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3 Dopamine Receptors 1 Au5 a0005 Au2 s0005 p0005 p0010 p0015 s0010 p0020 Dopamine Receptors P Seeman, University of Toronto,Toronto, ON, Canada ã 2008 Elsevier Ltd. All rights reserved. Introduction The discovery of dopamine receptors is intertwined with the discovery and development of antipsychotic drugs. The research in this area started with the development of antihistamines after World War II, particularly with H. Laborit using such compounds to enhance surgical analgesia. In patients receiving these medications, Laborit noticed a euphoric quietude, and that the patients were calm and somnolent, with a relaxed and detached expression. Of this series of Rhône-Poulenc compounds, RP4560, now known as chlorpromazine, was the most potent. Chlorpromazine was tested by many French physicians for use in various medical illnesses. Although Sigwald and Bouttier were the first to use it as the sole medication for a psychotic patient, their work was not reported until 1953, after a 1952 report by Delay and colleagues that chlorpromazine alleviated hallucinations and stopped internal voices in eight patients. A significant aspect of the action of chlorpromazine was that it was effective within 3 days. This rapid improvement, especially during the first week of antipsychotic treatment, has been observed in many studies and summarized in reviews by S. Kapur and O. Agid. The clinically successful action of chlorpromazine stimulated the search to identify chlorpromazine s mode of action. The assumption then, as now, was that the discovery of such a mode of action would open the avenue to uncovering the biochemical cause of psychosis and possibly of schizophrenia. Early Days: Before Discovery of Dopamine Receptors In searching for the mechanism of chlorpromazine action in the 1960s and 1970s, many types of electrophysiological and biochemical experiments were done. Because high doses of chlorpromazine and other antipsychotics (or neuroleptics, as they were then called) also elicited parkinsonism as a side effect, the basic science quickly focused on the action of antipsychotics on dopamine pathways in the brain. The rationale for examining brain dopamine regions was based on the finding by H. Ehringer and O. Horniekiewicz that the parkinsonism of Parkinson s disease was associated with a massive loss of brain dopamine. It was felt, therefore, that the unwanted side effect of chlorpromazine-induced parkinsonism, as well as the antipsychotic action itself, might arise by antipsychotics interfering with dopamine neurotransmission. The working assumption was that if the brain targets for antipsychotics could be found, then perhaps it could be determined whether these sites were overactive or underactive in psychosis or schizophrenia. A variety of mechanisms were explored for the mode of action of chlorpromazine, including its action on mitochondrial enzymes, sodium potassium- ATPase, and related enzymes, and its membranestabilizing action, such as its strong potency to inhibit membrane action potentials and to stabilize cellular and subcellular membranes from releasing their contents. It also became clear in 1963 that all antipsychotics were surface active, readily explaining their hydrophobic affinity for membranes. Some of these non-receptor-related findings, such as the surface activities of the antipsychotics, showed an astonishingly excellent correlation with clinical antipsychotic potencies. Therapeutic Concentrations of Antipsychotics All of the early experiments in the 1960s revealed that the in vitro active concentrations of the antipsychotics were generally between 20 and 1000 nm. These concentrations, however, were found in 1971 to be far in excess of the nanomolar concentrations (e.g., 1 2 nm for haloperidol) that exist in the spinal fluid in patients being successfully treated with these medications. In vivo Experiments In parallel with the in vitro experiments, there were many in vivo experiments by F. Bloom, by G. Aghajanian, and by B. Bunney, showing that dopamine agonists can excite or inhibit neurons in the nigrostriatal dopamine pathway. Moreover, other workers (W. D. Heiss, J. Hoyer) showed that direct application of dopamine on neurons also stimulated or inhibited snail neurons, and that haloperidol or fluphenazine could block these actions (H. Struyker Boudier). These studies provided evidence for the existence of distinct dopamine receptors on neurons. Additional work in vivo showed that chlorpromazine and haloperidol increased the production of normetanephrine and methoxytyramine, metabolites of epinephrine and dopamine, respectively. To explain the increased production of these metabolites, Carlsson and Linqvist suggested that the most likely [mechanism] appears to be that chlorpromazine and p0025 s0015 p0030 s0020 p0035 p0040

4 2 Dopamine Receptors s0025 haloperidol block monoaminergic receptors in brain; as is well known, they block the effects of accumulated 5-hydroxytryptamine.... In other words, they proposed that antipsychotics might block all three types of receptors for noradrenaline, dopamine, and serotonin, but they did not identify which receptor was selectively blocked or how to identify or test any of these receptors directly in vitro. This study in 1963 by Carlsson and Lindqvist is often mistakenly cited as discovering the dopamine receptor and that antipsychotics are selectively acting on this receptor. However, N.-E. Andén, a student of A. Carlsson, had a different view, and proposed that chlorpromazine and haloperidol delay the elimination of the (metabolites).... Moreover, 7 years later Andén reported that antipsychotics increased the turnover of both dopamine and noradrenaline, but he could not show that the antipsychotics were selective in blocking dopamine; for example, chlorpromazine enhanced the turnover of noradrenaline and dopamine equally. Therefore, it remained for in vitro radioreceptor assays to detect the dopamine receptor directly and to demonstrate antipsychotic selectivity for the dopamine receptor. The Dopamine D1 Receptor p0045 With the advent of assays for adenylate cyclase in the 1960s, J. Kebabian found that dopamine stimulated adenylate cyclase in the superior cervical ganglion. This receptor was later named the dopamine D1 receptor, selectively labeled by [ 3 H]SCH23390, and subsequently cloned by three research groups in p0050 The dissociation constants at D1 for dopamine agonists and antagonists of medical therapeutic interest are given in Tables 1 and 2. There is no correlation between the antipsychotic clinical doses and the dissociation constants of the antipsychotic antagonists at D1, as illustrated in Figure 1. These data suggested that D1 was not the major or common target for antipsychotics, in addition to the fact that the antipsychotic molarities at D1 are between 10 and nm, far in excess of the therapeutic concentrations in the spinal fluid of treated patients. p0055 In addition to the lack of targeting D1 receptors by clinical doses of the common antipsychotics, D1- selective compounds have not been found to be effective as antipsychotics (Figure 2). Au3 s0030 p0060 Discovery of the Antipsychotic Dopamine Receptor, or the Dopamine D2 Receptor In 1974 and 1975, in order to detect and discover the dopamine receptors on which the antipsychotics presumably acted, it was essential to label a receptor with a ligand, such as radioactive haloperidol, having an affinity (or dissociation constant) of 1 nm, because this was the haloperidol therapeutic concentration found in the spinal fluid or plasma water of treated patients. For this to occur, the specific activity of [ 3 H]haloperidol would have to be at least 10 Ci mmol 1. Although the [ 3 H]haloperidol donated in 1971 by Janssen Pharmaceutica (J. Heykants, J. Brugmans) had a low specific activity of mci mmol 1, I.R.E. Belgique (M. Winand) custom synthesized [ 3 H]haloperidol at even lower specific activity (10.5 Ci mmol 1 ) for Seeman s laboratory by June Specific binding of this new [ 3 H]haloperidol to brain striatal tissue was readily detected in 1975, and the following concentrations of compounds were found to inhibit the binding of [ 3 H]haloperidol by 50%: 2 nm for haloperidol, 20 nm for chlorpromazine, 3 nm for (þ)butaclamol, and nm for ( ) butaclamol. The stereoselective action of butaclamol and the good correlation between the IC 50% values and the clinical doses, as shown in Figure 3, indicated that the antipsychotic receptor had finally been discovered. Equally important, of the endogenous compounds tested, dopamine was the most potent in inhibiting the binding of [ 3 H]haloperidol, indicating that the antipsychotic receptor was actually a dopamine receptor (see Table 1). Using sequences related to the b-adrenoceptor, the antipsychotic/dopamine receptor, now named the dopamine D2 receptor, was finally cloned in 1988; its amino acid sequence is shown in Figure 4. Using the cloned D2 receptor, values for the dissociation constants for agonists and antagonists can be determined (Table 1). It is now known that therapeutic levels of antipsychotic drugs occupy 60 80% of brain D2 receptors, as shown by L. Farde and colleagues. In fact, using the antagonist dissociation constants in Table 1, the concentrations necessary to occupy 75% of D2 receptors can be calculated and are essentially identical to the free molarities of the antipsychotics found in either the plasma water or the spinal fluid of treated patients, as shown in Figure 5. Because radiolabeled raclopride is less tightly bound to D2 receptors than radiolabeled spiperone, which in turn is less tightly bound to D2 receptors than radiolabeled raclopride, the dissociation constant for any antagonist at D2 receptors is lowest when using radiolabeled raclopride, but rises when using radiolabeled spiperone, or rises higher still when using radiolabeled nemonapride (see Table 1). This dependence of the dissociation constant on the ligand is also seen in positron emission tomography studies, where it has been found, for example, when monitoring patients with [ 11 C]raclopride, that therapeutic doses of clozapine occupy 50% of D2 p0065 p0070 p0075 Au4

5 t0005 Table 1 Agonist potencies at dopamine receptors (in 120 mm NaCl) Tissue [Ref.]: Rat striatum or human D1 [f] D1 D2 D3 D4 D5 Human D2Long clone [b] Human D3 clone [b] Human D4 clone Human D5 clone Radioligand: [ 3 H]SCH [ 3 H]spiperone [ 3 H]SCH K High K 50 K High K Low K High K 50 K High K 50 K 50 Neurotransmitters nm nm nm nm nm nm nm nm Dopamine [g] 2.4 D 1400 D 25 D 25 I[a] 28 [f] 148 [f] 228 [g] 1.4 R;1.3 A 729 R 3.9 I [a] 22 S Noradrenaline No high D n.i. n.i. n.i. n.i [f] [g] Adrenaline No high 1700 No high 805 D n.i. n.i. n.i. n.i. n.i. Serotonin No high 9700 [g] No high 4300 D n.i. n.i. n.i [f] 3000 [g] Dopamine agonists N-propyl-norapomorphine-R-(-) [g] 0.14 D 54 D rat 0.25 D 0.8 n.i. 4 f 1,136 [g] (þ)phno 39 n.i. 0.4 D 62 D 0.6 D n.i. n.i. 47 f n.i. ()Quinagolide.HCl, or CV No high D 1400 D 3.2 D n.i. n.i. n.i. n.i. Bromocriptine.base No high 672 [g] 0.8 D 20 D 1.3 D 7.4 I[a] n.i. 250 [e] 720 [e] Apomorphine-R-(-).HCl [e] 0.75 D 140 D 2.6 D 73 I[a] 4.1 f 5 [e] 13 [e] Pergolide mesylate [LY 127,809] No high 330 [e] 1.3 D 19 I[a] 0.9 D 2.3 I[a] n.i. 62 [e] 39 [e] ( )Quinpirole, or (-)-LY 171,555.HCl No high D 202 R[d] 0.45 R[d] 256R d;50 I[e] n.i. 31 [e] >1000 [g] Pramipexole monohydrate No high D 2600 D 9 D 11 I[e] n.i. 120 [e] >1000 [e] SKF [c] 380 [c] 150 S[c] 8800 Sc No high 5000 [a] n.i [f] 100 [g] (þ)-7-oh-dipropylaminotetralin 10 [c] 4700 [c] 1. 1 R[d] 42 R[d] 0.06 R[d] 0.2 R[d] n.i. 650 [a] n.i. [a], Sokoloff et al. (1992); [b], Seeman et al. (2005); [c], Seeman and Niznik (1988); [d], Malmberg and Mohell (1995); [e], Millan et al. (2002); [f] P. Seeman; [g], Sunahara et al. (1991); K50 (dissociation constant), C50% (conc. to inhibit 50% of binding)/(1 þ C*/K d ); C*, concentration of radioligand in competition with agonist; K d ¼ dissociation constant obtained by saturation with radioligand (Scatchard analysis); A [ 3 H]dopamine (K d ¼ 1.3 nm); D [ 3 H]domperidone (Ref. b or P. Seeman, unpublished; K d ¼ 0.43 nm); I [ 125 I]Iodosulpride (K d ¼ 0.6 nm); R [ 3 H]raclopride (Kd ¼ 1.9 nm); S [ 3 H]spiperone (Kd ¼ 65 pm); rat rat striatum; no high no high-affinity state recognized by competing compound; n.i. no information available. Dopamine Receptors 3

6 4 Dopamine Receptors t0010 Table 2 K values (dissociation constants) Human clone: M1 D1 D2 D2 D2 D3 D4 D4 nm nm nm nm nm nm nm nm [3H]ligand used: QNB Sch. Raclo. Spip. Raclo. Spip. p0080 [3H]Amisulpride Amoxapine Aripiprazole 1.8 Butaclamol-(þ) Chlorpromazine [3H] Chlorpromazine Clozapine [3H]Clozapine 51 2 Clozapine-iso Cyproheptadine 24 Droperidol Epidepride Flupentixol-cis Flupentixol-trans 151 Fluphenazine Haloperidol [3H]Haloperidol Iloperidone (HP873) Loxapine Loxapine-iso Melperone Molindone Norclozapine Olanzapine [3H]Olanzapine Perphenazine Pimozide Prochlorperazine Quetiapine [3H]Quetiapine 104 Raclopride [3H]Raclopride Remoxipride >10 mm Risperidone >10 mm Risperidone-9-OH 1.6 Sertindole [3H]Sertindole Spiperone [3H]Spiperone Sulpiride-S Trifluperazine Ziprasidone Blank boxes indicate not done. Sch Schering 23390; Raclo. Raclopride; Spip. spiperone. receptors but that there is much less occupancy of D2 receptors when using [ 11 C]methylspiperone or [ 18 F] fluorethylspiperone, both of which bind more tightly to D2 receptors than raclopride (see Table 2). The correlations in Figures 3 and 5 remain a cornerstone of the dopamine hypothesis of psychosis or schizophrenia, and the dopamine hypothesis is still the major contender for an explanatory theory of schizophrenia etiology. Two Classes of Dopamine Receptors The D1 site and the [ 3 H]haloperidol/dopamine receptor binding site were soon considered as distinct, because B. Roufogalis found that the sulpiride antipsychotic did not block dopamine-stimulated adenylate cyclase. Two general classes of dopamine receptors were recognized, therefore, coupled or uncoupled to adenylate cyclase. These two classes were named D1 and D2 by J. Kebabian and D. Calne. s0035 p0085

7 Dopamine Receptors D 1 Clebopride MolindoneSulpiride K(mol l 1 ) on 3 H-SCH23390 binding Spiperone Clozapine Haloperidol Chlorpromazine Thioridazine Fluphenazine Trifluperazine Flupenthixol Range and average clinical dose for controlling schizophrenia (mg d 1 ) f0005 Figure 1 There is no correlation between the clinical antipsychotic doses and the antipsychotic dissociation constants (or concentrations) that inhibit the binding of a D1 ligand ([ 3 H]SCH23390) at dopamine D1 receptors in homogenized striatal tissue. The high concentrations inhibiting the D1 receptor are far higher than those found clinically in the plasma water or spinal fluid. Adapted from Seeman P (1967). f0010 Figure 2 Amino acid sequence of the dopamine D1 receptor. The two hydroxyls of dopamine are presumed to be associated with the two serine residues, while the tertiary nitrogen atom is presumed to be associated with the aspartic acid residue (D in transmembrane 3). Drawing by P. Seeman.

8 6 Dopamine Receptors 10 7 IC 50 (mol l 1 ) Spiroperidol Haloperidol Droperidol Fluphenazine Pimozide Trifluperidol Benperidol Nature, 1976 Promazine Chlorpromazine Trazodone Clozapine Thioridazine Molindone Prochlorperazine Moperone Trifluperazine Thiothixene P. Seeman T. Lee M. Chau-Wong K. Wong Range and average clinical dose for controlling schizophrenia (mg d 1 ) f0015 Figure 3 The clinical antipsychotic doses correlate with the concentrations that inhibit by 50% the specific binding of [ 3 H]haloperidol in homogenized caudate nucleus tissue (calf). These concentrations are similar to those found in the plasma water or spinal fluid in patients treated with antipsychotic drugs. Adapted from Seeman P, Lee T, Chau-Wong M, et al. (1976) Antipsychotic drug doses and neuroleptic/ dopamine receptors. Nature (London) 261: f0020 Figure 4 Amino acid sequence of the dopamine D2 receptor. The variants or polymorphisms of D2 include an alanine instead of a valine at position 96 (1% of population), an insertion of a 29-amino-acid polypeptide (D2Long) at the position shown by the lower left arrow, a serine instead of a proline at position 310, and a cysteine instead of a serine at position 311. Dopamine is presumed to be associated with the two serines (S) in transmembrane 5 and the aspartic acid (D) in transmembrane 3 (see Figure 2). Drawing by P. Seeman.

9 Dopamine Receptors 7 Concentration needed to occupy 75% of D2, nm Line for identical values 75% occupancy Chlorpromazine Raclopride Thioridazine Haloperidol cis-flupentixol Perphenazine Clozapine Remoxipride S-Sulpiride Molindone Olanzapine Therapeutic free neuroleptic (nm) in spinal fluid or plasma water f0025 Figure 5 The therapeutic antipsychotic concentrations in the spinal fluid or in the plasma water in treated patients are essentially identical to the antipsychotic concentrations that occupy approximately 75% of the D2 receptors in vitro. The concentrations in the plasma water were obtained by correcting for the amount bound to the plasma proteins. The concentrations to occupy 75% of D2 were calculated as being three times higher than the dissociation constant at D2. Adapted from Seeman P (2002) Atypical antipsychotics: Mechanism of action. Canadian Journal of Psychiatry 47: D1 site (= dopamine-sensitive adyenylate cyclase) Dopamine: 3000 nm Spiperone: 2000 nm D2 receptor Dopamine: 5000 nm Spiperone: 0.3 nm D4 site Dopamine: 3 nm Haloperidol: 1 nm D3 site Dopamine: 3 nm Spiperone: 1500 nm f0030 Figure 6 Early version of dopamine receptors before clones of receptors became available. The D1 receptor (dopamine-stimulated adenylate cyclase) was stimulated by 3000 nm dopamine and inhibited by high concentrations of butyrophenones such as 2000 nm spiperone. The D2 receptor, or the [ 3 H]haloperidol binding site, was highly sensitive to spiperone, but required 5000 nm dopamine to inhibit adenylate cyclase. The D3 site was a site labeled by [ 3 H]dopamine and, therefore, very sensitive to dopamine at 3 nm, but required very high concentrations of antipsychotics to be inhibited. The D4 site was defined as being sensitive to both the agonists and the antipsychotic antagonists. While these definitions for the D3 and D4 sites are no longer used, the D1 and D2 properties are still valid for the cloned D1 and D2 receptors, with the additional point being that both D1 and D2 have high- and low-affinity states Drawing by P. Seeman. s0040 p0090 Nomenclature of Dopamine Receptors The data for the pattern of binding of [ 3 H]haloperidol identifying the antipsychotic/dopamine D2 receptor were very different from those for the pattern of [ 3 H] dopamine binding described by studies in the mid- 1970s. For example, the binding of [ 3 H]haloperidol was inhibited by 5000 nm dopamine, while that of [ 3 H]dopamine was inhibited by 3 nm dopamine, as summarized in Figure 6. For several years, this latter [ 3 H]dopamine binding site was termed the D3 site, a term which is not to be confused with the later discovery of the dopamine D3 receptor.

10 8 Dopamine Receptors s0045 Dopamine D2 Receptor Variants p0095 As noted in Figure 4, there are several variants of D2, the most important of which are the short form and the long form of D2, the latter having an additional 29 amino acids. There is also a D2Longer form where a dipeptide, valine glutamine, is inserted into the intracellular loop, as shown in Figure 7. p0100 There are at least three polymorphisms in D2 (Figure 4): alanine replaces valine at position 96 in about 0.8 1% of some populations, serine replaces proline at position 310 in 0.4% of people, and cysteine replaces serine at position 311 in approximately 3 4% of the population. The variants at 310 and 311 are markedly less effective in inhibiting the synthesis of cyclic AMP than is the more common form of D2. Silent polymorphisms have also been found in the DNA code for D2, but there is no change in the amino acid (leucine at 141, histidine at 313, and proline at 319). p0105 There are also polymorphisms in the noncoding regions of D2, including an A-to-G at position 241, a C insert at position 141, an A-to-G before transmembrane 1 (Taq1B polymorphism), an A-to-G in the intron within transmembrane 2 (Taq1D polymorphism), an A-to-C in the intron before transmembrane 4, a G-to-A in the intron before transmembrane 6, and an A-to-G situated 10 kb beyond transmembrane 7 (Taq1A polymorphism). A further mutation in D2 occurs in individuals with hereditary autosomal dominant myoclonus dystonia, with valine replaced by isoleucine at position 154 at the beginning of the fourth transmembrane segment of D2 (see Figures 4 and 7). D2 Function and Distribution A wide variety of psychomotor functions, including biochemical, physiological, and pathological, have been attributed to D2. Specifically, D2 inhibits action potentials by eliciting a prolonged inhibitory postsynaptic potential (IPSP), inhibits adenylate cyclase, and inhibits the entry of calcium ions into cells, thereby inhibiting many aspects of stimulus response coupling in a variety of neurons and cells. D2 receptors are located on presynaptic terminals and, therefore, can readily inhibit the release of dopamine. These presynaptic receptors appear to be predominantly D2Short, while D2Long receptors are mostly postsynaptically located on dendritic spines. The genetic absence of D2 receptors leads to animals that are akinetic and Parkinson-like. In alcoholics, it has been reported that the prevalence of the Taq1A polymorphism is about twofold s0050 p0110 p0115 f0035 Figure 7 Amino acid sequence of D2Longer. Compared to D2Short and D2Long (Figure 4), D2Longer has an extra valine glutamine dipeptide (as indicated by the arrow) that is usually spliced out in D2Short and D2Long. Drawing by P. Seeman.

11 Dopamine Receptors 9 p0120 higher than in control subjects. In schizophrenia, it has been found in 27 studies, comprising 3707 patients and 5363 controls, that the serine311cysteine polymorphism was significantly associated with schizophrenia. Furthermore, the number of D2 receptors in the caudate-putamen is elevated in schizophrenia, as measured in vivo (Corripio) or in postmortem samples in vitro (Figure 8). Although Figure 8 shows that the density of D2 receptors in postmortem human schizophrenia tissues is elevated, some of this elevation may have resulted from the antipsychotic administered during the lifetime of the patient. The postmortem tissues from half of the patients with schizophrenia revealed elevated densities of [ 3 H]spiperone-labeled D2 receptors in the caudate-putamen tissue. The other half of the postmortem schizophrenia tissues were normal in D2 density, even though most of the patients were known to have been treated with antipsychotics during their lifetime. Such findings have long been controversial, because the D2 density is not elevated in schizophrenia when using [ 11 C]raclopride. It should be noted, however, that the number of D2 receptors is significantly elevated in healthy identical co-twins of individuals with schizophrenia, suggesting that an elevation of Control striata pmol g y Schizophrenia D2 may be a necessary but not sufficient requirement for schizophrenia. The distribution of D2 receptors within the various brain regions is reflected in the gene expression pattern of D2, as shown in Figure 9. An additional unique feature is that D2 receptors are organized in bands in the normal human temporal cortex, but these bands are not found in brains of patients with Alzheimer s disease. Finally, the density of D2 is not constant over one s lifetime, but slowly falls by about 2% per decade, as shown in the postmortem human tissues in Figure 10. There is a sharp transient rise during ages 1 3, but this is followed by a gradual pruning of neurons with D2 receptors. The Dopamine D3 Receptor Using methods similar to those used for cloning the D2 receptor, the D3 receptor was cloned in 1990; its sequence is shown in Figure 11. D3 has several polymorphisms, including a serine replacing glycine at position 9 in 28% of the population (see Figure 11). In addition, there are nonfunctional forms of D3, where the amino acid chain stops after transmembrane 2, after transmembrane 3, or before transmembrane 6, the latter being found in, but not diagnostic for, Alzheimer s disease and schizophrenia. Although BP897 is a partial agonist at D3, with a selectivity for D3 of about 100-fold higher than that for D2, this drug (at 10 mg day 1 ) did not appear effective against schizophrenia symptoms. Other drugs moderately selective for D3, such as S33138 and A437,203, are currently being tested in schizophrenia patients. The highly D3-selective drug, FAUC 365, has not yet been tested in this disease. It is possible that the D3-selective compounds may be helpful in treating drug abuse. The Dopamine D4 Receptor The dopamine D4 receptor was cloned in 199; its sequence is shown in Figure 12. The D4 receptor probably has more polymorphisms than any other protein in the body. For example, the intracellular loop contains repeat forms of a 16-amino acid polypeptide, the number of repeats varying from person to person. Most people have four such repeats, but up to 10 repeats are known. Moreover, the precise sequence within each repeat usually varies from person to person, with at least 20 different types of repeat units known, thereby resulting in a massive number of polymorphisms in the human population. An unusual polymorphism in D4 occurs at position 194, where glycine replaces valine in 13% of Africans and Caribbeans (Figure 13), but not in Caucasians D2 density pmol g 1 f0040 Figure 8 Bimodal pattern of D2 receptors in postmortem striata from individuals who had schizophrenia during life. The control individuals had died of non-neurological disorders. The bimodal pattern was found in the schizophrenia tissues, regardless of whether the individuals had received antipsychotics or not. Adapted from Seeman P and Niznik HB (1990) Dopamine receptors and transporters in Parkinson s disease and schizophrenia. FASEB Journal 4: Au6. p0125 s0055 p0130 p0135 s0060 p0140 p0145

12 10 Dopamine Receptors f0045 Figure 9 Anatomical location for gene expression of dopamine receptor genes in human brain. Abbreviations: CX, cerebral cortex; L, lateral ventricles; C, caudate nucleus; P, putamen; G, globus pallidus; AC, nucleus accumbens; O, olfactory tubercle; H, hypothalamus; AM, amygdala; Hipp, hippocampus; VTA, ventral tegmental area; SN, substantia nigra. Drawing by P. Seeman. p0150 This mutant form of D4 markedly reduces its sensitivity to dopamine. One young man in the Caribbean was found to be homozygous for this V194G polymorphism, but no medical abnormalities were found. Another D4 polymorphism occurs when the GASA sequence is missing at position 21. Interestingly, clozapine has a higher affinity at D4 than at D2, as shown in Table 2. Nevertheless, despite clozapine s selectivity for D4, clozapine occupies the necessary 60 70% of brain D2 receptors at clinical doses (400 mg day 1 ), compatible with the idea that D2 is the therapeutic target for clozapine, as with all the other antipsychotics. It may be noted that isoclozapine causes catalepsy, in contrast to clozapine, which does not elicit catalepsy. Both drugs have dentical affinity for D4, but isoclozapine has higher affinity for D2 (see Table 2), and, therefore, causes catalepsy. Although the gene expression of D4 was found to be elevated in the frontal cortex of schizophrenia tissues, selective D4 antagonists, such as sonepiprazole and L-745,870, did not have any antipsychotic action. Some evidence suggests that the longer forms of D4, such as D4.7, with seven repeats, or D4.9, are found in hyperactive individuals or in those persons who take unusual risks, but this is controversial. The Dopamine D5 Receptor The dopamine D5 receptor was cloned in 1991 (Sunahara), and its sequence is shown in Figure 14. There are two pseudogenes of D5, where the amino acid p0155 s0065 p0160 Au7

13 Dopamine Receptors D2 control striata (m and f) 2.2% loss per decade (p < 0.002) s0070 p0165 D2 density, pmol g 1 sequence stops at position 154. Although D5 is essentially D1-like in sequence and function, the characteristic feature of D5 is that it is more sensitive to dopamine than D1 is, as indicated in Table 1 for the K50 values. Regulation of Dopamine Receptors Each of the five dopamine receptors has a state of high affinity and a state of low affinity for dopamine, an example of which is shown in Figure 15 for the Age, years Upper limit for adults f0050 Figure 10 Postmortem human D2 densities in the striatum. After an initial rapid growth period in the first three years of life, the D2 receptor density is rapidly pruned before age 10, and thereafter decays by 2.2% every 10 years. Adapted from Seeman et al. (1987). f0055 Figure 11 Amino acid sequence of the human cloned dopamine D3 receptor. A polymorphism occurs at position 9, where glycine replaces serine. When a frame shift occurs in the cytoplasmic loop, as shown, the receptor is nonfunctional. Drawing by P. Seeman. D2 receptor in anterior pituitary tissue. Dopamine receptors belong to a group of more than 1000 receptors known to be associated with G proteins. The binding of an agonist to such a G-linked receptor occurs in two concentration ranges. Low nanomolar concentrations of the agonist binds to the high-affinity state of the receptor, while high micromolar concentrations bind to the low-affinity state of the receptor. Generally, it is the high-affinity state of the receptor that is the functionally active state of the receptor, because the agonist affinities for the high-affinity state Au8

14 12 Dopamine Receptors f0060 Figure 12 Amino acid sequence of the human dopamine D4 receptor and its polymorphisms. The cytoplasmic loop has repeat units of 16 amino acid polypeptides. Different humans have different numbers of repeats. Shown are four repeats (D4.4), the most common, and seven repeats (D4.7). Drawing by P. Seeman. p0170 are usually identical to the concentrations that elicit the physiological action of the agonists. This holds for many neurotransmitter receptors, including dopamine D2 receptors, cholinergic muscarinic receptors, a 2 -adrenoceptors, and b 2 -adrenoceptors. D2 High is the functional state in the anterior pituitary, upon which dopamine and other dopamine-like drugs (bromocriptine) act to inhibit the release of prolactin. D2 High is presumably also functional on the terminals of the dopamine-containing terminals, and these receptors are usually referred to as presynaptic receptors. Although it has been reported that 90% of the D2 receptors in brain slices are in the D2 High state, the proportion of D2 receptors in the high-affinity state in homogenized striatum in vitro is generally between 15% and 20%. The D2 High state can be quickly converted into the D2 Low state by guanine nucleotide, as illustrated in Figure 15 for anterior pituitary tissue and in Figure 16 for the striatum. Furthermore, as shown in Figure 16, the high-affinity state of D2 is most readily detected by dopamine competing with [ 3 H]domperidone, but not [ 3 H]raclopride or [ 3 H]spiperone, which are less sensitive to the competitive action of dopamine. In fact, it is known that the physiological concentration of dopamine in the synaptic space (between neurons) is 2 4 nm, matching the known dissociation constant of 2 nm for dopamine at the D2 High receptor. This latter value of 2 nm is obtained from the dopamine/[ 3 H]domperidone competition curve in Figure 16, using the standard Cheng Prusoff equation to correct for the ligand concentration and the [ 3 H]domperidone K d. There are at least two views of the physical existence of the high-affinity state. The traditional view is that the high-affinity state of the receptor exists when the receptor, R, is associated with the G protein, and the agonist, D, binds to this high-affinity state to form the ternary complex, namely DRG. This view of the receptor proposes that the low-affinity state occurs when the G protein is not associated with the p0175

15 Dopamine Receptors 13 D4 Valine G T C Repeat ASAG (8%, Italy) D4 Gly194 G G Glycine C 13% Africans and caribbeans 0% Caucasians f0065 Figure 13 Additional polymorphisms of the human dopamine D4 receptor. Approximately 13% of Africans and Caribbeans have a glycine replacing valine at position 194. Approximately 8% of Italians have an additional ASAG peptide inserted at the position shown. Drawing by P. Seeman. D5 Au9 f0070 Figure 14 Amino acid sequence of the human dopamine D5 receptor. Two polymorphisms of D5 exist where the sequence abruptly stops to create a sequence of 154 amino acids instead of the full-length sequence of 477 amino acids. Drawing by P. Seeman. receptor. However, there are many significant shortcomings with this view of the high-affinity state of the receptor in the ternary complex model. For example, the ternary complex suggests that RG should have a transient existence. This is the not the case, however, because it has been found that the purified muscarinic RG is stable. Moreover, the purified muscarinic receptor, free of G and GDP, clearly shows high-affinity and low-affinity states. An alternate view of the high-affinity state of the receptor is the cooperativity model, as worked out by J. Wells and colleagues. The cooperative model p0180

16 14 Dopamine Receptors 100 D2 p0185 s0075 p0190 [ 3 H]Spiperone bound, (% specific) D2 High proposes that the receptor cooperates with other receptors to form either a dimer, a tetramer, or a larger oligomer. The receptor is in the high-affinity state when it is vacant and unoccupied by the agonist. However, when the agonist binds to the vacant receptor, the occupied receptor interacts or cooperates with the other receptors (within the tetramer) such that the affinity of the other receptors for the agonist is markedly reduced. This reduced affinity for the agonist is a result of negative cooperativity between the receptors, and corresponds to the low-affinity state of the receptor. In other words, if there is very strong negative cooperativity, then the second, third, and fourth receptors (within the tetramer) would hardly bind the agonist, and only the high-affinity sites would be observed in the competition between, say, dopamine and [ 3 H]domperidone, all taking place at the first receptor. These events are depicted in a diagram in Figure 17. D2 Interactions with Other Receptors The D2 receptor is known to interact with the D1 receptor as well as with other receptors, such as the adenosine A2 receptor. Many D1/D2 interactions occur at all levels, including the molecular level, where D1 and D2 can form functional heterodimers Control D2 Low Dopamine, mol l 1 With guanine nucleotide (+)BTC f0075 Figure 15 The high-affinity state of the dopamine D2 receptor, or D2 High, occurs at low nanomolar concentrations of dopamine in inhibiting the binding of [ 3 H]spiperone to anterior pituitary tissue (Ant. Pit.; porcine). The low-affinity state of the D2 receptor, or D2 Low, occurs at high concentrations of dopamine, as shown. However, the presence of 200 mm guanilylimidodiphosphate converts all the D2 High receptors into the D2 Low form. Drawing by P. Seeman. with one another. Such interaction also occurs at the cellular level, where the block of D1 (by SCH23390) unmasks the high-affinity state of the D2 receptor, D2 High, as shown in Figure 18. This experiment shows that D1 normally inhibits or suppresses the high-affinity state of D2. Psychosis and the D2 High Basis of Dopamine Supersensitivity The dopamine hypothesis of psychosis or schizophrenia was first outlined by J. Van Rossum in 1967: The hypothesis that neuroleptic drugs may act by blocking dopamine receptors in the brain has been substantiated by preliminary experiments with a few selective and potent neuroleptic drugs. There is an urgent need for a simple isolated tissue that selectively responds to dopamine so that less-specific neuroleptic drugs can also be studied and the hypothesis further tested.... When the hypothesis of dopamine blockade by neuroleptic agents can be further substantiated it may have fargoing consequences for the pathophysiology of schizophrenia. Overstimulation of dopamine receptors could then be part of the aetiology. As noted earlier, it has not been clearly established that D2 receptors are elevated in psychosis or schizophrenia, although studies using brain imaging of healthy co-twins of schizophrenia individuals, as well s0080 p0195 p0200

17 Dopamine Receptors 15 f0080 Figure 16 Low nanomolar concentrations of dopamine readily inhibit the binding of [ 3 H]domperidone at the high-affinity state of dopamine D2 receptors (between 1 and 100 nm), in contrast to [ 3 H]raclopride and [ 3 H]spiperone, which are less sensitive to competition by dopamine. The high-affinity state, D2 High, is converted to the low-affinity state, D2 Low, by guanine nucleotide. Adapted from Seeman P, Tallerico T, and Ko F (2003) Dopamine displaces [ 3 H]domperidone from high-affinity sites of the dopamine D2 receptor, but not [ 3 H] raclopride or [ 3 H]spiperone in isotonic medium: Implications for human positron emission tomography. Synapse 49: p0205 as single-photon brain imaging of nonmedicated psychotic patients in at least one study, have shown significant elevations of D2. At present, the most promising direction in this field is to examine the molecular basis of dopamine supersensitivity, because up to 70% of patients are supersensitive to either methylphenidate or amphetamine at doses which do not affect controls. Moreover, a wide variety of brain alterations in animals (lesions, birth injury by C-section, amphetamine or phencyclidine treatment, knockouts of a variety of receptors) all lead to the final common finding of behavioral dopamine supersensitivity and elevated proportions of D2 receptors in the D2 High state in the striatum. For example, repeated administration of amphetamine to animals or humans leads to behavioral dopamine supersensitivity. While the density of D2 receptors in the striatum does not change in such studies, it is remarkable that the density of D2 High receptors increases dramatically by several fold, as shown in Figure 19. A similar situation occurs in animals that receive hippocampal lesions neonatally. Such animals, as adults, reveal behavioral dopamine supersensitivity, and the striatum contains a marked increase in the proportion of D2 receptors in the high-affinity state, as shown in Figure 20.Therefore, the molecular control of the high-affinity state of D2 is emerging as a central problem in this field. At present, there is uncertainty as to whether this high-affinity state of D2 is controlled through G o or one of the G i proteins, because this varies from cell to cell. p0210

18 16 Dopamine Receptors f0085 Figure 17 (a) The cooperativity model for the dopamine D2 receptor, according to J. Wells and colleagues. The receptor is proposed to exist as an oligomer of D2 receptors, such as a tetramer of D2. Each of the vacant receptors is in the high-affinity state, D2 High. When dopamine first attaches to one of the vacant D2 receptors in the tetramer, the occupied D2 then interacts with the other three receptors within the tetramer to reduce their affinity for dopamine (i.e., negative cooperativity). (b) One molecular explanation for dopamine supersensitivity is that an unknown factor may reduce the negative interaction between the D2 receptors, thereby allowing more dopamine to occupy more D2 High receptors. Drawing by P. Seeman. Bound [ 3 H]raclopride, DPM per filter Block of D1 reveals high states for D2 in rat striatum Control 1000 a With 100nM SCH Control With 500 nm ketamine With 30nM L745,870 With 30 nm ketanserin 500 b Dopamine, nm µm S-sulpiride f0090 Figure 18 Regulation of D2 High by the D1 receptor. (a) Dopamine inhibited the binding of [ 3 H]raclopride to D2 receptors at dopamine concentrations higher than 100 nm. However, in the presence of SCH23390 to block D1 receptors, the binding of [ 3 H]raclopride was readily inhibited by nm, corresponding to the presence of D2 High. These data suggest that D1 actively suppresses the existence of the functional D2 High state. (b) In comparison to 100 nm SCH23390 unmasking the D2 High state (a), other drugs do not lead to such unmasking of D2 High. L745,870 is a dopamine D4 receptor antagonist. Adapted from Seeman P and Tallerico T (2003) Link between dopamine D1 and D2 receptors in rat and human striatal tissues. Synapse 47:

19 Dopamine Receptors 17 p0215 According to the negative cooperativity model (Figure 17), the increased number of D2 receptors in the high-affinity state, D2 High, found in the striata of supersensitive animals, may be attributed to a f0095 Figure 19 Repeated administration of amphetamine to rats leads to behavioral dopamine supersensitivity. While the total density of D2 receptors was normal in the striata of such supersensitive rats (about 26 pmol g 1 ), the density of D2 High receptors was markedly elevated by 355%, from a control value of 2.9 pmol g 1 to the elevated level of 10.3 pmol g 1. Nonspecific binding of [ 3 H] raclopride was done in the presence of 10 mm S-sulpiride. In the presence of 200 mm guanilylimidodiphosphate (G.N.), all the D2 High receptors were converted to D2 Low. Adapted from Seeman P, Tallerico T, Ko F, et al. (2002) Amphetamine-sensitized animals show a marked increase in dopamine D2 High receptors occupied by endogenous dopamine Even in the absence of acute challenges. Synapse 46: % Specific binding of [ 3 H]domperidone reduction in the overall negative cooperativity between the receptors, as illustrated in Figure 17. Thus, in order to determine the molecular mechanism of dopamine supersensitivity, it will be essential to determine the factors that reduce negative cooperativity among the D2 receptors or that alter the association of the receptor with its G protein. The role of guanine nucleotides in regulating the overall sensitivity of the dopamine D2 receptors would be to alter the extent of the receptor receptor negative cooperativity. Current Clinical and Basic Research on Dopamine Receptors Of the five dopamine receptors and their many variants, the D2 receptor and its properties continue to be most actively investigated, because D2 is the main clinical target of antipsychotics and of dopamine agonist treatment of Parkinson s disease. The D1 receptor, however, also has an important clinical role in treating Parkinson s disease because the stimulation of D1 synergizes with the stimulation of D2, possibly via D1/D2 heterodimers or cell cell interactions. A current active area of clinical research on dopamine receptors is to measure the occupancy of D2 receptors both in the striatum and outside the striatum in individuals taking antipsychotic medications. Some researchers find that the same D2 occupancy occurs in both striatal and limbic regions, while others find a lower occupancy in the limbic regions. As previously noted, therapeutic doses of antipsychotics occupy 60 80% of the D2 receptors in psychotic patients, while D2 occupancies higher than Neonatal hippocampus lesion increases D2 High states Dopamine, nm D2 High = 10% D2 High = 37% Sham lesion Hippocampus lesion f0100 Figure 20 Rats with lesions made neonatally reveal behavioral dopamine supersensitivity when they become adults. Such supersensitive animals reveal a marked increase of 3.7-fold in the proportion of D2 receptors in the high-affinity state, D2 High. Nonspecific binding of [ 3 H]domperidone was done in the presence of 10 mm S-sulpiride. Adapted from Seeman P, Weinshenker D, Quirion R, et al. (2005) Dopamine supersensitivity correlates with D2 High states, implying many paths to psychosis. Proceedings of the National Academy of Sciences USA 102: s0085 p0220 p0225