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D R Sibley

Publications and source records attributed to D R Sibley.

At least 19 recordsLinked to original sources
PubMed2001

The role of phosphorylation/dephosphorylation in agonist-induced desensitization of D1 dopamine receptor function: evidence for a novel pathway for receptor dephosphorylation.

Exposure of D1 dopamine receptors to agonists results in rapid desensitization of the receptor-stimulated accumulation of cAMP. It is believed that agonist-induced phosphorylation of the receptor plays a critical role in the processes that underlie this phenomenon. To investigate the role of agonist-induced receptor phosphorylation, a FLAG epitope was added to the amino terminus of the rat D1 dopamine receptor and this construct was stably expressed in C6 glioma cells. It was found that the D1 receptor was stoichiometrically phosphorylated under basal conditions and that its phosphorylation state was increased by 2- to 3-fold upon exposure of the cells to dopamine for 10 min. The dopamine-induced receptor phosphorylation could be blocked by D1-selective antagonists but was unaffected by inhibitors of either protein kinase A or protein kinase C. The incorporation of phosphate into the receptor was rapid but transient, despite the continued presence of dopamine. A comparison of the rates of receptor phosphorylation approximately ion (t(1/2) < 1 min) and dopamine-induced desensitization (t(1/2) approximately 7 min) revealed that receptor phosphorylation was not the rate limiting step for receptor desensitization. Upon removal of dopamine, the receptor was rapidly dephosphorylated (t(1/2) approximately 10 min) and this was not blocked by agents (i.e., concanavalin A or hypertonic sucrose) that inhibit D1 receptor internalization. Using specific inhibitors, the phosphatase involved in D1 receptor dephosphorylation was shown not to correlate with the recently identified "G protein-coupled receptor phosphatase" (Proc Natl Acad Sci USA 92:8343-8347, 1995). These results suggest that the phosphorylated D(1) receptor is processed through a novel recovery pathway and that internalization is not required for receptor dephosphorylation.

Animals↗
PubMed2001

The binding-site crevice of the D4 dopamine receptor is coupled to three distinct sites of allosteric modulation.

Most biogenic amine G protein-coupled receptors contain a conserved aspartic acid residue positioned near the intracellular side of the second transmembrane-spanning (TMS) domain that is the primary site of allosteric modulation by sodium ions and pH. Recently, zinc ions and amiloride derivatives were found to allosterically modulate antagonist binding to dopamine receptors. In the current study, the wild-type D4 dopamine receptor showed an 8-fold decrease in zinc affinity in the presence of 120 mM NaCl, but the binding of zinc to the neutral TMS2 D4-D77N mutant was completely sodium-insensitive. In contrast to zinc, methylisobutylamiloride (MIA) binding to the wild-type D4 receptor was virtually unaffected by sodium. In addition, the binding affinity for MIA was essentially unchanged in the presence of an IC(50) concentration of zinc and vice versa. Furthermore, MIA binding affinity was decreased 4-fold for the D4-D77N mutant and increased 30-fold for the TMS3 mutant D4-M107V, even though the binding affinity for zinc was similar to the wild-type D4 background for both mutants. These findings demonstrate for the first time the existence of three distinct sites of allosteric modulation within a G protein-coupled receptor.

Amiloride↗
PubMed2001

Behavioral characterization of dopamine D5 receptor null mutant mice.

To study behavioral functions of the D5 subtype, mice were generated with null mutations in the D5 gene. This 1st behavioral characterization of D5 null mutant mice (D5-/-) indicated normal general health, sensory abilities, and neurological reflexes. Under basal conditions, D5-/- mice were generally normal on locomotor activity, the rotarod test, acoustic startle response, prepulse inhibition, elevated plus-maze, light <--> dark exploration, Morris water maze, and cued and contextual fear conditioning. In the Porsolt forced swim test for antidepressant activity, male D5-/- mice showed lower levels of immobility. D5-/- mice showed some evidence of reduced responses to the hyperactivity-inducing effects of the D1/D5 receptor agonist SKF 81297. The ability of SKF 81297 to disrupt acoustic startle and prepulse inhibition appeared to be attenuated in D5-/- mice. These results suggest that the D5 receptor is not essential for many dopamine-mediated behaviors but may contribute to the pharmacological activation of dopaminergic pathways relevant to exploratory locomotion, startle, and prepulse inhibition.

Animals↗
PubMed2000

Tandem sulfur-containing amino acids are epicritical determinants of dopamine D(2) receptor pharmacology.

The conserved aspartic acid that is required for ligand binding to the dopamine D(2) receptor is followed by three tandem sulfur-containing amino acids. While previous point mutation studies did not reveal any single one of these residues as being critical for ligand binding, we now show that simultaneously substituting all three with isovolumetric, non sulfur-containing amino acids results in large decreases in the binding affinity for dopamine, (-)-raclopride and 7-(-4(4-(2, 3-dichlorophenyl)-1-piperazinyl)butyloxy)-3, 4-dihydro-2(1H)-quinolinone (aripiprazole), but not for methylspiperone or allosteric modulators.

Amino Acids, Sulfur↗
PubMed2000

Nonconserved residues in the second transmembrane-spanning domain of the D(4) dopamine receptor are molecular determinants of D(4)-selective pharmacology.

The molecular determinants that govern selective ligand binding to the rat D(4) dopamine receptor were investigated by substituting D(2) dopamine receptor sequences into a D(4) dopamine receptor background. The resulting mutant D(4) dopamine receptors were then screened with a panel of 10 selective and nonselective ligands, which included two allosteric modulators as sensitive measures of protein conformational changes. Mutation of a phenylalanine at position 88 in the second transmembrane-spanning domain (TMS2) of the D(4) receptor to the corresponding valine in the D(2) receptor D(4)-F88V resulted in an approximately 100-fold decrease in the affinity of the highly D(4)-selective drug 3-([4-(4-iodophenyl) piperazin-1-yl]methyl)-1H-pyrrolo[2,3-b]pyridine (L-750,667) without substantially affecting the binding of the other ligands. Mutations at the extracellular side of D(4)-TMS3 produced moderate decreases in L-750,667 binding affinities with concomitant increases in binding affinity for the D(2)/D(3)-selective antagonist (-)-raclopride. However, the binding affinities of these same D(4)-TMS3 mutants for the allosteric modulator isomethylbutylamiloride also were an anomalous 6- to 20-fold higher than either wild-type receptor. In the combined D(4)-F88V/TMS3 mutants, L-750,667 binding affinity was further decreased, but this decrease was not additive. More importantly, the combined D(4)-F88V/TMS3 mutants had (-)-raclopride and isomethylbutylamiloride binding properties that reverted back to those of the wild-type D(4)-receptor. In contrast to the D(4)-F88V mutant, the adjacent D(4)-L87W mutant had an increased affinity for ligands with extended structures, but had essentially no effect on ligands with compact structures. These findings demonstrate that two residues near the extracellular side of D(4)-TMS2 are critical molecular determinants for the selective binding of L-750,667 and ligands with extended structures.

Animals↗
PubMed2000

Altered regulation of the D(1) dopamine receptor in mutant Chinese hamster ovary cells deficient in cyclic AMP-dependent protein kinase activity.

To investigate the role of the cAMP-dependent protein kinase (PKA) in the desensitization and down-regulation of the D(1) dopamine receptor, we stably expressed the rat cDNA for this receptor in mutant Chinese hamster ovary (CHO) cell lines deficient in PKA activity. The 10260 mutant CHO cell line has been characterized as expressing less than 10% of type I and type II PKA activities relative to the parental 10001 CHO cell line. The 10248 mutant CHO line lacks type II PKA activity and expresses a defective type I PKA. The transfected parental and mutant cell lines were found to express approximately 1 pmol/mg D(1) receptor binding activity (B(max)) as determined using [(3)H]SCH-23390 binding assays. All three cell lines demonstrated similar levels of dopamine-stimulated adenylyl cyclase activity. Pretreatment of all three CHO cells with dopamine resulted in desensitization of the adenylyl cyclase response, although the maximum desensitization was attenuated by 20 and 40% in the 10260 and 10248 cell lines, respectively. Dopamine also promoted, in a time- and dose-dependent fashion, a >90% down-regulation of D(1) receptors in the parental cell line but only a 50 and 30% decrease in the 10260 and 10248 cells, respectively. Similarly, treatment of the cells with the membrane-permeable cAMP analog 8-(4-chlorophenylthio)-cAMP induced functional desensitization and down-regulation of the D(1) receptor, although it was not as great as that observed with agonist pretreatment. As with the agonist pretreatments, the 8-(4-chlorophenylthio)-induced responses were attenuated in the mutant cells with the 10248 line exhibiting the least desensitization/down-regulation. Our results suggest that PKA significantly contributes to the desensitization and down-regulation of D(1) receptors in CHO cells and that type II PKA may be the more relevant isoform with respect to regulating D(1) receptor function.

Animals↗
PubMed1999

Spatial learning deficit in dopamine D(1) receptor knockout mice.

Dopamine D(1) receptors are expressed in the hippocampus and prefrontal cortex, suggesting a role in cognition. Dopamine D(1) receptor-deficient mice (D(1)-/-) were used to investigate the role of this receptor in spatial learning and memory. Using the Morris water maze, mice were trained to locate a hidden platform. Subsequently, the platform was removed from the maze and mice were scored for the percentage of time spent in the target quadrant and the number of crossings through the target position. D(1)-/- mice had significantly longer escape latencies compared to wild-type (D(1)+/+) and heterozygous (D(1)+/-) littermates and showed absence of spatial bias during the probe trials. In a visually cued task, D(1)-/- mice performed better than on the hidden platform trials, but maintained slightly higher escape latencies than D(1)+/+ and D(1)+/- mice. Naive D(1)-/- mice exposed only to the cued task eventually acquired identical escape latencies as the D(1)+/+ and D(1)+/- mice. Sensorimotor reflexes, locomotor activity, spontaneous alternation and contextual learning were not different among the groups. These results indicate that D(1)-/- mice have a deficit in spatial learning without visual or motor impairment, suggesting that dopamine D(1) receptors are involved in at least one form of the cognitive processes.

Animals↗
PubMed1999

D2S, D2L, D3, and D4 dopamine receptors couple to a voltage-dependent potassium current in N18TG2 x mesencephalon hybrid cell (MES-23.5) via distinct G proteins.

We utilized the approach of stably expressing different dopamine (DA) receptors into identified cell lines in an attempt to better understand the coupling of these receptors to membrane ion channels via second messenger systems. Recently, we examined the N18TG2 x mesencephalon (MES-23.5) cell line that is phenotypically similar to mesencephalic dopamine-containing neurons. Whole-cell voltage-clamp methods were used to investigate a voltage-dependent K+ current present in these cells. Untransfected MES-23.5 cells displayed a voltage-dependent slow-onset, slowly inactivating outward current which was not altered by bath application of either the D2 DA receptor agonist quinpirole (QUIN; 10-100 microM) or the D1 DA receptor agonist SKF38393, indicating that these cells were devoid of DA receptors. The K+ current studied was activated upon depolarization from a holding potential of -60 mV to a level more positive than -20 mV and was observed to be sensitive to bath application of tetraethylammonium. When MES-23.5 cells were transfected to stably express the D2S, D2L, D3, and D4 receptors, the same current was observed. In cells expressing D2L, D2S, and D3 receptors, application of the DA receptor agonists QUIN (1-80 microM), 7-hydroxy-dipropylaminoteralin (7-OH-DPAT, 1-80 microM), and dopamine (DA, 1-80 microM), increased the peak outward current by 35-40%. In marked contrast, cells stably expressing the D4 receptor demonstrated a significant DA agonist-induced reduction of the peak K+ current by 40%. For all four receptor subtypes, the D2-like receptor antagonist sulpiride (SUL 5 microM), when coapplied with QUIN (10 microM), totally abolished the change in K+ current normally observed, while coapplication of the D1-like receptor antagonist SCH23390 was without effect. The modulation of K+ current by D2L, D3, and D4 receptor stimulation was prevented by pretreatment of the cells with pertussis toxin (PTX, 500 ng/ml for 4 h). In addition, the intracellular application of a polyclonal antibody which specifically recognizes Goalpha completely blocked the ability of D2L, D3, and D4 receptors to modulate outward K+ currents. In contrast, the intracellular application of an antibody directed against Goalpha was without effect, whereas intracellular application of an antibody recognizing Gsalpha abolished the ability of the D2S receptor to enhance K+ current. These findings demonstrate that different members of the D2 DA receptor family may couple in a given cell to a common effector in dramatically different ways.

Animals↗
PubMed1999

Conservation of behavioural topography to dopamine D1-like receptor agonists in mutant mice lacking the D1A receptor implicates a D1-like receptor not coupled to adenylyl cyclase.

Though D1-like dopamine receptors [D1A/B] are defined in terms of linkage to the stimulation of adenylyl cyclase, with D1A assumed to be the functionally prepotent subtype, evidence suggests the existence of another, novel D1-like receptor without such coupling. To investigate these issues we challenged mutant mice having targeted gene deletion of the D1A receptor with selective agonists and used an ethologically-based assessment technique to resolve resultant behavioural topography. D1-like-dependent behaviour was substantially conserved in D1A-null mice relative to wild-types following challenge with each of two selective D1-like agents: A 68930 (0.068-2.0 mg/kg s.c.) which exhibits full efficacy to stimulate adenylyl cyclase, and SKF 83959 (0.016-2.0 mg/kg s.c.) which fails to stimulate adenylyl cyclase, and indeed inhibits the stimulation of adenylyl cyclase induced by dopamine. Furthermore, responsivity to the selective D2-like agonist RU 24213 (0.1-12.5 mg/kg s.c.) was conserved in D1A-null mice, indicating the integrity of D1-like:D2-like interactions at the level of behaviour. These data are consistent with behavioural primacy of a D1-like receptor other than D1A [or D1B] that is coupled to a transduction system other than/additional to adenylyl cyclase.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1999

Interactions of the novel antipsychotic aripiprazole (OPC-14597) with dopamine and serotonin receptor subtypes.

OPC-14597 {aripiprazole; 7-(-4(4-(2,3-dichlorophenyl)-1-piperazinyl) butyloxy)-3,4-dihydro-2(1H)-quinolinone} is a novel candidate antipsychotic that has high affinity for striatal dopamine D2-like receptors, but causes few extrapyramidal effects. These studies characterized the molecular pharmacology of OPC-14597, DM-1451 (its major rodent metabolite), and the related quinolinone derivative OPC-4392 at each of the cloned dopamine receptors, and at serotonin 5HT6 and 5HT7 receptors. All three compounds exhibited highest affinity for D2L and D2S receptors relative to the other cloned receptors examined. Both OPC-4392 and OPC-14597 demonstrated dual agonist/antagonist actions at D2L receptors, although the metabolite DM-1451 behaved as a pure antagonist. These data suggest that clinical atypicality can occur with drugs that exhibit selectivity for D2L/D2S rather than D3 or D4 receptors, and raise the possibility that the unusual profile of OPC-14597 in vivo (presynaptic agonist and postsynaptic antagonist) may reflect different functional consequences of this compound interacting with a single dopamine receptor subtype (D2) in distinct cellular locales.

Animals↗
PubMed1999

Analysis of neuroleptic binding affinities and potencies for the different human D2 dopamine receptor missense variants.

Neuroleptics, or antipsychotics, are widely used for the treatment of psychotic symptoms such as hallucinations and delusions in schizophrenia and other psychiatric disorders. Pharmacotherapy of these diseases is frequently complicated by a great variability in the clinical response to neuroleptics and by the development of serious and potentially life-threatening side-effects. Brain D2 dopamine receptors are one of the major targets of neuroleptic treatment. The human D2 dopamine receptor (DRD2) gene has three variants predicting the amino acid substitutions Ser311Cys, Pro310Ser and Val96Ala in the receptor protein. We show that several typical and atypical neuroleptics commonly used in the treatment of psychotic disorders have differences in binding affinities and potencies for the D2 dopamine receptor variants. Functional differences between dopamine receptor variants might be related to genetically determined differences in response to neuroleptic treatment.

Animals↗
PubMed1999

New insights into dopaminergic receptor function using antisense and genetically altered animals.

Dopaminergic receptors are widespread throughout the central and peripheral nervous systems, where they regulate a variety of physiological, behavioral, and endocrine functions. These receptors are also clinically important drug targets for the treatment of a number of disorders, such as Parkinson's disease, schizophrenia, and hyperprolactinemia. To date, five different dopamine receptor subtypes have been cloned and characterized. Many of these subtypes are pharmacologically similar, making it difficult to selectively stimulate or block a specific receptor subtype in vivo. Thus, the assignment of various physiological or behavioral functions to specific dopamine receptor subtypes using pharmacological tools is difficult. In view of this, a number of investigators have--in order to elucidate functional roles--begun to use highly selective genetic approaches to alter the expression of individual dopamine receptor subtypes in vivo. This review discusses recent studies involving the use of genetic approaches for the study of dopaminergic receptor function.

Animals↗
PubMed1999

Electrophysiological and morphological analyses of cortical neurons obtained from children with catastrophic epilepsy: dopamine receptor modulation of glutamatergic responses.

The present study examined the electrophysiological effects produced by activation of specific dopamine (DA) receptors and the distribution of DA receptor subtypes and glutamate receptor subunits [N-methyl-D-aspartate (NMDAR1) and GluR1] in cortical tissue samples obtained from children (ages 3 months to 16 years) undergoing epilepsy surgery. DA receptor activation produced differential effects depending on the receptor subtype that was activated. D1 receptor family agonists generally enhanced cortical excitability and favored the emergence of epileptogenic activity. In contrast, D2 receptor family agonists had more variable effects on cortical excitability and the expression of epileptiform discharges. Activation of D1 or D2 receptors decreased the amplitude of non-NMDA-mediated excitatory postsynaptic potentials. In contrast, DA and D1 agonists increased the amplitude of NMDA-mediated potentials. Immunohistochemical analysis showed that the DA receptor subtypes and glutamate receptor subunits examined were present in all cortical layers and areas throughout development. Whole-cell voltage clamp recordings of pyramidal neurons visualized with differential interference contrast optics and infrared videomicroscopy indicated that these neurons displayed a persistent Na(+) current, followed by an outward current. DA reduced the outward current but had little effect on the persistent Na(+) current. These results suggest a dual role for DA's actions in the human cerebral cortex. Activation of D2 receptors or antagonism of D1 receptors may help control seizures in children.

Adolescent↗
PubMed1999

Zinc modulates antagonist interactions with D2-like dopamine receptors through distinct molecular mechanisms.

Recently, zinc has been shown to modulate antagonist drug interactions with the D1 dopamine receptor (Schetz and Sibley, 1997) and the dopamine transporter (Norregaard et al., 1998). We now demonstrate that zinc also reversibly and dose-dependently modulates the specific binding of the butyrophenone antagonist [3H]methylspiperone to all D2-like dopamine receptors: D2L, D3, and D4. The molecular mechanisms of zinc regulation of these D2-like receptor subtypes are distinct because zinc inhibition of [3H]methylspiperone binding to the D4 receptor is noncompetitive by both equilibrium and kinetic measures (lower Bmax and essentially no change in koff), whereas the corresponding inhibition of zinc at D2L and D3 receptors is primarily characterized by competitive allosterism (increases in KD and koff). Interestingly, thermodynamic measurements reveal that the macroscopic properties of zinc binding are entropy-driven for all receptor subtypes, despite their having distinct molecular mechanisms. Zinc also reduces the binding affinity of the D2L receptor for [3H]raclopride, a structurally different antagonist of the substituted benzamide class. Sodium ions negatively modulate zinc inhibition of both sodium-insensitive [3H]methylspiperone binding and sodium-sensitive [3H]raclopride binding. In addition to its demonstrated effects on antagonist binding in membrane preparations, zinc also retards the functional effects of antagonist at the D2L receptor in intact cells. These findings suggest that synaptic zinc may be a factor influencing the effectiveness of therapies that rely on dopamine receptor antagonists.

Algorithms↗
PubMed1999

Regulation of D(1) dopamine receptors with mutations of protein kinase phosphorylation sites: attenuation of the rate of agonist-induced desensitization.

Investigations of D(1) receptor regulation have suggested a role for cAMP-dependent protein kinase (PKA) in agonist-induced desensitization and down-regulation of receptor expression. Given the presence of at least four possible consensus recognition sites for PKA on the D(1) receptor protein, a reasonable hypothesis is that some of these PKA-mediated effects are caused by phosphorylation of the receptor. As an initial test of this hypothesis, we used site-directed mutagenesis to create a mutant D(1) receptor with substitutions at each of its four potential PKA phosphorylation sites. The modified amino acids are as follows: Thr135 to Val, Ser229 to Ala, Thr268 to Val, and Ser380 to Ala. Characterization of the wild-type and mutant receptors stably expressed in C6 glioma cells suggests that the mutations have no effect on receptor expression, antagonist or agonist affinities, or on functional coupling with respect to cAMP generation. Similarly, dopamine preincubation of the stably transfected C6 cells expressing either the wild-type or mutated D(1) receptors results in an agonist-induced loss of ligand binding activity (down-regulation) in an identical fashion. In contrast, the time of onset of dopamine-induced desensitization is greatly attenuated in the quadruple mutant receptor. After 1 h of dopamine pretreatment, the wild-type receptor exhibits approximately 80% desensitization of the cAMP response, whereas the mutant receptor is desensitized by only approximately 20%. Further analyses of single mutated receptors, in which only one of the four putative phosphorylation sites is modified, reveals that Thr268 in the third cytoplasmic loop of the receptor protein is primarily responsible for regulating the desensitization kinetics. These results are consistent with the hypothesis that phosphorylation of the D(1) receptor on Thr268 is important for rapid agonist-induced homologous desensitization.

Amino Acid Sequence↗
PubMed1998

Disruption of dopamine D1 receptor gene expression attenuates alcohol-seeking behavior.

The role of the dopamine D1 receptor subtype in alcohol-seeking behaviors was studied in mice genetically deficient in dopamine D1 receptors (D1 -/-). In two-tube free choice limited (1-5 h) and continuous (24 h) access paradigms, mice were exposed to water and increasing concentrations of ethanol (3%, 6% and 12% w/v). Voluntary ethanol consumption and preference over water were markedly reduced in D1 -/- mice as compared to heterozygous (D1 +/-) and wild-type (D1 +/+) controls, whereas overall fluid consumption was comparable. When offered a single drinking tube containing alcohol as their only source of fluid for 24 h, D1 -/- mice continued to drink significantly less alcohol than D1 +/+ and D1 +/- mice. Dopamine D2 receptor blockade with sulpiride caused a small but significant reduction in alcohol intake and preference in D1 +/+ mice and attenuated residual alcohol drinking in D1 -/- mice. Dopamine D1 receptor blockade with SCH-23390 very effectively reduced alcohol intake in D1 +/+ and D1 +/- mice to the level seen in untreated D1 -/- mice. These findings suggest involvement of both dopamine D1 and D2 receptor mechanisms in alcohol-seeking behavior in mice; however, these implicate D1 receptors as having a more important role in the motivation for alcohol consumption.

Alcohol Drinking↗
PubMed1998

Topographical evaluation of the phenotype of spontaneous behaviour in mice with targeted gene deletion of the D1A dopamine receptor: paradoxical elevation of grooming syntax.

The phenotype of spontaneous behaviour in mice with targeted gene deletion of the DIA dopamine receptor was investigated topographically. Via direct visual observation, individual elements of behaviour were resolved and quantified using an ethologically-based, rapid time-sampling behavioural check-list procedure. Relative to wildtypes (D1A+/+), D1A-null (-/-) mice evidenced over initial exploration significant reductions in rearing free, sifting and chewing, but significant increases in locomotion, grooming and intense grooming. Sniffing and rearing to a wall habituated less readily in D1A-null mice such that these behaviours occurred subsequently to significant excess: increases in locomotion were persistent. The ethogram of spontaneous behaviour in D1A-null mice was characterised by neither 'hypoactivity' or 'hyperactivity' but, rather, by prominent topographical shifts between individual elements of behaviour that could not be encapsulated by either term. Given the substantial body of evidence that grooming and particularly intense grooming constitute the most widely accepted behavioural index of D1-like receptor function, the elevation of such behaviour in D1A-null mice was paradoxical; it may reflect (over)compensatory processes subsequent to developmental absence of D1A receptors and/or the involvement of a D1-like receptor other than/additional to the D1A subtype.

Animals↗