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1.
J Biol Chem ; 294(14): 5604-5615, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30670597

RESUMO

Most clinically available antipsychotic drugs (APDs) bind dopamine D2 receptors (D2R) at therapeutic concentrations, and it is thought that they suppress psychotic symptoms by serving as competitive antagonists of dopamine at D2R. Here, we present data that demonstrate that APDs act independently of dopamine at an intracellular pool of D2R to enhance transport of D2R to the cell surface and suggest that APDs can act as pharmacological chaperones at D2R. Among the first- and second-generation APDs that we tested, clozapine exhibited the lowest efficacy for translocating D2R to the cell surface. Thus, our observations could provide a cellular explanation for some of the distinct therapeutic characteristics of clozapine in schizophrenia. They also suggest that differential intracellular actions of APDs at their common G protein-coupled receptor (GPCR) target, D2R, could contribute to differences in their clinical profiles.


Assuntos
Antipsicóticos/farmacologia , Clozapina/farmacologia , Receptores de Dopamina D2/metabolismo , Dopamina/metabolismo , Células HEK293 , Humanos , Transporte Proteico/efeitos dos fármacos , Receptores de Dopamina D2/genética
2.
Front Biosci (Elite Ed) ; 10: 175-196, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-28930612

RESUMO

The interaction of neurotransmitters and genes that control the release of dopamine is the Brain Reward Cascade (BRC). Variations within the BRC, whether genetic or epigenetic, may predispose individuals to addictive behaviors and altered pain tolerance. This discussion authored by a group of concerned scientists and clinicians examines the Genetic Addiction Risk Score (GARS), the first test to accurately predict vulnerability to pain, addiction, and other compulsive behaviors, defined as Reward Deficiency Syndrome (RDS). Innovative strategies to combat epidemic opioid, iatrogenic prescription drug abuse and death, based on the role of dopaminergic tone in pain pathways, are proposed. Sensitivity to pain may reside in the mesolimbic projection system, where genetic polymorphisms associate with a predisposition to pain vulnerability or tolerance. They provide unique therapeutic targets that could assist in the treatment of pain, and identify risk for subsequent addiction. Pharmacogenomic testing of candidate genes like CB1, mu receptors, and PENK might result in pharmacogenomic, personalized solutions, and improved clinical outcomes. Genetically identifying risk for all RDS behaviors, especially in compromised populations, may be a frontline tool to assist municipalities to provide better resource allocation.


Assuntos
Predisposição Genética para Doença , Transtornos Relacionados ao Uso de Opioides/genética , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/efeitos adversos , Dopamina/metabolismo , Humanos , Transtornos Relacionados ao Uso de Opioides/complicações , Dor/genética , Manejo da Dor/métodos , Farmacogenética , Estresse Psicológico/complicações
3.
Org Biomol Chem ; 13(9): 2537-40, 2015 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-25592739

RESUMO

The development of a ß-CCT-lanthanide conjugate that binds the dopamine transporter (DAT) with high affinity (K(d) = 303 nM) is described. Contrast agents such as the one described herein could be used as molecular probes to directly study the binding of small molecules to receptors such as DAT via MRI, PET or SPECT.


Assuntos
Carbolinas/química , Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Elementos da Série dos Lantanídeos/química , Compostos Organometálicos/química , Sítios de Ligação , Células HEK293 , Humanos , Estrutura Molecular , Compostos Organometálicos/síntese química
4.
PLoS One ; 9(8): e105791, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25162404

RESUMO

G beta 5 (Gbeta5, Gß5) is a unique G protein ß subunit that is thought to be expressed as an obligate heterodimer with R7 regulator of G protein signaling (RGS) proteins instead of with G gamma (Gγ) subunits. We found that D2-dopamine receptor (D2R) coexpression enhances the expression of Gß5, but not that of the G beta 1 (Gß1) subunit, in HEK293 cells, and that the enhancement of expression occurs through a stabilization of Gß5 protein. We had previously demonstrated that the vast majority of D2R either expressed endogenously in the brain or exogenously in cell lines segregates into detergent-resistant biochemical fractions. We report that when expressed alone in HEK293 cells, Gß5 is highly soluble, but is retargeted to the detergent-resistant fraction after D2R coexpression. Furthermore, an in-cell biotin transfer proximity assay indicated that D2R and Gß5 segregating into the detergent-resistant fraction specifically interacted in intact living cell membranes. Dopamine-induced D2R internalization was blocked by coexpression of Gß5, but not Gß1. However, the same Gß5 coexpression levels had no effect on agonist-induced internalization of the mu opioid receptor (MOR), cell surface D2R levels, dopamine-mediated recruitment of ß-arrestin to D2R, the amplitude of D2R-G protein coupling, or the deactivation kinetics of D2R-activated G protein signals. The latter data suggest that the interactions between D2R and Gß5 are not mediated by endogenously expressed R7 RGS proteins.


Assuntos
Dopamina/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Receptores de Dopamina D2/metabolismo , Arrestinas/metabolismo , Proteínas de Transporte/metabolismo , Detergentes/farmacologia , Células HEK293 , Humanos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular , Octoxinol/farmacologia , Estabilidade Proteica , Receptores Opioides mu/metabolismo , beta-Arrestinas
5.
J Immunol ; 191(11): 5524-41, 2013 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-24184556

RESUMO

How autoantibodies target the brain and lead to disease in disorders such as Sydenham chorea (SC) is not known. SC is characterized by autoantibodies against the brain and is the main neurologic manifestation of streptococcal-induced rheumatic fever. Previously, our novel SC-derived mAb 24.3.1 was found to recognize streptococcal and brain Ags. To investigate in vivo targets of human mAb 24.3.1, VH/VL genes were expressed in B cells of transgenic (Tg) mice as functional chimeric human VH 24.3.1-mouse C-region IgG1(a) autoantibody. Chimeric human-mouse IgG1(a) autoantibody colocalized with tyrosine hydroxylase in the basal ganglia within dopaminergic neurons in vivo in VH 24.3.1 Tg mice. Both human mAb 24.3.1 and IgG1(a) in Tg sera were found to react with human dopamine D2 receptor (D2R). Reactivity of chorea-derived mAb 24.3.1 or SC IgG with D2R was confirmed by dose-dependent inhibitory signaling of D2R as a potential consequence of targeting dopaminergic neurons, reaction with surface-exposed FLAG epitope-tagged D2R, and blocking of Ab reactivity by an extracellular D2R peptide. IgG from SC and a related subset of streptococcal-associated behavioral disorders called "pediatric autoimmune neuropsychiatric disorder associated with streptococci" (PANDAS) with small choreiform movements reacted in ELISA with D2R. Reaction with FLAG-tagged D2R distinguished SC from PANDAS, whereas sera from both SC and PANDAS induced inhibitory signaling of D2R on transfected cells comparably to dopamine. In this study, we define a mechanism by which the brain may be altered by Ab in movement and behavioral disorders.


Assuntos
Coreia/imunologia , Neurônios Dopaminérgicos/metabolismo , Receptores de Dopamina D2/metabolismo , Febre Reumática/imunologia , Infecções Estreptocócicas/imunologia , Animais , Antígenos de Bactérias/imunologia , Autoanticorpos/genética , Autoanticorpos/metabolismo , Gânglios da Base/patologia , Criança , Coreia/etiologia , Reações Cruzadas , Dopamina/metabolismo , Neurônios Dopaminérgicos/imunologia , Gangliosídeo G(M1)/análogos & derivados , Gangliosídeo G(M1)/imunologia , Células HEK293 , Humanos , Imunoglobulina G/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Dopamina D2/genética , Proteínas Recombinantes de Fusão/genética , Febre Reumática/etiologia , Transdução de Sinais , Infecções Estreptocócicas/complicações , Transgenes/genética
6.
J Neurochem ; 127(1): 57-65, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23815307

RESUMO

We reconstituted D2 like dopamine receptor (D2R) and the delta opioid receptor (DOR) coupling to G-protein gated inwardly rectifying potassium channels (K(ir)3) and directly compared the effects of co-expression of G-protein coupled receptor kinase (GRK) and arrestin on agonist-dependent desensitization of the receptor response. We found, as described previously, that co-expression of a GRK and an arrestin synergistically increased the rate of agonist-dependent desensitization of DOR. In contrast, only arrestin expression was required to produce desensitization of D2R responses. Furthermore, arrestin-dependent GRK-independent desensitization of D2R-K(ir)3 coupling could be transferred to DOR by substituting the third cytoplasmic loop of DOR with that of D2R. The arrestin-dependent GRK-independent desensitization of D2R desensitization was inhibited by staurosporine treatment, and blocked by alanine substitution of putative protein kinase C phosphorylation sites in the third cytoplasmic loop of D2R. Finally, the D2R construct in which putative protein kinase C phosphorylation sites were mutated did not undergo significant agonist-dependent desensitization even after GRK co-expression, suggesting that GRK phosphorylation of D2R does not play an important role in uncoupling of the receptor.


Assuntos
Arrestina/fisiologia , Quinases de Receptores Acoplados a Proteína G/metabolismo , Receptores de Dopamina D2/metabolismo , Animais , Arrestinas/fisiologia , Clonagem Molecular , Citoplasma/metabolismo , DNA Complementar/biossíntese , DNA Complementar/genética , Fenômenos Eletrofisiológicos , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Oócitos/metabolismo , RNA Complementar/biossíntese , RNA Complementar/genética , Estaurosporina/farmacologia , Xenopus , beta-Arrestinas
7.
J Biol Chem ; 288(18): 12554-68, 2013 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-23493394

RESUMO

Plasma membrane microcompartments could allow different signaling pathways to operate more efficiently and prevent cross-talk. We utilized a novel in-cell biotin transfer assay to demonstrate that the majority of plasma membrane-expressed D2 dopamine receptor (D2R) is microcompartmentalized within detergent-resistant structures. Conversely, a minority of D2R existed in a detergent-soluble form and interacted in a relatively unrestricted manner with other cellular proteins. The microcompartmentalization of D2R had functional consequences because dopamine-induced internalization of D2R was largely restricted to the compartmentalized receptor. The D2R-containing microcompartments did not correspond to putative detergent-resistant lipid raft structures. First, the detergent-insoluble D2R structures were significantly denser than detergent-resistant membrane fragments containing flotillin, a widely utilized lipid raft marker protein. Second, the detergent solubility of D2R was unaffected by treatment of cells with the cholesterol chelating agent, methyl-ß-cyclodextrin, that is thought to disrupt lipid rafts. Finally, the in-cell biotinylation assay did not provide any evidence for the membrane compartmentalization of peptide motifs thought to target to lipid rafts. Thus, our observations form one of the first demonstrations, in living cells, of plasma membrane microcompartments defined by the ability of the compartment structure to broadly restrict the interaction of resident molecules with other cellular proteins.


Assuntos
Microdomínios da Membrana/metabolismo , Receptores de Dopamina D2/metabolismo , Motivos de Aminoácidos , Animais , Células HEK293 , Humanos , Microdomínios da Membrana/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Receptores de Dopamina D2/genética , beta-Ciclodextrinas/farmacologia
8.
Islets ; 4(5): 343-8, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23018249

RESUMO

Adenylyl cyclase 3 (AC3) is expressed in pancreatic islets of the Goto-Kakizaki (GK) rat, a spontaneous animal model of type 2 diabetes (T2D), and also exerts genetic effects on the regulation of body weight in man. In addition to pancreatic islets, the central nervous system (CNS) plays an important role in the pathogenesis of T2D and obesity by regulating feeding behavior, body weight and glucose metabolism. In the present study, we have investigated AC3 expression in pancreatic islets, striatum and hypothalamus of GK rats to evaluate its role in the regulation of glucose homeostasis. GK and Wistar rats at the age of 2.5 mo were used. A group of GK rats were implanted with sustained insulin release chips for 15 d. Plasma glucose and serum insulin levels were measured. AC3 gene expression levels in pancreatic islets, striatum and hypothalamus were determined by using real-time RT-PCR. Results indicated that plasma glucose levels in Wistar rats were found to be similar to insulin-treated GK rats, and significantly lower compared with non-treated GK rats. AC3 expression levels in pancreatic islets, striatum and hypothalamus of GK rats were higher compared with Wistar rats, while the levels were intermediate in insulin-treated GK rats. The AC3 expression display patterns between pancreatic islets and striatum-hypothalamus were similar. The present study thus provides the first evidence that AC3 is overexpressed in the regions of striatum and hypothalamus of brain, and similarly in pancreatic islets of GK rats suggesting that AC3 plays a role in regulation of glucose homeostasis via CNS and insulin secretion.


Assuntos
Adenilil Ciclases/fisiologia , Encéfalo/enzimologia , Diabetes Mellitus Tipo 2/enzimologia , Glucose/metabolismo , Homeostase , Ilhotas Pancreáticas/enzimologia , Adenilil Ciclases/genética , Animais , Insulina/metabolismo , Secreção de Insulina , Masculino , Proteínas RGS/genética , Proteínas RGS/fisiologia , RNA Mensageiro/análise , Ratos , Ratos Wistar
9.
J Neurochem ; 120(1): 56-69, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22035199

RESUMO

Detergent-resistant membranes (DRM) are thought to contain structures such as lipid rafts that are involved in compartmentalizing cell membranes. We report that the majority of D(2)-dopamine receptors (D(2)R) expressed endogenously in mouse striatum or expressed in immortalized cell-lines is found in DRM. In addition, exogenous co-expression of D(2)R in a cell line shifted the expression of regulator of G protein signaling 9-2 (RGS9-2) into DRM. RGS9-2 is a protein that is highly enriched in the striatum and specifically regulates striatal D(2)R. In the striatum, RGS9-2 is mostly associated with DRMs but when expressed in cell lines, RGS9-2 is present in the soluble cytoplasmic fraction. In contrast, the majority of mu opioid receptors and delta opioid receptors are found in detergent-soluble membrane and there was no shift of RGS9-2 into DRM after co-expression of mu opioid receptor. These data suggest that the targeting of RGS9-2 to DRM in the striatum is mediated by D(2)R and that DRM is involved in the formation of a D(2)R signaling complex. D(2)R-mediated targeting of RGS9-2 to DRM was blocked by the deletion of the RGS9-2 DEP domain or by a point mutation that abolishes the GTPase accelerating protein function of RGS9-2.


Assuntos
Membrana Celular/efeitos dos fármacos , Detergentes/farmacologia , Proteínas RGS/fisiologia , Receptores de Dopamina D2/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Western Blotting , Química Encefálica , Linhagem Celular , Corpo Estriado/metabolismo , DNA Complementar/genética , Subunidades beta da Proteína de Ligação ao GTP/genética , Humanos , Imuno-Histoquímica , Proteínas de Membrana/química , Mutação/genética , Octoxinol , Proteínas RGS/genética , Receptores de Dopamina D2/genética , Receptores Opioides delta/genética , Receptores Opioides mu/genética , Receptores Opioides mu/fisiologia , Solubilidade , Transfecção
10.
PLoS One ; 6(11): e27984, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22132185

RESUMO

Regulator of G protein signaling 9-2 (RGS9-2) is a protein that is highly enriched in the striatum, a brain region that mediates motivation, movement and reward responses. We identified a naturally occurring 5 nucleotide deletion polymorphism in the human RGS9 gene and found that the mean body mass index (BMI) of individuals with the deletion was significantly higher than those without. A splicing reporter minigene assay demonstrated that the deletion had the potential to significantly decrease the levels of correctly spliced RGS9 gene product. We measured the weights of rats after virally transduced overexpression of RGS9-2 or the structurally related RGS proteins, RGS7, or RGS11, in the nucleus accumbens (NAc) and observed a reduction in body weight after overexpression of RGS9-2 but not RGS7 or 11. Conversely, we found that the RGS9 knockout mice were heavier than their wild-type littermates and had significantly higher percentages of abdominal fat. The constituent adipocytes were found to have a mean cross-sectional area that was more than double that of corresponding cells from wild-type mice. However, food intake and locomotion were not significantly different between the two strains. These studies with humans, rats and mice implicate RGS9-2 as a factor in regulating body weight.


Assuntos
Peso Corporal/genética , Estudos de Associação Genética , Proteínas RGS/genética , Adipócitos/metabolismo , Adipócitos/patologia , Tecido Adiposo/patologia , Animais , Sequência de Bases , Índice de Massa Corporal , Feminino , Genes Reporter/genética , Humanos , Gordura Intra-Abdominal/metabolismo , Gordura Intra-Abdominal/patologia , Íntrons/genética , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Atividade Motora , Núcleo Accumbens/metabolismo , Processamento de RNA/genética , Ratos , Deleção de Sequência/genética , Perda de Peso/genética
11.
Neurosci Lett ; 502(3): 123-8, 2011 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-21616123

RESUMO

In this study we demonstrate up-regulation of mRNA for Regulator of G protein Signaling (RGS) 6, 7, 9 and 11, R7 family RGS binding protein (R7BP) and RGS9 anchor protein (R9AP) during neuronal differentiation of mouse embryonic stem cells (mESCs). This expression pattern was most robust for RGS9 whose transcript level was low in undifferentiated mESCs but increased over 125 fold when differentiating mESCs began to exhibit a neuronal precursor cell (NPC) phenotype. In addition, we demonstrate that RGS9 mRNA is expressed in neuronal stem cells isolated from embryonic mouse cortex. The expression of RGS9 in two distinct populations of NPCs suggests that RGS9 and its accessory proteins may play an important role in neuron development.


Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células-Tronco Neurais/metabolismo , Proteínas RGS/genética , RNA Mensageiro/biossíntese , Animais , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Células-Tronco Embrionárias/citologia , Camundongos , Células-Tronco Neurais/citologia , Proteínas RGS/biossíntese , Regulação para Cima/genética
12.
J Neurochem ; 114(3): 739-49, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20477943

RESUMO

Regulator of G protein signaling 9-2 (RGS9-2), a member of the RGS family of GTPase accelerating proteins, is expressed specifically in the striatum, a brain region involved in controlling movement, motivation, mood and addiction. RGS9-2 can be found co-localized with D(2)-class dopamine receptors in medium spiny striatal neurons and altered functioning of both RGS9-2 and D(2)-like dopamine receptors have been implicated in schizophrenia, movement disorders and reward responses. Previously we showed that RGS9-2 can specifically co-localize with D(2)-dopamine receptors (D2R). Here we provide further evidence of the specificity of RGS9-2 for regulating D2R cellular functions: the expression of RGS9-2 inhibits dopamine-mediated cellular internalization of D2R, while the expression of another RGS protein, RGS4, had no effect. In addition, the agonist-mediated internalization of the G protein coupled delta opioid receptor was unaffected by RGS9-2 expression. We utilized mutant constructs of RGS9-2 to show that the RGS9-2 DEP (for Disheveled, EGL-10, Pleckstrin homology) domain and the GTPase accelerating activity of RGS9-2 were necessary for mediating specific inhibition of D2R internalization.


Assuntos
Agonistas de Dopamina/farmacologia , Antagonistas dos Receptores de Dopamina D2 , Endocitose/fisiologia , Neurônios/metabolismo , Proteínas RGS/fisiologia , Receptores de Dopamina D2/metabolismo , Animais , Linhagem Celular , Dopamina/fisiologia , Endocitose/efeitos dos fármacos , Feminino , Humanos , Neurônios/efeitos dos fármacos , Oócitos , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Proteínas RGS/química , Proteínas RGS/metabolismo , Xenopus laevis
13.
Mol Pharmacol ; 77(5): 836-45, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20159941

RESUMO

Substitution of arginine-137 of the vasopressin type 2 receptor (V2R) for histidine (R137H-V2R) leads to nephrogenic diabetes insipidus (NDI), whereas substitution of the same residue to cysteine or leucine (R137C/L-V2R) causes the nephrogenic syndrome of inappropriate antidiuresis (NSIAD). These two diseases have opposite clinical outcomes. Still, the three mutant receptors were shown to share constitutive beta-arrestin recruitment and endocytosis, resistance to vasopressin-stimulated cAMP production and mitogen-activated protein kinase activation, and compromised cell surface targeting, raising questions about the contribution of these phenomenons to the diseases and their potential treatments. Blocking endocytosis exacerbated the elevated basal cAMP levels promoted by R137C/L-V2R but not the cAMP production elicited by R137H-V2R, demonstrating that substitution of Arg137 to Cys/Leu, but not His, leads to constitutive V2R-stimulated cAMP accumulation that most likely underlies NSIAD. The constitutively elevated endocytosis of R137C/L-V2R attenuates the signaling and most likely reduces the severity of NSIAD, whereas the elevated endocytosis of R137H-V2R probably contributes to NDI. The constitutive signaling of R137C/L-V2R was not inhibited by treatment with the V2R inverse agonist satavaptan (SR121463). In contrast, owing to its pharmacological chaperone property, SR121463 increased the R137C/L-V2R maturation and cell surface targeting, leading to a further increase in basal cAMP production, thus disqualifying it as a potential treatment for patients with R137C/L-V2R-induced NSIAD. However, vasopressin was found to promote beta-arrestin/AP-2-dependent internalization of R137H/C/L-V2R beyond their already elevated endocytosis levels, raising the possibility that vasopressin could have a therapeutic value for patients with R137C/L-V2R-induced NSIAD by reducing steady-state surface receptor levels, thus lowering basal cAMP production.


Assuntos
Diabetes Insípido Nefrogênico/genética , Síndrome de Secreção Inadequada de HAD/genética , Receptores de Vasopressinas/genética , Substituição de Aminoácidos , Arginina/genética , Arginina Vasopressina/farmacologia , Arrestinas/genética , Linhagem Celular , AMP Cíclico/metabolismo , Histidina/genética , Humanos , Rim , Microscopia de Fluorescência , Mutagênese , Mutação , Plasmídeos , Transfecção , beta-Arrestinas
14.
J Biol Chem ; 285(7): 4781-7, 2010 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-20007977

RESUMO

The R7 subfamily of RGS proteins critically regulates neuronal G protein-signaling pathways that are essential for vision, nociception, motor coordination, and reward processing. A member of the R7 RGS family, RGS11, is a GTPase-accelerating protein specifically expressed in retinal ON-bipolar cells where it forms complexes with the atypical G protein beta subunit, Gbeta(5), and transmembrane protein R9AP. Association with R9AP has been shown to be critical for the proteolytic stability of the complex in the retina. In this study we report that R9AP can in addition stimulate the GTPase-accelerating protein activity of the RGS11 x Gbeta(5) complex at Galpha(o). Single turnover GTPase assays reveal that R9AP co-localizes RGS11 x Gbeta(5) and Galpha(o) on the membrane and allosterically potentiates the GTPase-accelerating function of RGS11 x Gbeta(5). Reconstitution of mGluR6-Galpha(o) signaling in Xenopus oocytes indicates that RGS11 x Gbeta(5)-mediated GTPase acceleration in this system requires co-expression of R9AP. The results provide new insight into the regulation of mGluR6-Galpha(o) signaling by the RGS11 x Gbeta(5) x R9AP complex and establish R9AP as a general GTPase-accelerating protein activity regulator of R7 RGS complexes.


Assuntos
Subunidades beta da Proteína de Ligação ao GTP/imunologia , Proteínas de Membrana/metabolismo , Proteínas RGS/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transdução de Sinais/fisiologia , Animais , Bovinos , Linhagem Celular , Subunidades beta da Proteína de Ligação ao GTP/genética , Proteínas de Membrana/genética , Camundongos , Ligação Proteica , Proteínas RGS/genética , Receptores de Glutamato Metabotrópico/genética , Transdução de Sinais/genética , Spodoptera , Xenopus
15.
J Agric Food Chem ; 58(7): 3965-9, 2010 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-20014760

RESUMO

Recent data suggest that ellagitannins (ETs), a class of hydrolyzable tannins found in some fruits and nuts, may have beneficial effects against colon cancer. In the stomach and gut, ETs hydrolyze to release ellagic acid (EA) and are converted by gut microbiota to urolithin A (UA; 3,8-dihydroxy-6H-dibenzopyran-6-one) type metabolites, which may persist in the colon through enterohepatic circulation. However, little is known about the mechanisms of action of either the native compounds or their metabolites on colon carcinogenesis. Components of Wnt signaling pathways are known to play a pivotal role in human colon carcinogenesis, and inappropriate activation of the signaling cascade is observed in 90% of colorectal cancers. This study investigated the effects of UA, EA, and ET-rich fruit extracts on Wnt signaling in a human 293T cell line using a luciferase reporter of canonical Wnt pathway-mediated transcriptional activation. The ET extracts were obtained from strawberry (Fragaria annassa), Jamun berry (Eugenia jambolana), and pomegranate (Punica granatum) fruit and were all standardized to phenolic content (as gallic acid equivalents, GAEs, by the Folin-Ciocalteu method) and to EA content (by high-performance liquid chromatography methods): strawberry = 20.5% GAE, 5.0% EA; Jamun berry = 20.5% GAE, 4.2% EA; pomegranate = 55% GAE, 3.5% EA. The ET extracts (IC(50) = 28.0-30.0 microg/mL), EA (IC(50) = 19.0 microg/mL; 63 microM), and UA (IC(50) = 9.0 microg/mL; 39 microM) inhibited Wnt signaling, suggesting that ET-rich foods have potential against colon carcinogenesis and that urolithins are relevant bioactive constituents in the colon.


Assuntos
Colo/metabolismo , Cumarínicos/farmacologia , Ácido Elágico/farmacologia , Frutas/química , Taninos Hidrolisáveis/metabolismo , Extratos Vegetais/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Wnt/metabolismo , Linhagem Celular Tumoral , Colo/efeitos dos fármacos , Cumarínicos/metabolismo , Ácido Elágico/metabolismo , Fragaria/química , Humanos , Taninos Hidrolisáveis/farmacologia , Lythraceae/química , Extratos Vegetais/farmacologia
16.
J Neurosci ; 25(8): 2157-65, 2005 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-15728856

RESUMO

Regulator of G-protein signaling 9-2 (RGS9-2), a member of the RGS family of G GTPase accelerating proteins, is expressed specifically in the striatum, which participates in antipsychotic-induced tardive dyskinesia and in levodopa-induced dyskinesia. We report that RGS9 knock-out mice develop abnormal involuntary movements when inhibition of dopaminergic transmission is followed by activation of D2-like dopamine receptors (DRs). These abnormal movements resemble drug-induced dyskinesia more closely than other rodent models. Recordings from striatal neurons of these mice establish that activation of D2-like DRs abnormally inhibits glutamate-elicited currents. We show that RGS9-2, via its DEP domain (for Disheveled, EGL-10, Pleckstrin homology), colocalizes with D2DRs when coexpressed in mammalian cells. Recordings from oocytes coexpressing D2DR or the m2 muscarinic receptor and G-protein-gated inward rectifier potassium channels show that RGS9-2, via its DEP domain, preferentially accelerates the termination of D2DR signals. Thus, alterations in RGS9-2 may be a key factor in the pathway leading from D2DRs to the side effects associated with the treatment both of psychoses and Parkinson's disease.


Assuntos
Antipsicóticos/toxicidade , Dopamina/fisiologia , Transtornos dos Movimentos/genética , Proteínas RGS/fisiologia , Receptores de Dopamina D2/metabolismo , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/farmacologia , Animais , Antiparkinsonianos/farmacologia , Antiparkinsonianos/uso terapêutico , Antiparkinsonianos/toxicidade , Antipsicóticos/farmacologia , Apomorfina/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/fisiopatologia , Dopaminérgicos/farmacologia , Dopaminérgicos/uso terapêutico , Antagonistas de Dopamina/farmacologia , Antagonistas de Dopamina/toxicidade , Discinesia Induzida por Medicamentos/fisiopatologia , Feminino , Haloperidol/farmacologia , Haloperidol/toxicidade , Humanos , Camundongos , Camundongos Knockout , Transtornos dos Movimentos/fisiopatologia , Doença de Parkinson/fisiopatologia , Técnicas de Patch-Clamp , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Quimpirol/farmacologia , Proteínas RGS/deficiência , Proteínas RGS/genética , Receptores de Dopamina D1/genética , Receptores de Dopamina D2/genética , Receptores Acoplados a Proteínas-G/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Reserpina/farmacologia , Reserpina/toxicidade , Frações Subcelulares/química , Sulpirida/farmacologia , Transfecção
17.
Neuron ; 38(6): 941-52, 2003 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-12818179

RESUMO

Regulators of G protein signaling (RGS) modulate heterotrimeric G proteins in part by serving as GTPase-activating proteins for Galpha subunits. We examined a role for RGS9-2, an RGS subtype highly enriched in striatum, in modulating dopamine D2 receptor function. Viral-mediated overexpression of RGS9-2 in rat nucleus accumbens (ventral striatum) reduced locomotor responses to cocaine (an indirect dopamine agonist) and to D2 but not to D1 receptor agonists. Conversely, RGS9 knockout mice showed heightened locomotor and rewarding responses to cocaine and related psychostimulants. In vitro expression of RGS9-2 in Xenopus oocytes accelerated the off-kinetics of D2 receptor-induced GIRK currents, consistent with the in vivo data. Finally, chronic cocaine exposure increased RGS9-2 levels in nucleus accumbens. Together, these data demonstrate a functional interaction between RGS9-2 and D2 receptor signaling and the behavioral actions of psychostimulants and suggest that psychostimulant induction of RGS9-2 represents a compensatory adaptation that diminishes drug responsiveness.


Assuntos
Dopamina/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Proteínas RGS/fisiologia , Transdução de Sinais , Animais , Comportamento Animal/efeitos dos fármacos , Estimulantes do Sistema Nervoso Central/farmacologia , Cocaína/farmacologia , Corpo Estriado/fisiologia , Condutividade Elétrica , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Expressão Gênica , Masculino , Camundongos , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Núcleo Accumbens/química , Oócitos/metabolismo , Canais de Potássio/fisiologia , Proteínas RGS/análise , Proteínas RGS/deficiência , Proteínas RGS/genética , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D2/fisiologia , Transfecção , Xenopus
18.
Neuron ; 33(1): 6-8, 2002 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-11779473

RESUMO

G protein-activated potassium channels (GIRKs), monitored with the temporal and molecular resolution of electrophysiology, play a key role in the study of signal transduction. GIRKs are activated primarily by the G(beta)(gamma) subunits, but a paper by Peleg et al. (2002 [this issue of Neuron]) demonstrates a role for G(alpha) subunits in suppressing basal activity and supports the idea of a macromolecular complex of G protein, GIRK, and perhaps RGS protein.


Assuntos
Membrana Celular/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Sistema Nervoso/metabolismo , Neurônios/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/metabolismo , Transdução de Sinais/fisiologia , Transmissão Sináptica/fisiologia , Animais , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Humanos , Proteínas RGS/metabolismo
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