Proc. Nati. Acad. Sci. USA Vol. 89, pp. 8155-8159, September 1992 Neurobiology Identification, characterization, and localization of the dopamine D3 receptor in rat brain using 7-[3H]hydroxy-NN-di-n-propyl- 2-aminotetralin (guanine nucleotide-binding regulatory protein-coupled receptor/antipsychotics/islands of Calleja/cerebellum/nucleus accumbens) DANIEL LtVESQUE*, JORGE DIAZt, CATHERINE PILON*, MARIE-PASCALE MARTRES*, BRUNO GIROS*, EVELYNE SOUILt, DOMINIQUE SCHOTTt, JEAN-LOUIS MORGATt, JEAN-CHARLES SCHWARTZ*§, AND PIERRE SOKOLOFF* *Unite de Neurobiologie et Pharmacologie (U. 109) de l'Institut National de la Sante et de la Recherche Medicale, Centre Paul Broca, 75014 Paris, France; tLaboratoire de Physiologie, Facultd de Pharmacie, Universite Rene Descartes, 75006 Paris, France; and tD6partement de Biologie, Centre d'Etudes Nucleaires, 91191 Saclay, France Communicated by Jean-Pierre Changeux, May 20, 1992 ABSTRACT We have identified 7-[3Hlhydroxy-N,N-di-n- Therefore, there is an obvious interest in studying the propyl-2-aminotetralin ([3H]7-OH-DPAT) as a selective probe native receptor protein in brain, particularly in the case ofthe for the recently cloned dopamine D3 receptor and used it to D3 receptor, whose pharmacology in Chinese hamster ovary assess the presence ofthis receptor and establish its distribution (CHO) cells and cerebral localization of its mRNA suggest and properties in brain. In transfected Chinese hamster ovary that it may represent an important target for antipsychotics (CHO) cells, it binds to D3 receptors with subnanomolar (8, 12, 13). affinity, whereas its affinity is approximately 100-, 1000-, and Starting with the idea that designing a selective radioactive 10,000-fold lower at D2, D4, and D, receptors, respectively. probe for the D3 receptor would help to settle these issues, we Specific [3H]7-OH-DPAT binding sites, with aKd of0.8 nM and have screened a series of dopamine agonists and thereby a pharmacology similar to those at reference D3 receptors of identified 7-hydroxy-N, N-di-n-propyl-2-aminotetralin (7- CHO cells, were identified in rat brain. D3 receptors differ OH-DPAT) (14-16) as fulfilling this purpose. from D2 receptors in brain by their lower abundance (2 orders of magnitude) and distribution, restricted to a few mainly phylogenetically ancient areas-e.g., paleostriatum and ar- MATERIALS AND METHODS chicerebellum-as evidenced by membrane binding and auto- Brain Membranes. Male Wistar rats (IFFA Credo, Orldans, radiography studies. Native D3 receptors in brain are charac- France) weighing 180-200 g were used. They were exposed terized by an unusually high nanomolar affinity for dopamine to an artificial light cycle of 12 h/day and had free access to and a low modulatory influence of guanyl nucleotides on food and water. After decapitation, brain tissues were rapidly agonist binding. These various features suggest that D3 recep- dissected out, frozen on dry ice, and kept at -70°C until use. tors are involved in a peculiar mode of neurotransmission in a Tissues were homogenized with a Polytron (setting 7 for 10 restricted subpopulation of dopamine neurons. sec) in 10 mM Tris-HCl (pH 7.5) containing 1 mM EDTA and centrifuged at 2500 x g for 30 sec; the supernatant was Dopamine is an important neurotransmitter in brain, being centrifuged at 35,000 x g for 15 min. The pellet was resus- involved physiologically in the control of cognitive, motor, pended and centrifuged again, and this washing procedure and endocrine processes and pathologically in Parkinson and was repeated twice to eliminate endogenous dopamine. The possibly mental diseases. final was sonication in a buffer con- Until recently, it was largely believed that its various pellet resuspended by actions were mediated by two receptor subtypes, termed D1 taining 50 mM NaHepes, 1 mM EDTA, 50 ,uM 8-hydroxy- and D2 (1, 2). Molecular biology approaches have led, how- quinoleine, 0.005% ascorbic acid, and 0.1% bovine serum ever, to the identification and cloning of the genes corre- albumin (pH 7.5) (incubation buffer). sponding not only to these two receptor subtypes (3-7) but Membranes from Cell Lines. CHO cell lines expressing rat also to additional and less expected ones, termed D3 (8), D4 D2 or D3 dopamine receptors (CHO-D2 or CHO-D3) have (9), and D5 (10, 11). For the latter receptors, the information been described (12). CHO-D1 were created by transfecting so far available derives from molecular biology approaches; pCD-BS plasmid containing the human D1 receptor gene (6), their pharmacology and signaling system have only been a generous gift of P. Seeman. COS-7 cells transiently ex- studied in transfected cells, generally fibroblasts, and their pressing the human D4 receptor were obtained by transfect- distribution has been indirectly approached by studies of ing pCD-PS plasmid, a generous gift of H. H. M. Van Tol, mRNA localization. Nevertheless, these otherwise valuable containing the human D4 receptor gene (9). Cells were grown approaches suffer from some limitations. When expressed in in Dulbecco's modified Eagle's medium containing 10% fetal fibroblasts, receptors might find a membrane environment, bovine serum. Cells were harvested by trypsin treatment which could modify their pharmacological specificity, and a (0.25%) for 4-5 min and centrifugation at 2000 x g for 5 min. repertoire of cellular components, particularly GTP-binding They were homogenized with a Polytron in 10 mM Tris HCl proteins (G proteins), which may differ from that found in (pH 7.5) containing 1 mM EDTA and were centrifuged at neurons. Regarding distribution, mRNA detection reveals 35,000 x g for 15 min. The pellet was then resuspended by the sites of receptor synthesis rather than receptor localiza- sonication in the NaHepes buffer described above. tion, which may be different. Abbreviations: 7-OH-DPAT, 7-hydroxy-N,N-di-n-propyl-2-amino- The publication costs of this article were defrayed in part by page charge tetralin; p[NH]ppG, 5'-guanylyl imidodiphosphate; G protein, GTP- payment. This article must therefore be hereby marked "advertisement" binding protein. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 8155 Downloaded by guest on October 2, 2021 8156 Neurobiology: Ldvesque et al. Proc. Natl. Acad. Sci. -USA 89 (1992) A 20 B [3H]70H-OPAT [ 125I]IODOSULPRIDE I-I. CD3 60 - D3 D2 D04 D 010 cm o -11 -10 -9 -8 -7 -6 -5 -4 -3 0 100 200 300 400 500 0 200 400 600 8 1000 LOG [70H-OPAT] (M) BOUND (fuol.ag protein-') BOUND (fmol.mg protein-1) FIG. 1. (A) Inhibition by 7-OH-DPAT of ligand binding to D1, D2, D3, and D4 receptors in transfected CHO cells. The ligands were 0.3 nM [3H]SCH23390 at the D1 receptor, 0.1 and 0.2 nM (125I]iodosulpride at the D2 and D3 receptors, respectively, and 0.1 nM [3H]spiperone at the D4 receptor. Results are means from two experiments. (B) Scatchard analysis of [3H]7-OH-DPAT (Left) and [125Iliodosulpride (Right) specific binding to D2 and D3 receptors in transfected CHO cells. This is a representative example of two such experiments. Note that [3H]7-OH-DPAT binding to D2 receptors was not measurable. (Inset) Chemical structure of 7-OH-DPAT; *, tritium atom. Binding Assays. Membranes from brain (150-300 pg of 32P-labeled complementary RNA probe specific for the D3 protein) or cell lines (15-25 ,ug of protein) were added to receptor (13). polypropylene test tubes containing [3H]7-OH-DPAT for the Drugs. 7-OH-DPAT and its precursor 7-hydroxy-N,N- D3 receptor assay, [1251]iodosulpride for D2 and D3 receptors, diallylaminotetralin were custom synthesized at the Institut de [3H]spiperone for the D4 receptor, or [3H]SCH23390 for the Chimie des Substances Naturelles (Gif-sur-Yvette, France). D1 receptor. Competing drugs were dissolved in incubation [3H]7-OH-DPAT was prepared by catalytic reduction, with buffer, the final volume being 1 ml, except for [125I]iodosul- pure 3H gas, of the double bond located on the N,N-diallyl pride binding, for which it was 0.4 ml. Tubes were incubated precursor. The tritiated molecule was then purified by high- in triplicate for 1 h at room temperature. The incubations performance liquid chromatography through a preparative were stopped by rapid filtration under reduced pressure ,uBondapak column in a 0-50%o methanol gradient in water through Whatman GF/C glass filters coated with 0.1% bovine containing 0.05% triethanolamine. The specific activity was serum albumin (except for the [125I]iodosulpride binding estimated to be 158 Ci/mmol (1 Ci = 37 GBq). [M2IJIodosul- assay, for which the filters were Whatman GF/B-coated with pride (2000 Ci/mmol), [N-methyl-3H]SCH23390 (80 Ci/ 0.3% polyethyleneimine), followed by three rinses with 3-4 mmol), and [phenyl-4-3H]spiperone (60 Ci/mmol) were pur- ml of ice-cold buffer (50 mM TrisHCl, pH 7.5/120 mM chased from Amersham. Dopamine, apomorphine, and 5'- NaCl). Nonspecific binding was measured in the presence of guanylyl imidodiphosphate (p[NHflppG) were purchased from 1 ,uM dopamine (for [3H]7-OH-DPAT), 5 ,M (-)-sulpiride Sigma. (-)-Sulpiride was generously donated by Delagrange (for [1251]iodosulpride), 0.5 ,uM YM-09151-2 (for [3H]spiper- Laboratoires (Paris), haloperidol and domperidone were sup- one), and 1 ,uM SCH23390 (for [3H]SCH23390). Radioactiv- plied by Janssen Laboratoires (Paris), and SCH23390 was ity was counted by y scintigraphy for the 1251 ligand or by from Schering. scintillation spectrometry, at an efficiency of 41%, for 3H ligands. Saturation curves were analyzed by computer non- RESULTS linear regression using a one-site cooperative model to obtain equilibrium dissociation constants (Kd) and maximal density 7-OH-DPAT Binding to Ceil Line Membranes.