RESUMEN
Endopeptidase 24.15 (ep24.15: EC3.4.24.15), a secreted protein involved in peptide metabolism, is unusual in that it does not contain a signal peptide sequence. In this work, we describe the physical interaction between ep24.15 and 14-3-3 epsilon, one isoform of a family of ubiquitous phosphoserine/threonine-scaffold proteins that organizes cell signaling and is involved in exocytosis. The interaction between ep24.15 and 14-3-3 epsilon increased following phosphorylation of ep24.15 at Ser(644) by protein kinase A (PKA). The co-localization of ep24.15 and 14-3-3 epsilon was increased by exposure of HEK293 cells (human embryonic kidney cells) to forskolin (10 microm). Overexpression of 14-3-3 epsilon in HEK293 cells almost doubled the secretion of ep24.15 stimulated by A23187 (7.5 microm) from 10%[1.4 +/- 0.24 AFU/(min 10(6) cells)] to 19%[2.54 +/- 0.24 AFU/(min 10(6) cells)] (p < 0.001) of the total intracellular enzyme activity. Treatment with forskolin had a synergistic effect on the A23187-stimulated secretion of ep24.15 that was totally blocked by the PKA inhibitor KT5720. The ep24.15 point mutation S644A reduced the co-localization of ep24.15 and 14-3-3 in stably transfected HEK293 cells. Indeed, secretion of the ep24.15 S644A mutant from these cells was only slightly stimulated by A23187 and insensitive to forskolin, in contrast to that of the wild type enzyme. Together, these data suggest that prior interaction with 14-3-3 is an important step in the unconventional stimulated secretion of ep24.15.
Asunto(s)
Proteínas 14-3-3/metabolismo , Metaloendopeptidasas/metabolismo , Proteínas 14-3-3/fisiología , Animales , Western Blotting/métodos , Encéfalo/metabolismo , Clonación Molecular/métodos , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Humanos , Inmunohistoquímica/métodos , Inmunoprecipitación/métodos , Microscopía Confocal , Mutagénesis Sitio-Dirigida/fisiología , Fosforilación/efectos de los fármacos , ARN Mensajero/biosíntesis , Ensayo de Unión Radioligante/métodos , Ratas , Proteínas Recombinantes de Fusión , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Tasa de Secreción/efectos de los fármacos , Transfección/métodos , Azul de Tripano , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
The human dopamine D4 receptor (D4R) has received considerable attention because of its high affinity for the atypical antipsychotic clozapine and the unusually polymorphic nature of its gene. To clarify the in vivo role of the D4R, we produced and analyzed mutant mice (D4R-/-) lacking this protein. Although less active in open field tests, D4R-/- mice outperformed wild-type mice on the rotarod and displayed locomotor supersensitivity to ethanol, cocaine, and methamphetamine. Biochemical analyses revealed that dopamine synthesis and its conversion to DOPAC were elevated in the dorsal striatum from D4R-/- mice. Based on these findings, we propose that the D4R modulates normal, coordinated and drug-stimulated motor behaviors as well as the activity of nigrostriatal dopamine neurons.
Asunto(s)
Depresores del Sistema Nervioso Central/farmacología , Cocaína/farmacología , Dopaminérgicos/farmacología , Etanol/farmacología , Metanfetamina/farmacología , Narcóticos/farmacología , Receptores de Dopamina D2/genética , Ácido 3,4-Dihidroxifenilacético/metabolismo , Secuencia de Aminoácidos , Animales , Antipsicóticos/farmacología , Conducta Animal/efectos de los fármacos , Clozapina/farmacología , Cuerpo Estriado/anatomía & histología , Cuerpo Estriado/química , Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Genotipo , Humanos , Levodopa/análisis , Levodopa/farmacocinética , Locomoción/efectos de los fármacos , Conducta Materna/efectos de los fármacos , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Actividad Motora/efectos de los fármacos , Mutagénesis Sitio-Dirigida/fisiología , Núcleo Accumbens/química , Núcleo Accumbens/metabolismo , Receptores de Dopamina D2/deficiencia , Receptores de Dopamina D4 , Sensibilidad y Especificidad , Sustancia Negra/anatomía & histología , Sustancia Negra/química , Sustancia Negra/metabolismo , Transcripción Genética/genéticaRESUMEN
Chemical modification is usually employed to study enzyme active sites. Valuable information can also be obtained, however, when this technique is used to probe allosteric sites. This approach is discussed in this article, and it is exemplified in chemical modification studies of the allosteric enzyme phosphofructokinase.
Asunto(s)
Regulación Alostérica/fisiología , Sitio Alostérico/fisiología , Activación Enzimática/fisiología , Ligandos , Mutagénesis Sitio-Dirigida/fisiología , Fosfofructoquinasa-1/química , Conformación Proteica , Compuestos de Sulfhidrilo/química , Adenosina Trifosfato/química , Citratos/química , Fructosa/químicaRESUMEN
Chemical modification is usually employed to study enzyme active sites. Valuable information can also be obtained, however, when this technique is used to probe allosteric sites. This approach is discussed in this article, and it is exemplified in chemical modification studies of the allosteric enzyme phosphofructokinase