Antagonist Binding Characteristics of the Ser311→Cys Variant of Human Dopamine D2Receptorin Vivoandin Vitro (original) (raw)
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Human dopamine receptor subtypes?in vitro binding analysis using3H-SCH 23390 and3H-raclopride
Journal of Neural Transmission, 1988
Affinities and regional densities of the D1-and D2-dopamine receptor subtypes were studied in the human post-mortem brain in vitro using the two selective radioligands 3H-SCH 23390 and 3H-raclopride. 3H-Raclopride binding was confined to the caudate nucleus, the putamen and the substantia nigra, while 3H-SCH23390 bound to cortical regions as well. The binding of 3H-SCH 23390 was reduced by a low concentration of ketanserin, indicating binding to 5-HT2 receptors in addition to the D~-dopamine receptors. The endogenous neurotransmitter dopamine interacted potently both with the D~-dopamine receptor and the D2-dopamine receptor, displaying two affinity states for each subtype. The distribution of the dopamine receptor subtypes obtained in the present in vitro investigation is in agreement with data obtained with 1~C-SCH23390 and 11C-raclopride in positron emission tomographic studies in human volunteers.
Site-directed mutagenesis of the human dopamine D2 receptor
European Journal of Pharmacology: Molecular Pharmacology, 1992
Based on "mlim) acid sequence and computer modeling, two conflicting three-dimensional mndcls of die dopamine l)~ receptor have beth proposed. One m~del (Dahl et al., 1991, Prec. Natl. Acad. Sci. USA 88, 8111) stlggcsts that dopamine interacts with asp'~"latc 80 of transmcmbranc (TM) 2 and asparaginc 390 of TM6 with the transmenlbranes arranged in a clockwise manner, white a second m,,del (Hiberl et al., 1991, Mol. Pharmacol. 40, 8) suggests that dopaminc interacts with aspartatc 114 of TM3 and the serines of TM5 (194 and 197) with the transmcmbranes arranged m a countcrclockwise manner when viewed from the cxtracellular space. The present study tests the latter model by selectively mutating aspartate 114 and serines 194 and !c-~7 ,af the t;uman &~pamine D, receptor by site-directed rnutagcnesis. In addition, two methioeincs (116 and 117) were mutated to evaluate whether residues near aspartate (1i4) of the dopaminc D 2 receptor are critical in differentiating dopamine receptor agonists from adrenoceptor agonists. Removal of the negative charge with the mutation of aspartatc (114) to either asparagine or glycinc led to a to!al loss ¢~f both agonist and antagonist binding, lndNidual or dual methinnine mutations in positions 116 and 117, to make the dopamine D 2 binding pocket more closely resemble the/,12-adrenoceptor, did not result ill a change in selectivity toward noradrenergic ag~mists or anla[,,onists. Tile scrine mutations revealed interesting differences between the dopamine D 2 receptor and the adrenoceptors. In particular serine 197 appeared more imp,~rtant than serine 194 for agonist binding. In addition, the binding of Olle agonist (NdH.37) was un,J'fcctcd by individual scrinc nlutathms, white the binding of some antagonists, such as raclopride and spip,.'rone, was significantly al,.cred. These fi,ldings are discussed in relation to ligand slrucltlrc and their interactions with the putative binding pocket.
Genetic Aspects of Dopamine Receptor Binding in the Mouse and Rat Brain: An Overview
Journal of Neurochemistry, 1992
Remarkable advances during the past few years have sparked new interest in the molecular identification of receptor types and in the spatiotemporal regulation of their expression. Because dopamine (DA) receptors have been implicated in major neuropsychiatric illnesses, such as schizophrenia and affective disorders, both of which have significant heritable components, new research is now aimed at testing hypotheses which deal with the genetic regulation of DA receptor expression and with linkage of such expression to behavioral disorders. The purpose of this article, which is not intended to be an exhaustive review, is to examine the findings of two decades of genetic studies on DA receptor expression in terms of radioligand binding and inbred mouse and rat strains, as well as to discuss some of the pitfalls in past research and new strategies in current work. NEW DA RECEPTOR SUBTYPES AND ISOFORMS Before the advent of molecular genetic technology, definitions of receptor types had been based primarily on the rank order of receptor agonist and antagonist potencies (Seeman and Grigoriadis, 1987). Although the DA D, and D, receptors were discovered in the early 1970s (Kebabian et al., 1972; Seeman et al., 1974), it was not until 1979 that the terms D, and D, were used to refer to them (Kebabian and Calne, 1979). Later, the existence of DA D, and D4 receptor subtypes was also suggested (Seeman, 1983). The four receptor subtypes, however, were shown subsequently to be reducible to the known D, and D, receptors in high-or low-affinity states (Seeman and Grigoriadis, 1987). The DA D2 receptor was first cloned, isolated, and sequenced by Bunzow et al. (1988). Subsequently, it was shown that, in both rats and humans, there is a short and a long isoform derived by alternative splicing of exon 6 of the transcript (Dal Toso et al., 1989
European Journal of Pharmacology: Molecular Pharmacology, 1992
Since [3H]cmonapridc ([3H]yMo09151-2), a benzamidc ncuroleptic, consistently detects more dopaminc 11, receptors than [3H]spiperonc in the same tissue, wc tested whether this property was inherent in the cloned dopaminc D e receptor. Wc found that thc density of dopamine D e rcccptors labellcd by [SH]cmonapridc was 1.5-fold to 2-lklld (mean of 1.S-fold) highcr than the density of dc,paminc D, receptors labelled by [~H]spipcrone in cells expressing cloned dopaminc D z rcccplors (either the short form (from rat} or tile Ring form (from human)), matching similar findings in anterior pituitaly tissue (rat or pig) or in post-mortcm human caudatc nucleus tissuc. The situation was similar for another bcnzamide, [3H]raclopridc, which revealed 1.3-fold to 1.8-fold (mean of 1.5-fold) more binding sites than that for [3H]spipcronc in cell membranes containing cloned dopamine D: receptors. The apparently different dopaminc D: receptor densities revealed by these two typcs of 31ot-ligands (i.e. [3H]spipcronc and the [3H]bcnzamides), thereh)rc, arise from an inherent property of tile dopaminc D, receptor protein. These findings for the cloned dopaminc D~ receptor, therefore, partly explain the higher dopaminc D, receptor density measured in human brain (by positron emission tomography) when using radioactive raclopridc compared to rcsu!!s using radioactive mcthylspiperone. Hence, the apparent density of dopamine D e receptors measured by radioactive raclopridc nil) '.:c lower or higher than thai revealed by radioactive spipcrone or methylspipcrone, depending on the magnitude of cndogcn(ms dopamine (which inhibits tile binding of radioactive raclopridc) and the inherent properly of dopaminc 11, receptor protcir~s to exhibit more binding sites (D 2 monomers, possibly) for radioactive benzamidcs than for radioactive spipcronc or nacthylspipcrone (D 2 directs, possibly).
Journal of Neural Transmission, 1999
Competitive inhibition of 3 H-raclopride (RAC) and 3 H-Nmethylspiperone (NMSP) binding against haloperidol, raclopride and NMSP was measured in the mouse striatum. 3 H-RAC binding was more sensitive to competitive inhibition by all three compounds compared with 3 H-NMSP. For example, 0.3 mg/kg of haloperidol resulted in 95% inhibition of 3 H-RAC binding, however only 60% of inhibition of 3 H-NMSP binding was found at the same dose of haloperidol. The cross-inhibition experiments using nonradioactive RAC or NMSP as competitors indicated different binding sites for 3 H-RAC and 3 H-NMSP in mouse striatum. Specifically, about 40% of 3 H-NMSP binding was not displaced by treatment with a very high dose of raclopride (3 mg/kg). The time course of inhibition of the specific binding of 3 H-RAC and 3 H-NMSP were measured following i.p. injection of 0.5 mg/kg of haloperidol. No significant differences in the kinetics of haloperidol inhibition were observed between two radioligands.
Progress in Neuro-Psychopharmacology and Biological Psychiatry, 2009
The C957T (rs6277) single nucleotide polymorphism (SNP) of the human dopamine D2 receptor (DRD2) gene (DRD2) affects DRD2 mRNA stability and has been shown to predict striatal DRD2 availability (B(max)/K(D)) in vivo in man. Specifically, the C/C genotype is associated with low striatal DRD2 availability (C/C<C/T<T/T). It is not known, however, whether this pattern of genetic regulation of DRD2 expression also applies to low density DRD2 populations in extrastriatal regions. We analyzed extrastriatal DRD2 availability (indexed by binding potential, BP(ND)) measured in 38 healthy male volunteers with 3D-PET and the high-affinity DRD2 radioligand [(11)C]FLB457. The subjects were genotyped for the C957T as well as for two other widely studied DRD2 SNPs, the TaqIA (rs1800497) and the -141C Ins/Del (rs1799732). Statistical analyses showed that the C957T C/C genotype was associated with high extrastriatal DRD2 BP(ND) throughout the cortex and the thalamus (C/C>C/T>T/T). Also the TaqIA A1 allele carriers (p=0.101) tended to have higher extrastriatal DRD2 BP(ND) compared to non-carriers whereas the -141C Ins/Del genotype did not influence extrastriatal DRD2 BP(ND). Our findings indicate that the DRD2 SNPs regulate DRD2 availability in the human cortex and in the thalamus in vivo. However, the regulation pattern is different from that observed previously for striatal DRD2 availability in vivo, which may reflect distinct functional roles of dopamine and DRD2 in the cortex versus the striatum. The results provide useful information for the interpretation of genetic studies exploring the role of the DRD2 in normal physiology as well as in psychiatric and neurological diseases.
Neurochemistry International, 1998
A simple and rapid in vitro method for qualitative and quantitative estimation of the Ga!subunits interaction with the third intracellular loop of human D 1s dopamine receptor has been developed[ For this purpose\ D 1s !CL2 was cloned in pGEX!1T vector and expressed in E[ coli BL10 DE2 as a fusion protein with glutathione!S!transferase "D 1s !CL2!GST#[ The resulting soluble construct was puri_ed by a.nity chromatography on glutathione!Sepharose[ Ga!subunits were expressed and puri_ed as His!tagged proteins[ For the assay of Ga:D 1s !CL2!GST interactions\ varying concentrations of pure His!tagged Ga!proteins were immobilized on His!Bind Resin and titrated with D 1s !CL2!GST fusion protein[ Ga:D 1s !CL2!GST interactions were quanti_ed by GST activity determination assay[ It was shown that the fusion protein interacts speci_cally with di}erent Ga proteins\ especially with Ga i proteins[ Based on saturation binding analyses\ Kd values were determined revealing the highest a.nity of His!Ga i\1 binding to the fusion protein[ The a.nities for Ga i :D 1s !CL2!GST protein interactions estimated in this way were in nanomolar range of concentrations[ Þ 0887 Elsevier Science Ltd[ All rights reserved[