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1.
Neuroscience ; 248: 479-87, 2013 Sep 17.
Article in English | MEDLINE | ID: mdl-23811070

ABSTRACT

D2 receptor null mutant (Drd2(-/-)) mice have altered responses to the rewarding and locomotor effects of psychostimulant drugs, which is evidence of a necessary role for D2 receptors in these behaviors. Furthermore, work with mice that constitutively express only the D2 receptor short form (D2S), as a result of genetic deletion of the long form (D2L), provides the basis for a current model in which D2L is thought to be the postsynaptic D2 receptor on medium spiny neurons in the basal forebrain, and D2S the autoreceptor that regulates the activity of dopamine neurons and dopamine synthesis and release. Because constitutive genetic deletion of the D2 or D2L receptor may cause compensatory changes that influence functional outcomes, our approach is to identify aspects of the abnormal phenotype of a Drd2(-/-) mouse that can be normalized by virus-mediated D2 receptor expression. Drd2(-/-) mice are deficient in basal and methamphetamine-induced locomotor activation and lack D2 receptor agonist-induced activation of G protein-regulated inward rectifying potassium channels (GIRKs) in dopaminergic neurons. Here we show that virus-mediated expression of D2L in the nucleus accumbens significantly restored methamphetamine-induced locomotor activation, but not basal locomotor activity, compared to mice receiving the control virus. It also restored the effect of methamphetamine to decrease time spent in the center of the activity chamber in female but not male Drd2(-/-) mice. Furthermore, the effect of expression of D2S was indistinguishable from D2L. Similarly, virus-mediated expression of either D2S or D2L in substantia nigra neurons restored D2 agonist-induced activation of GIRKs. In this acute expression system, the alternatively spliced forms of the D2 receptor appear to be equally capable of acting as postsynaptic receptors and autoreceptors.


Subject(s)
Locomotion/drug effects , Neurons/metabolism , Nucleus Accumbens/cytology , Receptors, Dopamine D2/metabolism , Animals , Behavior, Animal/drug effects , Dopamine Agonists/pharmacology , Dopaminergic Neurons/drug effects , Dopaminergic Neurons/metabolism , Female , G Protein-Coupled Inwardly-Rectifying Potassium Channels/metabolism , Gene Expression Regulation/drug effects , Gene Transfer Techniques , Male , Methamphetamine/pharmacology , Mice , Mice, Knockout , Neurons/drug effects , Receptors, Dopamine D2/agonists , Receptors, Dopamine D2/deficiency
2.
Mol Pharmacol ; 60(6): 1168-72, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11723222

ABSTRACT

Whereas acute stimulation of Galphai/o-coupled receptors inhibits the activity of adenylyl cyclase, a delayed consequence of persistent activation of the receptors is heterologous sensitization, an enhanced responsiveness of adenylyl cyclase to activators such as forskolin or agonists of Galphas-coupled receptors. Galphas-insensitive mutants of adenylyl cyclase type V were used to test the hypothesis that heterologous sensitization requires Galphas-dependent activation of adenylyl cyclase. When adenylyl cyclase was stably expressed in human embryonic kidney (HEK) 293 cells with the D2L dopamine receptor, basal, forskolin-stimulated, and isoproterenol-stimulated cyclic AMP accumulation were all enhanced by 2-h pretreatment with the D2 receptor agonist quinpirole. Transient expression of wild-type adenylyl cyclase and three Galphas-insensitive mutants (F379L, R1021Q, and F1093S) in HEK293 cells stably expressing the D2L receptor demonstrated that all three mutants had little or no responsiveness to beta-adrenergic receptor-mediated activation of Galphas but that the mutants retained sensitivity to forskolin and to D2L receptor-mediated inhibition. Transiently expressed adenylyl cyclase V was robustly sensitized by 2-h pretreatment with quinpirole. In contrast, the Galphas-insensitive mutants displayed no sensitization of forskolin-stimulated cyclic AMP accumulation, indicating that responsiveness to Galphas is required for the expression of heterologous sensitization.


Subject(s)
Adenylyl Cyclases/metabolism , Heterotrimeric GTP-Binding Proteins/metabolism , Isoenzymes/metabolism , Receptors, Dopamine D2/metabolism , Adenylyl Cyclases/genetics , Adrenergic beta-Agonists/pharmacology , Cells, Cultured , Heterotrimeric GTP-Binding Proteins/physiology , Humans , Isoenzymes/genetics , Isoproterenol/pharmacology , Mutation
3.
Mol Endocrinol ; 15(8): 1306-17, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11463855

ABSTRACT

Melatonin is a pineal hormone that regulates seasonal reproduction and has been used to treat circadian rhythm disorders. The melatonin 1a receptor is a seven- transmembrane domain receptor that signals predominately via pertussis toxin-sensitive G-proteins. Point mutations were created at residue N124 in cytoplasmic domain II of the receptor and the mutant receptors were expressed in a neurohormonal cell line. The acidic N124D- and E-substituted receptors had high-affinity (125)I-melatonin binding and a subcellular localization similar to the neutral N124N wild-type receptor. Melatonin efficacy for the inhibition of cAMP by N124D and E mutations was significantly decreased. N124D and E mutations strongly compromised melatonin efficacy and potency for inhibition of K(+)-induced intracellular Ca(++) fluxes and eliminated control of spontaneous calcium fluxes. However, these substitutions did not appear to affect activation of Kir3 potassium channels. The hydrophobic N124L and N124A or basic N124K mutations failed to bind (125)I-melatonin and appeared to aggregate or traffic improperly. N124A and N124K receptors were retained in the Golgi. Therefore, mutants at N124 separated into two sets: the first bound (125)I-melatonin with high affinity and trafficked normally, but with reduced inhibitory coupling to adenylyl cyclase and Ca(++) channels. The second set lacked melatonin binding and exhibited severe trafficking defects. In summary, asparagine-124 controls melatonin receptor function as evidenced by changes in melatonin binding, control of cAMP levels, and regulation of ion channel activity. Asparagine-124 also has a unique structural effect controlling receptor distribution within the cell.


Subject(s)
Asparagine , Potassium Channels, Inwardly Rectifying , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/physiology , Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Cytoplasmic and Nuclear/physiology , Signal Transduction , Adenylyl Cyclases/metabolism , Animals , Calcium/metabolism , Calcium Channels/metabolism , Cyclic AMP/metabolism , Electrophysiology , Fluorescent Antibody Technique , G Protein-Coupled Inwardly-Rectifying Potassium Channels , Golgi Apparatus/metabolism , Iodine Radioisotopes , Melatonin/metabolism , Melatonin/pharmacology , Mice , Mutagenesis, Site-Directed , Pituitary Neoplasms , Potassium/pharmacology , Potassium Channels/drug effects , Potassium Channels/physiology , Receptors, Cell Surface/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Melatonin , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured
4.
Mol Pharmacol ; 60(2): 373-81, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11455025

ABSTRACT

A homology model of the dopamine D2 receptor was constructed based on the crystal structure of rhodopsin. A putative sodium-binding pocket identified in an earlier model (PDB ) was revised. It is now defined by Asn-419 backbone oxygen at the apex of a pyramid and Asp-80, Ser-121, Asn-419, and Ser-420 at each vertex of the planar base. Asn-423 stabilizes this pocket through hydrogen bonds to two of these residues. Highly conserved Asn-52 is positioned near the sodium pocket, where it hydrogen-bonds with Asp-80 and the backbone carbonyl of Ser-420. Mutation of three of these residues, Asn-52 in helix 1, Ser-121 in helix 3, and Ser-420 in helix 7, profoundly altered the properties of the receptor. Mutants in which Asn-52 was replaced with Ala or Leu or Ser-121 was replaced with Leu exhibited no detectable binding of radioligands, although receptor immunoreactivity in the membrane was similar to that in cells expressing the wild-type D2L receptor. A mutant in which Asn-52 was replaced with Gln, preserving hydrogen-bonding capability, was similar to D2L in affinity for ligands and ability to inhibit cAMP accumulation. Mutants in which either Ser-121 or Ser-420 was replaced with Ala or Asn had decreased affinity for agonists (Ser-121), but increased affinity for the antagonists haloperidol and clozapine. Interestingly, the affinity of these Ser-121 and Ser-420 mutants for substituted benzamide antagonists showed little or no dependence on sodium, consistent with our hypothesis that Ser-121 and Ser-420 contribute to the formation of a sodium-binding pocket.


Subject(s)
Receptors, Dopamine D2/chemistry , Sodium/metabolism , Amino Acid Sequence , Asparagine/genetics , Binding Sites , Cells, Cultured , DNA Mutational Analysis , Humans , Models, Molecular , Molecular Sequence Data , Protein Conformation , Receptors, Dopamine D2/genetics , Receptors, Dopamine D2/metabolism , Serine/genetics
5.
Curr Protoc Neurosci ; Chapter 4: Unit 4.5, 2001 May.
Article in English | MEDLINE | ID: mdl-18428488

ABSTRACT

This overview describes issues that must be considered before attempting to express neural cDNAs in mammalian cells, including the choice of expression vector and cell type. Considerations for introducing recombinant vectors into cells are discussed along with a comparison of achieving stable or transient expression. Finally, the appropriate promoter is crucial and must be chosen to fit the design of the expression system.


Subject(s)
Gene Expression Regulation/genetics , Neurons/physiology , Animals , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Genetic Vectors/biosynthesis , Genetic Vectors/genetics , Humans
6.
J Med Chem ; 43(16): 3005-19, 2000 Aug 10.
Article in English | MEDLINE | ID: mdl-10956209

ABSTRACT

Agonist affinity changes dramatically as a result of serine to alanine mutations (S193A, S194A, and S197A) within the fifth transmembrane region of D2 dopamine receptors and other receptors for monoamine neurotransmitters. However, agonist 2D-structure does not predict which drugs will be sensitive to which point mutations. Modeling drug-receptor interactions at the 3D level offers considerably more promise in this regard. In particular, a comparison of the same test set of agonists across receptors differing minimally (point mutations) offers promise to enhance the understanding of the structural bases for drug-receptor interactions. We have previously shown that comparative molecular field analysis (CoMFA) can be applied to comparisons of affinity at recombinant D1 and D2 dopamine receptors for the same set of agonists, a differential QSAR. Here, we predicted agonist K(L) for the same set of agonists at wild type D2 vs S193A, S194A, and S197A receptors using CoMFA. Each model used bromocriptine as the template. ln(1/K(L)) values for the low-affinity agonist binding conformation at recombinant wild type and mutant D2 dopamine receptors stably expressed in C6 glioma cells were used as the target property for the CoMFA of the 16 aligned agonist structures. The resulting CoMFA models yielded cross-validated R(2) (q(2)) values ranging from 0.835 to 0.864 and simple R(2) values ranging from 0.999 to 1.000. Predictions of test compound affinities at WT and each mutant receptor were close to measured affinity values. This finding confirmed the predictive ability of the models and their differences from one another. The results strongly support the idea that CoMFA models of the same training set of compounds applied to WT vs mutant receptors can accurately predict differences in drug affinity at each. Furthermore, in a "proof of principle", two different templates were used to derive the CoMFA model for the WT and S193A mutant receptors. Pergolide was chosen as an alternate template because it showed a significant increase in affinity as a result of the S193A mutation. In this instance both the bromocriptine- and pergolide-based CoMFA models were similar to one another but different from those for the WT receptor using bromocriptine- or pergolide- as templates. The pergolide-based S193A model was more strikingly different from that of the WT receptor than was the bromocriptine-based S193A model. This suggests that a "dual-template" approach to differential CoMFA may have special value in elucidating key differences across related receptor types and in determining important elements of the drug-receptor interaction.


Subject(s)
Alanine/genetics , Dopamine Agonists/chemistry , Receptors, Dopamine D2/chemistry , Serine/genetics , Amino Acid Substitution , Animals , Bromocriptine/chemistry , Combinatorial Chemistry Techniques , Dopamine Agonists/chemical synthesis , Dopamine Agonists/metabolism , Models, Molecular , Pergolide/chemistry , Point Mutation , Radioligand Assay , Rats , Receptors, Dopamine D2/genetics , Receptors, Dopamine D2/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structure-Activity Relationship , Tumor Cells, Cultured
7.
Clin Exp Pharmacol Physiol ; 27(1-2): 9-13, 2000.
Article in English | MEDLINE | ID: mdl-10696522

ABSTRACT

1. Decline in beta-adrenoceptor (beta-AR)-mediated function occurs with increasing age, as well as in multiple disease conditions. The mechanisms responsible for this decline include alterations in beta-AR itself, beta-AR coupling proteins, such as G-proteins, or other beta-AR-linked proteins, such as G-protein receptor kinases and/or phosphatases. 2. The present study examines the physiological effects of in vitro transfer of constitutively activated G alpha s (G alpha s-Q227L) to both cultured vascular smooth muscle cells (VSMC) and whole aortic tissue of 6-month-old (adult) animals via a replication-deficient Herpes simplex virus (HSV) vector. These studies were conducted to provide a model for future examination of the role of G alpha s in the age-related decline in beta-AR-mediated vasorelaxation. 3. Gene transfer was confirmed by western blotting for specific proteins. Aortic tissue infected with HSV-G alpha s-Q227L had reduced phenylephrine-induced contraction and enhanced isoproterenol-stimulated vasorelaxation. Infection of cultured VSMC with HSV-G alpha s-Q227L increased both basal- and isoproterenol-stimulated cAMP accumulation, whereas forskolin-stimulated cAMP production was unchanged. 4. These results implicate G alpha s as a target for further investigation in age-related changes in vascular reactivity and support the use of viral-mediated gene transfer as an effective tool to study adrenergic signal transduction and physiology in vascular tissue.


Subject(s)
Cyclic AMP/metabolism , GTP-Binding Proteins/metabolism , Gene Transfer Techniques , Muscle, Smooth, Vascular/metabolism , Vasodilation/physiology , Animals , Aorta/metabolism , Cells, Cultured , Cyclic AMP/genetics , GTP-Binding Proteins/genetics , Genetic Vectors/genetics , Male , Rats , Rats, Inbred F344 , Simplexvirus/genetics , Vasodilation/genetics
8.
J Pharmacol Exp Ther ; 289(2): 877-85, 1999 May.
Article in English | MEDLINE | ID: mdl-10215666

ABSTRACT

We characterized the effects of drugs on the uptake of [3H]neurotransmitter by and the binding of [125I](3beta-(4-iodophenyl)tropane-2beta-carboxylic acid methyl ester ([125I]RTI-55) to the recombinant human dopamine (hDAT), serotonin (hSERT), or norepinephrine (hNET) transporters stably expressed in human embryonic kidney 293 cells. RTI-55 had similar affinity for the hDAT and hSERT and lower affinity for hNET (Kd = 1. 83, 0.98, and 12.1 nM, respectively). Kinetic analysis of [125I]RTI-55 binding indicated that the dissociation rate (k-1) was significantly lower for hSERT and the association rate (k+1) was significantly lower for hNET compared with the hDAT. The potency of drugs at blocking [3H]neurotransmitter uptake was highly correlated with potency at blocking radioligand binding for hDAT and hSERT. Substrates were more potent at the inhibition of [3H]neurotransmitter uptake than radioligand binding. The potency of drugs was highly correlated between displacement of [3H]nisoxetine (Kd = 6.0 nM) and [125I]RTI-55 from the hNET, suggesting that these radioligands recognize similar sites on the transporter protein. The correlation observed between inhibitory potency for uptake and binding of either ligand at the hNET was lower than correlations between uptake and binding for hDAT and hSERT. The present results indicate that the cocaine analog [125I]RTI-55 has unique binding properties at each of the transporters and that the use of recombinant transporters expressed by a single cell type can provide a powerful screening tool for drugs interacting with biogenic amine transporters, such as possible cocaine antagonists.


Subject(s)
Carrier Proteins/metabolism , Dopamine/metabolism , Membrane Glycoproteins/metabolism , Membrane Transport Proteins , Nerve Tissue Proteins , Norepinephrine/metabolism , Pharmaceutical Preparations/metabolism , Serotonin/metabolism , Symporters , Carrier Proteins/genetics , Cell Line , Cocaine/analogs & derivatives , Cocaine/metabolism , Dopamine Plasma Membrane Transport Proteins , Fluoxetine/analogs & derivatives , Fluoxetine/metabolism , Humans , Iodine Radioisotopes , Kidney/cytology , Kidney/metabolism , Kinetics , Membrane Glycoproteins/genetics , Norepinephrine Plasma Membrane Transport Proteins , Protein Binding , Radioligand Assay , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Serotonin Plasma Membrane Transport Proteins , Stereoisomerism
9.
Psychopharmacology (Berl) ; 141(1): 83-92, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9952069

ABSTRACT

The D4 dopamine receptor, a member of the D2-like dopamine receptor family, may be important in the etiology and treatment of schizophrenia. The present study was designed to examine the effects of dopamine agonist exposure on adenylate cyclase activity in HEK293 cells stably expressing recombinant-D4 receptors. Two hour pretreatment with dopamine receptor agonists resulted in heterologous sensitization of forskolin-stimulated cyclic AMP accumulation in intact cells expressing the D4.2, D4.4, or D4.7 dopamine receptor variant. The potency and efficacy of dopamine for sensitization of cyclic AMP accumulation was comparable at all D4 receptor variants. D4 dopamine receptor-mediated sensitization was blocked by the D4 antagonist, clozapine, and prevented by overnight pretreatment with pertussis toxin, implying a role for Gi/Go proteins in heterologous sensitization. Further, long-term (18 h) agonist exposure resulted in a greater degree of sensitization of forskolin-stimulated cyclic AMP accumulation in both intact cells and membrane preparations of cells expressing the D4 receptor, compared to 2 h agonist exposure, without altering the density of the receptors. In addition, long-term agonist exposure decreased the abundance of Gialpha without altering the abundance of Gsalpha, whereas short-term agonist treatment had no effect on the immunoreactivity of either G protein. In summary, long-term agonist-induced sensitization of adenylate cyclase by the D4 receptor may involve mechanisms that do not contribute to short-term sensitization.


Subject(s)
Adenylyl Cyclases/metabolism , Receptors, Dopamine D2/metabolism , Cells, Cultured , Colforsin/pharmacology , Cyclic AMP/metabolism , Dopamine Agonists/pharmacology , GTP-Binding Proteins/biosynthesis , Humans , Receptors, Dopamine D2/agonists , Receptors, Dopamine D4
10.
J Med Chem ; 41(22): 4385-99, 1998 Oct 22.
Article in English | MEDLINE | ID: mdl-9784114

ABSTRACT

We have previously shown that using agonist affinity at recombinant receptors selectively expressed in clonal cells as the dependent variable in three-dimensional quantitative structure-activity relationship studies (3D-QSAR) presents a unique opportunity for accuracy and precision in measurement. Thus, a comparison of affinity's structural determinants for a set of compounds at two different recombinant dopamine receptors represents an attainable goal for 3D-QSAR. A molecular database of bound conformations of 16 structurally diverse agonists was established by alignment with a high-affinity template compound for the D1 receptor, 3-allyl-6-bromo-7,8-dihydroxy-1-phenyl-2,3,4, 5-tetrahydro-1H-benzazepin. A second molecular database of the bound conformations of the same compounds was established against a second template for the D2 receptor, bromocriptine. These aligned structures suggested three-point pharmacophore maps (one cationic nitrogen and two electronegative centers) for the two dopamine receptors, which differed primarily in the height of the nitrogen above the plane of the catechol ring and in the nature of the hydrogen-bonding region. The ln(1/KL) values for the low-affinity agonist binding conformation at recombinant D1 and D2 dopamine receptors stably expressed in C6 glioma cells were used as the target property for the CoMFA (comparative molecular field analysis) of the 16 aligned structures. The resulting CoMFA models yielded cross-validated R2 (q2) values (standard error of prediction) of 0. 879 (1.471, with five principal components) and 0.834 (1.652, with five principal components) for D1 and D2 affinity, respectively. The simple R2 values (standard error of the estimate) were 0.994 (0.323) and 0.999 (0.116), respectively, for D1 and D2 receptor. F values were 341 and 2465 for D1 and D2 models, respectively, with 5 and 10 df. The predictive utility of the CoMFA model was evaluated at both receptors using the dopamine agonists, apomorphine and 7-OH-DPAT. Predictions of KL were accurate at both receptors. Flexible 3D searches of several chemical databases (NCI, MDDR, CMC, ACD, and Maybridge) were done using basic pharmacophore models at each receptor to determine the similarity of hit lists between the two models. The D1 and D2 models yielded different lists of lead compounds. Several of the lead compounds closely resembled high-affinity training set compounds. Finally, homology modeling of agonist binding to the D2 receptor revealed some consistencies and inconsistencies with the CoMFA-derived D2 model and provided a possible rationale for features of the D2 CoMFA contour map. Together these results suggest that CoMFA-homology based models may provide useful insights concerning differential agonist-receptor interactions at related receptors. The results also suggest that comparisons of CoMFA models for two structurally related receptors may be a fruitful approach for differential QSAR.


Subject(s)
Dopamine Agonists/chemistry , Models, Molecular , Receptors, Dopamine D1/agonists , Receptors, Dopamine D2/agonists , Animals , Binding Sites , Databases, Factual , Dopamine Agonists/metabolism , Dopamine Agonists/pharmacology , Humans , Ligands , Macaca mulatta , Molecular Conformation , Protein Structure, Secondary , Rats , Receptors, Dopamine D1/biosynthesis , Receptors, Dopamine D1/chemistry , Receptors, Dopamine D1/metabolism , Receptors, Dopamine D2/biosynthesis , Receptors, Dopamine D2/chemistry , Receptors, Dopamine D2/metabolism , Recombinant Proteins/agonists , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Structure-Activity Relationship , Tumor Cells, Cultured
11.
J Neurosci ; 18(21): 8692-9, 1998 Nov 01.
Article in English | MEDLINE | ID: mdl-9786976

ABSTRACT

D2L dopamine receptor activation results in rapid inhibition and delayed heterologous sensitization of adenylate cyclase in several host cell types. The D2L dopamine receptor was stably transfected into NS20Y neuroblastoma cells to examine inhibition and sensitization in a neuronal cell environment and to identify the particular G-proteins involved. Acute activation of D2L receptors with the selective D2 agonist quinpirole inhibited forskolin-stimulated cAMP accumulation, whereas prolonged incubation (2 hr) with quinpirole resulted in heterologous sensitization (more than twofold) of forskolin-stimulated cAMP accumulation in NS20Y-D2L cells. To unambiguously identify the pertussis toxin (PTX)-sensitive G-proteins responsible for inhibition and sensitization, we used viral-mediated gene delivery to assess the ability of genetically engineered PTX-resistant G-proteins (Galphai1*, Galphai2*, Galphai3*, and Galphao*) to rescue both responses after PTX treatment. The expression and function of individual recombinant G-proteins was confirmed with Western blotting and inhibition of GTPgammaS-stimulated adenylate cyclase, respectively. To assess the specificity of D2L-Galpha coupling, cells were infected with herpes simplex virus (HSV) recombinants expressing individual PTX-resistant G-protein alpha subunits and treated with PTX, and quinpirole-induced responses were measured. Infection of NS20Y-D2L cells with HSV-Galphao* rescued both inhibition and sensitization in PTX-treated cells, whereas infection with HSV-Galphai1*, HSV-Galphai2*, or HSV-Galphai3* failed to rescue either response. In summary, the current study provides strong evidence that the D2L dopamine receptor couples to Galphao in neuronal cells, and that this coupling is responsible for both the acute and subacute effects of D2 receptor activation on adenylate cyclase activity.


Subject(s)
GTP-Binding Proteins/physiology , Neurons/physiology , Receptors, Dopamine D2/physiology , Adenylate Cyclase Toxin , Animals , Colforsin/pharmacology , Cyclic AMP/metabolism , Enzyme Activation , GTP-Binding Proteins/metabolism , Genetic Vectors , Mice , Neuroblastoma , Pertussis Toxin , Quinpirole/pharmacology , Receptors, Dopamine D2/chemistry , Receptors, Dopamine D2/genetics , Simplexvirus/genetics , Transfection , Tumor Cells, Cultured , Virulence Factors, Bordetella/pharmacology
12.
Synapse ; 30(1): 97-106, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9704886

ABSTRACT

Drug-induced efflux of substrates was characterized in C6 rat glioma cells stably expressing a recombinant human dopamine (DA) or serotonin (5-HT) transporter (C6-hDAT and C6-hSERT, respectively). In the absence of Ca2+, these cells spontaneously and rapidly released preloaded [3H]DA or [3H]5-HT, respectively, but maintained constant levels of [3H]N-methy-4-phenylpyridinium (MPP+) for up to 90 minutes. In C6-hSERT cells, transporter substrates such as methamphetamine, amphetamine, and dopamine induced relatively rapid release of [3H]MPP+, with t1/2 values of approximately 15 minutes, while the t1/2 value for serotonin was about 30 minutes. Similar results were obtained with C6-hDAT cells. Uptake blockers that are not substrates at the transporters had considerably greater t1/2 values, as compared to substrates, suggesting different mechanisms for altering transporter function. Dose-response curves for each drug, conducted at each drug's t1/2, indicated considerable differences in potency (EC50) at stimulating [3H]MPP+ release from C6-hSERT cells [3beta-(4-iodophenyl)tropane-2beta-carboxylic acid methyl ester (RTI-55) > imipramine > 1-[2-diphenylmethoxy]ethyl-4-(3-phenylpropyl)-piperazine (GBR-12935) threo-(+/-)-methylphenidate > cocaine > mazindol > 2-beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT) > (+)methamphetamine > amphetamine > DA > fenfluramine > norepinephrine (NE) > 5-HT]. A different rank order of potency was observed for the effects of drugs on [3H]MPP+ release from C6-hDAT cells [imipramine > RTI-55 > cocaine > mazindol > CFT > GBR-12935 > threo-(+/-)-methylphenidate > amphetamine > (+)methamphetamine > fenfluramine > DA > NE > 5-HT]. Based on efficacies for stimulating [3H]MPP+ release from C6-hDAT cells, drugs could be grouped into three categories, with substrates causing release of approximately 75% of loaded [3H]MPP+, cocaine analogues causing approximately 50% release, and other drugs causing an average release of approximately 25% of loaded [3H]MPP+. The results, taken together with results from previous reports, suggest that the transfected cell type contributes to the characteristics of transporter-mediated release, that drugs interact with different sites on the transporters in the uptake and release process, and that the mechanism of transporter-mediated release may not be a simple reversal of substrate uptake.


Subject(s)
Carrier Proteins/metabolism , Dopamine Uptake Inhibitors/pharmacology , Dopamine/metabolism , Membrane Glycoproteins/metabolism , Membrane Transport Proteins , Nerve Tissue Proteins , Selective Serotonin Reuptake Inhibitors/pharmacology , Serotonin/metabolism , 1-Methyl-4-phenylpyridinium/metabolism , Animals , Biological Transport , Calcium/metabolism , Cloning, Molecular , Cocaine/pharmacology , Dopamine/pharmacology , Dopamine Plasma Membrane Transport Proteins , Glioma , Half-Life , Humans , Imipramine/pharmacology , Kinetics , Methamphetamine/pharmacology , Rats , Recombinant Proteins/metabolism , Serotonin/pharmacology , Serotonin Plasma Membrane Transport Proteins , Transfection , Tritium
13.
Mol Pharmacol ; 54(2): 435-44, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9687586

ABSTRACT

Dopamine D2 receptors contain a cluster of serine residues in the fifth transmembrane domain that contribute to activation of the receptor as well as to the binding of agonists. We used rat D2S dopamine receptor mutants, each containing a serine-to-alanine substitution (S193A, S194A, S197A), to investigate the mechanism through which these residues affect activation of the receptor. Activation of the mutant receptor S194A was abolished in an agonist-dependent manner, such that dopamine no longer inhibited cAMP accumulation in C6 glioma cells or activated G protein-regulated K+ channels in Xenopus laevis oocytes, whereas the efficacy of several other agonists was unaffected. Dihydrexidine did not inhibit cAMP accumulation at either S193A or S194A. The decreased efficacy of dihydrexidine at S193A and S194A and dopamine at S194A was associated with a decreased ability to detect a GTP-sensitive high affinity binding state for these agonists. The ability of dopamine to stimulate [35S]guanosine-5'-O-(3-thio)triphosphate binding via S194A also was decreased by approximately 50%. Finally, constitutive stimulation of [35S]guanosine-5'-O-(3-thio)triphosphate binding and inhibition of adenylate cyclase by the D2S receptor was reduced by mutation of either S193 or S194. These data support the existence of multiple active receptor conformations that are differentially sensitive to mutation of serine residues in the fifth-transmembrane domain.


Subject(s)
Receptors, Dopamine D2/metabolism , Serine/metabolism , Signal Transduction , Animals , Cyclic AMP/metabolism , Female , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Guanosine Triphosphate/metabolism , Isoproterenol/pharmacology , Mutagenesis , Oocytes/metabolism , Potassium Channels/agonists , Potassium Channels/metabolism , Protein Conformation , Rats , Receptors, Dopamine D2/agonists , Receptors, Dopamine D2/genetics , Sulfur Radioisotopes , Tumor Cells, Cultured , Xenopus laevis
14.
J Neurochem ; 71(3): 980-90, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9721723

ABSTRACT

Dopamine D2 receptors are members of the G protein-coupled receptor superfamily and are expressed on both neurons and astrocytes. Using rat C6 glioma cells stably expressing the rat D2L receptor, we show here that dopamine (DA) can activate both the extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) pathways through a mechanism involving D2 receptor-G protein complexes and the Ras GTP-binding protein. Agonist binding to D2 receptors rapidly activated both kinases within 5 min, reached a maximum between 10 and 15 min, and then gradually decreased by 60 min. Maximal activation of both kinases occurred with 100 nM DA, which produced a ninefold enhancement of ERK activity and a threefold enhancement of JNK activity. DA-induced kinase activation was prevented by either (+)-butaclamol, a selective D2 receptor antagonist, or pertussis toxin, an uncoupler of G proteins from receptors, but not by (-)-butaclamol, the inactive isomer of (+)-butaclamol. Cotransfection of RasN17, a dominant negative Ras mutant, prevented DA-induced activation of both ERK and JNK. PD098059, a specific MEK1 inhibitor, also blocked ERK activation by DA. Transfection of SEK1 (K --> R) vector, a dominant negative SEK1 mutant, specifically prevented DA-induced JNK activation and subsequent c-Jun phosphorylation without effect on ERK activation. Furthermore, stimulation of D2 receptors promoted [3H]thymidine incorporation with a pattern similar to that for kinase activation. DA mitogenesis was tightly linked to Ras-dependent mitogen-activated protein kinase (MAPK) and JNK pathways. Transfection with RasN17 and application of PD098059 blocked DA-induced DNA synthesis. Transfection with Flag delta169, a dominant negative c-Jun mutant, also prevented stimulation of [3H]thymidine incorporation by DA. The demonstration of D2 receptor-stimulated MAPK pathways may help to understand dopaminergic physiological functions in the CNS.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , GTP-Binding Proteins/physiology , Mitogen-Activated Protein Kinases , Mitosis/physiology , Pertussis Toxin , Receptors, Dopamine D2/physiology , Virulence Factors, Bordetella/pharmacology , Animals , Dopamine/pharmacology , Enzyme Activation/drug effects , Enzyme Activation/physiology , Flavonoids/pharmacology , GTP-Binding Proteins/drug effects , GTPase-Activating Proteins , JNK Mitogen-Activated Protein Kinases , Mutation , Proteins/physiology , Rats , Recombinant Proteins , Thymidine/metabolism , Tumor Cells, Cultured , ras GTPase-Activating Proteins , ras Proteins/genetics , ras Proteins/pharmacology , ras Proteins/physiology
15.
J Neurosci ; 18(11): 4119-32, 1998 Jun 01.
Article in English | MEDLINE | ID: mdl-9592092

ABSTRACT

The modulation of motor behavior by protein kinase C (PKC) signaling pathways in nigrostriatal neurons was examined by using a genetic intervention approach. Herpes simplex virus type 1 (HSV-1) vectors that encode a catalytic domain of rat PKCbetaII (PkcDelta) were developed. PkcDelta exhibited a constitutively active protein kinase activity with a substrate specificity similar to that of rat brain PKC. As demonstrated in cultured sympathetic neurons, PkcDelta caused a long-lasting, activation-dependent increase in neurotransmitter release. In the rat brain, microinjection of HSV-1 vectors that contain the tyrosine hydroxylase promoter targeted expression to dopaminergic nigrostriatal neurons. Expression of pkcDelta in a small percentage of nigrostriatal neurons (approximately 0.1-2%) was sufficient to produce a long-term (>/=1 month) change in apomorphine-induced rotational behavior. Nigrostriatal neurons were the only catecholaminergic neurons that contained PkcDelta, and the amount of rotational behavior was correlated with the number of affected nigrostriatal neurons. The change in apomorphine-induced rotational behavior was blocked by a dopamine receptor antagonist (fluphenazine). D2-like dopamine receptor density was increased in those regions of the striatum innervated by the affected nigrostriatal neurons. Therefore, this strategy enabled the demonstration that a PKC pathway or PKC pathways in nigrostriatal neurons modulate apomorphine-induced rotational behavior, and altered dopaminergic transmission from nigrostriatal neurons appears to be the affected neuronal physiology responsible for the change in rotational behavior.


Subject(s)
Behavior, Animal/physiology , Gene Transfer Techniques , Herpesvirus 1, Human , Neurons/enzymology , Protein Kinase C/genetics , Animals , Apomorphine , Behavior, Animal/drug effects , Catecholamines/metabolism , Corpus Striatum/cytology , DNA, Viral/analysis , Dopamine Agonists , Fibroblasts/physiology , Gene Expression Regulation, Enzymologic , Mesencephalon , Point Mutation , RNA, Messenger/analysis , Rabbits , Rats , Recombinant Proteins/genetics , Rotation , Substantia Nigra/cytology , Substrate Specificity , Synaptic Transmission/drug effects
16.
Mol Pharmacol ; 52(6): 1137-49, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9396784

ABSTRACT

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.


Subject(s)
Receptors, Dopamine D1/physiology , Receptors, Dopamine D2/physiology , Recombinant Fusion Proteins/physiology , Cell Line , Cyclic AMP/metabolism , DNA, Complementary/genetics , Dopamine/metabolism , Dopamine/pharmacology , Dopamine Agonists/metabolism , Dopamine Agonists/pharmacology , Dopamine Antagonists/metabolism , Dopamine Antagonists/pharmacology , Humans , Isomerism , Kinetics , Ligands , Mutation , Protein Conformation , Receptors, Dopamine D1/drug effects , Receptors, Dopamine D1/genetics , Receptors, Dopamine D2/drug effects , Receptors, Dopamine D2/genetics , Recombinant Fusion Proteins/genetics
17.
J Neurochem ; 69(4): 1459-66, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9326274

ABSTRACT

To determine if catechol-O-methyltransferase (COMT) metabolizes catecholamines within cell lines used for heterologous expression of plasmalemmal transporters and alters the measured characteristics of 3H-substrate transport, the uptake of monoamine transporter substrates was assessed in three cell lines (C6 glioma, L-M fibroblast, and HEK293 cells) that had been transfected with the recombinant human transporters. Uptake and cellular retention of 3H-catecholamines was increased by up to fourfold by two COMT inhibitors, tropolone and Ro 41-0960, with potencies similar to those for inhibition of COMT activity, whereas the uptake of two transporter substrates that are not substrates for COMT, [3H]serotonin and [3H]MPP+, was unaffected. Direct measurement of monoamine substrates by HPLC confirmed that tropolone (1 mM) increased the retention of the catecholamines dopamine and norepinephrine, but not the retention of serotonin in HEK293 cells. Saturation analysis of the uptake of [3H]dopamine by C6 cells expressing the dopamine transporter demonstrated that tropolone (1 mM) decreased the apparent Km of transport from 0.61 microM to 0.34 microM without significantly altering the maximal velocity of transport. These data suggest that endogenous COMT activity in mammalian cells may alter neurotransmitter deposition and thus the apparent kinetic characteristics of transport.


Subject(s)
Carrier Proteins/metabolism , Catechol O-Methyltransferase/metabolism , Catecholamines/metabolism , Membrane Transport Proteins , Catechol O-Methyltransferase Inhibitors , Catecholamine Plasma Membrane Transport Proteins , Cell Line , Chromatography, High Pressure Liquid , Dopamine/pharmacokinetics , Electrochemistry/methods , Enzyme Inhibitors/pharmacology , Humans , Norepinephrine/pharmacokinetics , Recombinant Proteins/metabolism , Time Factors
18.
Mol Pharmacol ; 52(2): 181-6, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9271339

ABSTRACT

The D2-like dopamine receptors couple to a variety of signal transduction pathways, including inhibition of adenylate cyclase, mitogenesis, and activation of potassium channels. Although these effects are mediated via pertussis toxin-sensitive G proteins, G(i/o), it is likely that some of these effects are influenced by the release of G protein betagamma subunits. Type II adenylate cyclase (ACII) is highly regulated by multiple biochemical stimuli, including protein kinase C, forskolin, G protein alpha subunits, and G protein betagamma subunits. The ability of betagamma subunits to activate this enzyme in the presence of activated alpha(s) has been particularly well characterized. Although stimulation by betagamma subunits has been described as conditional on the presence of activated alpha(s), betagamma subunits also potentiate ACII activity after activation of protein kinase C. We created stable cell lines expressing ACII and the D2L receptor, the D3 receptor, or the D4.4 receptor. Activation of D2L or D4.4 receptors, but not D3 receptors, potentiated beta-adrenergic receptor/Gs-stimulated activity of ACII, as measured by the intracellular accumulation of cAMP. Similarly, stimulation of D2L or D4.4 receptors potentiated phorbol ester-stimulated ACII activity in the absence of activated alpha(s), whereas stimulation of D3 receptors did not. The effect of D2-like receptor stimulation was blocked by pretreatment with pertussis toxin and by inhibition of protein kinase C. We propose that activation of both D2L and D4.4 dopamine receptors potentiated phorbol-12-myristate-13-acetate-stimulated ACII activity through the release of betagamma subunits from pertussis toxin-sensitive G proteins. In contrast, the lack of D3 receptor-mediated effects suggests that stimulation of D3 receptors does not result in an appreciable release of betagamma subunits.


Subject(s)
Adenylyl Cyclases/metabolism , Receptors, Dopamine D2/physiology , Cell Line , Cyclic AMP/metabolism , Dopamine/pharmacology , Drug Synergism , Enzyme Activation , Humans , Isoproterenol/pharmacology , Protein Kinase C/physiology , Quinpirole/pharmacology , Receptors, Dopamine D3 , Receptors, Dopamine D4 , Tetradecanoylphorbol Acetate/pharmacology , Transfection
19.
Mol Pharmacol ; 50(4): 966-76, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8863843

ABSTRACT

Prolonged stimulation of Gi-coupled receptors often sensitizes adenylate cyclase to subsequent activation by forskolin or Gs-coupled receptors. To identify mechanisms of heterologous sensitization, we characterized sensitization of cAMP accumulation that was induced by activation of recombinant dopamine D2 receptors expressed in C6 glioma and human embryonic kidney (HEK)293 cells. Pretreatment with dopamine or other agonists for 2 hr induced heterologous sensitization that was blocked by the D2 antagonist spiperone but not by the beta-adrenergic receptor antagonist propranolol. Sensitization was evident after 15 min of treatment with dopamine and persisted for at least 2 hr after washout. The EC50 value for sensitization by dopamine in HEK-D2L cells was 100 nM(r) approximately 80-fold higher than the IC50 value for dopamine inhibition of cAMP accumulation. The D2 receptor agonists quinpirole, 7-hydroxy-dipropylamin-otetralin, and pergolide also induced sensitization, whereas the high affinity ergot agonists bromocriptine and lisuride did not. Stimulation of either D2L or D2S receptors sensitized cAMP accumulation to similar extents, but stimulation of D3 receptors did not. In C6-D21 cells, sensitization of isoproterenol-stimulated activity was manifested as a > 100% increase in maximal response, with no change in potency. In contrast, the potency for forskolin-stimulated activity was increased 4-fold, with no apparent change in maximal response. Overnight treatment with pertussis toxin (25 ng/ml) had little effect on isoproterenol or forskolin activation of adenylate cyclase per se but prevented D2 receptor-mediated sensitization in both C6-D2L and HEK-D2L cells, indicating an involvement of one or more of the pertussis toxin-sensitive G proteins, Gi/Gzero. D2 receptor stimulation also sensitized type I and type II adenylate cyclases, each expressed in HEK293 cells together with D2L dopamine receptors. Rapid D2 receptor-mediated heterologous sensitization may be the result of enhanced interaction of G6 with adenylate cyclase and may represent a novel mechanism for modulation of neural activity by D2 receptors.


Subject(s)
Adenylyl Cyclases/physiology , Isoenzymes/physiology , Receptors, Dopamine D2/physiology , Adenylyl Cyclases/metabolism , Adenylyl Cyclases/pharmacology , Cells, Cultured , Colforsin/pharmacology , Cyclic AMP/metabolism , Dopamine Agonists/pharmacology , Dopamine Antagonists/pharmacology , Dopamine D2 Receptor Antagonists , Humans , Isoproterenol/pharmacology , Kinetics , Receptors, Dopamine D2/agonists , Recombinant Proteins/pharmacology , Second Messenger Systems/drug effects , Second Messenger Systems/physiology , Sensitivity and Specificity , Spiperone/pharmacology , Stimulation, Chemical , Tetrahydronaphthalenes/pharmacology
20.
J Med Chem ; 39(4): 850-9, 1996 Feb 16.
Article in English | MEDLINE | ID: mdl-8632409

ABSTRACT

Determination of quantitative structure-activity relationship (QSAR) for affinity at particular dopamine (DA) receptors has become an even greater priority with the cloning of five DA receptor subtypes. The use of agonist affinity at recombinant receptors selectively expressed in clonal cells as the dependent variable in QSAR presents a unique opportunity for accuracy and precision in measurement of biological values. Bound conformations of 11 agonists (for which both affinity data at the recombinant D1A DA receptor and stereochemical configurations were available) were determined by alignment with a template compound, SKF38393, which shows high affinity and selectivity for D1A receptors and is fairly rigid in structure. These aligned structures suggested a 3-point pharmacophore map (one cationic nitrogen and two electronegative centers) of the D1A DA receptor. This map shows both similarities and differences when compared with a previously reported D2 DA receptor pharmacophore map based on biological data from rat brain and with a recently published map of the native D1 DA receptor using several semirigid compounds. Log(1/K(d)) values at recombinant D1A DA receptors were used as the target property for a CoMFA (comparative molecular field analysis) of the 11 aligned structures. The resulting CoMFA model yielded a cross-validated r(2)(q(2)) value of 0.829 and a simple r(2) = 0.96. In contrast, when a CoMFA model was developed for 10 of these compounds using striatal D1 K(d) values, the q(2) value was reduced to 0.178. These results are consistent with the idea that drug affinity data obtained from clonal cells expressing recombinant receptors may be superior to that obtained using heterogeneous mixtures of native receptors prepared from brain membranes. The predictive utility of the CoMFA model was evaluated using several high-affinity dopamine agonists and m- and p-tyramine, two compounds with a single hydroxyl group on the aromatic ring. Predictions were fairly accurate for all compounds but the two tyramines.


Subject(s)
Dopamine Agonists/chemistry , Dopamine Agonists/metabolism , Dopamine/analogs & derivatives , Dopamine/metabolism , Receptors, Dopamine D1/metabolism , Recombinant Proteins/metabolism , Animals , Cell Line , Chlorocebus aethiops , Computer Graphics , Dopamine/chemistry , Glioma , Humans , Kinetics , Models, Molecular , Structure-Activity Relationship , Tumor Cells, Cultured
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