title: Comparison of in vitro binding properties of a series of dopamine antagonists and agonists for cloned human dopamine D2S and D2L receptors and for D2 receptors in rat striatal and mesolimbic tissues, using [125I] 2'-iodospiperone.
abstract: We investigated the ligand binding properties in vitro of two splice variants of the cloned human dopamine D2 receptor (the 443 and 414 amino acids long forms called D2L and D2S, respectively), expressed in 293 human kidney cells, in comparison with those of the dopamine D2 receptors in rat striatum, nucleus accumbens and tuberculum olfactorium. The new radioligand, [125I]2'-iodospiperone, showed a similar high binding affinity (KD:0.056-0.122 nM) for cloned human D2S and D2L receptors and for the D2 receptors in the three rat brain areas. Binding affinities of 25 dopamine antagonists and of 10 dopamine agonists belonging to different chemical classes were measured. The IC50 values of the antagonists were virtually identical in the five preparations: spiperone was the most potent compound (pIC50 approximately 9.9), remoxipride the least potent one (pIC50 approximately 5.7). The agonists showed similar IC50 values for the cloned human D2S and D2L receptors but their affinity for rat brain D2 receptors was 2- to 5-fold higher. Dopamine showed shallow inhibition curves, the high affinity binding was 10-fold lower for the cloned human D2 receptors than for the rat brain D2 receptors. Addition of stable guanosine-5'-triphosphate (GTP) analogues shifted the D2 receptors in the rat brain tissues to the "low" affinity state, the low affinity binding of dopamine was equal to the affinity for the cloned human receptor. None of the dopamine antagonists or agonists could differentiate between the two splice forms of the cloned human D2 receptors or between the D2 receptors in rat striatal and mesolimbic tissues. The lower apparent affinity of some agonists and of dopamine in the absence of stable GTP analogues suggests a less appropriate receptor G-protein coupling for the cloned human D2 receptors expressed in the 293 human kidney cells. Unexpectedly, guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S) reduced the [125I]2'-iodospiperone binding to the D2 receptors by 20-35% in the rat brain tissues and the cloned human D2L receptor, and by 75% to the cloned human D2S receptor. The inhibition in the last case could be prevented partly by submicromolar concentrations of dopamine. The GTP-gamma-S effect is suggested to be due to reduction of disulphide bonds in the receptor. Recent molecular modelling studies indicated an important role of the disulphide bridge between Cys107 at the start of transmembrane domain three and Cys182 in the third extracellular loop, for the binding of dopamine to the D2 receptor.
authors.affiliation: Department of Biochemical Pharmacology, Janssen Research Foundation, Beerse, Belgium.
authors.forename: J E
authors.initials: JE
authors.lastname: Leysen
authors.forename: W
authors.initials: W
authors.lastname: Gommeren
authors.forename: J
authors.initials: J
authors.lastname: Mertens
authors.forename: W H
authors.initials: WH
authors.lastname: Luyten
authors.forename: P J
authors.initials: PJ
authors.lastname: Pauwels
authors.forename: M
authors.initials: M
authors.lastname: Ewert
authors.forename: P
authors.initials: P
authors.lastname: Seeburg
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D018491:Dopamine Agonists / Q000378:metabolism*; D018492:Dopamine Antagonists / Q000378:metabolism*; D005260:Female; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D007457:Iodine Radioisotopes; D008024:Ligands; D008032:Limbic System / Q000187:drug effects / Q000378:metabolism*; D017072:Neostriatum / Q000187:drug effects / Q000378:metabolism*; D009714:Nucleus Accumbens / Q000187:drug effects / Q000378:metabolism; D009830:Olfactory Bulb / Q000187:drug effects / Q000378:metabolism; D010902:Pituitary Gland / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017448:Receptors, Dopamine D2 / Q000378:metabolism*; D013134:Spiperone / Q000031:analogs & derivatives* / Q000493:pharmacokinetics
chemical_list: D018491:Dopamine Agonists; D018492:Dopamine Antagonists; D007457:Iodine Radioisotopes; D008024:Ligands; D017448:Receptors, Dopamine D2; C053751:2'-iodospiperone; D013134:Spiperone; D006160:Guanosine Triphosphate