RESUMEN
Serum amyloid P component (SAP) is a universal constituent of human amyloid deposits including those in Alzheimer's disease. SAP has been observed to be elevated in patients with depression, and higher SAP levels are associated with better response to the antidepressant escitalopram. The mechanisms underlying these clinical observations remain unclear. We examined the effect of SAP on serotonin transporter (SERT) expression and localization using Western blot, confocal microscopy, and positron emission tomography with the radioligand [11C]DASB. We also investigated the effect of SAP on treatment response to escitalopram in mice with the forced swim test (FST), a classical behaviour paradigm to assess antidepressant effects. SAP reduced [11C]DASB binding as an index of SERT levels, consistent with Western blots showing decreased total SAP protein because of increased protein degradation. In conjunction with the global decrease in SERT levels, SAP also promotes VAMP-2 mediated SERT membrane insertion. SAP levels are correlated with behavioural despair and SSRI treatment response in mice with FST. In MDD patients, the SAP and membrane SERT levels are correlated with response to SSRI treatment. SAP has complex effects on SERT levels and localization, thereby modulating the effect of SSRIs, which could partially explain clinical variability in antidepressant treatment response. These results add to our understanding of the mechanism for antidepressant drug action, and with further work could be of clinical utility.
Asunto(s)
Proteínas de Transporte de Serotonina en la Membrana Plasmática , Componente Amiloide P Sérico , Humanos , Ratones , Animales , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Componente Amiloide P Sérico/metabolismo , Escitalopram , Antidepresivos/farmacología , Inhibidores Selectivos de la Recaptación de Serotonina/farmacologíaRESUMEN
The retromer complex is an evolutionarily conserved protein complex involved in the endosomal recycling of various cargo proteins. It is ubiquitously expressed in all tissue and is found in both invertebrate as well as mammalian nervous systems, where it recycles various synaptic membrane proteins including the dopamine transporter and dopamine D1 receptor, two proteins implicated in dopamine homeostasis and neurotransmission. The involvement of the retromer complex in dopamine neurobiology is further underscored by its links to Parkinson's disease, a neurodegenerative disorder of the dopamine system. In this article, the existing literature linking the retromer complex to synaptic function and dopamine homeostasis is reviewed. Additional possible links are highlighted by exploring the retromer and other Parkinson's disease-associated proteins and possible relationships to synaptic function and dopamine transmission.
Asunto(s)
Enfermedad de Parkinson , Animales , Dopamina/metabolismo , Endosomas/metabolismo , Mamíferos , Enfermedad de Parkinson/metabolismo , Transporte de ProteínasRESUMEN
Using an in vitro model of ischemic stroke we treated differentiated SH-SY5Y cells to oxygen-glucose deprivation (OGD) followed by a reperfusion period where normal growth conditions were restored. Cells undergoing OGD exhibited significant cell death as measure by propidium iodide staining. However, cells treated with exogenous extracellular DJ-1 during reperfusion exhibited significant rescue from OGD-induced cell death.
Asunto(s)
Glucosa/deficiencia , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Oxígeno/metabolismo , Proteína Desglicasa DJ-1/farmacología , Hipoxia de la Célula , Línea Celular Tumoral , Humanos , Neuronas/metabolismo , Estrés OxidativoRESUMEN
Loss of tumor suppressor proteins, such as the retinoblastoma protein (Rb), results in tumor progression and metastasis. Metastasis is facilitated by low oxygen availability within the tumor that is detected by hypoxia inducible factors (HIFs). The HIF1 complex, HIF1α and dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT), is the master regulator of the hypoxic response. Previously, we demonstrated that Rb represses the transcriptional response to hypoxia by virtue of its association with HIF1. In this report, we further characterized the role Rb plays in mediating hypoxia-regulated genetic programs by stably ablating Rb expression with retrovirally-introduced short hairpin RNA in LNCaP and 22Rv1 human prostate cancer cells. DNA microarray analysis revealed that loss of Rb in conjunction with hypoxia leads to aberrant expression of hypoxia-regulated genetic programs that increase cell invasion and promote neuroendocrine differentiation. For the first time, we have established a direct link between hypoxic tumor environments, Rb inactivation and progression to late stage metastatic neuroendocrine prostate cancer. Understanding the molecular pathways responsible for progression of benign prostate tumors to metastasized and lethal forms will aid in the development of more effective prostate cancer therapies.
Asunto(s)
Biomarcadores de Tumor/genética , Diferenciación Celular , Hipoxia/genética , Células Neuroendocrinas/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Proteína de Retinoblastoma/metabolismo , Apoptosis , Movimiento Celular , Proliferación Celular , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Masculino , Invasividad Neoplásica , Células Neuroendocrinas/metabolismo , Neoplasias de la Próstata/metabolismo , Proteína de Retinoblastoma/genética , Células Tumorales CultivadasRESUMEN
The regulation of the dopamine transporter (DAT) impacts extracellular dopamine levels after release from dopaminergic neurons. Furthermore, a variety of protein partners have been identified that can interact with and modulate DAT function. In this study we show that DJ-1 can potentially modulate DAT function. Co-expression of DAT and DJ-1 in HEK-293T cells leads to an increase in [3H] dopamine uptake that does not appear to be mediated by increased total DAT expression but rather through an increase in DAT cell surface localization. In addition, through a series of GST affinity purifications and co-immunoprecipitations, we provide evidence that the DAT can be found in a complex with DJ-1, which involve distinct regions within both DAT and DJ-1. Using in vitro binding experiments we also show that this complex can be formed in part by a direct interaction between DAT and DJ-1. Co-expression of a mini-gene that can disrupt the DAT/DJ-1 complex appears to block the increase in [3H] dopamine uptake by DJ-1. Mutations in DJ-1 have been linked to familial forms of Parkinson's disease, yet the normal physiological function of DJ-1 remains unclear. Our study suggests that DJ-1 may also play a role in regulating dopamine levels by modifying DAT activity.
Asunto(s)
Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Dopamina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Oncogénicas/metabolismo , Enfermedad de Parkinson/genética , Membrana Celular/metabolismo , Dopamina/genética , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Neuronas Dopaminérgicas/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/genética , Mutación , Proteínas Oncogénicas/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Unión Proteica , Proteína Desglicasa DJ-1RESUMEN
Both the cannabinoid CB1 receptor (CB1) and dopamine D2 receptor (D2R) are G protein-coupled receptors that are linked to inhibitory Gαi/o protein, whereby activation of the receptor leads to the inhibition of cAMP production. Moreover, previous findings have shown evidence of cross-talk between the dopamine and endocannabinoid systems. In this report, we confirm the interaction of CB1 and D2R with co-immunoprecipitation experiments using human embryonic kidney 293T (HEK-293T) cells co-expressing both receptors. We also generated GST and His-tagged fusion proteins of the D2R and CB1 and conducted affinity purification assays and in vitro binding experiments to show that the CB1-D2R complex can be formed by a direct protein-protein interaction. This interaction is mediated by the carboxyl terminus of the CB1 receptor and the third intracellular loop of the D2 receptor. Co-transfection of an inhibitory mini-gene resulted in decreased levels of the CB1-D2R complex. Using a cAMP biosensor, we show that activation of D2R or CB1 alone in HEK-293T cells co-expressing both receptors leads to an inhibition of forskolin-stimulated cAMP accumulation. However, co-activation of both receptors resulted in a loss of this inhibition on cAMP accumulation. Our findings characterize the physical interaction between CB1 and D2R as well as demonstrate the potential functional outcome of the receptor complex.
Asunto(s)
Receptor Cannabinoide CB1/metabolismo , Receptores de Dopamina D2/metabolismo , Células HEK293 , Humanos , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Receptor Cannabinoide CB1/química , Receptores de Dopamina D2/químicaRESUMEN
Over-activation of AMPARs (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid subtype glutamate receptors) is implicated in excitotoxic neuronal death associated with acute brain insults, such as ischemic stroke. However, the specific molecular mechanism by which AMPARs, especially the calcium-impermeable AMPARs, induce neuronal death remains poorly understood. Here we report the identification of a previously unrecognized molecular pathway involving a direct protein-protein interaction that underlies GluR2-containing AMPAR-mediated excitotoxicity. Agonist stimulation of AMPARs promotes GluR2/GAPDH (glyceraldehyde-3-phosphate dehydrogenase) complex formation and subsequent internalization. Disruption of GluR2/GAPDH interaction by administration of an interfering peptide prevents AMPAR-mediated excitotoxicity and protects against damage induced by oxygen-glucose deprivation (OGD), an in vitro model of brain ischemia.
Asunto(s)
Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Neurotoxinas/toxicidad , Subunidades de Proteína/metabolismo , Receptores AMPA/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Células Cultivadas , Endocitosis/efectos de los fármacos , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Glucosa/deficiencia , Células HEK293 , Humanos , Modelos Biológicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Oxígeno/metabolismo , Unión Proteica/efectos de los fármacos , Ratas , Ratas Wistar , Receptores AMPA/agonistas , TransfecciónRESUMEN
BACKGROUND: Although dopamine D1 receptors are involved in working memory, how D1 receptors contribute to this process remains unclear. Numerous studies have shown that D1 receptors have extensive functional interaction with N-methyl-D-aspartate (NMDA) receptor. Our group previously demonstrated that D1 receptors were able to regulate NMDA receptor functions through direct protein-protein interactions involving the carboxyl terminals of D1 receptors and NMDA receptor NR1a and NR2A subunits respectively. In this study, we explored the effects of the D1-NR1 interaction on NMDA receptor-dependent long-term potentiation (LTP) and working memory by using the TAT-conjugated interfering peptide (TAT-D1-t2). METHODS: Miniature excitatory postsynaptic currents are recorded in rat hippocampal primary cultures. Coimmunoprecipitation and calcium/calmodulin-dependent protein kinase II (CaMKII) activity are measured in hippocampal slices and hippocampal neurons under the specified experimental conditions, respectively. Working memory was assessed using a delayed match-to-place protocol in the Morris Water Maze following administration of the TAT-D1-t2 peptide. RESULTS: Electrophysiology experiments showed that activation of D1 receptor upregulates NMDA receptor-mediated LTP in a CaMKII-dependent manner. Furthermore, D1 receptor agonist stimulation promotes the NR1-CaMKII coupling and enhances the CaMKII activity; and the D1 receptor-mediated effects can be blocked by the application of the TAT-D1-t2 peptide. Interestingly, animals injected with TAT-D1-t2 peptide exhibited significantly impaired working memory. CONCLUSIONS: Our study showed a critical role of NMDA-D1 direct protein-protein interaction in NMDA receptor-mediated LTP and working memory and implicated the involvement of CaMKII in this process.
Asunto(s)
Agonistas de Aminoácidos Excitadores/farmacología , Potenciación a Largo Plazo/efectos de los fármacos , Memoria a Corto Plazo/efectos de los fármacos , N-Metilaspartato/farmacología , Receptores de Dopamina D1/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Benzazepinas/farmacología , Biofisica , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Células Cultivadas , Agonistas de Dopamina/farmacología , Estimulación Eléctrica/métodos , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Hipocampo/citología , Inmunoprecipitación/métodos , Técnicas In Vitro , Potenciación a Largo Plazo/fisiología , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Memoria a Corto Plazo/fisiología , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/fisiología , Técnicas de Placa-Clamp , Péptidos/farmacología , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Receptores de Dopamina D1/químicaRESUMEN
The molecular underpinnings of exploration and its link to learning and memory remain poorly understood. Here we show that inducible, modest overexpression of neuronal calcium sensor 1 (Ncs1) selectively in the adult murine dentate gyrus (DG) promotes a specific form of exploratory behavior. The mice also display a selective facilitation of long-term potentiation (LTP) in the medial perforant path and a selective enhancement in rapid-acquisition spatial memory, phenotypes that are reversed by direct application of a cell-permeant peptide (DNIP) designed to interfere with NCS-1 binding to the dopamine type-2 receptor (D2R). Moreover, the DNIP and the D2R-selective antagonist L-741,626 attenuated exploratory behavior, DG LTP, and spatial memory in control mice. These data demonstrate a role for NCS-1 and D2R in DG plasticity and provide insight for understanding how the DG contributes to the origin of exploration and spatial memory acquisition.
Asunto(s)
Giro Dentado/fisiología , Conducta Exploratoria/fisiología , Potenciación a Largo Plazo/fisiología , Memoria/fisiología , Proteínas Sensoras del Calcio Neuronal/metabolismo , Neuropéptidos/metabolismo , Percepción Espacial/fisiología , Animales , Membrana Celular/metabolismo , Giro Dentado/efectos de los fármacos , Antagonistas de Dopamina/farmacología , Antagonistas de los Receptores de Dopamina D2 , Conducta Exploratoria/efectos de los fármacos , Técnicas In Vitro , Indoles/farmacología , Aprendizaje/efectos de los fármacos , Aprendizaje/fisiología , Potenciación a Largo Plazo/efectos de los fármacos , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Memoria/efectos de los fármacos , Ratones , Ratones Transgénicos , Modelos Neurológicos , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Pruebas Neuropsicológicas , Piperidinas/farmacología , Ratas , Receptores de Dopamina D2/metabolismo , Reconocimiento en Psicología/efectos de los fármacos , Reconocimiento en Psicología/fisiología , Percepción Espacial/efectos de los fármacos , Factores de TiempoAsunto(s)
Encéfalo/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Receptores de Dopamina D2/metabolismo , Esquizofrenia/fisiopatología , Adulto , Anciano , Análisis de Varianza , Trastorno Bipolar/patología , Trastorno Bipolar/fisiopatología , Encéfalo/patología , Femenino , Humanos , Inmunoprecipitación/métodos , Masculino , Persona de Mediana Edad , Cambios Post Mortem , Esquizofrenia/patologíaRESUMEN
The fragile X mental retardation protein (FMRP) is an RNA-binding protein that controls translational efficiency and regulates synaptic plasticity. Here, we report that FMRP is involved in dopamine (DA) modulation of synaptic potentiation. AMPA glutamate receptor subtype 1 (GluR1) surface expression and phosphorylation in response to D1 receptor stimulation were reduced in cultured Fmr1(-/-) prefrontal cortex (PFC) neurons. Furthermore, D1 receptor signaling was impaired, accompanied by D1 receptor hyperphosphorylation at serine sites and subcellular redistribution of G protein-coupled receptor kinase 2 (GRK2) in both PFC and striatum of Fmr1(-/-) mice. FMRP interacted with GRK2, and pharmacological inhibition of GRK2 rescued D1 receptor signaling in Fmr1(-/-) neurons. Finally, D1 receptor agonist partially rescued hyperactivity and enhanced the motor function of Fmr1(-/-) mice. Our study has identified FMRP as a key messenger for DA modulation in the forebrain and may provide insights into the cellular and molecular mechanisms underlying fragile X syndrome.
Asunto(s)
Dopamina/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Potenciación a Largo Plazo/fisiología , Neuronas/metabolismo , Corteza Prefrontal/metabolismo , Animales , Células Cultivadas , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Masculino , Ratones , Ratones Noqueados , Corteza Prefrontal/citología , Desempeño Psicomotor/fisiología , Receptores AMPA/metabolismo , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/metabolismo , Transmisión Sináptica/fisiologíaRESUMEN
Dopamine is a major neurotransmitter in the mammalian central nervous system (CNS) that regulates neuroendocrine functions, locomotor activity, cognition and emotion. The dopamine system has been extensively studied because dysfunction of this system is linked to various pathological conditions including Parkinson's disease, schizophrenia, Tourette's syndrome, and drug addiction. Accordingly, intense efforts to delineate the full complement of signaling pathways mediated by individual receptor subtypes have been pursued. Dopamine D1-like receptors are of particular interest because they are the most abundant dopamine receptors in CNS. Recent work suggests that dopamine signaling could be regulated via dopamine receptor interacting proteins (DRIPs). Unraveling these DRIPs involved in the dopamine system may provide a better understanding of the mechanisms underlying CNS disorders related to dopamine system dysfunction and may help identify novel therapeutic targets.
Asunto(s)
Sistema Nervioso Central/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de Dopamina D1/metabolismo , Animales , Proteínas del Citoesqueleto/metabolismo , Humanos , Canales Iónicos/metabolismo , Chaperonas Moleculares/metabolismoRESUMEN
Parkinson's disease (PD) is a neurodegenerative disease characterized by a loss of nigrostriatal dopaminergic neurons. Recently, PD research has been stimulated by the identification of genes that are implicated in rare familial forms of PD. However, despite these discoveries, the primary cause of PD is still unclear. Various pathogenic mechanisms may be involved including mitochondrial dysfunction, proteasomal dysfunction/protein aggregation, oxidative damage, environmental factors and genetic disposition. Furthermore, dopamine has also been implicated in contributing to the pathogenesis of PD. This review will focus on the genes that have been identified to be associated with PD and how they may impair dopamine metabolism. Understanding the role of these PD-related genes in dopamine neurobiology may provide insight into the underpinning pathogenic mechanisms of PD.
Asunto(s)
Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Animales , Dopamina/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Proteínas de Neoplasias/genética , Proteínas del Tejido Nervioso , Proteínas Oncogénicas/genética , Proteína Desglicasa DJ-1 , Proteínas Quinasas/genética , Ubiquitina Tiolesterasa/genética , Ubiquitina-Proteína Ligasas/genética , alfa-Sinucleína/genéticaRESUMEN
Parkinson's disease is characterized by progressive neuronal degeneration of dopaminergic neurons in the substantia nigra. Many factors are thought to contribute to the neuronal cell death that occurs in Parkinson's disease, including alpha-synuclein-mediated toxicity. Previously, we have reported that alpha-synuclein directly couples to the carboxyl tail of the dopamine transporter (DAT) and that the alpha-synuclein/DAT protein complex formation accelerates DAT-mediated cellular dopamine (DA) uptake and DA-induced cellular apoptosis. In the present study, we report that parkin, an E2-dependent E3 protein ubiquitin ligase associated with recessive early onset Parkinson's disease, exerts a protective effect against DA-induced alpha-synuclein-dependent cell toxicity. Parkin impairs the alpha-synuclein/DAT coupling by interacting with the carboxyl-terminus of the DAT and blocks the alpha-synuclein-induced enhancement in both DAT cell surface expression and DAT-mediated DA uptake. Moreover, we have found that parkin protects against DA-induced cell toxicity in dopaminergic SK-N-SH cells. These findings will help identify the role of these proteins in the etiology and/or maintenance of Parkinson's disease.
Asunto(s)
Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/fisiología , Dopamina/toxicidad , Ubiquitina-Proteína Ligasas/fisiología , alfa-Sinucleína/fisiología , Sitios de Unión , Línea Celular , Membrana Celular/fisiología , Cocaína/análogos & derivados , Cocaína/metabolismo , Inhibidores de Captación de Dopamina/metabolismo , Humanos , Riñón , Cinética , Microscopía Confocal , TransfecciónRESUMEN
Altered synaptic dopamine levels have been implicated in several neurological/neuropsychiatric disorders, including drug addiction and schizophrenia. However, it is unclear what precipitates these changes in synaptic dopamine levels. One of the key presynaptic components involved in regulating dopaminergic tone is the dopamine transporter (DAT). Here, we report that the DAT is also regulated by the dopamine D2 receptor through a direct protein-protein interaction involving the DAT amino-terminus and the third intracellular loop of the D2 receptor. This physical coupling facilitates the recruitment of intracellular DAT to the plasma membrane and leads to enhanced dopamine reuptake. Moreover, mice injected with peptides that disrupt D2-DAT interaction exhibit decreased synaptosomal dopamine uptake and significantly increased locomotor activity, reminiscent of DAT knockout mice. Our data highlight a novel mechanism through which neurotransmitter receptors can functionally modulate neurotransmitter transporters, an interaction that can affect the synaptic neurotransmitter levels in the brain.
Asunto(s)
Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Receptores de Dopamina D2/metabolismo , Sinapsis/metabolismo , Animales , Membrana Celular/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoprecipitación , Ratones , Microscopía Confocal , Transporte de Proteínas/fisiologíaRESUMEN
Dopamine is a neurotransmitter that can regulate both excitatory and inhibitory fast synaptic transmission. The overlapping dopaminergic, glutamatergic, and GABAergic systems provide a basis for the interaction between these three neurotransmitters. Although there is considerable evidence for the involvement of second-messenger systems to mediate receptor cross-talk between these receptor systems, there is emerging evidence that receptors can interact through direct protein-protein interactions. The functional implications and overall significance of the dopamine/glutamate/GABA interactions will be examined.
Asunto(s)
Dopamina/farmacología , Receptor Cross-Talk/fisiología , Animales , Dopamina/fisiología , Ácido Glutámico/fisiología , Humanos , Neurotransmisores , Receptores Dopaminérgicos/fisiología , Receptores de GABA/fisiología , Receptores de Glutamato/fisiología , Ácido gamma-Aminobutírico/fisiologíaRESUMEN
here is considerable evidence that dopamine D2 receptors can modulate AMPA receptor-mediated neurotoxicity. However, the molecular mechanism underlying this process remains essentially unclear. Here we report that D2 receptors inhibit AMPA-mediated neurotoxicity through two pathways: the activation of phosphoinositide-3 kinase (PI-3K) and downregulation of AMPA receptor plasma membrane expression, both involving a series of protein-protein coupling/uncoupling events. Agonist stimulation of D2 receptors promotes the formation of the direct protein-protein interaction between the third intracellular loop of the D2 receptor and the ATPase N-ethylmaleimide-sensitive factor (NSF) while uncoupling the NSF interaction with the carboxyl tail (CT) of the glutamate receptor GluR2 subunit of AMPA receptors. Previous studies have shown that full-length NSF directly couples to the GluR2CT and facilitates AMPA receptor plasma membrane expression. Furthermore, the CT region of GluR2 subunit is also responsible for several other intracellular protein couplings, including p85 subunit of PI-3K. Therefore, the direct coupling of D2-NSF and concomitant decrease in the NSF-GluR2 interaction results in a decrease of AMPA receptor membrane expression and an increase in the interaction between GluR2 and the p85 and subsequent activation of PI-3K. Disruption of the D2-NSF interaction abolished the ability of D2 receptor to attenuate AMPA-mediated neurotoxicity by blocking the D2 activation-induced changes in PI-3K activity and AMPA receptor plasma membrane expression. Furthermore, the D2-NSF-GluR2-p85 interactions are also responsible for the D2 inhibition of ischemia-induced cell death. These data may provide a new avenue to identify specific targets for therapeutics to modulate glutamate receptor-governed diseases, such as stroke.
Asunto(s)
Agonistas de Aminoácidos Excitadores/toxicidad , Receptores AMPA/fisiología , Receptores de Dopamina D2/fisiología , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/toxicidad , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Western Blotting/métodos , Encéfalo/citología , Encéfalo/fisiología , Células Cultivadas , Agonistas de Dopamina/farmacología , Antagonistas de Dopamina/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática/métodos , Antagonistas del GABA/farmacología , Glucosa/deficiencia , Glutatión Transferasa/metabolismo , Humanos , Hipoxia , Inmunoprecipitación/métodos , Proteínas Sensibles a N-Etilmaleimida/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Picrotoxina/farmacología , Propidio , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Quinpirol/farmacología , Racloprida/farmacología , Ensayo de Unión Radioligante/métodos , Ratas , Ratas Wistar , Receptores AMPA/clasificación , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Functional interactions between dopamine D1-like receptors and NMDA subtype glutamate receptors have been implicated in the maintenance of normal brain activity and neurological dysfunction. Although modulation of NMDA receptor functions by D1 receptor activation has been the subject of extensive investigation, little is known as to how the activation of NMDA receptors alters D1 function. Here we report that NMDA receptors regulate D1 receptor function via a direct protein-protein interaction mediated by the carboxyl tail regions of both receptors. In both cotransfected cells and cultured hippocampal neurons the activation of NMDA receptors increases the number of D1 receptors on the plasma membrane surface and enhances D1 receptor-mediated cAMP accumulation via a SNARE-dependent mechanism. Furthermore, overexpression of mini-genes encoding either NR1 or D1 carboxyl tail fragments disrupts the D1-NR1 direct protein-protein interaction and abolishes NMDA-induced changes in both D1 cell surface expression and D1-mediated cAMP accumulation. Our results demonstrate that the D1-NR1 physical interaction enables NMDA receptors to increase plasma membrane insertion of D1 receptors and provides a novel mechanism by which the activation of NMDA receptors upregulates D1 receptor function. Understanding the molecular mechanisms by which D1 and NMDA receptors functionally interact may provide insight toward elucidating the molecular neurobiological mechanisms involved in many neuropsychiatric illnesses, such as schizophrenia.
Asunto(s)
Neuronas/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Células COS , Membrana Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , AMP Cíclico/metabolismo , Exocitosis/fisiología , Hipocampo/citología , Humanos , Fusión de Membrana/fisiología , N-Metilaspartato/farmacología , Neuronas/citología , Neuronas/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Estructura Terciaria de Proteína/fisiología , Ratas , Ratas Wistar , Receptores de Dopamina D1/genética , Receptores de N-Metil-D-Aspartato/genética , TransfecciónRESUMEN
The presenilins and nicastrin form high molecular mass, multimeric protein complexes involved in the intramembranous proteolysis of several proteins. Post-translational glycosylation and trafficking of nicastrin is necessary for the activity of these complexes. We report here that although there are differences in the post-translational processing of nicastrin in neurons and glia, both of the presenilins are required for the physiological post-translational modification and for the correct subcellular distribution of nicastrin. Absence of presenilin 1 (PS1) is associated with dramatic reductions in the level of mature glycosylated nicastrin and with redistribution of nicastrin away from the cell surface. In contrast, absence of presenilin 2 (PS2) is associated with only modest reductions in the levels of immature nicastrin. It is notable that these differential effects parallel the differential effects of null mutations in PS1 and PS2 on APP and Notch processing. Our data therefore suggest that the differential interactions of PS1 and PS2 with nicastrin reflect different functions for the PS1 and PS2 complexes.
Asunto(s)
Encéfalo/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/fisiología , Secretasas de la Proteína Precursora del Amiloide , Animales , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Presenilina-1 , Presenilina-2RESUMEN
Dopamine D1-like receptors, composed of D1 and D5 receptors, have been documented to modulate glutamate-mediated fast excitatory synaptic neurotransmission. Here, we report that dopamine D1 receptors modulate NMDA glutamate receptor-mediated functions through direct protein-protein interactions. Two regions in the D1 receptor carboxyl tail can directly and selectively couple to NMDA glutamate receptor subunits NR1-1a and NR2A. While one interaction is involved in the inhibition of NMDA receptor-gated currents, the other is implicated in the attenuation of NMDA receptor-mediated excitotoxicity through a PI-3 kinase-dependent pathway.