The molecular biological characterization of the dopamine receptors began with the cloning of the gene and cDNA for the hamster beta-2 adrenergic receptor in 1986 (1), followed closely by the isolation of cDNAs encoding the turkey erythrocyte beta-adrenergic receptor (2) and the porcine ml muscarinic receptor (3). The realization that G protein-coupled receptors form a family of proteins related by primary structure and predicted secondary structural features, a family big enough to include the light-activated receptor rhodopsin, opened up the possibility of exploiting this homology for the molecular cloning of additional members of the gene family. Indeed, all the dopamine receptors have been cloned because of their homology to other dopamine or monoamine receptors.
Using homologous probes for the cloning of related genes within the family of guanine nucleotide-binding protein-coupled receptors, we have cloned the gene for the rhesus macaque D1 dopamine receptor. By using the rat D1 receptor coding sequence as a probe under high stringency conditions, the rhesus D1 receptor gene was isolated from a lambda EMBL3 rhesus genomic DNA library. The rhesus D1 dopamine receptor gene is intronless and encodes a 446-amino acid protein that contains two consensus sites for asparagine-linked glycosylation (Asn-5 and Asn-176) and two consensus sites for cAMP-dependent protein kinase phosphorylation (Thr-136 and Thr-268). The primary amino acid sequence of the rhesus D1 dopamine receptor shows an extremely high degree of similarity (99.6%) to the human D1 receptor. Genomic DNA analyses conducted with high and reduced stringency hybridizations indicate that the rhesus macaque D1 receptor is a member of a large multigene family. Like the human D1 receptor mRNA, the rhesus D1 receptor mRNA is approximately 4 kilobases in size and is localized predominantly in the caudate, with lesser amounts in the hippocampus and cortex. The rhesus D1 receptor coding region was inserted into the cytomegalovirus promoter-driven expression vector pcDNA-1, and the recombinant (pcDNA-D1) was cotransfected with the selectable marker pRSVneo, conferring G418 resistance, into D1 receptor-deficient C6 glioma cells. Analyses of the selected transfectant demonstrate the expression of a high affinity, functional D1 dopamine receptor. The D1 receptor radioligand [3H]SCH 23390 bound transfectant membranes with an affinity (Kd), of 0.3 nM; the D2-selective ligand spiperone, the dopamine receptor ligand clozapine, and the serotonin receptor antagonist ketanserin bound with considerably lower affinities (102, 80, and 95 nM, respectively). Both dopamine and the D1-selective agonist SKF 38393 inhibited the binding of [3H]SCH 23390 to transfectant cell membranes; the binding of these agonists was sensitive to GTP. Dopamine potently stimulated the accumulation of cAMP in transfected C6 cells, whereas SKF 38393 was a partial agonist in these cells. Also, the density of recombinant D1 receptors on the transfectant cells was decreased 40% upon treatment with 10 microM dopamine, indicating that occupation of recombinant D1 receptors by agonists alters surface expression of the receptors.
The aim of this work is to evaluate a model of dopamine receptor function in which the short splice variant of the dopamine D2 receptor, D2S, is thought to be the autoreceptor that inhibits firing of dopamine neurons and dopamine release, and the long splice variant, D2L, is proposed to be the postsynaptic receptor that regulates the activity of medium spiny neurons (MSNs) in the basal forebrain. We obtained the results described here using two approaches. In the first, D2S and D2L were virally expressed in midbrain dopamine neurons of mice in which the D2 receptor was genetically deleted (D2‐KO) either globally or selectively in dopamine neurons. The second approach involved the use of mice constitutively expressing only D2S (D2L‐KO) or D2L (D2S‐KO) due to genetically preventing expression of one variant. Viral expression of either variant in dopamine neurons of D2‐KO mice restored inhibition of locomotor activity by a high dose of quinpirole (1 mg/kg) and quinpirole inhibition of electrically evoked dopamine release in neostriatal slices. Regulation of G protein‐regulated inward‐rectifying potassium channels (GIRKs) by the splice variants was indistinguishable when whole‐cell recordings were carried out using a strong calcium‐buffering solution (10 mM BAPTA) in slices containing the substantia nigra pars compacta (SNc), or in neostriatal slices from D2‐KO mice virally expressing both the D2 receptor and GIRK in MSNs. When recordings were made from SNC dopamine neurons virally or constitutively expressing only one splice variant and with a weak calcium‐buffering intracellular solution (0.1 mM EGTA), D2S desensitized more rapidly than D2L in the presence of bath‐applied quinpirole (10 μM). The D2S‐mediated GIRK current in midbrain slices was also found to have increased sensitivity to calcium buffering in the early phase of desensitization induced by a photoactivatable caged dopamine. Finally, D2‐GIRK IPSCs evoked with electrical stimulation (1/min) were studied in the receptor variants. Upon bath application of cocaine (30 μM), the IPSCs initially increase and then desensitize. The initial increase in amplitude was significantly less and the rate of desensitization significantly slower in slices from mice constitutively expressing D2L compared with mice expressing D2S. Beyond desensitization, this could also reflect splice variant differences in inhibition of dopamine release, dopamine transport, or activation of GIRKs. Overall, our results suggest that either variant can function as an autoreceptor, but that expression of D2S more closely models wild type D2 receptor under normal slice recording conditions. Support or Funding Information Merit Review Award BX003279 from the US Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
Chimeric D1/D2 receptors were constructed to identify structural determinants of drug affinity and efficacy. We previously reported that chimeras that had D1 receptor transmembrane domain VII together with amino-terminal sequence from the D2 receptor were nonfunctional. D2/D1 chimeras were constructed that contained D2 receptor sequence at the amino- and carboxyl-terminal ends and D1 receptor sequence in the intervening region. Chimeric receptors with D2 sequence from transmembrane domain 7 to the carboxyl terminus together with D2 receptor sequence from the amino terminus through transmembrane helix 4 (D2[1–4,7]) and 5 (D2[1–5,7]) bound [3H]spiperone with high affinity, consistent with the hypothesis that D2 receptor transmembrane domain I or II is incompatible with D1 receptor transmembrane domain VII. D2[1–4,7] and D2[1–5,7] had affinities similar to D1 and D2 receptors for most nonselective dopamine antagonists and had affinities for most of the selective antagonists that were intermediate between those of the parent receptors. D2[1–4,7] and D2[1–5,7]mediated dopamine receptor agonist-induced stimulation and inhibition, respectively, of cAMP accumulation. The more efficient coupling of D2[1–5,7] to inhibition of cAMP accumulation, compared with the coupling of D2[5–7] and D2[3–7], supports the view that multiple D2 receptor cytoplasmic domains acting in concert are necessary for receptor activation of Gi. In contrast, D2[1–4,7], which contains only one cytoplasmic loop (the third) from the D1 receptor, is capable of activating Gs. D2[1–4,7]exhibited several characteristics of a constitutively active receptor, including enhanced basal (unliganded) stimulation of cAMP accumulation, high affinity for agonists even in the presence of GTP, and blunted agonist-stimulated cAMP accumulation. A number of dopamine receptor antagonists were inverse agonists at D2[1–4,7], inhibiting basal cAMP accumulation. Some of these drugs were also inverse agonists at the D1 receptor. Interestingly, several antagonists also potentiated forskolin-stimulated cAMP accumulation via D2[1–5,7] and via the D2 receptor, which could reflect inverse agonist inhibition of native constitutive activity of this receptor.
Abstract: A diverse panel of monoclonal antibodies was obtained from BALB/c mice immunized with two haptens structurally related to spiroperidol (SPD). Bromoacetyl derivatives of aminospiroperidol (NH 2 SPD) and N ‐amino‐phenethylspiroperidol (NAPS) were synthesized to couple the haptens covalently to a protein carrier for immunization, thereby maintaining the butyrophenone portion of the immunogen. Hybridomas were selected based on their ability to secrete antibody that binds [ 3 H]SPD with high affinity. Equilibrium dissociation constants for these antibodies ranged from 0.2 to > 100 n M . The antigen binding sites of the anti‐NH 2 SPD and anti‐NAPS antibodies were characterized in studies of the inhibition of the binding of [ 3 H]‐SPD by a series of ligands that are either (a) structurally related to SPD or (b) structurally unrelated to the butyrophenones but known to be selective antagonists of the D2 subtype of dopamine receptor. Based on the patterns of inhibition of the binding of [ 3 H]SPD by these compounds, 12 classes of antibody combining sites were identified. Most of these antibodies bound butyrophenones with high affinity. One anti‐NH 2 SPD and four anti‐NAPS antibodies also bound domperidone, a nonbutyrophenone that has a high affinity for D2 receptors. None of the antibodies bound clebopride or sulpiride, D2‐selective antagonists of the benzamide class, or the agonist dopamine.
The distribution of dopamine D2 receptors in the rat brain was determined by quantitative autoradiography of the binding of [125I]epidepride and the effects of chronic drug administration on regulation of receptors in striatal and extrastriatal brain regions were characterized. [125I]Epidepride (2200 Ci/mmol) bound with high affinity to coronal tissue sections from the rat brain (Kd = 78 pM), and specific binding was detected in a number of discrete layers, nuclei or regions of the hippocampus, thalamus, cerebellum and other extrastriatal sites. Pharmacological analysis of radioligand binding to hippocampal and cerebellar membranes indicated binding to dopamine D2 receptors, and approximately 10% of the binding appeared to represent low affinity idazoxan-displaceable binding to alpha-2 adrenoceptors. The binding to extrastriatal regions resembled previously reported radioligand binding to dopamine D2 receptors in striatal and cortical membranes. Chronic (14 day) administration of two dopamine D2 receptor antagonists, either the typical neuroleptic haloperidol (1.5 mg/kg i.p.) or the atypical neuroleptic clozapine (30 mg/kg i.p.), caused a significant increase in the density of [125I]epidepride binding sites in the medial prefrontal cortex and parietal cortex. Only haloperidol caused a significant increase in the density of [3H]spiperone and [125I]epidepride binding sites in the striatum and a slight increase in [125I]epidepride binding sites in the hippocampus. Similar administration of amphetamine (5 mg/kg i.p.) had no significant effect on the density of dopamine D2 receptors in any brain region examined. In addition, no drug-induced changes in the characteristics of dopamine D2 receptors in discrete areas of the cerebellum were observed.(ABSTRACT TRUNCATED AT 250 WORDS)
S100B is a calcium‐binding protein with numerous regulatory activities in the mammalian brain. Using a bacterial two‐hybrid assay, we have identified a novel interaction between S100B and the third cytoplasmic loop of the dopamine D2 receptor (D2‐IC3). The binding of S100B to D2‐IC3 was confirmed using a polyHis pull‐down assay. The binding of heterologous S100B to full‐length D2 receptor in HEK293 cells, and of endogenous S100B to endogenous D2 receptor in the rat neostriatum, was demonstrated by co‐precipitation. A putative S100B binding motif is near the N‐terminus of D2‐IC3. Although S100B is considered a glial protein, S100B expression was detected in microtubule‐associated protein‐2 (MAP2) expressing neurons in neostriatal cultures. Expression of S100B in HEK293 cells also stably expressing the D2 receptor significantly increased D2 receptor stimulation of extracellular signal‐regulated kinases (ERKs), while causing little change in NGF‐stimulated activation of ERKs, and also potentiated D2 receptor inhibition of cyclic AMP accumulation. Taken together, these fundings suggest that the interaction of S100B with the D2 dopamine receptor enhances D2 receptor signaling. (MH045372 and VA Merit Review)
