RESUMEN
Mice lacking dopamine D2 receptors exhibit a significantly decreased agonist-promoted forebrain neocortical D1 receptor activation that occurs without changes in D1 receptor expression levels. This raises the possibility that, in brains of D2 mutants, a substantial portion of D1 receptors are uncoupled from their G protein, a phenomenon known as receptor desensitization. To test this, we examined D1-agonist-stimulated [35S]GTPgammaS binding (in the presence and absence of protein phosphatase inhibitors) and cAMP production (in the presence and absence of pertussis toxin) in forebrain neocortical tissues of wild-type mice and D2-receptor mutants. These studies revealed a decreased agonist-stimulated G-protein activation in D2 mutants. Moreover, whereas protein phosphatase 1/2A (PP1/2A) and 2B (PP2B) inhibitors decrease [35S]GTPgammaS binding in a concentration-dependent manner in wild type, they have either no (PP2B) or only partial (PP1/2A) effects in D2 mutants. Furthermore, for D2 mutants, immunoprecipitation experiments revealed increased basal and D1-agonist-stimulated phosphorylation of D1-receptor proteins at serine residues. Finally, D1 immunoprecipitates of both wild type and D2 mutants also contain protein kinase A (PKA) and PP2B immunoreactivities. In D2 mutants, however, the catalytic activity of the immunoprecipitated PP2B is abolished. These data indicate that neocortical D1 receptors are physically linked to PKA and PP2B and that the increased phosphorylation of D1 receptors in brains of D2 mutants is due to defective dephosphorylation of the receptor rather than increased kinase-mediated phosphorylation.
Asunto(s)
Calcineurina/metabolismo , Receptores de Dopamina D1/metabolismo , Adenilil Ciclasas/efectos de los fármacos , Adenilil Ciclasas/metabolismo , Animales , Unión Competitiva/efectos de los fármacos , Unión Competitiva/genética , Inhibidores de la Calcineurina , AMP Cíclico/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Agonistas de Dopamina/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas de Unión al GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacocinética , Sustancias Macromoleculares , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neocórtex/efectos de los fármacos , Neocórtex/metabolismo , Toxina del Pertussis/farmacología , Fosfoserina/análisis , Fosfoserina/metabolismo , Proteína Fosfatasa 1 , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/química , Receptores de Dopamina D2/deficiencia , Receptores de Dopamina D2/genéticaRESUMEN
Positron emission tomography studies in major depression show reduced serotonin (5-HT)1A receptor antagonist-binding potentials in many brain regions including occipital cortex. The functional meaning of this observation in terms of signal transduction is unknown. We used postmortem brain samples from depressed suicide victims to examine the downstream effectors of 5-HT1A receptor activation. The diagnosis was established by means of psychological autopsy using Diagnostic and Statistical Manual of Mental Disorders (DSM) III-R criteria. Measurements of [35S]GTPgammaS binding to Galphai/o in the occipital cortex of suicide victims and matched controls revealed a blunted response in suicide subjects and a decrease in the coupling of 5-HT1A receptor to adenylyl cyclase. No significant group differences were detected in the expression levels of Galphai/o, Galphaq/11 or Galphas proteins, or in the activity of cAMP-dependent protein kinase A. Studies of a parallel transduction pathway downstream from 5-HT1A receptor activation demonstrated a decrease in the activity of phosphatidylinositol 3-kinase and its downstream effector Akt, as well as an increase in PTEN (phosphatase and tensin homolog deleted on chromosome 10), the phosphatase that hydrolyzes phosphatidylinositol 3,4,5-triphosphate. Finally, the activation of extracellular signal-regulated kinases 1 and 2 was attenuated in suicide victims. These data suggest that the alterations in agonist-stimulated 5-HT1A receptor activation in depressed suicide victims are also manifest downstream from the associated G protein, affecting the activity of second messengers in two 5-HT1A receptor transduction pathways that may have implications for cell survival.
Asunto(s)
Encéfalo/metabolismo , Proteínas Serina-Treonina Quinasas , Receptores de Serotonina/metabolismo , Sistemas de Mensajero Secundario , Transducción de Señal , Suicidio , Adenilil Ciclasas/efectos de los fármacos , Adenilil Ciclasas/metabolismo , Adulto , Anciano , Química Encefálica , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Depresión/metabolismo , Femenino , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Lóbulo Occipital/química , Lóbulo Occipital/metabolismo , Fosfohidrolasa PTEN , Toxina del Pertussis/química , Fosfatidilinositol 3-Quinasas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Subunidades de Proteína/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Receptores de Serotonina 5-HT1 , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Elevation of extracellular Ca2+ (increase[Ca2+]e) stimulates the Ca2+ receptor (CaR) to induce secretion of 5-hydroxytryptamine (5-HT) from the calcium-sensing parafollicular (PF) cells. The CaR has been reported to couple to Galpha(q) with subsequent activation of protein kinase C-gamma (PKCgamma). We have identified a parallel transduction pathway in primary cultures of sheep PF cells by using a combinatorial approach in which we expressed adenoviral-encoded dominant-negative signaling proteins and performed in vitro kinase assays. The role of the CaR was established by expression of a dominant-negative CaR that eliminated calcium-induced 5-HT secretion but not secretion in response to KCl or phorbol esters. The calcium-induced secretion was inhibited by a dominant-negative p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3-K). PI3-K activity was also assayed using isoform-specific antibodies. The activity of p85/p110beta (PI3-Kbeta) immunocomplexes was elevated by increase[Ca2+]e and activated by Gbetagamma subunits. In addition, secretion of 5-HT was antagonized by the expression of a minigene encoding a peptide scavenger of Gbetagamma subunits (C-terminal fragment peptide of bovine beta-adrenergic receptor kinase). One target of PI3-K activity is phosphoinositide-dependent kinase-1 (PDK1), which in turn activated PKCzeta. Expression of a dominant-negative PKCzeta in PF cells reduced 5-HT secretion. Together, these observations establish that increase[Ca2+]e evokes 5-HT secretion from PF cells by stimulating both Galpha(q)- and Gbetagamma-signaling pathways downstream of the CaR. The betagamma cascade subsequently activates PI3-Kbeta-dependent signaling that is coupled to PDK1 and the downstream effector PKCzeta, and results in an increase in 5-HT release.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Serotonina/metabolismo , Transducción de Señal/fisiología , Glándula Tiroides/metabolismo , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Animales , Calcio/metabolismo , Calcio/farmacología , Proteínas de Unión al Calcio/genética , Separación Celular , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Espacio Extracelular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Expresión Génica , Genes Dominantes , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/genética , Fosfatidilinositol 3-Quinasas/genética , Fosforilación , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Ovinos , Transducción de Señal/efectos de los fármacos , Glándula Tiroides/citología , Glándula Tiroides/efectos de los fármacos , Transfección , Quinasas de Receptores Adrenérgicos betaRESUMEN
Serotonin 2C (5-HT2C) receptor pre-mRNA is a substrate for RNA editing enzymes that convert five adenosines (named A, B, C', C, and D editing sites) to inosines. Editing of two of these sites (C' and C) is crucial for decreasing the efficiency of the receptor to activate G-protein. Nucleotide sequence analysis of mouse forebrain neocortical 5-HT2C mRNA isoforms revealed that editing at these two sites is regulated in a serotonin-dependent manner. In serotonin-depleted mice, C'- and C-site editing is significantly decreased. This results in an increased expression of 5-HT2C mRNA isoforms encoding receptors with higher sensitivity to serotonin. In contrast, a 4 d treatment with the 5-HT2A/2C agonist (+/-)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane significantly increases the editing frequency at the C' site and leads to increased expression of 5-HT2C mRNA isoforms encoding receptors that activate G-protein least efficiently. None of the drug treatments led to alterations in cytoplasmic 5-HT2C mRNA levels. These data indicate that editing of 5-HT2C pre-mRNA is a mechanism that retains basic response properties of 5-HT2C receptors in the face of changing synaptic input to keep receptor activation within an optimal range for information processing. Key words: serotonin; 5.
Asunto(s)
Neocórtex/metabolismo , Edición de ARN , Precursores del ARN/metabolismo , ARN Mensajero/biosíntesis , Receptores de Serotonina/genética , Anfetaminas/farmacología , Animales , Citoplasma/genética , Fenclonina/farmacología , Masculino , Ratones , Neocórtex/efectos de los fármacos , Receptor de Serotonina 5-HT2C , Receptores de Serotonina/metabolismo , Serotonina/metabolismo , Antagonistas de la Serotonina/farmacología , Agonistas de Receptores de Serotonina/farmacologíaRESUMEN
Knockout mice deficient for dopamine D(2) and D(3) receptors exhibit blunted c-fos responses to D(1)-agonist stimulation. A single dose of methamphetamine (METH), however, leads to a long-term reversal of these blunted c-fos responses in both mutants, and the same effect is obtained with a single administration of a full D(1)-agonist. Consistent with the predominant c-fos expression in the neocortex induced by METH itself, METH pretreatment leads to the largest D(1)-agonist-stimulated c-fos responses in the neocortex of these mutants. For example, a pronounced blunting of neocortical c-fos responses is detected in the prefrontal cortex, a region in which D(1) receptors play a critical role in working memory. METH pretreated mutants, however, exhibit robust c-fos responses in this region that are indistinguishable from wild type. Recent studies indicate that different mechanisms operate in brains of D(2) and D(3) mutants to lead to decreased D(1)-receptor activity. For example, drug-naive D(2), but not D(3), mutants show significantly decreased G protein activation in response to D(1)-agonist stimulation, and METH pretreatment also rescues this abnormal molecular phenotype. Moreover, although the protein phosphatases (PP) 1/2A and 2B play a critical role in modulating G protein activation in wild type, their effect is either diminished (PP1/2A) or abolished (2B) in D(2) mutants. Interestingly however, METH pretreatment does not rescue the activities of these phosphatases in the mutants, suggesting that the long-term effects of a single dose of METH are mediated via effector systems that act downstream of G protein activation.