Chapter 2 Gene and Promoter Structures of the Dopamine Receptors

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1 Chapter 2 Gene and Promoter Structures of the Dopamine Receptors Ursula M. D Souza Abstract The dopamine receptors have been classified into two groups, the D 1 - like and D 2 -like dopamine receptors, respectively, based on molecular biology and pharmacological studies. The D 1 -like dopamine receptors comprise the D 1 and D 5 dopamine receptors and the D 2 -like dopamine receptors include the D 2, D 3 and D 4 dopamine receptors. The gene structures of these two classes of receptors are dissimilar with respect to the organization of their coding and regulatory regions. First, the D 2 -like dopamine receptor genes have revealed the presence of coding exons separated by introns whereas the D 1 -like dopamine receptor genes consist of a single exon and thus are intronless. Second, examination of the 5 - regulatory regions reveals the presence of non-coding exon(s) several kilobases upstream from their coding exons in the D 2 and D 3 dopamine receptor genes, while regulatory regions of the D 1 -like dopamine receptor genes have only one noncoding exon that is separated by a small intron from the coding exon. However, in general, characterization of the 5 -flanking regions of the dopamine receptor genes demonstrates that they lack TATA boxes or CCAAT boxes, are GC rich and have several consensus binding sites for the transcription factor Sp1. The regulatory region of the D 2 dopamine receptor gene is similar to that in the D 3 dopamine receptor gene as they both contain an initiator-like element suggesting transcription initiation from this position and are under strong negative regulation in mammalian cell cultures. Furthermore, amongst the dopamine receptor genes, the 5 -flanking regions of the D 3 and D 5 dopamine receptors have much lower GC content than those in the D 1, D 2 and D 4 dopamine receptor genes. Nevertheless, overall, the promoter regions of all the dopamine receptor genes are regulated in a cell-specific manner, including the additional promoter of the D 1 dopamine receptor gene located within intron 1. There are several studies that have identified transcription factors (DNA binding proteins) that regulate the dopamine receptor genes, U.M. D Souza (B) MRC Social, Genetic and Developmental Psychiatry (SGDP) Centre, Institute of Psychiatry, King s College, London, UK ursula.d souza@iop.kcl.ac.uk K.A. Neve (ed.), The Dopamine Receptors, 2nd Edition, The Receptors, DOI / _2, C Humana Press, a part of Springer Science+Business Media, LLC

2 24 U.M. D Souza with more experimental data generated for D 1 and D 2 compared to the D 3, D 4 and D 5 genes. Therefore, all the evidence suggests that the genes encoding the dopamine receptor subtypes have diverse transcriptional regulation mechanisms that result in cell-specific expression patterns that are coupled with different molecular functions. Keywords Dopamine receptors Promoters Transcriptional regulation Gene structure 2.1 Dopamine Receptors Dopamine is a catecholamine neurotransmitter that mediates several important physiological functions in both the central and peripheral nervous system. In the brain, it plays a major role in the control of motor function, reward, emotional expression, neuroendocrine release and behavioural homeostasis. Dopamine induces cellular and biochemical effects by interacting with its cell surface receptors [1, 2]. These receptors belong to the superfamily of G protein-coupled receptors having seven transmembrane domains and were first classified in the 1970s based primarily on pharmacological and biochemical studies, which included the rank order of receptor agonist and antagonist affinities [3], and the ability of dopamine to stimulate camp formation, by the activation of adenylyl cyclase in the central nervous system [4]. Later on in the 1980s four distinct dopamine receptor subtypes termed D 1, D 2, D 3 and D 4 were proposed on the basis of radioligand binding studies [5, 6]. However, this terminology was soon abandoned when the proposed D 3 and D 4 dopamine receptors were realized to be high-affinity states of the D 1 and D 2 dopamine receptors, respectively. In the 1990s, modification of this dopamine receptor classification was necessary after the development of molecular biology experimental techniques such as PCR cloning which revealed a much larger number of dopamine receptors than originally postulated (see [7] for a review). The current nomenclature for dopamine receptors is based on their structure, pharmacological specificity and effector responses. Consequently, the studies have divided the dopamine receptors into two groups called the D 1 -like and the D 2 -like dopamine receptors. The D 1 -like dopamine receptor group is composed of the D 1 and D 5 dopamine receptors, sometimes also referred to as D 1A and D 1B dopamine receptors, respectively. The D 2 -like dopamine receptors are the D 2, D 3 and D 4 dopamine receptors which include the two D 2 dopamine receptor isoforms, the different isoforms of D 3 and the polymorphic forms of the D 4 dopamine receptors. Molecular cloning of the dopamine receptor genes paved the way for the characterization of their 5 -flanking and promoter regions to understand their transcriptional control. This information further enabled the identification of key polymorphisms (single nucleotide and tandem repeats) within

3 2 Gene Structures of the Dopamine Receptors 25 these regulatory regions which have been found to be associated with several neuropsychiatric and behavioural disorders. Interestingly, these genetic variants within the regulatory regions of the dopamine receptor genes have been found to have functional effects at a molecular and cellular level (reviewed in [8 11]). Thus this provides plausible molecular mechanisms underlying the aetiology of psychiatric phenotypes. This chapter will describe the gene and promoter structures of the dopamine receptors under subheadings of the D 1 -like and D 2 -like dopamine receptor genes. The section on the D 1 -like dopamine receptor genes will be divided into D 1 and D 5 dopamine receptor genes. Similarly, the subdivision on the D 2 -like dopamine receptor genes will be separated into the D 2, D 3 and D 4 dopamine receptor genes. Every section for each gene will be further divided into two parts: one focusing on the topic of gene structure and organization and the other concentrating on the regulatory and promoter regions. All this information has been summarized in Table 2.1 and should be referred for the overall description of each of the dopamine receptor subtypes. However, specific details can be obtained from original references that have been cited in each section. The themes of transcriptional gene regulation and gene expression have been described concisely in several reports [12 17]. More recently, several other levels of gene regulation are currently being studied and include RNA interference which is important in gene silencing [18] and non-coding RNA (ncrna) that comprise a hidden layer of internal signals that control various levels of gene expression [19, 20]. These non-coding RNAs include rrna and trna involved in mrna translation, small nuclear RNA (snrna) implicated in splicing, small nucleolar RNA (snorna) involved in the modification of rrna and microrna (mirna) which function as repressors at the level of post-transcriptional control. Furthermore, since DNA is packaged into a nucleoprotein complex known as chromatin, it is becoming important to understand this structure together with histone modification and cytosine methylation in gene regulation [21]. In general the first stage in characterizing the 5 -flanking and promoter region(s) of a gene involves isolation of a genomic clone that harbours the transcription initiation site and contains the upstream sequence of the gene. This is followed by the determination of the exon/intron organization to identify any untranslated region and then measurement of the transcriptional activity of the upstream regulatory sequences [12]. These strategies involve the generation of serial 5 -deletion plasmid constructs fused with a reporter gene such as luciferase or chloramphenicol acetyltransferase (CAT). These constructs are transiently transfected into mammalian cell lines to determine transcriptional activity of the mutant fragments in vitro or they can be used in vivo to generate transgenic mice. Furthermore, electrophoretic mobility assays and yeast one-hybrid studies have also focused on identifying which DNA binding proteins (transcription factors) interact within the regulatory domains of the genes coding for the dopamine receptors. The findings generated from the methods described above are described and discussed below.

4 26 U.M. D Souza Table 2.1 Description of gene organization and transcriptional regulation of the dopamine receptor genes Gene organization [coding and 5 -regulatory region] Features in promoter region Upstream regulation D2-like dopamine receptors D2 dopamine receptor One non-coding exon separated from seven coding exons by large intron D3 dopamine receptor Two non-coding exons separated from six coding exons by large intron D4 dopamine receptor Four or five coding exons (depends on species) separated by introns D1-like dopamine receptors D1 dopamine receptor One non-coding exon separated from the coding exon by small intron D5 dopamine receptor One non-coding exon separated from the coding exon by small intron Two promoter regions, initiator-like element, no CCAAT or TATA boxes, 80% GC One promoter region, initiator-like element, no CCAAT or TATA boxes, 52% GC One promoter region, no CCAAT or TATA boxes, CpG island, over 50% GC Two promoter regions, no CCAAT or TATA boxes, 80% GC One promoter region, no CCAAT or TATA boxes, not GC rich Two silencer regions in mammalian cell lines Two silencer regions in neuroblastoma and hepatoblastoma cell lines Two silencer regions in neuroblastoma and retinoblastoma cell lines Two activator regions and one silencer region in neuroblastoma cell lines One activator and one silencer region in neuroblastoma cell lines Regulation by transcription factors AP1, retinoids Sp1/Sp3, Zif68, DRRF, nuclear factor-κb DRRF Sp1 Sp1, POU, Brn4, Meis2, TGIF, ZIC, Sp3, DRRF Not known to date

5 2 Gene Structures of the Dopamine Receptors D 2 -Like Dopamine Receptor Genes D 2 Dopamine Receptor Genes Gene Structure and Organization The cloning of the rat D 2 dopamine receptor was a major breakthrough in the neuroscience field [22]. The cloning strategy that was employed utilized the coding region of the hamster β 2 -adrenergic receptor [23]. This genomic DNA sequence was used to probe a rat genomic library for the presence of homologous fragments in Southern blot analysis. Under low-stringency hybridization conditions several clones were found, and one clone (clone RGB-2) was further characterized. This clone consisted of a 0.8 kb EcoRI PstI fragment that revealed a high degree of similarity to the nucleotide sequence of the putative transmembrane domain of the hamster β 2 -adrenergic receptor. The 0.8 kb EcoRI PstI fragment of RGB-2 was then used to probe a rat brain cdna library. A full-length cdna of 2,455 bases was isolated that encoded a protein of 415 amino acids. A hydrophobicity plot of this amino acid sequence indicated that it belonged to the family of G protein-coupled receptors, as it consisted of seven putative transmembrane domains [24]. Subsequently, the human pituitary cdna (hpitd 2 ) was cloned using rat brain D 2 dopamine receptor cdna as a hybridization probe [25]. The human and rat nucleotide sequences were found to be 90% identical and they indicated 96% homology at the amino acid level. When hpitd 2 cdna was expressed in mouse Ltk cells, the protein showed a pharmacological profile which was essentially identical to that obtained with the cloned rat D 2 dopamine receptor [22]. However, the human pituitary D 2 dopamine receptor encoded a protein of 444 amino acids, 29 amino acids longer than the rat D 2 dopamine receptor. DNA sequence analysis showed that the coding sequence had seven exons interrupted by six introns and that the additional amino acid sequence was encoded by a single exon (exon 5) of 87 base pairs, which was present in the putative third cytoplasmic loop of the receptor. These D 2 dopamine receptors of different sizes from the two species were referred to as the D 2L (long) and D 2S (short) forms and it was postulated that they were produced by alternative splicing of mrna [25]. The human D 2 dopamine receptor gene was found to localize to chromosome 11q23-24 [26]. The structure and organization of the rat D 2 dopamine receptor gene was subsequently further delineated when demonstrated that the gene contains eight exons and spans at least 50 kb [27]. This research group identified seven coding exons (numbered as exons 2 8), including the alternatively expressed exon (exon 6), clustered in approximately 13 kb of the genome which revealed a similar structure to the human D 2 dopamine receptor gene. Furthermore, they also identified a non-coding exon termed as exon 1, thus generating a different exon numbering system to that previously used for the human D 2 dopamine receptor gene [25]. Additionally, the same research group consequently analysed the structure of the human D 2 dopamine receptor [28]. Like the rat D 2 dopamine receptor gene, the human D 2 dopamine receptor gene was found to contain at least eight exons and spans at least 52 kb. The

6 28 U.M. D Souza coding exons 2 8 are clustered within 14 kb and the non-coding exon 1 is separated from exon 2 by at least 38 kb. Similarly, the mouse D 2 dopamine receptor gene was found to span at least 30 kb with the coding exons 2 8 clustered in 11 kb and the non-expressed exon 1 located at least 18 kb away from exon 2 revealing an analogous organization to the rat and human D 2 dopamine receptor genes [29]. Each intron/exon boundary was also sequenced in the mouse D 2 dopamine receptor gene and compared with the rat and human species [29]. The position of all boundaries was conserved in all three species except for intron 4 which contains a variant donor splice site (a GC dinucleotide instead of the canonical GT) in the mouse and rat but not in the human D 2 dopamine receptor gene [27]. Other studies also demonstrated that alternative splicing produces the expression of two rat D 2 dopamine receptor isoforms [27, 30 32]. Furthermore, similar investigations were performed on both the rat and human D 2 dopamine receptor isoforms [31], on the rat and bovine D 2 dopamine receptor isoforms [33 36] and for the mouse D 2 dopamine receptor isoforms [29]. In the literature the long form of the D 2 dopamine receptor was referred to as the D 2L, D 2(long), D 2A, D 2(444) or D 2-in, whereas the short form was termed D 2S, D 2(short), D 2B, D 2(415) or D 2-o Promoter Structure and Transcriptional Regulation A short fragment of 500 bp from the translational start site of the rat D 2 dopamine receptor gene was initially sequenced [27]. No transcriptional elements such as CCAAT or TATA boxes were found but the region was 78% GC rich and consisted of several Sp1-like binding sites. Subsequently, the analysis of the promoter region of the rat D 2 dopamine receptor gene was comprehensively determined [37]. This analysis included cloning of exon 1, identification of its 5 -end, determination of the transcription start sites and the ability of D 2 promoter deletion mutants to transcribe the reporter gene chloramphenicol acetyltransferase (CAT) in various cell lines. The rat D 2 dopamine receptor gene spans at least 50 kb with coding exons 2 7 clustered in approximately 13 kb of genome, revealing that intron 1 is very long and over 20 kb [27]. A 21-mer oligonucleotide probe consisting of exon 1 sequences [27] was used to screen a rat genomic library [37]. A 1.3 kb region including all of exon 1, its 5 -flanking region and part of intron 1 was sequenced. S1 nuclease analysis indicated three consecutive nucleotides as the main transcription start sites and several weaker sites also noted upstream from the 3 -end of exon 1. The results also reveal no exon further upstream to the non-coding exon 1 in the D 2 dopamine receptor gene. The +1 was designated as the adenine that corresponds to one of the strong S1 signals and to one of the 5 -cdna ends generated by RACE (rapid amplification of cdna ends). The promoter region of the D 2 dopamine receptor gene was found to lack TATA and CCAAT boxes and is rich in GC content (reaching 80% in some portions) with several putative binding sites for the transcription factor Sp1. An initiator-like sequence was sited between nucleotides 6 and +11, suggesting transcription initiation from this position. Transient expression assays using 5 -deletion mutant constructs controlling transcription of the CAT gene were determined in murine neuroblastoma cells (NB41A3) that endogenously express the

7 2 Gene Structures of the Dopamine Receptors 29 D 2 dopamine receptor gene. Strongest transcriptional activity was found between nucleotides 75 and 30 and silencing activity was present between nucleotides 217 and 76. DNase I footprinting studies using nuclear extract from NB41A3 cells suggested Sp1 binding to its consensus sequence at nucleotide 48 but inhibition of Sp1 binding at nucleotide 86 by the extract. The D 2 promoter showed no transcription activity of the heterologous CAT gene in rat glioma C6, mouse embryonal NIH 3T3 and human hepatoblastoma Hep G2 cells, indicating that it is regulated in a tissue-specific manner. Subsequently, another study demonstrated the transcription of the rat D 2 dopamine receptor gene from two promoter regions [38]. Using single-stranded ligation to single-stranded cdna (SLIC), the gene was found to contain two transcription start sites: the major one located about 320 bp upstream from the 3 -end of the first exon and a minor site 70 bp further upstream. Transient expression assays with fusion constructs consisting of fragments of the rat D 2 promoter region and the luciferase reporter gene confirmed the presence of two independent TATAlacking promoter regions. Both promoters independently induced transcription of the luciferase gene in C6 glioma cells, fibroblasts and GH3 and MMQ rat pituitary cell lines, although only the MMQ cells express the D 2 dopamine receptor. The transcriptional activity was enhanced in the presence of both promoters and modified by the upstream sequences. These data differ from that derived by [37] and were suggested to be due to the use of different reporter gene assays with varying sensitivities and/or the utilization of different cell lines [38]. The negative modulator of the rat D 2 dopamine receptor gene was further analysed [39]. In this study, a small deletion series within the negative modulator fused with the CAT reporter gene was used to transfect the D 2 -expressing cells, NB41A3. The results identified two cis-acting functional DNA sequences. The first is a 41 bp segment between nucleotides 116 and 76 (D 2 Neg-B) and the second is a 26 bp segment between nucleotides 160 and 135 (D 2 Neg-A). D 2 Neg-B decreased transcription from the D 2 promoter by 45%, whereas D 2 Neg-A in the presence of the downstream negative modulator reduced transcription down to the level of a promoterless vector. Furthermore, DNase I footprinting, gel mobility shift and competitive cotransfection experiments suggested that D 2 Neg-A functions without trans-acting factors, while D 2 Neg-B interacts with nuclear factors at its Sp1 binding sequences. Gel supershift assays with anti-sp1 antibody and UV crosslinking experiments revealed that a novel 130 kda factor as well as Sp1 interacts with D 2 Neg-B in NB41A3 cells. The novel protein that recognizes Sp1 binding sequences in the D 2 gene negative modulator was also found to be present in rat striatum nuclear extract. In the case of the human D 2 dopamine receptor gene only a small fragment of the 5 -flanking region was isolated and sequenced which enabled screening of genetic variants [40]. A significant polymorphism in the D 2 promoter is the 141C Ins/Del (insertion/deletion), where one or two cytosines are found as part of a putative binding site for the transcription factor Sp1. Constructs consisting of the 141C Del allele cloned into a plasmid with the luciferase reporter gene demonstrated lower transcriptional activity in human retinoblastoma Y-79 cells (that express D 2 ) and

8 30 U.M. D Souza human kidney 293 cells (D 2 non-expressing) compared to the 141C Ins allele [40]. Interestingly, it was additionally demonstrated in case control studies that the 141C Del allele was significantly lower in schizophrenic patients than in control subjects in Japanese and Swedish populations [40, 41]. The promoter region of the D 2 dopamine receptor has been found to be regulated by other transcription factors including retinoids [42, 43], AP1 [44], Sp1/Sp3 [45] and Zif268 in the rat [46], by nuclear factor-κb in human [47] and by dopamine receptor regulating factor (DRRF) in mouse [48]. The latter report showed that DRRF is a zinc finger transcription factor that binds to GC and GT boxes in the D 2 dopamine receptor promoters and effectively displaces Sp1 and Sp3 from these sequences. Highest levels of DRRF mrna were found in mouse brain in areas including olfactory bulb and tubercle, nucleus accumbens, striatum, hippocampus, amygdala and frontal cortex. Interestingly, these brain regions also express abundant levels of dopamine receptors, indicating the importance of DRRF in regulating dopaminergic neurotransmission. In the D 2 -expressing NB41A3 cells, DRRF potently inhibited transcription from the D 2 promoter, whereas it was found to activate the D 2 promoter in NS20Y and TE671 cells. In vivo experiments show that DRRF mrna is significantly altered in striatum and nucleus accumbens brain regions in mice treated with acute and chronic doses of cocaine and haloperidol. Furthermore, in situ hybridization studies in mice have shown that DRRF mrna is expressed uniquely during development with high levels observed at E12, E14 and E16 in various tissues [49]. DRRF expression during development is also found in particular brain regions such as the neopallial cortex, olfactory lobe and corpus striatum. This pattern of DRRF distribution during embryogenesis overlaps with that found in the adult brain and with the expression profile of dopamine receptors both in adult and during development. Additionally, the promoter region of murine DRRF was characterized, revealing tissue-specific activity, suggesting that it shares structural and functional similarities with the dopamine receptor genes that it regulates [50]. More recently, it has been found that DRRF auto-regulates its own promoter by competing with Sp1 and that both AP1 and AP2 modulate its expression [51]. Additionally a small segment of the rat D 2 promoter has been found to be regulated by corticosterone and oestrogen in NB41A3 cells in vitro [52, 53]. Moreover, DNA methylation has been demonstrated within the promoter region of the human D 2 dopamine receptor gene, suggesting that this transcription regulatory mechanism plays a role in controlling human D 2 dopamine receptor gene expression [54]. Lately, the 5 -regulatory region of the human D 2 dopamine receptor gene has been found to have methylated cytosines mainly in three clusters [55] D 3 Dopamine Receptor Genes Gene Structure and Organization The molecular cloning of the rat D 3 dopamine receptor gene was performed using reverse transcription polymerase chain reaction (RT-PCR) [56]. Genomic and cdna

9 2 Gene Structures of the Dopamine Receptors 31 libraries were screened using a probe derived from the D 2 dopamine receptor sequence published earlier [22]. The positive clone that was obtained coded for a protein of 446 amino acid residues, and hydrophobicity analysis of the clone indicated seven putative transmembrane regions characteristic of G protein-coupled receptors. The D 3 dopamine receptor gene contained introns similar to the D 2 dopamine receptor gene, and 75% homology existed between the rat D 2S and D 3 dopamine receptor genes within the transmembrane regions. Consistent with this high sequence homology, the pharmacological properties of the D 3 receptor were similar to but distinct from those of the D 2 dopamine receptor. The rat D 3 dopamine receptor gene contains six coding exons separated by five introns. In humans, this gene is located on chromosome 3, band 3q13.3 [57], with a coding region consisting of six exons over 53 kb and an open reading frame of only 400 amino acids. This difference of 46 amino acid residues between the rat and human D 3 dopamine receptors is located within the third cytoplasmic loop of the protein [58]. The polymerase chain reaction amplification of mrna from rat brain revealed the existence of two shorter isoforms of the D 3 dopamine receptor in addition to the D 3 dopamine receptor itself [59]. The isoforms were suggested to result from different processes of alternative splicing. One form was produced by splicing of an exon whose absence deletes the third transmembrane domain, resulting in a protein (termed D 3 (TM3-del)) having no dopaminergic ligand binding activity. The second isoform resulted from splicing at a receptor site that coded for half the second extracellular loop and part of the fifth transmembrane domain (referred to as D 3 (O2-del)). Other research groups have also demonstrated splice variants of the D 3 dopamine receptor in the rat and human brain [60 62], but none of the truncated proteins encoded by these variants has dopamine receptor activity. However, the alternatively spliced short isoform of the mouse D 3 dopamine receptor that lacks 63 nucleotides in the third cytoplasmic loop was found to bind dopaminergic ligands [63]. This alternative splicing reflects the presence of a sixth intron found in the mouse D 3 receptor gene [64, 65]. The functional and physiological role of the truncated forms of the D 3 dopamine receptors is not known, but it has been suggested that they could be formed for controlling the amount of active D 3 dopamine receptors [59]. Defects in the regulation of alternative splicing of the receptor could result in formation of inactive D 3 dopamine receptors and may be associated with psychiatric disorders Promoter Structure and Transcriptional Regulation The D 3 dopamine receptor gene has been implicated in neuropsychiatric disorders and found to be regulated following antipsychotic drug treatment [66, 67]. To begin with only a short segment of the 5 -untranslated region of the D 3 dopamine receptor gene was described in the mouse [65] and human [62]. However, a comprehensive investigation of the gene s transcriptional control was elucidated when the 5 -flanking region was characterized by isolating the 5 -end of its cdna as well as 4.6 kb of genomic sequence [68]. Analysis of this region revealed the presence of two new (untranslated) exons of 196 bp and 120 bp, designated exon 1 and exon

10 32 U.M. D Souza 2, that are separated by an 855-bp intron located several kilobases (at least 4 kb) upstream of the previously published coding exons. This evidence shows that the rat D 3 dopamine receptor gene is organized into eight exons and is comparable to the structure of the rat D 2 dopamine receptor gene. The rat D 2 dopamine receptor gene has a single non-coding exon located at least 35 kb upstream from its first of seven coding exons [27, 37, 38]. However, sequence comparison between the 5 - UTR and 5 -flanking regions of the rat D 2 and D 3 dopamine receptor genes shows substantial homology. On the other hand, the D 1A dopamine receptor gene has been found to have a different organization with a non-coding exon separated from a single coding exon by a small intron [69, 70], which is 116 bp in humans (see Section 2.3). There is no sequence homology between the 5 -flanking region of the D 1A and the rat D 3 dopamine receptor genes. The transcription initiation site of the rat D 3 dopamine receptor gene determined by primer extension analysis and repeated rounds of 5 -RACE (rapid amplification of cdna ends) was found to consist of a pyrimidine-rich consensus initiator sequence, similar to the rat D 2 dopamine receptor gene [37, 68]. The promoter region of the rat D 3 dopamine receptor gene did not reveal any TATA and CCAAT boxes but unlike that of D 1 and D 2 dopamine receptor genes has only 52% GC content. These results demonstrate that the rat D 3 dopamine gene has similarities with the rat D 2 dopamine receptor gene as both are transcribed from a TATAless promoter that has an initiator element. Functional studies of rat D 3 promoter deletion mutants fused to the CAT reporter gene were carried out in a human medulloblastoma cell line (TE671 cells) [68]. These cells endogenously express the D 3 dopamine receptor [71]. Strongest transcriptional activity was determined within 36 nucleotides upstream of the transcriptional start site and a potent silencer identified between bases 37 and 86 which extends to 537 as transcriptional activity is noticeably and gradually reduced with the addition of sequences between 36 and 537 until complete inhibition. There was a small recovery of reporter gene activity with the addition of nucleotides 538 to 782, suggesting the presence of a potential activator. Additionally, another weaker silencer is located between nucleotides 783 and 1,046. These data suggest that the rat D 3 dopamine receptor gene is under intense negative regulation and similar to that observed with the rat D 2 dopamine receptor gene. Interestingly, none of the D 3 deletion mutant constructs showed any transcriptional activity in COS-7 (African green monkey kidney) or C6 rat glioma cells, which are not known to express the D 3 dopamine receptor gene endogenously. However, the shortest construct having 36 nucleotides from the transcriptional start site also showed significant transcriptional activity in OK (opossum kidney) and HepG2 (human hepatoblastoma) cells even though the evidence suggests that these cells do not express the D 3 dopamine receptor mrna. However, unlike the potent silencing effect of the longer upstream regulatory regions of the D 3 gene in TE671 cells, these fragments showed only weak inhibition in OK cells or strong activation in HepG2 cells. Thus although the core promoter of the rat D 3 dopamine receptor gene is active in these three different cell types, its regulation by the upstream elements varies in D 3 - and non-d 3 -expressing cells. The presence of specific transcription factors in the different cell lines could help explain the complex differential

11 2 Gene Structures of the Dopamine Receptors 33 regulation of the rat D 3 dopamine receptor gene. Interestingly, the transcription factor dopamine receptor regulating factor (DRRF) was found to activate the regulatory region of the rat D 3 dopamine receptor gene in TE671 cells [48]. Subsequently, a 9 kb genomic fragment of the human D 3 dopamine receptor gene was isolated upstream from the translational start site [72]. Studies using 5 - RACE identified three additional exons, and transcriptional activity was found in two putative 500 bp 5 -regions derived from brain tissue and lymphoblast cells following transfection in human cell lines. However, interpretations of these findings were ambiguous as the transcriptional start site was not accurately determined and no series of deletion constructs tested for transcriptional activity. The main focus of this report appeared to be identification of single nucleotide polymorphisms in the 5 -region of the gene, but no association was found with schizophrenia D 4 Dopamine Receptor Genes Gene Structure and Organization The human D 4 dopamine receptor was cloned and characterized after screening various cell lines for other D 2 -like dopamine receptors [73]. The cloning strategy employed in the discovery of this receptor utilized the D 2 dopamine receptor sequence that encoded for the sixth and seventh putative transmembrane regions [22]. This fragment of the rat D 2 dopamine receptor served as a probe for screening genomic and cdna libraries under low- and high-stringency conditions. The genomic intron exon organization of the human D 4 dopamine receptor gene indicated the presence of five coding exons for a protein of 387 amino acids. Hydrophobicity analysis of the protein sequence indicated seven putative transmembrane regions, which suggested that it belonged to the family of G protein-coupled receptors. The pharmacological characteristics of the D 4 dopamine receptor resembled that of the D 2 and D 3 dopamine receptors. However, the atypical neuroleptic drug clozapine had higher affinity for D 4 than for D 2 and D 3 dopamine receptors. The rat analogue of the human D 4 dopamine receptor gene was cloned, also revealing high affinity for clozapine [74]. This rat gene was found to have only four coding exons which encoded a protein of 368 amino acids, suggesting an additional splice site in the human D 4 dopamine receptor gene. However, despite the differences in gene structure the rat gene shares a high homology of 73% and 77% with the human D 4 gene at the amino acid and nucleic acid level, respectively. Interestingly, the amino acid homology of the gene between the two species increased to 89 96% when only the transmembrane regions were considered. The majority of the differences observed amongst the rat and human D 4 dopamine receptor genes occurred within the third cytoplasmic loop, which have only 50% amino acid identity. In the human gene, this region contains an unusual splice site within intron 3 (a donor/acceptor site of TC/CT is present instead of the GT/AG). Additionally, the rat D 4 dopamine receptor mrna was detected in the cardiovascular system in addition to the brain, suggesting that this receptor is an important

12 34 U.M. D Souza dopamine receptor in the peripheral nervous system [74]. Similar to the rat D 4 gene, the murine D 4 dopamine receptor gene was found to have four coding exons that span over 30 kb [75]. The gene encodes a 387 amino acid protein displaying 80% and 95% homology with the human and rat D 4 dopamine receptors, respectively, at the amino acid level. Likewise, at the nucleotide level the mouse D 4 dopamine receptor gene revealed 79% and 93% homology with the human and rat D 4 dopamine receptor genes, respectively. The most conserved regions were seen within the transmembrane domains that are thought to form the ligand binding site. Three polymorphic forms of the D 4 dopamine receptor in humans were further discovered [76]. These were the three most common variants, having 2-, 4- or 7-fold imperfect repeats of a 48 bp sequence in the putative third cytoplasmic loop of the receptor, located in exon 3 of the gene. This was the first example of polymorphic variation observed in catecholamine receptors. Although the different forms of the receptor showed slightly different pharmacological profiles with drugs spiperone and clozapine, they all coupled to G proteins [76]. Similarly, the same group later found that the polymorphic repeat sequences conferred only small differences in pharmacological binding properties [77, 78] and also in functional properties to inhibit cyclic adenosine monophosphate [79]. Therefore, it was concluded from the evidence that there was no direct relationship between length of the polymorphism and changes in these activities. The D 4 dopamine receptor variants were also capable of coupling to several G protein (G i α ) subtypes, but no evidence of any quantitative difference in G protein coupling related to repeat length was observed [80]. However, transcriptional differences were observed when the repeat variants were cloned downstream from the luciferase gene in expression vectors and tested in a somatomammotrophic (GH4C1) cell line [81]. Constructs having 7 repeat sequences significantly suppressed expression of the reporter gene compared to those consisting of the 2 and 4 repeats, which was suggested to be via mechanisms involving RNA stability or translational efficiency. More recently, dopamine was found to be more potent at D 4 receptors having 2 and 7 repeats than those with 4 repeats, suggesting that the actions of dopamine and therapeutic drugs on D 4 dopamine receptors may vary amongst individuals depending on the variants they have [82]. At least 19 different repeat unit sequences, used in 25 different haplotypes that code for 18 different unique receptor variants, were identified in the human D 4 dopamine receptor gene [83]. More recently, an extra 35 different alleles have been detected in the population having single nucleotide polymorphisms [84]. The 7- repeat allele was initially reported to be associated with the personality of novelty seeking [85, 86] and then more lately with attention deficit hyperactivity disorder (ADHD) [87 90] Promoter Structure and Transcriptional Regulation The 5 -flanking region of the human D 4 dopamine receptor gene was isolated and sequenced, revealing a transcription initiation region located between 501 and 436 bp relative to the first nucleotide of the translational codon [91]. A CpG island

13 2 Gene Structures of the Dopamine Receptors 35 spanned the region from 900 to +500 bp (which is over 50% GC rich) but no TATA or CCAAT boxes were present in the 5 -flanking region. However, the region was found to have consensus binding sites for transcription factors including Sp1. These properties are similar in the 5 -regulatory sequences of the D 1, D 2, D 3 and D 5 dopamine receptor genes. Functional analysis of deletion constructs fused to the CAT reporter gene and transiently transfected into IMR32 (neuroblastoma) and Y-79 (retinoblastoma) cells demonstrated promoter activity in the region around 591 and 123 bp and the presence of a negative modulator between 770 and 679 bp. However, no transcriptional activity was observed in human epithelial (HeLa) cells, suggesting cell-specific regulation of the human D 4 dopamine receptor gene. The 5 -flanking region of the human D 4 dopamine receptor contains several polymorphisms [92]. One of these is a 521 C/T polymorphism that showed weak association to schizophrenia. Functional studies demonstrated that this promoter polymorphism had reduced transcriptional activity compared to the C allele in human neuroblastoma cells (Y-79) [93]. However, more lately the 521 C/T polymorphism showed no significant differences in transcriptional activity in three human neuroblastoma (SK-N-F1, IMR32 and Y-79) cell lines [94]. The discrepancy in results between the two studies was suggested to be due to the use of different reporter vectors and variation in the length of the cloned fragment that harboured the polymorphism. Interestingly, the transcriptional regulation of the human D 4 dopamine receptor gene was also further analysed in SK-N-F1, Y-79, IMR32 and HeLa cells [94]. The highest transcriptional activity was observed between nucleotides 668 and 389 from the translational start site and a putative silencer region was located from 1,571 to 800 bp. These results together with the previous findings on the regulatory region of the D 4 dopamine receptor gene [91] suggest that the gene may possess two negative modulators, one of which was not functional in the cell systems utilized in the study by [94]. A tandem duplication of 120 bp located 1.2 kb upstream from the initiation codon and approximately 850 bp upstream from the transcription start site was identified in the human D 4 dopamine receptor gene [95]. This polymorphism has been found to be associated with ADHD [96 98]. Transient transfection in human neuroblastoma cells and other human cell lines with coupled luciferase reporter gene assays demonstrated that the duplication had lower transcriptional activity in human neuroblastoma cells (SK-N-MC, SH-SY5Y), human embryonic kidney cells (HEK293) and HeLa cells compared to the non-duplicated form [99]. These data also suggested that the D 4 dopamine receptor gene may have an alternative promoter in an intron region, as the tandem duplication revealed promoter activity. This is yet to be confirmed, but this speculation is in agreement with a previous report that suggested that the promoter region characterized by [91] could be in an intron region due to the discrepancy in size of mrna from previous studies [100]. They also suggested that a large intron could separate the coding exons of the D 4 dopamine receptors from potential untranslated exons similar to that observed for the other D 2 -like receptors [100], and the interpretation of findings from the functional studies of the tandem duplication were discussed in light of these considerations [99].

14 36 U.M. D Souza A recent study has verified these functional data, also showing that the duplicated allele but this case in constructs comprising the major part of the 5 -regulatory region showed lower transcriptional activity in human cell lines Y-79, SK-N-F1 and HeLa compared to the non-duplicated form [101]. They also identified a 4-repeat allele of this polymorphism which showed dose-dependent functional effects with the lowest transcriptional activity in the cell lines tested. Capillary electrophoretic mobility shift assays were used to compare the binding capacity of the transcription factor Sp1 to the polymorphic 120 bp sequence in the human D 4 dopamine receptor gene [102]. The data suggest enhanced binding capacity of the transcription factor to the duplicated form using HeLa nuclear extracts. However, these data have not been verified independently using other cell culture systems and/or other techniques such as standard electrophoretic mobility shift assays (EMSA). 2.3 D 1 -Like Dopamine Receptor Genes Gene Structure and Organization of D 1 -Like Dopamine Receptors The human and rat D 1 dopamine receptors were cloned by several groups [ ]. This was achieved using a cloning strategy based on the nucleotide sequence of the D 2 dopamine receptor gene [22]. The cloned D 1 dopamine receptor encodes a protein of 446 amino acids and the gene consists of one coding exon and thus was found to be intronless. Hydrophobicity analysis of the amino acid sequence of the receptor indicated that it belonged to the family of G protein-coupled receptors, as it consists of the characteristic seven stretches of hydrophobic amino acid residues. The structural features of this receptor include a short third cytoplasmic loop and a long carboxyl-terminal tail, in contrast to the D 2 dopamine receptor with a long third cytoplasmic loop and short carboxyl-terminal tail. Furthermore, the cloned D 1 dopamine receptor when transfected into a cell line was able to stimulate adenylyl cyclase in response to dopamine. This clearly indicated that cellular physiological responses mediated at the D 1 dopamine receptor were different from those mediated at the D 2 dopamine receptor. A second member of the D 1 dopamine receptor subfamily was then cloned and referred to as the D 5 dopamine receptor as it had affinity for dopamine and lower expression in the brain compared to the D 1 dopamine receptor [107]. After the cloning of the D 5 dopamine receptor, the cloning of the rat D 1 dopamine receptor subtype was reported [108]. They referred to this receptor as the D 1B dopamine receptor, and the previously cloned D 1 dopamine receptors were termed D 1A dopamine receptor [ ]. The cloned rat D 1B dopamine receptor encoded a protein of 475 amino acids and belonged to the family of proteins that couple to G proteins. The distribution of the receptor was found to be similar to that of the D 5 dopamine receptor and it was suggested that the D 1B dopamine receptor is the rat ortholog of the D 5 dopamine receptor, and therefore the terms D 1B and D 5

15 2 Gene Structures of the Dopamine Receptors 37 were used interchangeably to describe the same receptor [109]. The current standard nomenclature for dopamine receptors refers to these subtypes as D 1 and D 5 ( Interesting developments arose when three genes related to the D 1 dopamine receptor were identified in the human genome [ ]. The authors demonstrated that one of the genes lacked introns and was found to function as human D 5 dopamine receptor. However, the other two genes were pseudogenes. The transcript of one of these pseudogenes is expressed in several brain regions and produces a protein of 154 amino acids [113]. The human D 1 dopamine receptor is located on chromosome 5 at q35.1 [114] and the functional human D 5 dopamine receptor gene is localized to chromosome 4p16.1 [115]. The first and second pseudogenes of the D 5 dopamine receptor genes demonstrated chromosome localization to 2p11.1-p11.2 and 1q21.1, respectively [115] Promoter Region of the D 1 Dopamine Receptor Gene The 5 -flanking region of the human D 1 dopamine receptor gene was characterized by sequencing a 2.3 kb genomic fragment from 2,571 to 236 bp relative to the adenosine of the first methionine codon [69]. S1 nuclease mapping and reverse transcription PCR revealed the presence of a small intron of 116 bp ( 599 to 484) and an exon of about 440 bases in the 5 -non-coding region upstream from the coding exon in the human D 1 dopamine receptor gene which was previously thought to be intronless [ ]. The rat D 1 dopamine receptor gene was also found to have a small intron in its 5 -untranslated region [70] and additional information on its 5 -flanking region has been described at the end of the section. The human D 1 dopamine receptor gene has multiple transcription initiation sites located between 1,061 and 1,040. The promoter region lacks a TATA box and a CCAAT box, is rich in G+C content (up to 80% in some regions) and has several putative binding sites for the general transcription factor Sp1 [69]. Thus the promoter region of D 1 dopamine receptor was similar to that in the D 2 gene having similar features. However, it also has consensus sequences for a putative camp response element and binding sites for the transcription factors AP1 and AP2. Transient expression assays suggested the presence of a positive modulator between nucleotides 1,340 and 1,102 and a negative modulator between 1,730 and 1,341 in the murine neuroblastoma cell line (NS20Y), with no or very low transcriptional activity in NB41A3, C6 and HepG2 cells which do not express the D 1 dopamine receptor gene, suggesting regulation in a tissue-specific manner. It was later demonstrated that the transcriptional activity of the intron within the human D 1 dopamine receptor gene is higher than the upstream promoter by 12-fold in SK-N-MC cells and by 5.5-fold in NS20Y cells in an orientation-dependent manner [116]. Studies in SK-N-MC cells additionally revealed that longer D 1 mrnas including exon 1 are degraded 1.8 times faster than the shorter D 1 transcripts. Thus all this evidence indicates that the human D 1 dopamine receptor gene is transcribed in neural cells from a second strong promoter that is located in the intron and generates shorter transcripts

16 38 U.M. D Souza deficient in exon 1. However, only this short D 1 dopamine receptor transcript was present in human and rat kidneys [117]. These data demonstrate that the upstream promoter is active only in neural cells, whereas the intron promoter is active in both neuronal and renal cells. However, the activator region (nucleotides 1,154 to 1,136) that enhances transcriptional activity of the upstream promoter in SK-N- MC and NS20Y could not activate this promoter in OK cells [117]. Furthermore, gel shift assays using nuclear extracts from either OK cells or rat kidney tissue showed no protein binding to the activator region. The results suggest that differential expression of long and short D 1 dopamine receptor transcripts is due to tissue-specific expression of the activator protein binding to the activator region promoting transcription from the upstream promoter. It was suggested that absence of this protein would result in a non-functional D 1 upstream promoter in the kidney. The activator region of the human D 1 dopamine receptor gene was further analysed and found to localize to two regions ( 1,154 to 1,137 and 1,197 to 1,154) which increased promoter activity [118]. In addition, both the 1,197 to 1,154 region and the core promoter located downstream to 1,102 resulted in cell-specific D 1 dopamine receptor gene expression. Additionally, DNase I footprinting and gel shift assays revealed DNA protein interactions mainly in the region between 1,197 and 1,116. Furthermore, functional significance of nuclear factors interacting with the activator regions of the human D 1 dopamine receptor gene was determined in the neuroblastoma cell line SK-N-MC using competitive cotransfections with different fragments of this region. Novel transcription factors interacted with the D 1 -Act-1 ( 1,197 to 1,152) sequence even though it does not have consensus binding sites for known transcription factors. DNA binding proteins interact with D 1 -Act-2 ( 1,154 to 1,116) sequence as a complex that includes Sp1 or Sp1-like protein as well as a novel factor. However, even though recombinant AP2 binds to some of its consensus sequences in the D 1 dopamine receptor gene, it was suggested that it is unlikely that AP2 has a significant role in positively modulating the basal expression of the gene in NS20Y cells as the AP2 consensus sequences in the D 1 dopamine activator could not bind to nuclear extracts of these cells [118]. Supplementary transcriptional regulation studies of the human D 1 dopamine receptor gene by camp revealed location of two camp responsive regions in exon 1, both of which interacted with nuclear proteins in D 1 -expressing cells SK-N-MC [119]. The segment of D 1 gene between these two regions strongly interacted with nuclear proteins following forskolin/ibmx which directly increases camp levels. A different study demonstrated that 6.4 kb upstream region on the human D 1 dopamine receptor gene is sufficient to confer tissue-specific expression in specific D 1 -expressing brain regions of transgenic mice in vivo and in neuroblastoma cells in vitro [120]. The regulatory regions of the rat and mouse D 1 dopamine receptor genes have additionally been studied. The promoter region of the rat D 1 dopamine receptor gene was initially characterized locating the transcription start site to 864 bp upstream from the translational start site [70]. Like the human gene, the 5 -flanking region of the rat gene does not have any TATA or CCAAT boxes but is G + C rich, with binding sites for the transcription factors Sp1, Ap1 and Ap2 and with potential