RESUMEN
It is well known that cocaine blocks the dopamine transporter. This mechanism should lead to a general increase in dopaminergic neurotransmission, and yet dopamine D(1) receptors (D(1)Rs) play a more significant role in the behavioral effects of cocaine than the other dopamine receptor subtypes. Cocaine also binds to σ-1 receptors, the physiological role of which is largely unknown. In the present study, D(1)R and σ(1)R were found to heteromerize in transfected cells, where cocaine robustly potentiated D(1)R-mediated adenylyl cyclase activation, induced MAPK activation per se and counteracted MAPK activation induced by D(1)R stimulation in a dopamine transporter-independent and σ(1)R-dependent manner. Some of these effects were also demonstrated in murine striatal slices and were absent in σ(1)R KO mice, providing evidence for the existence of σ(1)R-D(1)R heteromers in the brain. Therefore, these results provide a molecular explanation for which D(1)R plays a more significant role in the behavioral effects of cocaine, through σ(1)R-D(1)R heteromerization, and provide a unique perspective toward understanding the molecular basis of cocaine addiction.
Asunto(s)
Trastornos Relacionados con Cocaína/metabolismo , Cocaína/toxicidad , Receptores de Dopamina D1/efectos de los fármacos , Receptores de Dopamina D1/metabolismo , Receptores sigma/efectos de los fármacos , Receptores sigma/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Células CHO , Línea Celular , Trastornos Relacionados con Cocaína/etiología , Cricetinae , Cricetulus , Dimerización , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Estructura Cuaternaria de Proteína/efectos de los fármacos , Receptores de Dopamina D1/química , Receptores de Dopamina D1/genética , Receptores sigma/química , Receptores sigma/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección , Receptor Sigma-1RESUMEN
The adenosine A(2A)-dopamine D(2) receptor heteromer is one of the most studied receptor heteromers. It has important implications for basal ganglia function and pathology. Recent studies using Bioluminescence and Sequential Resonance Energy Transfer techniques shed light on the role of Ca(2+) in the modulation of the quaternary structure of the A(2A)-D(2) receptor heteromer, which was found to depend on the binding of calmodulin (CaM) to the carboxy-terminus of the A(2A) receptor in the A(2A)-D(2) receptor heteromer. Importantly, the changes in quaternary structure correlate with changes in function. A Ca(2+)/CaM-dependent modulation of MAPK signaling upon agonist treatment could be observed in cells expressing A(2A)-D(2) receptor heteromers. These studies provide a first example of a Ca(2+)-mediated modulation of the quaternary structure and function of a receptor heteromer.
Asunto(s)
Calcio/metabolismo , Receptor de Adenosina A2A/metabolismo , Receptores de Dopamina D2/metabolismo , Calmodulina/metabolismo , Transferencia de Energía , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Receptor de Adenosina A2A/química , Receptores de Dopamina D2/química , Transducción de SeñalRESUMEN
The Ca(2+)-binding protein calmodulin (CaM) has been shown to bind directly to cytoplasmic domains of some G protein-coupled receptors, including the dopamine D(2) receptor. CaM binds to the N-terminal portion of the long third intracellular loop of the D(2) receptor, within an Arg-rich epitope that is also involved in the binding to G(i/o) proteins and to the adenosine A(2A) receptor, with the formation of A(2A)-D(2) receptor heteromers. In the present work, by using proteomics and bioluminescence resonance energy transfer (BRET) techniques, we provide evidence for the binding of CaM to the A(2A) receptor. By using BRET and sequential resonance energy transfer techniques, evidence was obtained for CaM-A(2A)-D(2) receptor oligomerization. BRET competition experiments indicated that, in the A(2A)-D(2) receptor heteromer, CaM binds preferentially to a proximal C terminus epitope of the A(2A) receptor. Furthermore, Ca(2+) was found to induce conformational changes in the CaM-A(2A)-D(2) receptor oligomer and to selectively modulate A(2A) and D(2) receptor-mediated MAPK signaling in the A(2A)-D(2) receptor heteromer. These results may have implications for basal ganglia disorders, since A(2A)-D(2) receptor heteromers are being considered as a target for anti-parkinsonian agents.