Most central nervous system D2 dopamine receptors are coupled to their effectors by Go (original) (raw)
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Life Sciences, 1999
The dopamine receptor-mediated modulation of guanosine 5'-O-(gamma-[35S]thio)triphosphate ([35S]GTP gamma S) binding has been characterized in rat striatal membranes. In optimized experimental conditions, the potency of dopamine was 4.47 microM [3.02-6.61 microM] and a maximal response representing 54.8 +/- 4.5% increase above basal level was observed. Data obtained with different agonists and antagonists clearly revealed that the most important fraction of this response was reflecting D2 receptor activation. Further analysis with specific antagonists also supported evidence for the involvement of D1 dopamine receptors. The potencies of compounds interacting with D1 and D2 receptors were deduced from [35S]GTP gamma S binding experiments and compared with their binding affinities for these receptors measured in similar experimental conditions. A good correlation between these parameters was observed, supporting the applicability of this technique for the study of dopamine receptors in the central nervous system.
Stimulated D1 dopamine receptors couple to multiple Gα proteins in different brain regions
Journal of Neurochemistry, 2001
Previous studies have revealed that activation of rat striatal D 1 dopamine receptors stimulates both adenylyl cyclase and phospholipase C via G s and G q , respectively. The differential distribution of these systems in brain supports the existence of distinct receptor systems. The present communication extends the study by examining other brain regions: hippocampus, amygdala, and frontal cortex. In membrane preparations of these brain regions, selective stimulation of D 1 dopamine receptors increases the hydrolysis of phosphatidylinositol/phosphatidylinositol 4,5-biphosphate. In these brain regions, D 1 dopamine receptors couple differentially to multiple Ga protein subunits. Antisera against Ga q blocks dopamine-stimulated PIP 2 hydrolysis in hippocampal and in striatal membranes. The binding of [ 35 S]GTPgS or [a-32 P]GTP to Ga i was enhanced in all brain regions. Dopamine also increased the binding of [ 35 S]GTPgS or [a-32 P]GTP to Ga q in these brain regions: hippocampus amygdala . frontal cortex. However, dopamine-stimulated binding of [ 35 S]GTPgS to Gas only in the frontal cortex and striatum. This differential coupling pro®le in the brain regions was not related to a differential regional distribution of the Ga proteins. Dopamine induced increases in GTPgS binding to Ga s and Ga q was blocked by the D 1 antagonist SCH23390 but not by D 2 receptor antagonist l-sulpiride, suggesting that D 1 dopamine receptors couple to both Ga s and Ga q proteins. Co-immunoprecipitation of Ga proteins with receptor-binding sites indicate that in the frontal cortex, D 1 dopamine-binding sites are associated with both Ga s and Ga q and, in hippocampus or amygdala, D 1 dopamine receptors couple solely to Ga q . The results indicate that in addition to the D 1 /G s / adenylyl cyclase system, brain D 1 -like dopamine receptor sites activate phospholipase C through Ga q protein.
European Neuropsychopharmacology, 2004
The C-terminus domain of G protein-coupled receptors confers a functional cytoplasmic interface involved in protein association. By screening a rat brain cDNA library using the yeast two-hybrid system with the C-terminus domain of the dopamine D 3 receptor (D 3 R) as bait, we characterized a new interaction with the PDZ domain-containing protein, GIPC (GAIP interacting protein, C terminus). This interaction was specific for the dopamine D 2 receptor (D 2 R) and D 3 R, but not for the dopamine D 4 receptor (D 4 R) subtype. Pull-down and affinity chromatography assays confirmed this interaction with recombinant and endogenous proteins. Both GIPC mRNA and protein are widely expressed in rat brain and together with the D 3 R in neurons of the islands of Calleja at plasma membranes and in vesicles. GIPC reduced D 3 R signaling, cointernalized with D 2 R and D 3 R, and sequestered receptors in sorting vesicles to prevent their lysosomal degradation. Through its dimerization, GIPC acts as a selective scaffold protein to assist receptor functions. Our results suggest a novel function for GIPC in the maintenance, trafficking, and signaling of GPCRs.
GIPC Recruits GAIP (RGS19) To Attenuate Dopamine D2 Receptor Signaling
Molecular Biology of the Cell, 2004
Pleiotropic G proteins are essential for the action of hormones and neurotransmitters and are activated by stimulation of G protein-coupled receptors (GPCR), which initiates heterotrimer dissociation of the G protein, exchange of GDP for GTP on its G␣ subunit and activation of effector proteins. Regulator of G protein signaling (RGS) proteins regulate this cascade and can be recruited to the membrane upon GPCR activation. Direct functional interaction between RGS and GPCR has been hypothesized. We show that recruitment of GAIP (RGS19) by the dopamine D 2 receptor (D 2 R), a GPCR, required the scaffold protein GIPC (GAIP-interacting protein, C terminus) and that all three were coexpressed in neurons and neuroendocrine cells.
Molecular Biology of the Cell, 2003
The C-terminus domain of G protein-coupled receptors confers a functional cytoplasmic interface involved in protein association. By screening a rat brain cDNA library using the yeast two-hybrid system with the C-terminus domain of the dopamine D 3 receptor (D 3 R) as bait, we characterized a new interaction with the PDZ domain-containing protein, GIPC (GAIP interacting protein, C terminus). This interaction was specific for the dopamine D 2 receptor (D 2 R) and D 3 R, but not for the dopamine D 4 receptor (D 4 R) subtype. Pull-down and affinity chromatography assays confirmed this interaction with recombinant and endogenous proteins. Both GIPC mRNA and protein are widely expressed in rat brain and together with the D 3 R in neurons of the islands of Calleja at plasma membranes and in vesicles. GIPC reduced D 3 R signaling, cointernalized with D 2 R and D 3 R, and sequestered receptors in sorting vesicles to prevent their lysosomal degradation. Through its dimerization, GIPC acts as a selective scaffold protein to assist receptor functions. Our results suggest a novel function for GIPC in the maintenance, trafficking, and signaling of GPCRs.
Neuroscience Letters, 2001
The cross regulation of agonist binding to D 2 dopamine receptors and guanosine nucleotide binding to G proteins was studied using membranes of Chinese hamster ovary cells expressing rat D 2short dopamine receptors. All guanosine nucleotides studied caused a concentration-dependent loss of high-af®nity agonist binding sites of D 2 receptors with potencies corresponding to their af®nity to bind to G proteins in these membranes. On the other hand, the dopaminergic agonists, but not antagonists, decreased the af®nities of guanosine diphosphate and guanosine monophosphate, but not of guanosine 5 H -(g-thiotriphosphate). The cross regulation of ligand binding to D 2 dopamine receptors and G proteins suggests the existence of several conformational states of these proteins during the signal transduction and that the high-af®nity state of agonist binding is a transient state of the agonist-receptor-G protein complex, where no nucleotides are bound. q
Molecular pharmacology, 1999
Despite extensive study, the G protein coupling of dopamine D3 receptors is poorly understood. In this study, we used guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]-GTPgammaS) binding to investigate the activation of G proteins coupled to human (h) D3 receptors stably expressed in Chinese hamster ovary (CHO) cells. Although the receptor expression level was high (15 pmol/mg), dopamine only stimulated G protein activation by 1.6-fold. This was despite the presence of marked receptor reserve for dopamine, as revealed by Furchgott analysis after irreversible hD3 receptor inactivation with the alkylating agent, EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline). Thus, half-maximal stimulation of [35S]-GTPgammaS binding required only 11.8% receptor occupation of hD3 sites. In contrast, although the hD2(short) receptor expression level in another CHO cell line was 11-fold lower, stimulation by dopamine was higher (2.5-fold). G protein activation was increased at hD3 and, less pot...
Stimulated D(1) dopamine receptors couple to multiple Galpha proteins in different brain regions
Journal of Neurochemistry
Previous studies have revealed that activation of rat striatal D 1 dopamine receptors stimulates both adenylyl cyclase and phospholipase C via G s and G q , respectively. The differential distribution of these systems in brain supports the existence of distinct receptor systems. The present communication extends the study by examining other brain regions: hippocampus, amygdala, and frontal cortex. In membrane preparations of these brain regions, selective stimulation of D 1 dopamine receptors increases the hydrolysis of phosphatidylinositol/phosphatidylinositol 4,5-biphosphate. In these brain regions, D 1 dopamine receptors couple differentially to multiple Ga protein subunits. Antisera against Ga q blocks dopamine-stimulated PIP 2 hydrolysis in hippocampal and in striatal membranes. The binding of [ 35 S]GTPgS or [a-32 P]GTP to Ga i was enhanced in all brain regions. Dopamine also increased the binding of [ 35 S]GTPgS or [a-32 P]GTP to Ga q in these brain regions: hippocampus amygdala . frontal cortex. However, dopamine-stimulated binding of [ 35 S]GTPgS to Gas only in the frontal cortex and striatum. This differential coupling pro®le in the brain regions was not related to a differential regional distribution of the Ga proteins. Dopamine induced increases in GTPgS binding to Ga s and Ga q was blocked by the D 1 antagonist SCH23390 but not by D 2 receptor antagonist l-sulpiride, suggesting that D 1 dopamine receptors couple to both Ga s and Ga q proteins. Co-immunoprecipitation of Ga proteins with receptor-binding sites indicate that in the frontal cortex, D 1 dopamine-binding sites are associated with both Ga s and Ga q and, in hippocampus or amygdala, D 1 dopamine receptors couple solely to Ga q . The results indicate that in addition to the D 1 /G s / adenylyl cyclase system, brain D 1 -like dopamine receptor sites activate phospholipase C through Ga q protein.
Methods in molecular biology (Clifton, N.J.), 2013
In mammals, dopamine G protein-coupled receptors (GPCR) are segregated into two categories: D1-like (D1R and D5R) and D2-like (D2R(short), D2R(long), D3R, and D4R) subtypes. D1R and D5R are primarily coupled to stimulatory heterotrimeric GTP-binding proteins (Gs/olf) leading to activation of adenylyl cyclase and production of intracellular cAMP. D1R and D5R share high level of amino acid identity in transmembrane (TM) regions. Yet these two GPCR subtypes display distinct ligand binding and G protein coupling properties. In fact, our studies suggest that functional properties reported for constitutively active mutants of GPCRs (e.g., increased basal activity, higher agonist affinity and intrinsic activity) are also observed in cells expressing wild type D5R when compared with wild type D1R. Herein, we describe an experimental method based on mutagenesis and transfection of human embryonic kidney 293 (HEK293) cells to explore the molecular mechanisms regulating ligand affinity, agonis...