RESUMO
This Letter describes the discovery of GSK189254 and GSK239512 that were progressed as clinical candidates to explore the potential of H3 receptor antagonists as novel therapies for the treatment of Alzheimer's disease and other dementias. By carefully controlling the physicochemical properties of the benzazepine series and through the implementation of an aggressive and innovative screening strategy that employed high throughput in vivo assays to efficiently triage compounds, the medicinal chemistry effort was able to rapidly progress the benzazepine class of H3 antagonists through to the identification of clinical candidates with robust in vivo efficacy and excellent developability properties.
Assuntos
Benzazepinas/química , Antagonistas dos Receptores Histamínicos H3/química , Receptores Histamínicos H3/química , Animais , Benzazepinas/farmacocinética , Cães , Meia-Vida , Haplorrinos , Antagonistas dos Receptores Histamínicos H3/síntese química , Antagonistas dos Receptores Histamínicos H3/farmacocinética , Humanos , Masculino , Microssomos Hepáticos/metabolismo , Niacinamida/análogos & derivados , Niacinamida/química , Niacinamida/farmacocinética , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Receptores Histamínicos H3/metabolismo , Relação Estrutura-AtividadeRESUMO
This Letter describes the discovery of a novel series of H3 receptor antagonists. The initial medicinal chemistry strategy focused on deconstructing and simplifying an early screening hit which rapidly led to the discovery of a novel series of H3 receptor antagonists based on the benzazepine core. Employing an H3 driven pharmacodynamic model, the series was then further optimised through to a lead compound that showed robust in vivo functional activity and possessed overall excellent developability properties.
Assuntos
Benzazepinas/química , Antagonistas dos Receptores Histamínicos H3/química , Receptores Histamínicos H3/química , Animais , Benzazepinas/síntese química , Benzazepinas/farmacocinética , Citocromo P-450 CYP2D6/química , Citocromo P-450 CYP2D6/metabolismo , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Antagonistas dos Receptores Histamínicos H3/síntese química , Antagonistas dos Receptores Histamínicos H3/farmacocinética , Humanos , Microssomos Hepáticos/metabolismo , Ligação Proteica , Ratos , Receptores Histamínicos H3/genética , Receptores Histamínicos H3/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Relação Estrutura-AtividadeRESUMO
Asthma and chronic obstructive pulmonary disease (COPD) are characterized in their pathogenesis by chronic inflammation in the airways. Phosphoinositide 3-kinase δ (PI3Kδ), a lipid kinase expressed predominantly in leukocytes, is thought to hold much promise as a therapeutic target for such inflammatory conditions. Of particular interest for the treatment of severe respiratory disease is the observation that inhibition of PI3Kδ may restore steroid effectiveness under conditions of oxidative stress. PI3Kδ inhibition may also prevent recruitment of inflammatory cells, including T lymphocytes and neutrophils, as well as the release of proinflammatory mediators, such as cytokines, chemokines, reactive oxygen species, and proteolytic enzymes. In addition, targeting the PI3Kδ pathway could reduce the incidence of pathogen-induced exacerbations by improving macrophage-mediated bacterial clearance. In this review, we discuss the potential and highlight the unknowns of targeting PI3Kδ for the treatment of respiratory disease, focusing on recent developments in the role of the PI3Kδ pathway in inflammatory cell types believed to be critical to the pathogenesis of COPD.
Assuntos
Asma/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Asma/enzimologia , Células Epiteliais/metabolismo , Humanos , Inflamação/metabolismo , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Macrófagos/metabolismo , Terapia de Alvo Molecular , Neutrófilos/metabolismo , Estresse Oxidativo , Fosfatidilinositol 3-Quinases/metabolismo , Doença Pulmonar Obstrutiva Crônica/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T/metabolismoRESUMO
The human 5-hydroxytryptamine (5-HT(4)) receptor is encoded by a highly complex gene which gives rise to at least 10 distinct splice variants. However, the functional relevance of these variants is unknown. In rat, only three such variants have been identified, 5-HT(4a) (r5-HT(4a)), 5-HT(4b) (r5-HT(4b)) and 5-HT(4e) (r5-HT(4e)). In the current study we identify and characterise the pharmacology of a novel rat splice variant (r5-HT(4c1)) and present the first comprehensive analysis of 5-HT(4) splice variant mRNA expression levels throughout the rat gastrointestinal tract. In addition, we describe preliminary characterisation of the first 5-HT(4) splice variant specific antibodies. In transfected cells, r5-HT(4c1) receptor exhibited similar binding properties to r5-HT(4a) and r5-HT(4b). Functional studies showed that 5-HT(4) agonists prucalopride (4-amino-5-chloro-2,3-dihydro-N-[1-(3-methoxypropyl)-4-piperidinyl]-7-benzofuran carboxamide monohydrochloride and renzapride (+/-)-endo-4-amino-5-chloro-2-methoxy-N-(1-azabicyclo[3.3.1]non-4-yl)benzamide monohydrochloride) acted as partial agonists at r5-HT(4c1), but full agonists at r5-HT(4a) and r5-HT(4b). Moreover, in contrast to r5-HT(4a) and r5-HT(4b), r5-HT(4c1) was not constitutively active. TaqMan mRNA analysis showed that r5-HT(4a) expression in brain and dorsal root ganglion exceeded that in the gastrointestinal tract, whilst the reverse was true for r5-HT(4b) and r5-HT(4c1). mRNA expression of each variant also increased distally throughout the gastrointestinal tract with the highest levels in the colon. r5-HT(4a) and r5-HT(4b) specific immunoreactivity was abundant on enteric neurons in jejunum, ileum and colon as well as neurons and satellite cells of the dorsal root ganglion. Only r5-HT(4b) immunoreactivity was observed on endocrine cells in the duodenum. These data could have implications in rat models and aid understanding of 5-HT(4) splice variant function.
Assuntos
Processamento Alternativo , Anticorpos Monoclonais/farmacologia , Receptores 5-HT4 de Serotonina/genética , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Sequência de Bases , Ligação Competitiva , Linhagem Celular , Membrana Celular/metabolismo , Clonagem Molecular , AMP Cíclico/metabolismo , Feminino , Trato Gastrointestinal/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Dados de Sequência Molecular , Isoformas de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Receptores 5-HT4 de Serotonina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Agonistas do Receptor de Serotonina/farmacologia , TransfecçãoRESUMO
Consistent with their function in immune surveillance, natural killer (NK) cells are distributed throughout lymphoid and nonlymphoid tissues. However, the mechanisms governing the steady-state trafficking of NK cells remain unknown. The lysophospholipid sphingosine 1-phosphate (S1P), by binding to its receptor S1P1, regulates the recirculation of T and B lymphocytes. In contrast, S1P5 is detected in the brain and regulates oligodendrocyte migration and survival in vitro. Here we show that S1P5 was also expressed in NK cells in mice and humans and that S1P5-deficient mice had aberrant NK cell homing during steady-state conditions. In addition, we found that S1P5 was required for the mobilization of NK cells to inflamed organs. Our data emphasize distinct mechanisms regulating the circulation of various lymphocyte subsets and raise the possibility that NK cell trafficking may be manipulated by therapies specifically targeting S1P5.
Assuntos
Células Matadoras Naturais/fisiologia , Lisofosfolipídeos/metabolismo , Receptores de Lisoesfingolipídeo/fisiologia , Esfingosina/análogos & derivados , Linfócitos T/fisiologia , Animais , Humanos , Subpopulações de Linfócitos/imunologia , Camundongos , Receptores de Lisoesfingolipídeo/genética , Esfingosina/metabolismoRESUMO
Gamma-aminobutyric acid type B receptors (GABAB) are G-protein-coupled receptors that mediate GABAergic inhibition in the brain. Their functional expression is dependent upon the formation of heterodimers between GABAB1 and GABAB2 subunits, a process that occurs within the endoplasmic reticulum. However, the mechanisms that regulate GABAB receptor oligomerization at the plasma membrane remain largely unknown. We first characterized the functional cytoarchitecture of an organotypic co-culture model of rat dorsal root ganglia and spinal cord. Subsequently, we studied the interactions between GABAB subunits after chronic stimulation of sensory fibres with capsaicin. Surface labelling of recombinant proteins showed a decrease in subunit co-localization and GABAB2 labelling, after capsaicin treatment. In these conditions, fluorescence lifetime imaging measurements further demonstrated a loss of interactions between green fluorescent protein-GABAB1b and t-dimer discosoma sp red fluorescent protein-GABAB2 subunits. Finally, we established that the GABAB receptor undergoes clathrin-dependent internalization and rapid recycling to the plasma membrane following activation with baclofen, a GABAB agonist. However, in cultures chronically stimulated with capsaicin, the agonist-induced endocytosis was decreased, reflecting changes in the dimeric state of the receptor. Taken together, our results indicate that the chronic stimulation of sensory fibres can dissociate the GABAB heterodimer and alters its responsiveness to the endogenous ligand. Chronic stimulation thus modulates receptor oligomerization, providing additional levels of control of signalling.
Assuntos
Capsaicina/farmacologia , Neurônios Aferentes/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Receptores de GABA-B/metabolismo , Animais , Animais Recém-Nascidos , Bicuculina/farmacologia , Técnicas de Cocultura/métodos , Relação Dose-Resposta a Droga , Interações Medicamentosas , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Antagonistas GABAérgicos/farmacologia , Gânglios Espinais/citologia , Gânglios Espinais/fisiologia , Ionóforos/farmacologia , Monensin/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios Aferentes/fisiologia , Técnicas de Cultura de Órgãos , Transporte Proteico/efeitos dos fármacos , Ratos , Medula Espinal/citologia , Medula Espinal/fisiologia , Fatores de Tempo , Transfecção/métodosRESUMO
6-[(3-Cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride (GSK189254) is a novel histamine H(3) receptor antagonist with high affinity for human (pK(i) = 9.59 -9.90) and rat (pK(i) = 8.51-9.17) H(3) receptors. GSK189254 is >10,000-fold selective for human H(3) receptors versus other targets tested, and it exhibited potent functional antagonism (pA(2) = 9.06 versus agonist-induced changes in cAMP) and inverse agonism [pIC(50) = 8.20 versus basal guanosine 5'-O-(3-[(35)S]thio)triphosphate binding] at the human recombinant H(3) receptor. In vitro autoradiography demonstrated specific [(3)H]GSK189254 binding in rat and human brain areas, including cortex and hippocampus. In addition, dense H(3) binding was detected in medial temporal cortex samples from severe cases of Alzheimer's disease, suggesting for the first time that H(3) receptors are preserved in late-stage disease. After oral administration, GSK189254 inhibited cortical ex vivo R-(-)-alpha-methyl[imidazole-2,5(n)-(3)H]histamine dihydrochloride ([(3)H]R-alpha-methylhistamine) binding (ED(50) = 0.17 mg/kg) and increased c-Fos immunoreactivity in prefrontal and somatosensory cortex (3 mg/kg). Microdialysis studies demonstrated that GSK189254 (0.3-3 mg/kg p.o.) increased the release of acetylcholine, noradrenaline, and dopamine in the anterior cingulate cortex and acetylcholine in the dorsal hippocampus. Functional antagonism of central H(3) receptors was demonstrated by blockade of R-alpha-methylhistamine-induced dipsogenia in rats (ID(50) = 0.03 mg/kg p.o.). GSK189254 significantly improved performance of rats in diverse cognition paradigms, including passive avoidance (1 and 3 mg/kg p.o.), water maze (1 and 3 mg/kg p.o.), object recognition (0.3 and 1 mg/kg p.o.), and attentional set shift (1 mg/kg p.o.). These data suggest that GSK189254 may have therapeutic potential for the symptomatic treatment of dementia in Alzheimer's disease and other cognitive disorders.
Assuntos
Benzazepinas/farmacologia , Encéfalo/efeitos dos fármacos , Antagonistas dos Receptores Histamínicos/farmacologia , Niacinamida/análogos & derivados , Nootrópicos/farmacologia , Receptores Histamínicos H3/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Animais , Benzazepinas/metabolismo , Benzazepinas/farmacocinética , Ligação Competitiva , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular , Cães , Agonistas dos Receptores Histamínicos/metabolismo , Agonistas dos Receptores Histamínicos/farmacocinética , Agonistas dos Receptores Histamínicos/farmacologia , Antagonistas dos Receptores Histamínicos/metabolismo , Antagonistas dos Receptores Histamínicos/farmacocinética , Humanos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Pessoa de Meia-Idade , Neurotransmissores/metabolismo , Niacinamida/metabolismo , Niacinamida/farmacocinética , Niacinamida/farmacologia , Nootrópicos/metabolismo , Nootrópicos/farmacocinética , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Receptores Histamínicos H3/análise , Sus scrofaRESUMO
GSK207040 (5-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-2-pyrazinecarboxamide) and GSK334429 (1-(1-methylethyl)-4-({1-[6-(trifluoromethyl)-3-pyridinyl]-4-piperidinyl}carbonyl)hexahydro-1H-1,4-diazepine) are novel and selective non-imidazole histamine H(3) receptor antagonists from distinct chemical series with high affinity for human (pK(i)=9.67+/-0.06 and 9.49+/-0.09, respectively) and rat (pK(i)=9.08+/-0.16 and 9.12+/-0.14, respectively) H(3) receptors expressed in cerebral cortex. At the human recombinant H(3) receptor, GSK207040 and GSK334429 were potent functional antagonists (pA(2)=9.26+/-0.04 and 8.84+/-0.04, respectively versus H(3) agonist-induced changes in cAMP) and exhibited inverse agonist properties (pIC(50)=9.20+/-0.36 and 8.59+/-0.04 versus basal GTPgammaS binding). Following oral administration, GSK207040 and GSK334429 potently inhibited cortical ex vivo [(3)H]-R-alpha-methylhistamine binding (ED(50)=0.03 and 0.35 mg/kg, respectively). Functional antagonism of central H(3) receptors was demonstrated by blockade of R-alpha-methylhistamine-induced dipsogenia in rats (ID(50)=0.02 and 0.11 mg/kg p.o. for GSK207040 and GSK334429, respectively). In more pathophysiologically relevant pharmacodynamic models, GSK207040 (0.1, 0.3, 1 and 3mg/kg p.o.) and GSK334429 (0.3, 1 and 3mg/kg p.o.) significantly reversed amnesia induced by the cholinergic antagonist scopolamine in a passive avoidance paradigm. In addition, GSK207040 (0.1, 0.3 and 1mg/kg p.o.) and GSK334429 (3 and 10mg/kg p.o.) significantly reversed capsaicin-induced reductions in paw withdrawal threshold, suggesting for the first time that blockade of H(3) receptors may be able to reduce tactile allodynia. Novel H(3) receptor antagonists such as GSK207040 and GSK334429 may therefore have therapeutic potential not only in dementia but also in neuropathic pain.
Assuntos
Azepinas/uso terapêutico , Benzazepinas/uso terapêutico , Capsaicina , Antagonistas dos Receptores Histamínicos/uso terapêutico , Transtornos da Memória/tratamento farmacológico , Neuralgia/tratamento farmacológico , Pirazinas/uso terapêutico , Piridinas/uso terapêutico , Receptores Histamínicos H3/metabolismo , Escopolamina , Analgésicos/farmacocinética , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Azepinas/administração & dosagem , Azepinas/farmacocinética , Benzazepinas/farmacocinética , Benzazepinas/farmacologia , Sistema Nervoso Central/efeitos dos fármacos , Ingestão de Líquidos/efeitos dos fármacos , Agonistas dos Receptores Histamínicos/farmacocinética , Agonistas dos Receptores Histamínicos/farmacologia , Antagonistas dos Receptores Histamínicos/farmacocinética , Antagonistas dos Receptores Histamínicos/farmacologia , Humanos , Masculino , Transtornos da Memória/induzido quimicamente , Neuralgia/induzido quimicamente , Pirazinas/farmacocinética , Pirazinas/farmacologia , Piridinas/administração & dosagem , Piridinas/farmacocinética , Ratos , Ratos Sprague-DawleyRESUMO
GABA(B) receptors are heterodimeric G protein-coupled receptors composed of R1 and R2 subunits that mediate slow synaptic inhibition in the brain by activating inwardly rectifying K(+) channels (GIRKs) and inhibiting Ca(2+) channels. We demonstrate here that GABA(B) receptors are intimately associated with 5'AMP-dependent protein kinase (AMPK). AMPK acts as a metabolic sensor that is potently activated by increases in 5'AMP concentration that are caused by enhanced metabolic activity, anoxia, or ischemia. AMPK binds the R1 subunit and directly phosphorylates S783 in the R2 subunit to enhance GABA(B) receptor activation of GIRKs. Phosphorylation of S783 is evident in many brain regions, and is increased dramatically after ischemic injury. Finally, we also reveal that S783 plays a critical role in enhancing neuronal survival after ischemia. Together our results provide evidence of a neuroprotective mechanism, which, under conditions of metabolic stress or after ischemia, increases GABA(B) receptor function to reduce excitotoxicity and thereby promotes neuronal survival.
Assuntos
Monofosfato de Adenosina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptores de GABA-B/metabolismo , Animais , Encéfalo/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Sobrevivência Celular , Células Cultivadas , Hipocampo/metabolismo , Humanos , Hipóxia/induzido quimicamente , Hipóxia/metabolismo , Hipóxia/patologia , Hipóxia/fisiopatologia , Soros Imunes , Neurônios/metabolismo , Concentração Osmolar , Fosforilação , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Isoformas de Proteínas/imunologia , Ratos , Receptores de GABA-B/química , Receptores de GABA-B/imunologiaRESUMO
Dopamine receptor activation is thought to contribute adversely to several neuropathological disorders, including Parkinson's disease and schizophrenia. In addition, dopamine may have a neuroprotective role: dopamine receptor agonists are reported to protect nerve cells by virtue of their antioxidant properties as well as by receptor-mediated mechanisms. White matter injury can also be a significant factor in neurological disorders. Using real-time RT-PCR, we show that differentiated rat cortical oligodendrocytes express dopamine D2 receptor and D3 receptor mRNA. Oligodendrocytes were vulnerable to oxidative glutamate toxicity and to oxygen/glucose deprivation injury. Agonists for dopamine D2 and D3 receptors provided significant protection of oligodendrocytes against these two forms of injury, and the protective effect was diminished by D2 and D3 antagonists. Levels of oligodendrocyte D2 receptor and D3 receptor protein, as measured by Western blotting, appeared to increase following combined oxygen and glucose deprivation. Our results suggest that dopamine D2 and D3 receptor activation may play an important role in oligodendrocyte protection against oxidative glutamate toxicity and oxygen-glucose deprivation injury.
Assuntos
Agonistas de Dopamina/farmacologia , Ácido Glutâmico/metabolismo , Hipóxia-Isquemia Encefálica/metabolismo , Oligodendroglia/metabolismo , Estresse Oxidativo/fisiologia , Receptores Dopaminérgicos/genética , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Células Cultivadas , Antagonistas de Dopamina/farmacologia , Glucose/metabolismo , Hipóxia-Isquemia Encefálica/fisiopatologia , Hipóxia-Isquemia Encefálica/prevenção & controle , Oligodendroglia/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Oxigênio/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Dopaminérgicos/metabolismo , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/genética , Receptores de Dopamina D3/metabolismoRESUMO
GABAergic synaptic transmission plays an important role in the patterning of epileptiform activity. We have previously shown that global loss of GABA(B) receptor function due to transgenic deletion of the GABA(B1) receptor subunit exacerbates epileptiform activity induced by pharmacological manipulations in hippocampal slices. Here we show that a similar hyperexcitable phenotype is observed in hippocampal slices prepared from a transgenic mouse expressing a GABA(B2) receptor subunit lacking its C terminal tail (the DeltaGB2-Ct mouse); a molecular manipulation that also produces complete loss of GABA(B) receptor function. Thus, epileptiform bursts that are sensitive to NMDA receptor antagonists (induced by either the GABA(A) receptor antagonist bicuculline (10muM) or removal of extracellular Mg(2+)) were significantly longer in duration in DeltaGB2-Ct slices relative to WT slices. We now extend these observations to demonstrate that a stimulus train induced bursting (STIB) protocol also evokes significantly longer bicuculline sensitive bursts of activity in DeltaGB2-Ct slices compared to WT. Furthermore, synchronous GABA(A) receptor-mediated potentials recorded in the presence of the potassium channel blocker 4-aminopyridine (4-AP, 100muM) and the ionotropic glutamate receptor antagonists NBQX (20muM) and D-AP5 (50muM) were significantly prolonged in duration in DeltaGB2-Ct versus WT slices. These data suggest that the loss of GABA(B) receptor function in DeltaGB2-Ct hippocampal slices promotes depolarising GABA(A) receptor-mediated events, which in turn, leads to the generation of ictal-like events, which may contribute to the epilepsy phenotype observed in vivo.
Assuntos
Epilepsia/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/metabolismo , Receptores de GABA-B/metabolismo , 4-Aminopiridina/farmacologia , Animais , Bicuculina/farmacologia , Interações Medicamentosas , Estimulação Elétrica/métodos , Embrião de Mamíferos , Epilepsia/genética , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Antagonistas GABAérgicos/farmacologia , Deleção de Genes , Técnicas In Vitro , Magnésio/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Knockout , Ácidos Fosfínicos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Propanolaminas/farmacologia , Células Piramidais , Compostos de Quinolínio/farmacologia , Receptores de GABA-B/química , Receptores de GABA-B/deficiência , Fatores de Tempo , Valina/análogos & derivados , Valina/farmacologiaRESUMO
gamma-Aminobutyric acid type B receptors (GABA(B)) are G-protein-coupled receptors that mediate GABAergic inhibition in the brain. Their functional expression is dependent upon the formation of heterodimers between GABA(B)R1 and GABA(B)R2 subunits, a process that occurs within the endoplasmic reticulum (ER). However, the mechanisms that regulate receptor surface expression remain largely unknown. Here, we demonstrate that access to the cell surface for GABA(B)R1 is sequentially controlled by an RSR(R) motif and a LL motif within its cytoplasmic domain. In addition, we reveal that msec7-1, a guanine-nucleotide-exchange factor (GEF) for the ADP-ribosylation factor (ARF) family of GTPases, critical regulators of vesicular membrane trafficking, interacts with GABA(B)R1 via the LL motif in this subunit. Finally, we establish that msec7-1 modulates the cell surface expression of GABA(B) receptors, a process that is dependent upon the integrity of the LL motif in GABA(B)R1. Together, our results demonstrate that the cell surface expression of the GABA(B)R1 subunit is regulated by multiple motifs, which act at distinct checkpoints in the secretory pathway, and also suggest a novel role for msec7-1 in regulating the membrane trafficking of GABA(B)R1 subunits.
Assuntos
Hipocampo/metabolismo , Receptores de GABA-B/metabolismo , Motivos de Aminoácidos/fisiologia , Sequência de Aminoácidos , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Dados de Sequência Molecular , Transporte Proteico/fisiologia , Ratos , Receptores de GABA-B/genética , Transdução de Sinais/fisiologiaRESUMO
gamma-Hydroxybutyric Acid (GHB) is thought to be a weak partial agonist at the gamma-aminobutyric acid(B) Receptor (GABA(B)R), but the precise relationship of the GHB receptor (GHBR) to the GABA(B)R remains unclear. In order to test the hypothesis that the GHBR is not identical to the GABA(B)R, we conducted two groups of experiments. First, GABA(B)R subtype 1 (R1) and/or subtype 2 (R2) were over expressed in HEK 293 cells and membrane binding studies on the transfected cells done using [(3)H]GHB and [(3)H] (2E)-(5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[a][7]annulen-6-ylidene) ethanoic acid ([(3)H]NCS-382). The latter is a specific antagonist at the GHB binding site. Second, [(3)H]GHB and [(3)H]NCS-382 autoradiographic binding studies were done on the brains of mice in which the gene for GABA(B)R1a was deleted. Such mice do not have a functioning GABA(B)R. There was no detectable specific [(3)H]GHB or [(3)H]NCS-382 binding in HEK 293 cells transfected with GABA(B)R1, R2, or R1/R2. Binding to [(3)H]CGP54626A, a high affinity GABA(B)R antagonist, was absent in GABA(B)R1a(-/-) mice. There was no difference in [(3)H]NCS-382 binding observed in the brains of GABA(B)R1a(-/-), GABA(B)R1a(+/-) or GABA(B)R1a(+/+) mice. Specific [(3)H]GHB binding was observed in the brain of GABA(B)R1a(-/-) mice but was significantly lower than in wild type mice. These data support the hypothesis that the GHB binding site is separate and distinct from the GABA(B)R.
Assuntos
Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores de GABA-B/genética , Receptores de GABA-B/metabolismo , Animais , Sítios de Ligação/fisiologia , Encéfalo/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Knockout , Receptores de GABA-B/deficiência , Oxibato de Sódio/metabolismoRESUMO
GABA(B) receptors are the cornerstone receptors in the modulation of inhibitory signalling in the central nervous system and continue to be targets for the amelioration of a number of neuropsychiatric and neurological disorders. Unravelling the molecular identity of this receptor has spurred much research over the past five or so years and generated a renewed interest and excitement in the field. Many questions are being answered and lessons learnt, not only about GABA(B) receptor function but also about general mechanisms of G-protein-coupled receptor signalling. However, as questions are being answered as many new questions are being raised and many GABA(B)-related conundrums continue to remain unanswered. In this report, we review some of the most recent work in the area of GABA(B) receptor research. In particular, we focus our attentions on the emerging mechanisms thought to be important in GABA(B) receptor signalling and the growing complex of associated proteins that we consider to be part of the GABA(B) receptor "signalosome."
Assuntos
Receptores de GABA-B/fisiologia , Transdução de Sinais/fisiologia , Animais , Membrana Celular/metabolismo , Sistema Nervoso Central/metabolismo , Dimerização , Humanos , Camundongos , Camundongos Transgênicos , Fosforilação , Receptores de GABA-B/metabolismoRESUMO
Studies in heterologous systems have demonstrated that heterodimerisation of the two GABA(B) receptor subunits appears to be crucial for the trafficking and signalling of the receptor. Gene targeting of the GABA(B1) gene has demonstrated that the expression of GABA(B1) is essential for GABA(B) receptor function in the central nervous system (CNS). However, the contribution of the GABA(B2) subunit in the formation of native GABA(B) receptors is still unclear, in particular whether other proteins can substitute for this subunit. We have created a transgenic mouse in which the endogenous GABA(B2) gene has been mutated in order to express a C-terminally truncated version of the protein. As a result, the GABA(B1) subunit does not reach the cell surface and concomitantly both pre- and post-synaptic GABA(B) receptor functions are abolished. Taken together with previous gene deletion studies for the GABA(B1) subunit, this suggests that classical GABA(B) function in the brain is exclusively mediated by GABA(B1/2) heteromers.
Assuntos
Receptores de GABA-B/genética , Receptores Pré-Sinápticos/metabolismo , Sinapses/metabolismo , Animais , Encéfalo , Corpo Estriado/metabolismo , Deleção de Genes , Camundongos , Mutação , Fenótipo , Transporte Proteico , Receptores de GABA-B/imunologia , Receptores de GABA-B/metabolismoRESUMO
GABA(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. The dynamic control of the cell surface stability of GABA(B) receptors is likely to be of fundamental importance in the modulation of receptor signaling. Presently, however, this process is poorly understood. Here we demonstrate that GABA(B) receptors are remarkably stable at the plasma membrane showing little basal endocytosis in cultured cortical and hippocampal neurons. In addition, we show that exposure to baclofen, a well characterized GABA(B) receptor agonist, fails to enhance GABA(B) receptor endocytosis. Lack of receptor internalization in neurons correlates with an absence of agonist-induced phosphorylation and lack of arrestin recruitment in heterologous systems. We also demonstrate that chronic exposure to baclofen selectively promotes endocytosis-independent GABA(B) receptor degradation. The effect of baclofen can be attenuated by activation of cAMP-dependent protein kinase or co-stimulation of beta-adrenergic receptors. Furthermore, we show that increased degradation rates are correlated with reduced receptor phosphorylation at serine 892 in GABA(B)R2. Our results support a model in which GABA(B)R2 phosphorylation specifically stabilizes surface GABA(B) receptors in neurons. We propose that signaling pathways that regulate cAMP levels in neurons may have profound effects on the tonic synaptic inhibition by modulating the availability of GABA(B) receptors.
Assuntos
Membrana Celular/metabolismo , Receptores de GABA-B/química , Animais , Arrestina/metabolismo , Baclofeno/farmacologia , Biotinilação , Células COS , Cálcio/metabolismo , Linhagem Celular , Células Cultivadas , Córtex Cerebral/citologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , DNA Complementar/metabolismo , Dimerização , Endocitose , Ativação Enzimática , Agonistas GABAérgicos/farmacologia , Agonistas dos Receptores de GABA-B , Hipocampo/citologia , Humanos , Microscopia de Fluorescência , Neurônios/metabolismo , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Ratos , Receptores Adrenérgicos beta/metabolismo , Receptores de GABA-B/metabolismo , Temperatura , Fatores de TempoRESUMO
GABA(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. Whereas heterodimerization between GABA(B) receptor GABA(B)R1 and GABA(B)R2 subunits is essential for functional expression, how neurons coordinate the assembly of these critical receptors remains to be established. Here we have identified Marlin-1, a novel GABA(B) receptor-binding protein that associates specifically with the GABA(B)R1 subunit in yeast, tissue culture cells, and neurons. Marlin-1 is expressed in the brain and exhibits a granular distribution in cultured hippocampal neurons. Marlin-1 binds different RNA species including the 3'-untranslated regions of both the GABA(B)R1 and GABA(B)R2 mRNAs in vitro and also associates with RNA in cultured neurons. Inhibition of Marlin-1 expression via small RNA interference technology results in enhanced intracellular levels of the GABA(B)R2 receptor subunit without affecting the level of GABA(B)R1. Together our results suggest that Marlin-1 functions to regulate the cellular levels of GABA(B) R2 subunits, which may have significant effects on the production of functional GABA(B) receptor heterodimers. Therefore, our observations provide an added level of regulation for the control of GABA(B) receptor expression and for the efficacy of inhibitory synaptic transmission.
Assuntos
Neurônios/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores de GABA-B/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Células COS , Hipocampo/citologia , Humanos , Camundongos , Dados de Sequência Molecular , Neurônios/citologia , RNA Mensageiro/metabolismo , Ratos , Homologia de Sequência de Aminoácidos , Gânglio Cervical Superior/citologia , Transmissão Sináptica/fisiologiaRESUMO
There is increasing evidence for a role of 5-hydroxytrypta-mine-6 (5-HT6) receptors in cognitive function. In the rat and human brain, 5-HT6 receptors are widely expressed and highly enriched in the basal ganglia. However, in the mouse brain, only very low levels of 5-HT6 receptor mRNA and receptor protein, measured by TaqMan reverse transcriptase-polymerase chain reaction and selective radioligand binding, could be detected, with no evidence of enrichment in the basal ganglia. The mouse receptor was cloned and transiently expressed in human embryonic kidney 293 cells to characterize its pharmacological profile. Despite significant sequence homology between human, rat, and mouse 5-HT6 receptors, the pharmacological profile of the mouse receptor was significantly different from the rat and human receptors. Four amino acid residues, conserved in rat and human and divergent in mouse receptors, were identified, and various mutant receptors were generated and their pharmacologies studied. Residues 188 (tyrosine in mouse, phenylalanine in rat and human) in transmembrane region 5 and 290 (serine in mouse, asparagine in rat and human) in transmembrane region 6 were identified as key amino acids responsible for the different pharmacological profiles. Molecular modeling of the receptor and docking of selective and nonselective compounds was undertaken to elucidate the ligand receptor interactions. The binding pocket was predicted to be different in the mouse compared with rat and human 5-HT6 receptors, and the models were in excellent agreement with the observed mutation results and have been used extensively in the design of further selective 5-HT6 antagonists.
Assuntos
Sistema Nervoso Central/metabolismo , Mutagênese Sítio-Dirigida/genética , Ensaio Radioligante/métodos , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismo , Idoso , Animais , Linhagem Celular , Sistema Nervoso Central/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Modelos Moleculares , Ligação Proteica/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores de Serotonina/química , Antagonistas da Serotonina/química , Antagonistas da Serotonina/metabolismo , Antagonistas da Serotonina/farmacologia , Agonistas do Receptor de Serotonina/química , Agonistas do Receptor de Serotonina/metabolismo , Agonistas do Receptor de Serotonina/farmacologia , Especificidade da EspécieRESUMO
The regulation and control of plasma membrane Ca(2+) fluxes is critical for the initiation and maintenance of a variety of signal transduction cascades. Recently, the study of transient receptor potential channels (TRPs) has suggested that these proteins have an important role to play in mediating capacitative calcium entry. In this study, we have isolated a cDNA from human brain that encodes a novel transient receptor potential channel termed human TRP7 (hTRP7). hTRP7 is a member of the short TRP channel family and is 98% homologous to mouse TRP7 (mTRP7). At the mRNA level hTRP7 was widely expressed in tissues of the central nervous system, as well as some peripheral tissues such as pituitary gland and kidney. However, in contrast to mTRP7, which is highly expressed in heart and lung, hTRP7 was undetectable in these tissues. For functional analysis, we heterologously expressed hTRP7 cDNA in an human embryonic kidney cell line. In comparison with untransfected cells depletion of intracellular calcium stores in hTRP7-expressing cells, using either carbachol or thapsigargin, produced a marked increase in the subsequent level of Ca(2+) influx. This increased Ca(2+) entry was blocked by inhibitors of capacitative calcium entry such as La(3+) and Gd(3+). Furthermore, transient transfection of an hTRP7 antisense expression construct into cells expressing hTRP7 eliminated the augmented store-operated Ca(2+) entry. Our findings suggest that hTRP7 is a store-operated calcium channel, a finding in stark contrast to the mouse orthologue, mTRP7, which is reported to enhance Ca(2+) influx independently of store depletion, and suggests that human and mouse TRP7 channels may fulfil different physiological roles.
Assuntos
Cálcio/metabolismo , Canais Iônicos/química , Proteínas de Membrana , Sequência de Aminoácidos , Encéfalo/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular , Sistema Nervoso Central/embriologia , Clonagem Molecular , DNA Complementar/metabolismo , Inibidores Enzimáticos/farmacologia , Epitopos , Éxons , Feminino , Biblioteca Gênica , Humanos , Imidazóis/farmacologia , Canais Iônicos/metabolismo , Rim/metabolismo , Masculino , Manganês/metabolismo , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/farmacologia , Filogenia , Hipófise/metabolismo , Ligação Proteica , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Canais de Cátion TRPM , Tapsigargina/farmacologia , Fatores de Tempo , Distribuição Tecidual , TransfecçãoRESUMO
The mammalian homologues of the Drosophila transient receptor potential (TRP) channel are plasma membrane proteins involved in the regulation of cellular Ca(2+) influx. These ion channels can be activated subsequent to either depletion of Ca(2+) from internal stores or through receptor-mediated processes. The mRNA expression patterns of several individual mammalian short transient receptor potential channels (TRPCs) have been described. Cross-comparisons between these data, however, are at best difficult predominantly due to the non-quantitative methods used. Furthermore there is limited data on the expression of TRPC family members in human tissues. In the present study we used a single technique, namely TaqMan real-time quantitative RT-PCR, to investigate the mRNA distribution of human TRPC1, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7 (hTRPCs) in discrete human brain areas, peripheral tissues as well as a panel of cell-lines. All hTRPCs studied were widely expressed within CNS and significant peripheral expression was often observed. Despite this, each channel exhibited a distinctive hallmark distribution profile. hTRPC1 was widely expressed in CNS and peripheral tissues, whereas hTRPC3 and hTRPC5 were predominantly expressed in tissues of CNS. hTRPC4 mRNA was detected in CNS and certain peripheral tissues such as bone, heart and prostate. hTRPC6 was homogeneously expressed throughout the CNS and peripheral tissues with the highest levels in placenta and lung. hTRPC7 mRNA was also broadly expressed in CNS as well as some peripheral tissues. The pattern of expression of the TRPCs was quite different in the various cell lines examined. TRPC3 and TRPC6 were selectively present in HEK-293 cells whilst TRPC1 was broadly distributed in the cell lines analyzed. In contrast TRPC4 and TRPC5 mRNAs were predominantly expressed in HK-2 and HEK-293 cell lines respectively. TRPC7 was selectively expressed in COS-1, COS-7 and HK-2 cell lines. These results show tissue- and cell-specific co-expression of multiple TRPC forms indicating widespread potential for formation of heteromeric channels. These data will be useful in the complex task of relating channel subunit composition to function in native cells.