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Molecular biology of dopamine receptors David R. Sibley ar;ti Frederick J. Monsma, Jr The application of modern molecular biological methods has had an increasing and dramatic impact upon the discipline of molecular neuropharmacology. This is particularly true for the study of neurotransmitfer receptors, where the use-of recombinant DNA techniques has resulted in the cloning of multiple and sometimes unexpected receptor subtypes for a given neurotransmifter and, in some cases, the cloning of receptors for which no neurotransmitter is known. Within fhe past couple of years, it has become readily apparent that dopamine receptors will be no exception to this trend. Five different dopamine receptors have now been cloned and identified using molecular biological techniques, while only a few years ago only two receptor subtypes were thought to exist. David Sibley and Frederick Monsma review the molecular characteristics of the recently cloned dopamine receptors and discuss prospects for the cloning and identification of additional subtypes in this receptor family. Although proposals for multiple types of dopamine receptors have waxed and waned over the past decade, until recently only two subvges had been definitively shown to exist. These were the Dr receptors, which activate the enzyme adenylyl cyclase and inintracellular levels of crease CAMP, and the Da receptors, which exert an inhibitory influence on this enzymel. D2 receptors may also be linked to additional second messenger systems including inhibition of phosphatidylinositol turnover, activation of K+ channels and inhibition of Ca*+ channel activiv. Both of these receptor subtypes belong to a large superfamily of neurotransmitter and hormone receptors that are coupled to their specific effector functions via guanine nucleotide regulatory proteins (G proteins)3. A recent TiPS review4, however, has summarized the inadequacy of the D1/D2 receptor classification scheme (see Box) and recent isolation of cDNAs and/ or genes encoding novel receptor subtypes has confirmed that the dopamine receptor family is more D. R. Sibley is Chief of the Molecular PharmacologyUnit, and F. 1. Monsma, jr is a Senior Staff Fellow in the Exuerimental Therapeutics‘branch, National I&We of Neurological Disorders and Stroke, Building 20, RoomK-108, Bethesda,MD 20892, USA.

diverse than previously imagined. Five pharmacologically distinct dopamine receptors have now been defined through molecular cloning techniques, and identification of additional family members is a likely event. In the light of these recent developments, this article summarizes the progress that has been made in understanding the molecular biology of dopamine receptors. D2 receptor subfamily The first dopamine receptor to be cloned, and consequently the most studied to date, was the Dz receptor. Exploiting the known homologies among the G proteinlinked receptor family, Bunzow et al. used a B-adrenoceptor probe to isolate clones encoding putatively novel receptors from a rat genomic librarys. One clone containing a partial genomic fragment with significant homology to the B-adrenoceptor was used to isolate a corresponding fulllength cDNA from a rat brain library. The cDNA encoded a protein of 415 amino acids which, when expressed in mouse fibroblast cells, exhibited appropriate radioligand binding activity with the pharmacological characteristics expected for a 4 receptor. The human homolog of the rat D2 receptor was subsequently cloned and shown to exhibit a protein

sequence that is 96% identical to the rat receptor, with one amino acid deletion6*7. Figure 1 shows a schematic diagram of the Dz receptor as it is believed to be organized in the plasma membrane. Hydropathy analysis of this protein predicts the presence of seven transmembrane (TM) domains where the N-terminus is localized to the extracellular surface and the C-terminus projects into the cytosol. This overall membrane topography has been suggested for all of the G protein-linked receptors that have been cloned. It should be emphasized, however, that biochemical evidence supporting this model has been generated for only two members of this family, namely rhodopsin and the Bz-adrenoceptora. Several other structural features of the Dz receptor are particularly noteworthy. First, the N-terminus lacks an apparent signal sequence and contains consensus sequences for three potential N-linked glycosylation sites. This is in keeping with previous biochemical data indicating that the 4 receptor is, in fact, a glycoprotein. Secondly, the predicted size of the C-terminus is rather small and it possesses a Cys residue that is conserved among most G protein-linked receptors and may serve as a site for palmitoylation3. Thirdly, the cytoplasmic loop between TM regions 5 and 6 is quite large and contains one potential site for phosphorylation by the CAMP-dependent protein kinase. This feature of having a large third cytoplasmic loop and short C-terminus is a characteristic of most, but not all, receptors that inhibit adenylyl cyclase activity3. The regional localization Of mBNA for this receptor has been determined, initially by northern blot analysis and, more recently, by in situ hybridization histochemistrys-rs. In general, the distribution of D2 receptor mENA correlates well with previous information regarding the presence of receptor protein in various tissues and brain regions. The areas of highest expression in the brain correspond to major dopaminergic projection areas Such as the caudate putamen, nucleus accumbens and olfactory tuber&. Receptor mRNA is also found in dopaminergic cell bodies within

Q 1992. ELsevlerSciencePublishers Ltd (UK)

TiPS - February 1992 IVol. 131

62 the substantia nigra pars compacta and ventral tegmental areas, indicating a prqTklptk, aS Wd as a postsynaptic, role for the 4 reCeptor. CeRuku localization of the D2 receptor mRNA has also been investigated in the striatum, where about 50% ci the medium-sized cells express receptor ~IRNA”~*‘~. _ . pr&mmry evidence indicates that some, if not most, of these are enkephalinergic neurons14*15, Da receptor mRNA has also been observed in large-diameter cells in the striatum, the majority of which appear to be cholinergic intemeurons16. Recently, investigations using antibodies against the Da receptor protein have provided a similar cellular localization pattern in the striatum17 as that determined by mRNA analysis. Expression of the cloned 4 receptor in various mammalian cell lines has resulted in some interesting observations. In the original publication, Bunzow et al. could not obtain evidence for receptor-G protein interactions in stably transfected LtIC- fibroblasts5. %G was sub~qu~~y determined to be dependent on assay conditions as a later report demonstrated guanine nucleotide-sensitive agonist binding to the D2 receptor in these cells as well as pharmacologically specific - .I ;$$~-;~;~$JY,c-Y~~ a;; receptor in LtK- ceRs stimulates gzfphoinositide [email protected] and mobilization , although these events have not previously been seen in tissues that endogenously express this receptor2. This may be related to the relatively high, non-physiological level of Dz receptor expression in the LtK- cells (105 copies per cell). When expressed in the GH& pituitary cell line, the D2 receptor inhibits adenylyl cyclase activity and prolactin release in addition to reducing CaZf levels and hyperpolarizing the ceIlszo. All of these events are known to occur in response to D2 receptor stimuWon in normal lactotroph cehs2. fnterestingly, when expressed in Chinese hamster ovary (Cl-LO) cells, the Dz receptor also potentiates ATP-mediated arachidonic acid releaseal, a property now known for several receptors that are linked to their effector systems through Gi proteins22. This poten-

4=. EXTRACELLULAR

D2 Da= D21D4=

INTRACELLULAR

Propoead membrane topography of human & dopamine receptor and its reMionship to human D3 and D4 receptors. Membrane-spanning regions defined on basis of hydropathyanalysis.CHO. potantial N-linkedglycosytationsites.Heavyline indicates tha attematiwiy spliced exon of the 4 receptor. From Ref. 8; Selbie, L. A. eta1 (1989) DNA 8,683-66Q; Grandy,D. K. etal. (1989) Proc. ~adAc~. E&i. USA 88. 9762-9786; Monsma, F. J., Jr et at. (1989) Nature 342,926929; Giros,B. et al. (1989) Nature342,923-926; Eidne,K. A. et al. (198s) Nature 342,865; Chio,C. L. etal. (1990) Nature343,266-269: Rao, D. D. et a/. (1990) FEBS Lett.263, 18-22; O’Dowd.8. F. et al. (3990) FfBS Left. 262,8-12; Miller,J. C. et al. (1990) Biochem. Biophys. Res. in. lQ8, IO%1 12; M~tmayeu~, J-P. et at. (1991) FfBS iett. 278,23!&243.

Fig. 1.

was convincingly shown to occur independently from changes in intracellular cAMP~~*~. A recent study has also reported this observation for the Dz receptora3, however, in this case, elevation of CAMP (either artificially or by Dr receptor stimulation) was shown to augment the Dz response. This D1-mediated augmentation of the Dz response was hypothesized to be a possible mechanism for Dr/Dz receptor synergism. Additional work will thus be necessary to resolve these discrepant findings.

tiation

Two protein isoforms Shortly after the initial cloning of the Da receptor, it was determined that this receptor exists in two protein isoforms that differ in length by 29 amino acids and are derived from the same gene by alternative RNA splicing (Fig. 1). The location of this splice variation occurs within the third cytoplasmic loop of the receptor protein, approximately 30 residues from the fifth TM domain. Both Dz receptor isoforms are generated in human, rat, bovine and mouse tissues and both are present in all

tissues and regions where D2 receptors are expressed. Interestingly, the larger isofonn appears to be expressed predominantly in all regions, although the exact ratio of the two isoforms can vary si~~~ntl~~O‘ This might suggest that the two isoforms differ functionally in some fashion, but no pharmacological differences have been observed between them (see Fig. 1). This is not surprising as the intracellular loops of the G proteincoupled receptors do not appear to be involved in ligand binding3”‘. Conversely, mutagenesis studies with other catecholamine receptors have suggested that the third cytoplasmic loop is important for G protein coupling and effector regulatior?. Thus far, however, both of the D2 receptor isoforms have been shown to inhibit adenylyl cyclases~, activate K+ channels33 , potentiate arachidonic acid releaseZ1, and undergo agonist-induced desensitizati.m32 with approximately equal efficacy. By contrast, a recent report has claimed that the shorter D2 receptor isoform is more efficiently coupled to adenylyl cyclase inhibition in

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Dopamine receptor nomenclature Along with the unexpected heterogeneity of the dopamine receptor system has come the problem of receptor nomenclature. Before the advent of molecular cloning techniques, receptor subtypes were defined primarily by their pharmacological profiies in eliciting a physiological or biochemical response. In some cases, this information was coupled with what was known of the signal transduction pathway linked to the receptor subtype. For instance, D1 r&eptors were defineci as being linked to the stimulation of adenvlvl cvclase act&y, while Dz receptors were either unli&;?d o>they inhibited this enzyme. This system is important and useful and should be continued, but can be hampered when there is a limited number of drugs available for defining a distinct pharmacological profile. Thus, when a new receptor subtype is cloned with little or no apparent pharmacological differences from previously characterized receptors, its terminology is not straightforward. This is occurring quite frequently now, and various classification schemes, both hierarchical and non-hierarchical, have been developed to address this problem. An example of a non-hierarchical approach can be found in the muscarinic receptor family, for which there were two pharmacologically defined subtypes, the M1 and M2 receptors. Subsequent to the cloning of these receptors, three more muscarinic receptors were cloned and named simply in order of identification, MJ to Ms. This is similar to what has occurred during the cloning of additional dopamine receptor subtypes. [This has also been suggested by the IUPHAR receptor nomenclature committee - Ed.] It shoi’d be noted, however, that the muscarinic classification scheme is fortuitous in that all of the even-numbered receptors couple to adenylyl cyclase (inhibition), and all the odd-numbered receptors to phospholipase C (activation). Such a division is not the case with the dopamine receptors. An alternative solution, hierarchical in approach, uses primary sequence information of the receptor along with information on its pharmacology. The adrenocep tor family provides an example of this approach. This family of receptors is clearly divisible into three separate subfamilies, each ccmprised of three, receptor subtypes. Thus, the? are the QT~_CI(Y~A-c,and &$J

cells that were co-transfected with a reporter plasmid containing a CAMP-responsive elemenP. Since CAMP levels were not directly examined in these experiments, the authors’ conclusions could be premature, therefore additional experimentation will be required to determine whether functional differences exist between the D2 receptor isoforms. The D2 receptor gene is unique in that it represents the first example among the G proteinlinked receptors (many of which lack introns in the coding region) to generate alternative transcripts resulting in different functional protein isoforms. Chromosomal analysis has indicated that the D2

1

receptors. The members of each subfamily are structurally homologous to one another, particularly within the membrane-spanning regions where the sequence homologies are greater than 50% in every instance. Conversely, the TM homologies are always less than 50% when receptors are compared between subfamilies. Each subfamily also exhibits a similar pharmacology that can be defined using prototypic ligands. ‘Ihis is, perhaps, not surprising, as the TM regions are believed to comprise the ligand-binding domains andthus form a structural basis for the receptor pharmacology. The adrenoceptor nomenclature clearly reflects the hierarchical organization of this receptor family. We would like to propose that a similar hierarchical system of nomenclature be applied to the dopamine receptor family. The basis of Gs nomenclat& would be the well-defined D1 and DV subtwes for which prototypic receptors h&e been cloiid. Additional receptor subtypes that are identified and cloned should be compared both structurally and pharmacok+ally with the Dl and Dz receptors. If the pharmacology is similar and the TM sequence homology is greater than 50% compared with a previously cloned subtype, then it should be designated as a member of that subfamily using an *,s,= etc. nomenclature. Using this method, the D1 and Ds receptors would be designated Du and Dls, and the Da D:, and D4 recepton designated Dm, D2s, and D20 respectively. It might also be helpful if the D1 receptor subfamily is defined pharmacologically as exhibiting nanomolar affinity for the prototypic antagonist ligand XX23390 while the I& subfamily should exhibit picomolar to nanomolar affinity for the antagonist spiperone. If a novel dopamine receptor is cloned that does not meet these criteria, then it will constitute the prototypic member of a new subfamily of dopamine recetptors, with the designation 4,

D, etc.

A major advantage of this classification scheme would be to maintain consistency with all of the previous bio-

chemical, physiological, behavioral and clinical investigations of ‘D,’ and ‘4 receptors, which may have involved any or all of the members within these particular subfamilies.

receptor gene is found on human chromosome 11 (Ref. 35). Detailed investigation of this gene has revealed the presence of at least eight exons, one of which codes for the 29 amino acid insert sequencezPz6. The presence of a large intron(s) in the 5’ flanking region of the gene has thus far precluded its additional characterization, including the definition of its promoter site. D3 receptor The second receptor within the D2 subfamily to be identified and. cloned was the ‘DJ’ receptor. Using probes derived from the D2 receptor sequence, and rat libraries, Sokoloff et al. cloned a

novel dopamine receptor cDNA and gene encoding a protein of 446 amino acids in length36. The amino acid sequence as well as the proposed membrane topography of the D3 receptor is very similar to that of the D2 receptor (Fig. 1). Both receptors exhibit relatively large third cytoplasmic loops and short C-termini. Overall, the D3 receptor is 52% homologous with the D2 receptor; however this homology increases to about 75% if only the TM regions are considered. As with the D2 receptor, the D3 receptor contains consensus sequences for N-linked glycosylation, two of which are in the N-terminus and one is in the first extracellular loop. Other similar-

TiPS - February 2992 [Vol. 231

64

ities include a cAMP-dependent phosphorylation site in the third cytoplasmic loop and a conserved Cys residue at the C-terminus. The human gene and cDNAs for the Ds receptor have recently been characterized37. Surprisingly, the human receptor has 46 fewer amino acids in the third cytoplasmic loop, resulting in a protein of 400 residues in length. I!.xcluding this deletion, the human receptor is 88% homologous with the rat protein but this increases to 97% within the TM regions. Chromosomal analysis of the human gene has assigned its location to chromosome 3. As with the Dz receptor, the coding region in the gene for the Ds receptor is interrupted by multiple introns, four of which are in identical or similar locations in the two genes. Interestingly, a recent report has described the existence of two apparently nonfunctional RNA splice variants of the rat Da receptor gene%. One of these alternative transcripts is completely lacking e;con 2, which encodes the first extracellular loop and the third TM domain. This results in a shift of the open reading frame, introducing a downstream stop codon. The resulting putative protein would be 100 residues in length and possess only the first two TM regions. The second RNA splice variant arises through the use of an alternative internal acceptor site within the fourth exon. This results in a deletion of most of the second extracellular loop and about a third of the fifth membrane-spanning domain, giving rise to a 428 residue protein. These two splice variants appear to comprise about 40% and lo%, respectively, of the total D3 receptor mRNA in brain. Giros et al. attempted to express the less abundant receptor variant in CHO cells but did not observe any specific radioligand binding activitys8. A likely possibility is that neither of these Ds receptor splice variants undergoes appropriate protein folding and insertion into the plasma membrane. Regional analysis of D3 receptor mRNA in the brain has indicated that it is much less abundant and more narrowly distributed than that for the Dz recepto?. In general, the Ds receptor is expressed predominantly in limbic

brain areas including the olfactory accumbens, nucleus tubercle, islands of Calleja and hypothalamus. Some expression of mRNA is also observed in the caudate putamen and certain cerebral cortical regions. This pattern of localization has led to the hypothesis that the 4 receptor mediates dopaminergic control of cognitive and emotional functions and thus may be relevant to antipsychotic involving dopamine therapy antagonists. Importantly, the Ds receptor also appears to be expressed in dopaminergic neurons within the substantia nigra, indicating that it may serve an autoreceptor or presynaptic function as has been suggested for the D2 recepto?. pharmacological Preliminary characterization of the Da receptor is consistent with its role as an autoreceptor. Radioligand binding experiments in transfected CHO cells indicate that the Ds receptor pharmacology is similar to, yet distinct from, that of the Dz receptos6. Most notable is the fact that the only antagonists found to exhibit greater affinity for the Da receptor (by about fourfold) were the autoreceptor-selective agents AJ76 and UH232. Other antagonists examined were between twoand 30-fold more selective for Dz than for D3 receptors. Conversely, agonist ligands demonstrated either equal or greater affinity for the Da receptor. Dopamine was about X-fold Ds-selective while the most selective agonist (loofold) was quinpirole. A proposed characteristic of dopamine autoreceptors is a higher affinity for agonists compared with postsynaptic D2 receptors. Curiously, binding of agonist to the D3 receptor expressed in the CHO cells was found to be insensitive to guanine nucleotide36. In addition, Ds receptor activation was reported to have no effect on adenylyl cyclase activity. These observations suggest the lack of appropriate Da receptor-G protein coupling in the CHO cells. Additional experimentation using different cell types will thus be necessary to define the signal transduction pathway(s) associated with the Ds receptor although inhibition of adenylyl cyclase activity is a likely possibility given its structural similarity to the D2 receptor.

D4 receptor The ‘Da’ receptor represents the latest receptor in the Dz subfamily to be identified and cloned39. This was accomplished by low-stringency screening of a human neuroblastoma cell cDNA library using the rat D2 receptor cDNA as a probe. One of the clones represented a partial-length cDNA with a single open reading frame protein sequences encoding highly homologous to TM regions 5, 6 and 7 of the D2 receptor. This cDNA was subsequently used to isolate the corresponding gene from a human genomic library. Characterization of the gene revealed the presence of the cDNA sequence which was preceded by sequences potentially encoding the putative N-terminus through the fourth TM domain of the receptor. The coding region of the gene is interrupted by four introns in TM regions 1, 3 and 6, as well as the third cytoplasmic loop. These are in equivalent positions as those in the D2 and Ds receptors. Chromosomal analysis has indicated that this gene is located on chromosome 11, as is that for the D2 receptor. The D4 receptor amino acid sequence deduced from the genomic and cDNA sequences would comprise a protein of 387 residues in length with seven putative membrane-spanning domains. The proposed membrane topography is similar to that seen with the Dz and D3 receptors but, as with the human Ds receptor, the D4 receptor has a slightly smaller third cytoplasmic loop. The overall homology of the D4 receptor to the Dz and Ds receptors is 41% and 39%, respectively, but the homology increases to about 56% for both receptors within the membrane-spanning domains (Fig. 1). The D4 receptor contains one potential s,ite for N-linked glycosylation in the N-terminus and one consensus cAMP-dependent phosphorylation site in the third cytoplasmic loop. As with the D2 and D3 receptors, the C-terminal amino acid is a conserved Cys residue (Fig. 1). As attempts to obtain a fulllength D4 receptor cDNA either by additional !ibrary screening or by the polymerase chain reaction were unsuccessful, a hybrid genecDNA was constructed for expression in mammalian cells. This

TiPS - February 2992 I&l. 131 construct contained the 5’ region of the gene, including the first two exons and introns, but was spliced to the cDNA about midway through the third exon at a site corresponding to the end of the fifth TM region. When transfected into COS-7 cells, this construct displayed saturable and specific [3H]spiperone binding with an affinity similar to that seen for the Dz and D3 receptors3’. Detailed characterization of the binding activity revealed a pharmacological profile similar to those of the D2 or D3 receptors. In general, the D4 receptor displayed similar or lower affinities for doparnine receptor antagonists and agonists compared with the Dz receptor. Importantly, however, the atypical antipsychotic clozapine, and its congener clorotepine (octoclothepin), exhibited about tenfold higher affinity for the D4 receptor. The affinity constant of clozapine, in fact, is similar to the concentration of clozapine measured in plasma water during antipsychotic therapy. This would seem to suggest that clozapine might exert its antipsychotic activity primarily by blocking the D4 receptor. The interaction of dopamine with the D4 receptor was sensitive to guanine nucleotides, indicating effective receptor coupling to G proteins endogenous to the COS-7 cells3’. Functional activity of the D4 receptor, such as regulation of adenylyl cyclase activity, was not examined. The distribution .of D4 receptor mRNA in the brain was investigated by northern blot analysis using either the partial-length cDNA or a gene fragment containing the fifth exon as a probe3g. (It should be noted that without northern data using probes from the first two exons, which encode the putative N-terminus through the third TM domain but were not found in the cDNA, there is no evidence that this region of the gene is actually expressed.) As with the D3 receptor, the D4 receptor appears to be expressed at a lower level than the D2 receptor. The areas of highest D4 expression included the frontal cortex, midbrain, amygdala and medulla, with lower levels observed in the striatum and olfactory tubercle. This distribution profile may partly explain the lack of extrapyramidal side effects observed

INTRACELLULAR

Fig. 2. Proposed membrane topography of the human 0, dopamine receptor and its relationship to the human D5 receptor. Membrane-spanning domains are definadon the basis of hydropathy analysis. CHO. potential N-linked gljcosytatkm sites.

with clozapine treatment. Obviously, it will be important in future experiments to find other drugs with selective and high affinity for the D4 receptor as well as to examine for genetic linkage between the Da receptor gene and schizophrenia. Dr receptor subfamily In 1990, four laboratory groups the simultaneously reported cloning of cDNAs and/or genes encoding a Dr receptor linked to the activation of adenylyl cyclase activity from either rat or human tissues4m3. Three of the groups employed the polymerase chain reaction while one group used a probe derived from the Dz receptor for library screening. The translational start sites for these clones are somewhat ambiguous as there are two potential initiator methionines in the human sequence4W3 and three in the rat40. Preliminary evidence from expression of the rat cDNA, however, suggests that the first Met residue in ’ this sequence is not used for translational start (Sibley et al., unpublished). If it is thus assumed that translation begins at the first Met residue in the human and the second Met in the rat, then both receptors would be 446 residues in length and contain seven potential membrane-span-

ning domains. Overall, the human and rat receptors are 91% homologous but this increases to 96% in the TM domains. The gene for the human receptor has been localized to chromosome 5 (Ref. 43) and lacks introns, at least within the coding region. Figure 2 shows a diagram of the proposed membrane organization of the human D1 receptor. In contrast to the Dz receptor subfamily, the Dr receptor has a small third cytoplasmic loop and a long C-terminus. This seems to be a characteristic of receptors that are coupled to G, and activate adenylyl cyclase, such as the & adrenoceptor. There are two potential sites for N-linked glycosylation, one on the N-terminus and a second on the second extracellular loop. Previous experimentation had indicated that the Dr receptor is indeed a glycoprotein. In addition, there is one consensus site for cAMPdependent phosphorylation in the third cytoplasmic loop and there is a conserved Cys in the carboxyl tail. The C-terminus also contains numerous Ser and Thr residues that may serve as additional sites of regulatory phosphorylation. Expression of the cloned rat and human Dr receptors in various mammalian cells has confirmed the pharmacological identities of

TiPS - Februa y 1992 [Vol. 131

56 these receptor subtypes. Saturable and high-affinity binding of either [3H1SCH23390 or [1251]SCH23982, Dr receptor-selective antagonists with the appropriate pharmacological specificity, was demontransfected cell strated usin membranes 40.& Importantly, the Di receptors were also shown to mediate stimulation of adenylyl cyclase activity with a pharmacology identical to that seen in endogenous receptor-expressing tissue systems. The tissue distribution of mRNA for the Or receptor has been determined by Northern blot analysis and in situ hybridization hi~tochemistry~~~~‘. In general, the localization of D1 receptor mRNA correlates well with previous information on the regional distribution of Di receptor binding and/or dopamine-stimulated adenyIy1 cyclase activities. The areas of highest expression are the caudat+putamen, nucleus accumbens, and olfactory tubercIe. D1 receptor rnRNA is also observed in the cerebral cortex, limbic system, hypothalamus and thalamus. Within the caudateputamen, about 50% of the medium-sized neurons exhibit labeling*~~, although a small number of the large-sized neurons may also express a low level of Dr mRNA&. Preliminary evidence indicates that the majority of the medium-sized neurons that express DZreceptor mRNA belong to the striatonigral projection system and also express substance P14sM. Since there is a wealth of behavioral, physiological and bio&micaI evidence for ‘Dr’ and ‘Dz’ receptor interactions including both opposition and synergismu, it will be important to determine, at both the mRNA and protein levels, which neurons may coexpress the various members of the D1 and 4 receptor subfamilies. D5 receptor A second member of the D1 receptor subfamily, termed the ‘I&,’ receptor, has recently been isolated and cloned. Using a fragment of the human Dr receptor DNA to probe a human genomic library, Sunahara et al. isolated a clone encoding a putative 477 amino acid protein with hi h homology to the D1 receptor 99. [email protected] analysis suggests

the presence of seven TM regions with a membrane topography similar to that of the Di receptor. Overall, the level of homology is about 50% between the Dr and D5 receptors, but this homology increases to about 80% within the membrane-spanning regions (Fig. 2). Other similarities include consensus N-linked glycosylation sites in the N-terminus and second extracellular loops, a cAMP-dependent phosphorylation site in the third cytoplasmic loop, and a conserved Cys residue in the C-terminus. The coding region in this gene also lacks introns. Interestingly, the human genome also appears to contain two pseudogene sequences that are 95% to 982 homologous to the Ds receptor genomic sequence5’. Relative to the Ds sequence, both pseudogenes contain insertions and deletions that result in inframe stop codons, thus precluding the expression of a functional receptor. Expression of the D5 receptor in G&C1 cells indicates that it is linked to stimulation of adenylyl cyclase activity with a D1 receptorlike pharmacology49. More detailed pharmacological analysis in transfected COS-7 cells using radioligand binding methods showed that various agonist and antagonist ligands exhibit similar affinities for the D1 and D5 receptors, with the notable exception of dopamine, which is about tenfold more potent at D5 than at D1 receptors. This has led to the hypothesis that the D5 receptor may be important in maintaining dopaminergic tone and arousal. The affinity for dopamine in the COS-7 cells was not modulated by guanine nucleotides, suggesting the absence of appropriate G-protein coupling in these cells. The distribution of the D5 receptor mRNA in the brain was preliminarily characterized by northern blot and in situ hybridization analyses4’. Highest expression is seen in the hippocampus and hypothalamus, with lower amounts in the striatum and frontal cortex. In general, the level of expression of the D5 receptor is much lower than that for the Dr receptor. In situ hybridization analysis in rat brain also suggested that the Ds receptor is significantly expressed in the caudate-putamen, nucleus accum-

bens and olfactory tubercle, as is the D1 receptor49. Unfortunately, two of the in situ hybridization probes used in this study exhibit high (71% and 83%) homology to the rat D1 receptor suggesting that D1 receptor mRNA may have been additionally identified. This requires clarification. Recently, two groups have cloned what appears to be the rat homolog of the D5 receptor, which, in this case, has been termed Drs (Refs 51, 52). Both groups used the polymerase chain reaction approach, wfth either getzoyo; DNA’l or kidney mRNA initial amplification. The rat receptor is 475 amino acids in length and is 83% homologous overall, but 95% homologous in the TM regions, with the human Ds receptor. Expression of the rat D1s receptor exhibits a similar pharmacology to the D5 receptor, including a high affinity for dopamine. With the exception of the questionable in situ hybridization data reported for the D5 receptor, the regional distribution of the DrB receptor mRNA agrees quite well with that for the Ds with the areas of highest expression seen in the hippocampus and hypothalamus, and little or no mRNA in the striatum. In addition, the D1s receptor was found to be expressed in high levels in the mammillary and pretectal nuclei. The gene encoding the human homolog of this receptor (the Ds receptor) has been localized to chromosome 4 (Ref. 51). Sequence comparisons Recently, extensive mutagenesis studies on the Pa-adrenoceptor have mapped the ligandbinding domains to the TM regions of the receptor. As these regions are the most conserved among the dopamine receptors and may be responsible for generating the specific subtype pharmacology, their comparison would be interesting. Figure 3 shows an alignment of the approximated TM domains for each of the cloned dopamine receptors. As can be seen, there are 64 amino acids in these regions that are common to all of the dopamine receptors. The third TM domain has the highest concentration of these shared residues. Similarly, there are 66 amino acids that are shared by, and unique to, the Dr receptor

TiPS - February 1992 [Vol. 231

I

II

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67

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C-IDSNTSLNPIIYA-FN C -VSETTFDWVWFmS SLNPVIYA-FN CNIPPVLYSAFTWLGYVNSAVNPIIYTTFM CRVSPELYSATTWLGYVNSAVIYTTFN CsvPPRLvsAvTwLGYvNsAY~

Fig. 3. Alignment of amino a&Is approximating the seven (t-W) Tfb4regions and adjacent residues of the human dopamine receptor subtypes. TM regions were initially estimated by hydmpathy analysis. Red residues represent amino acids present in all five human dopamine receptors.Omen residues representamino acids that are unique to and shared by the 0, and D5receptors.Blueresiduesrepresentamino acids that are unique to and shared by the 4,&, and D4 receptors.

subfamily with the highest number of these residues occurring within the fifth and sixth membrane-spanning domains. The Dz receptor subfamily exhibits 28 shared and unique residues, the seventh TM domain containing the highest number of these. The

smallest number of subtypespecific residues are found in the first TM domain. In general, the number of subtype-specific residues increases from the first to the seventh TM domains srrggesting that these latter regions may be more important in defining the

ligand-binding pharmacology_ There are a number of suecific amino acids within the TM regions that are additionally noteworthy (Fig. 3). One is the conserved Asp residue located in the second TM region. This amino acid has been found in almost all G protein-linked receptors cloned, and shown to be critical for maintaining a functional conformation of the receptors3s1. Recently, Neve et al. have used site-directed mutagenesis to show that this residue is also important for the binding of substituted benzamide antagonists to the DZreceptors, as well as the regulation of these receptors by Na+ and H+ (Ref. 53). Another Asp residue in about the middle of the third TM region has been found to be conserved among all biogenic amine receptors. Mutagenesis studies with the gz-adrenoceptor have implicated this amino acid as the counterion in the binding of protonated amines to the receptors’. Other important residues that may comprise the ligand-binding site include a pair of Ser residues, separated by two additional amino acids, within TM domain 5. These have been postulated to form hydrogen bonds with the catechol hydroxyl groups during binding of the catecholamine to the fiz-adrenocepto9’. The dopamine receptors also possess several Cys residues in their extracellular loops (Figs 1 and 2) that are conserved among many G protein-linked receptors. Mutagenesis studies with rhodopsin and the gz-adrenoceptor have suggested these are important in stabilizing the receptor in a functional conformation. It will be important to determine if the analogous residues in the dopamine receptors perform similar types of functions.

How manymore dopamine receptors? Table I summarizes the proper-

ties of all the dopamine receptors

cloned to date. The identification of five separate genes encoding pharmacologically distinct receptors indicates a heterogeneity within this receptor system that was unexpected as of a few years ago. It is thus logical to ask if even more dopamine receptor subtypes might yet be identified. This seems likely, because the cloned

TPS - Februay 1992 iVol. 131 TABLE 1. F%operties of cloned dopamine receptor subtypes 4

Altemativenomenctature

Aminoacids Human Hat lntronsingene Hurnanct~rom~e

subtypes do not exhibit all the properties of some dopamine receptors that have previously been chamcterized. For instmce, evidence has been accumulating that there exist ‘Di-like’ dopamine receptors that are linked to activation of phospholipase C (PLC) and to Ca2+ mobilization. Dopamine, as well as some Di-selective agonists, have been shown to stimulate phosphatidylinositol (Pi) turnover in both brain slices and kidney membranes5455 and injection of striatal mRNA into Xenopus oocytes leads to a dopaminestimulated PI response56. This dopamine receptor(s) might be analogous to the cri-adrenoceptors that stimulate PLC activity. There is also evidence for additional ‘D&ike’ receptors. Using genetransfer methods, a receptor with D&ke pharmacology has been identified and expressed but not yet sequenceds7 Also, a D2related receptor has recently been characterized. in kidney inner medulla membranes5s. Finally, amino acid sequence information from a ‘I&’ receptor purified from the pituitary has suggested that this receptor may differ from any of the cloned subtypes4. It appears that there may be many dopamine receptor subtypes yet to come. 0

cl

DW

446 446

477 475

No

No

5

4

caudate-putamen nucleusaccumbens olfactory tubercle

mRNAdistributionin brain

q

It is reasonable to wonder why there are so many dopamine receptors. Although the answer may not be found immediately, multiple receptor subtypes are an emerging phenomenon in most receptor systems. Receptor heterogeneity provides an opportunity for developing subtypeselective agonists and antagonists for use in clinical therapeutics. Compounds interacting with the

9

D,A

hippocampus hypothalamus

4

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D4

D28

D2c

414/443 4151444

400 446

387

Yes

Yes

Yes

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11

3

11

J CAMP t K’channel j Ca*+ channel

J CAMP?

4 CAMP?

caudate-putamen olfactory tubercle nucleusaccumbens hypothalamus olfactory tubercle nucleus accumbens

dopamine system are already used extensively in the treatment of numerous psychiatric, neurok&c and endocrine disorders. However, as with many other drugs, their utility is limited by sideeffects, which may be due to multiple receptor interactions. The cloning, characterization and expression of individual dopamine receptor subtypes will assist in the development of more selective drugs for modulating dopamine receptor function in various disease states. References 1 Kebabian, J. W. and C&e, D. 8. (1979) Nature 277.93-96 2 VaIIar, L. and Meldolesi, J. (1989) Trends Phawnacol. Sci. 10,7&77 3 Dohlman, H. G., Thorner, J., Caron, M. G. and Lefkowitz, R. J. (1991) Annu. Rev. Biochem.60,653688 4 Andersen, P. H. et al. (1990) Trends Pharmacol. Sci. 11,231-236 5 Bunzow, J. R. et al. (1988) Nature 336, 783-787 6 Dal Toso, R. et al. (1989) EM&I 1. 8, 402.54034 7 Stormann. T. M., Gdula, D. C., Weiner, D. M. and Brann, M. R. (1990) Mol. Pha~acol. 37.1-6 8 Weiner, D. M. and Brann, M. R. (1989) FEBS Lett. 253,207-X3 9 Meador-Woodruff, J. H. et a[. (1989) Proc. Nat1 Acad. Sci. USA 86, 762.5-7628 10 Najlemhim, A., Barton, .A. J. L., Harrison, P. J., Heffeman, J. and Pearson, R. C. A. (1989) FEBS Lett. 255, 335-339 11 Mengod, G., Martinez-Mb, M. I., Vifarci, M. T. and Palacios. J. M. f19891 Froc. Nat1 Aced. Sci. USA &, 8S6GG 12 Mansour, A. et al. (1990) 1. Neurosd. 10, 2587-2600 13 Weiner, D. M., Levey, A. 1. and Brann, M. R. (1990) Proc. Nat1 Acad. Sci. USA 87,7058-7054 14 Gerfen, C. R. et al. (1990) Science 250, 1429-1432 15 Le Moine, C. et al. (1990) Proc. Nat1 Acad. Sci. USA 87,230-234 16 Le Moine, C., Tison, F. and Bloch, B. (1990) Neurusci. L&t. 117,24&252 17 McVittie, L. D., Ariano, M. A. and Sibley, D. R. (1991) Pmt. Nat1 Acad. Sci. USA 88,1441-1445

frontal cortex medulla midbrain

18 Neve, K. A., Henningsen, R. A., Bunzow, J. R. and CiveIIi, 0. (1989) Mol. Pharmacol. 36,446-451 19 Valtar. L. et al. (1990) J. Biol. Chem. 265, 1032&10326 . 20 Albert, P. R., Neve, K. A., Bunzow, J. R. and Civelli, 0. (1990) I. Biol. Chem. 265, 2098-2104 21 Kantennan. R. Y. et al. (1991) Mol. ~ha~acol. &I,366369 22 Felder, C. C., WiIIiams, H. L. and Axelrod. 1. (1991) Proc. Nat1 Acad. Ski. USA 88; 6477-6480 23 Piomehi, D. et al. (1991) Nature 353, 164-167 24 O’MaIley, K. L., Mack, K. J., Gandelman, K. Y. and Todd, R. D. (1990) Biochemisfty 29, 1367-1371 25 Gandelman, K-Y., Harmon, S., Todd, R. D. and O’MalIey, K. L. (1991) J. Nearochem. 56,102~1029 26 Mack, K. J., Todd, R. D. and O’MalIey, K. L. (1991) J. Neurochem. 57,795-801 27 MacLennan, A. J., Frantz, G. D., Weatherwax, R. C., Tillakaratne, N. J..K. and Tobin, A. J. (1990) Mol. Cell. Neurosci. 1,151-160 28 Snyder, L. A., Roberts, J. L. and SeaIfon, S. C. (1991) Neurosci. Lett. 122, 37-40 29 Neve, K. A., Neve, R. L., Fidel, S., Janowsky, A. and Higgins, G. A. (1991) Froc. Nati Acad. Sci. USA 88,28~-2~ 30 Le Maine, C. and Bloch, B. (1991) Mof. Brain Res. 10, 283-289 31 Strader, C., Sigal, I. S. and Dixon, R. A. F. (1989) FASEB 1.3, 1825-1832 32 Rinando, M. S., Monsma, F. J., Jr, Black, L. E., Mahan, L. C. and Sibley, D. R. (1990) Sot. Neurosci. Abstr. 16, 209 33 Binhorn, L. C., Falardeau, P., Caron, M. G. and Oxford, G. S. (1990) Sot. Neurosci. Absfr. 16, 382 34 Mon~ay~r, J-P. and Borrelli, E. (1991) Proc. Nat1 Acad. Sci. USA 88,313s3139 35 Grandy, D. K. et al. (1989) Am. 1. Hum. Genet. *45, 778-785 36 Sokoloff, I’., Giros, B., Martres, M-P., Bouthenet, M. L. and Schwartz, J. C. (1990) Nature 347,146-l% 37 Giros, B., Mar&es, M-F., Sokoloff, I?. and Schwartz, J-C. (1990) C. R. Acad. Sci. Ser. Ill 311,501-508 38 Giros, B., Martres, M-P., PiIon, C., Sokoloff, P. and Schwartz, J-C. (1991) Biochem. Biophys. Res. Commun. 176, 1584-1592 39 Van Tol, H. H. M. et al. (1991) Nature 350,6&l-614 40 Monsma, F. J., Jr, Mahan, L. C., McVittie, L. D., Gerfen, C. R. and Sibley, D. R. (1990) Proc. NatI Acad. Sci.

USA 87,67234727

TiPS - February 1992 [Vol. 131 41 Deany, A. et al. (1990) Nature347,72-76 42 Zhou, Q. Y. et al. (1990) Nature 347, 76-80 43 Sunahara, R. K. et al. (1990) Nature 347, 80-83 44 Weiner, D. M. et al. (1991) Pm. Nat1 Acad. Sci. USA 88,1859-1863 45 Fremeau, R. T. et al. 119911 Prof. Nat1 Acad. SC;. USA 88,377%3776 46 Le Moine, C., Normand, E. and Bloch, B. (1991) Pm. Natl Acad. Sci. USA 88, 4205-4209 47 Mengod, G. et al. (1991) Mol. Brain Res. 10,185-191 48 Clark, D. and White, F. J. (1987) Synapse 1,347X%88 49 Sunahara, R. K. et al. (1991) Nature 350,

69 614-619 50 Grandy, D. et al. (1991) Proc. Nat1 Acad. Sci. USA 88,9175-9179 51 Tiberi, M. et al. (1991) Proc. Natl Acad. Sci. USA 88,7491-7495 52 Monsma, F. J., Jr et al. (1991) Sot. Neurosci. Abstr. 17,85 53 Neve, K. A. et al. (1991) Mol. Pharmacol. 39,733-739 54 Undie, A. S. and Friedman, E. (1990) J. Pharmacol. Exp. Ther. 253,987-992 55 Felder, R. A., Felder, C. C., Eisner, G. M. and Jose, P. A. (1989) Am. J. Physiol. 275, F315-F327 56 Mahan, L. C., Burch, R. M., Monsma, F. J., Jr and Sibley, D. R. (19?n) Pror. Nat1 Acad. Sci. USA 87,219~2200

5HT and alcohol abuse Edward M. Sellers, Guy A. Higgins and Mark B. Sobell Recent experimental data, both in animals and the clinic, suggest that drugs selectively interacting with the 5-HT system may reduce alcohol intake. Although the precise mechanisms underlying these drug effects are unknown, it seems that there are at least two pharmacological strategies available, described in this review by Edward Sellers and colleagues. The first is enhancement of 5-HT neuronal activity using compounds that will release 5-HT, block 5-HT reuptake, or act as selective 5-HT receptor agonists. A second approach involves selective 5-HT3 receptor antagonists. lf the initial research findings with these drugs are confirmed and extended, they may present useful therapies for the treatment of alcohol abuse, especially if used in conjunction with psychosocial therapy. Substantial progress in the treatment of mental disorders over the past 40 years can be ascribed to the development of psychotropic medications. To date, most of these have been developed for their anxiolytic, sedativehypnotic, antidepressant or antipsychotic therapeutic potential. However, from a public health perspective, the social, health and economic consequences of alcohol abuse and dependence constitute a much greater and yet seemingly preventable problem. For example, it is estimated that in 1990 in the USA, the economic cost of alcohol abuse and dependence E. M. Selle.vs is Professor, Departments of Pharmacolo~u. Medicine and Psuchiattv. University of Toronto, and Director; Clinic2 Research and Treatment Institute, Addiction Research Foundation, Toronto, Onfario, Canada; G. A. Higgins is a Research Fellow, Clinical Research and Treatment Institute, Addiction Research Foundation, and Department of Pharmacology, University of Toronto; M. B. Sobell is Professor, Departments of Psychology and Behavioural Science, University of Toronto, and Associate Director, Clinical Research and Treatmenf Insfitute, Addiction Research Foundafion. “r-

@ 1992, Elsevier

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Publishen

Ltd(UK)

was $136.3 billionl. Little effort has been directed to develop medications for the treatment of this disorder. This is surprising since the aldehyde dehydrogenase inhibitor disulfiram (‘Antabuse’),

57 Todd, R. D., Khurana, T. S., Sajovic, p., Stone, K. R. and O’MaUev, K_ L. (1989) Proc. Nat1 Acad. SC;. USA‘ 86; 10134-10138 58 Huo, T., Ye, M. Q. and Healy, D. P. (1991: Proc. Nat1 Acad. Sci. USA 88, 3170-3174 AJ76z cis-(+)-(ls,2u)-5 methoxy-l-methyl2-(n-propylamino) tetrah scH23390: 7-chloro-2,3.4.5-tetrahvdro-3methyl-5-phenyl-lH-gb. enzazepi&-7-01 SCHZ3Y82: (+)-8-iodo-2,3,4,5-tetrahvb 3-methyl-5-phcnyl-lH-3-b enzazepi&-7-ol UHZ332:cis-( +) (Is, 2R)-5-methoxy-lmethyl-2-(di-n-propylamino) tetraiin

the most commonly prescribed medication, is without therapeutic efficacy (although it can play an adjunctive role as part of a broader treatment progran& Lack of drug development in the area of alcohol dependence may relate, at least in part, to attitudes and beliefs regarding the nature of alcoholism, which has been variously perceived as genetically determined, irreversible, uncontrollable and incurable. While this may be true ‘or certain cases, the mast conm_on pattern of alcohol abuse is for an individual to move into and out of periods of drinking problems separated by spells of abstinence or of limited drinking without Furthermore, an problems3”. implication of the belief that alcohol problems, once present, are irreversible, is that abstinence is the only appropriate treatment goal for anyone who has an

TABLE I. Targets and mechanisms by which drugs could reduce alcohol consumption Target

behaviour

Mood, motivation or cognition

Mechanism -Antagonize the reinfordIng effec%, e.g. ethanol-selective antagonist Substitute for the reinforcing effecis., s.g. medication with ethanol-like properties Provoke an unconditioned aversive or dysphoric reaction by pairing behaviour with shock or any other aversive conditioning stimulus Provide an alternative and dissimilar reinforcer, e.g. stimulant Pmvoke a conditioned aversive or dysphoric physiological reaction, e.g. apomorphine or possibly disulfiram Induce dvsohoric svmotoms that produce mild malaise (in other drugs this is . someimbs a sid&e&ct) Modify ethanol biodisposition. e.g. accelerate elimination from body Treatment of primary or secondary mental disorders associated with alcohol abuse/deoendence, such as major depression or chronic anxiety Suppress t&get symptoms, such as anxiety, that may pmmpt or sustain alcohol use or that may prevent reduction or cessation of use Facilitate the learning or retention of a new behaviour, e.g. coping skills Augment self-efficacy by providing cues that active medication is pti of treatment Threatened punishment, e.g. disulfiram Decrease the desire to drink (no examples known) Increase patient control over initiation and continuation . .. . of.drinking, which may be the product of several of the other approacnes wea

Accelerate or modify the conditioned cues associated with alcchol use

Molecular biology of dopamine receptors.

The application of modern molecular biological methods has had an increasing and dramatic impact upon the discipline of molecular neuropharmacology. T...
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