xpendence of the inhibition constant on the H ligand

w3 xresults from the higher solubility of the H ligand in thewmembrane, as extensively described elsewhere see Ref. of

w xxSeeman and Van Tol, 1995, in Ref. 39 .Moreover, the binding of dopamine to the dopamine

D2 receptor was sensitive to guanilylimidodiphos-Longer

phate, suggesting that a portion of the dopamine D2 Longer

receptors were in the functional high-affinity state forw xdopamine 40 .

Despite the low prevalence of the dopamine D2 Longer

receptor in the tissues examined, it was possible to amplifythis receptor selectively from cDNA prepared from post-mortem human striatum and frontal cortex tissues, usingthe PCR, and using primers 253 and 863. This is shown inFig. 3, where 40 cycles of PCR were sufficient to amplifythe D2 cDNA from the striatum, while an additionalLonger

25 cycles of PCR were required to amplify the D2 Longer

cDNA from the frontal cortex. The selectivity of theconditions for amplifying D2 are illustrated by theLonger

Ž .lack of amplification of the cloned D2 cDNA Fig. 3 ,Long

despite the 65 cycles of PCR.

4. Discussion

The new variant, D2 , was found in brain striataLongerŽ .from both control and psychotic individuals Table 2 . The

proportion of the D2 variant among the population ofLonger

D2 transcripts examined in all the striata was about 2.3%,Žcompared to 18% for D2 and 79% for D2 TableShort Long

.2 , the latter two values generally agreeing with thosew xfound by others in human striata 31 and rat striata

w x12,26,45 . Although no cellular location of D2 wasLonger

done in the present study, such work will be important todo, considering that D2 predominates in dopamine cellShort

bodies axons while D2 is more strongly expressed byLongw xneurons in the striatum and nucleus accumbens 18 .

A unique feature of the dopamine D2 receptor isLonger

that it arises from an aberrant splicing pattern of the RNA.Virtually all known introns have an AG sequence at their

X Ž . w x3 ends the acceptor site 25 , including all the intronsw xspliced out in D2 7,9,13 . However, intron 5 forLong

D2 ends with a unique TG sequence, as illustrated inLonger

Fig. 2. Of the many thousands of known genes, no otherintron has a 3X acceptor site which ends in TG. There areonly two other proteins where the normal AG acceptor sitehas mutated into a TG. In these instances, however, the

Žmutation results in either the exon being skipped as inhypoxanthine phosphoribosyl-transferase in the Lesch–

.Nyhan syndrome or the appearance of an abnormal spliceŽsite occurring within the exon as in ornithine transcar-

.bamylase deficiency , resulting in abnormal proteins beingw xexpressed, and resulting in disease in both cases 25 .

Although the normal splicing reaction for introns ending inAG is done by the spliceosome with its 70 proteins andfive small nuclear RNAs, the different ending of TG inintron 5 for D2 would presumably be spliced byLonger

w xdifferent small nuclear RNAs 46 .Although the present variant could reflect an abnormal-

ity associated with psychosis, the D2 splice variantLonger

was also found in a control brain, indicating that psychosisis not uniquely associated with the splice variant. There isa possibility, however, that the ‘‘normal’’ control brainrevealing the D2 variant may have come from anLonger

Ž .individual T1292 with subclinical symptoms of psy-chosis. Moreover, too few individuals were examined toindicate anything for or against linking a psychotic condi-tion to the prevalence of the D2 variant in the brain.Longer

The prevalence of the transcript variant described heremay be different in different brain regions in health anddisease. For example, in the present study the dopamineD2 splice variant was found five times in 219 tran-Longer

Žscripts examined in the striatal tissues. Although fourother brain striata did not reveal the D2 variant, asLonger

noted in Table 3, only a few D2 transcripts were examined.in these four tissues. Because the abundance and propor-

tions of both D2 and D2 vary from region toShort Longw xregion in the brain 12,18,26,48 , the prevalence of the

D2 variant could be significantly higher or lower inLonger


other brain regions expressing dopamine receptors in healthor in psychosis.

The low prevalence of this transcript variant is similarto the low prevalence of one transcript variant in 4000normal transcripts for b-amyloid precursor protein and

w xubiquitin-B in Alzheimer’s disease 47,48 .The prevalence or expression of the transcript variants,

moreover, may vary from time to time, depending on avariety of factors which affect gene expression. This wouldbe consistent with the natural undulating course of psy-chotic symptoms in schizophrenia, where it has been ob-served that the symptomatology waxes and wanes for noapparent reason. Moreover, it is possible that the preva-lence or expression of such transcript variants may be

w xage-dependent 48 , explaining why age is an importantfactor in the onset of psychosis.

There are several molecular mechanisms whereby tran-script variants can result in abnormal neurotransmission.For example, the transcript variant can lead to the synthe-sis of an inactive receptor, as in the case of the dopamine

w xD4 variant 21,44 . Second, the transcriptValine194Glycine

variant can produce an inherently active receptor in theŽabsence of any activating ligand i.e., a ‘‘constitutively

. w xactive’’ receptor 35 . Third, the mRNA transcript variantsof a receptor can lead to the production of variant receptorproteins which can interfere with the function of thenormal receptor. Dopamine D1, D2 and D3 receptors, for

w xexample, exist in the form of monomers or dimers 27,50 .

Fig. 4. The dopamine D2 receptor variant has two extra amino acids, compared to D2 , in the third cytoplasmic loop involved in coupling toLonger LongŽ . Ž .intracellular proteins mediating the actions of dopamine. The amino moiety of dopamine in red attaches to aspartic acid labeled D , while the two

Ž .hydroxyls of dopamine attach to the two serines labeled S . Yellow: Phospholipid bilayer region of the cell membrane, with yellow spheres representingthe head groups. Blue: Extracellular side of the membrane. Pink: Cytoplasmic side of the cell membrane. The amino acid chain of this D2 receptor proteinstarts on the extracellular side and traverses the cell membrane seven times, as indicated by the hydrophobic nature of the amino acids within theyellow–white hydrophobic region. The four cytoplasmic loops of amino acids serve to relay the receptors interactions with various intracellular messengermolecules such as G proteins and kinases. Amino acid code: A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H,histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; A, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W,

Ž .tryptophan; Y, tyrosine Copyright of main template, Seeman, 1990, 1991, 1996 .


Thus, a dimer consisting of D2 and D2 may notShort Longer

be fully active. Fourth, the transcript variant can producereceptor variants which can result in abnormal coupling toother proteins, such as G proteins or other proteins whichbind to the SH domains of receptors. Such physiologicalcoupling generally occurs between the third cytoplasmicloop of the receptor and other proteins, especially the Gproteins. In fact, the receptor variant reported here har-bours a variation in the third cytoplasmic loop between theregions of transmembrane 5 and 6, as illustrated moreclearly in Fig. 4.

In addition to the question of regional abundance of thetranscript variant, the expression of the D2 receptorLonger

protein may not quantitatively parallel the level of thetranscript. For example, although the frontal cortex of therat brain contains a relatively high level of dopamine D4receptor mRNA, there is an absence of any D4-like spe-

w xcific binding in that region 36 . Moreover, although long-term antipsychotic medication generally elevates the den-sity of dopamine D2 receptors by 30–50% in the rat

w xstriatum 38 , the density of the dopamine D2 receptorw xmRNA usually exhibits little or no elevation 8,49 ; in

those cases where D2 mRNA has been found to be ele-w xvated, the elevation is short-lived 15 . Hence, despite the

low prevalence of the dopamine D2 receptor tran-Longer

script variant found in the present study, the variant recep-tor protein itself may actually be higher in density thanwould be indicated by the low level of the variant mRNA.

Future research needs to examine the prevalence ofsuch transcript and receptor variants in different brain

Ž w x.regions such as in Ref. 18 , the age-dependence of thisprevalence, and, as well, the regulatory factors which maybe responsible for creating transcript variants. In particular,because most of the known aberrant splicing patterns are

w xassociated with disease 1,25 , future research must explorewhether the splicing mechanisms in producing D2 Longer

may sometimes be altered and associated with neuropsy-chiatric disease.

Acknowledgements

We thank Dr. Hubert H.M. Van Tol for advice. Wethank Dr. Mary V. Seeman for discussion and clinicalexpertise, and Dr. H.-C. Guan and Ms. Ivy Qian, MSc, forexcellent assistance. We thank the following individualsand agencies for their support: the Medical ResearchCouncil of Canada, the Ontario Mental Health Foundation,the Schizophrenia Society of Canada, the National Al-liance for Research in Schizophrenia and DepressionŽ .NARSAD , Mr. and Mrs. Robert Peterson of The Peter-

Ž .son Foundation University Park, FL , the Stanley Founda-tion of the Research Institute of the National Alliance for

Ž . Žthe Mentally Ill NAMI , the Medland Family J. Aubreyand Helen Medland, Pamela and Desmond O’Rorke, Janet

.Marsh and David Medland , and the National Institute on

Ž .Drug Abuse NIDA, USA . We thank Dr. John Wherrettand Ms. Maria Pataki of the Canadian Brain Tissue BankŽ .cro the Center for Addiction and Mental Health, Toronto ,Dr. W.W. Tourtellotte, Mr. R.S. Riehl, and Ms. J. Sonen-shein of the National Neurological Research Specimen

Ž .Bank Wadsworth hospital, Los Angeles, CA , and Dr. J.Xuereb and Mr. R.E.W. Hills of the Cambridge Brain

Ž .Bank Laboratory University of Cambridge, Englandfor providing post-mortem human brain tissue samples.Dr. Ivy S.C. Liu and Dr. T. Tallerico were supportedby Post-doctoral Fellowships jointly sponsored by theSchizophrenia Society of Canada and the Medical Re-search Council of Canada.

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