RESUMEN
Melatonin, a neuro-hormone released by the pineal gland, has multiple effects in the central nervous system including the regulation of dopamine (DA) levels, but how melatonin accomplishes this task is not clear. Here, we show that melatonin MT1 and MT2 receptors co-immunoprecipitate with the DA transporter (DAT) in mouse striatal synaptosomes. Increased DA re-uptake and decreased amphetamine-induced locomotor activity were observed in the striatum of mice with targeted deletion of MT1 or MT2 receptors. In vitro experiments confirmed the interactions and recapitulated the inhibitory effect of melatonin receptors on DA re-uptake. Melatonin receptors retained DAT in the endoplasmic reticulum in its immature non-glycosylated form. In conclusion, we reveal one of the first molecular complexes between G protein-coupled receptors (MT1 and MT2) and transporters (DAT) in which melatonin receptors regulate the availability of DAT at the plasma membrane, thus limiting the striatal DA re-uptake capacity in mice.
Asunto(s)
Membrana Celular/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Dopamina/metabolismo , Receptor de Melatonina MT1/metabolismo , Receptor de Melatonina MT2/metabolismo , Animales , Cuerpo Estriado/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/genética , Unión Proteica , Receptor de Melatonina MT1/genética , Receptor de Melatonina MT2/genética , Sinaptosomas/metabolismoRESUMEN
G-protein-coupled receptors (GPCRs) are the largest family of integral membrane receptors with key roles in regulating signaling pathways targeted by therapeutics, but are difficult to study using existing proteomics technologies due to their complex biochemical features. To obtain a global view of GPCR-mediated signaling and to identify novel components of their pathways, we used a modified membrane yeast two-hybrid (MYTH) approach and identified interacting partners for 48 selected full-length human ligand-unoccupied GPCRs in their native membrane environment. The resulting GPCR interactome connects 686 proteins by 987 unique interactions, including 299 membrane proteins involved in a diverse range of cellular functions. To demonstrate the biological relevance of the GPCR interactome, we validated novel interactions of the GPR37, serotonin 5-HT4d, and adenosine ADORA2A receptors. Our data represent the first large-scale interactome mapping for human GPCRs and provide a valuable resource for the analysis of signaling pathways involving this druggable family of integral membrane proteins.
Asunto(s)
Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas , Receptores Acoplados a Proteínas G/metabolismo , Membrana Celular/metabolismo , Humanos , Receptor de Adenosina A2A/metabolismo , Receptores de Serotonina 5-HT4/metabolismo , Transducción de Señal , Técnicas del Sistema de Dos HíbridosRESUMEN
In mammals, the hormone melatonin is mainly produced by the pineal gland with nocturnal peak levels. Its peripheral and central actions rely either on its intrinsic antioxidant properties or on binding to melatonin MT1 and MT2 receptors, belonging to the G protein-coupled receptor (GPCR) super-family. Melatonin has been reported to be involved in many functions of the central nervous system such as circadian rhythm regulation, neurotransmission, synaptic plasticity, memory, sleep, and also in Alzheimer's disease and depression. However, little is known about the subcellular localization of melatonin receptors and the molecular aspects involved in neuronal functions of melatonin. Identification of protein complexes associated with GPCRs has been shown to be a valid approach to improve our understanding of their function. By combining proteomic and genomic approaches we built an interactome of MT1 and MT2 receptors, which comprises 378 individual proteins. Among the proteins interacting with MT1 , but not with MT2 , we identified several presynaptic proteins, suggesting a potential role of MT1 in neurotransmission. Presynaptic localization of MT1 receptors in the hypothalamus, striatum, and cortex was confirmed by subcellular fractionation experiments and immunofluorescence microscopy. MT1 physically interacts with the voltage-gated calcium channel Cav 2.2 and inhibits Cav 2.2-promoted Ca(2+) entry in an agonist-independent manner. In conclusion, we show that MT1 is part of the presynaptic protein network and negatively regulates Cav 2.2 activity, providing a first hint for potential synaptic functions of MT1.
Asunto(s)
Encéfalo/metabolismo , Canales de Calcio Tipo N/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Terminales Presinápticos/metabolismo , Receptor de Melatonina MT1/metabolismo , Canales de Calcio Tipo N/genética , Células HEK293 , Humanos , Proteínas del Tejido Nervioso/genética , Receptor de Melatonina MT1/genéticaRESUMEN
Inasmuch as the neurohormone melatonin is synthetically derived from serotonin (5-HT), a close interrelationship between both has long been suspected. The present study reveals a hitherto unrecognized cross-talk mediated via physical association of melatonin MT2 and 5-HT2C receptors into functional heteromers. This is of particular interest in light of the "synergistic" melatonin agonist/5-HT2C antagonist profile of the novel antidepressant agomelatine. A suite of co-immunoprecipitation, bioluminescence resonance energy transfer, and pharmacological techniques was exploited to demonstrate formation of functional MT2 and 5-HT2C receptor heteromers both in transfected cells and in human cortex and hippocampus. MT2/5-HT2C heteromers amplified the 5-HT-mediated Gq/phospholipase C response and triggered melatonin-induced unidirectional transactivation of the 5-HT2C protomer of MT2/5-HT2C heteromers. Pharmacological studies revealed distinct functional properties for agomelatine, which shows "biased signaling." These observations demonstrate the existence of functionally unique MT2/5-HT2C heteromers and suggest that the antidepressant agomelatine has a distinctive profile at these sites potentially involved in its therapeutic effects on major depression and generalized anxiety disorder. Finally, MT2/5-HT2C heteromers provide a new strategy for the discovery of novel agents for the treatment of psychiatric disorders.
Asunto(s)
Melatonina/metabolismo , Multimerización de Proteína , Receptor de Melatonina MT2/química , Receptor de Serotonina 5-HT2C/química , Serotonina/metabolismo , Transducción de Señal , Acetamidas/farmacología , Arrestinas/metabolismo , Sinergismo Farmacológico , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Células HeLa , Humanos , Melatonina/farmacología , Multimerización de Proteína/efectos de los fármacos , Estructura Cuaternaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Receptor de Melatonina MT1/metabolismo , Receptor de Melatonina MT2/genética , Receptor de Melatonina MT2/metabolismo , Receptor de Serotonina 5-HT2C/genética , Receptor de Serotonina 5-HT2C/metabolismo , Serotonina/farmacología , Transducción de Señal/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética , Fosfolipasas de Tipo C/metabolismo , beta-ArrestinasRESUMEN
G protein-coupled receptors (GPCRs), also known as seven transmembrane domain-spanning proteins (7TM), play an important role in tissue homeostasis and cellular and hormonal communication. GPCRs are targeted by a large panel of natural ligands such as photons, ions, metabolites, lipids and proteins but also by numerous drugs. Research efforts in the GPCR field have been rewarded in 2012 by the Nobel Price in Chemistry. The present article briefly summarizes our current knowledge on GPCRs and discusses future challenges in terms of fundamental aspects and therapeutic applications.
Asunto(s)
Receptores Acoplados a Proteínas G/fisiología , Animales , Diseño de Fármacos , Quimioterapia , Variación Genética , Humanos , Ligandos , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Transducción de SeñalRESUMEN
The formation of G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) heteromers enables signaling diversification and holds great promise for improved drug selectivity. Most studies of these oligomerization events have been conducted in heterologous expression systems, and in vivo validation is lacking in most cases, thus questioning the physiological significance of GPCR heteromerization. The melatonin receptors MT1 and MT2 exist as homomers and heteromers when expressed in cultured cells. We showed that melatonin MT1/MT2 heteromers mediated the effect of melatonin on the light sensitivity of rod photoreceptors in mice. This effect of melatonin involved activation of the heteromer-specific phospholipase C and protein kinase C (PLC/PKC) pathway and was abolished in MT1(-/-) or MT2(-/-) mice, as well as in mice overexpressing a nonfunctional MT2 mutant that interfered with the formation of functional MT1/MT2 heteromers in photoreceptor cells. Not only does this study establish an essential role of melatonin receptor heteromers in retinal function, it also provides in vivo support for the physiological importance of GPCR heteromerization. Thus, the MT1/MT2 heteromer complex may provide a specific pharmacological target to improve photoreceptor function.
Asunto(s)
Proteínas del Ojo/metabolismo , Multimerización de Proteína/fisiología , Receptor de Melatonina MT1/metabolismo , Receptor de Melatonina MT2/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Animales , Proteínas del Ojo/genética , Ratones , Ratones Noqueados , Mutación , Receptor de Melatonina MT1/genética , Receptor de Melatonina MT2/genética , Células Fotorreceptoras Retinianas Bastones/citología , Fosfolipasas de Tipo C/genética , Fosfolipasas de Tipo C/metabolismoRESUMEN
Recent proteomic and biochemical evidence indicates that cellular -signaling is organized in protein modules. G protein-coupled receptors (GPCRs) are privileged entry points for extracellular signals that are transmitted through the plasma membrane into the cell. The adequate cellular response and signaling specificity is regulated by GPCR-associated protein modules. The composition of these modules is dynamic and might depend on receptor stimulation, the proteome of a given cellular context, the subcellular localization of receptor-associated modules, the formation of GPCR oligomers and the variation of expression levels of components of these modules under physiological, for example circadian rhythm, or pathological conditions. The current article will highlight the importance of GPCR-associated protein modules as a biochemical basis for signaling specificity.
Asunto(s)
Receptores Acoplados a Proteínas G/metabolismo , Animales , Humanos , Multimerización de Proteína , Transporte de Proteínas , Transducción de SeñalRESUMEN
G protein-coupled receptors (GPCRs) are, with approximately 800 members, among the most abundant membrane proteins in humans. They are responding to a plethora of ligands and are involved in the transmission of extracellular signals inside the cell. GPCRs are synthesized in the endoplasmatic reticulum and are then transported to the cell surface where they are typically activated. Receptor activation triggers several processes such as signaling and receptor endocytosis. Along their life cycle, GPCRs are accompanied by a range of specialized GPCR-interacting proteins (GIPs) to assist nascent receptors in proper folding, to target them to the appropriate subcellular compartments and to fulfill their signaling tasks. Differential expression of GIPs and rapid alterations of GPCR/GIP interaction networks are efficient means to regulate GPCR function in a tissue-specific and spatiotemporal manner to trigger appropriate cellular responses. Interfering with a GPCR/GIP interaction might become a new strategy for specific therapeutic intervention. This chapter will focus on the importance of GIPs along the GPCR life cycle and discuss the dynamics and molecular organization of GPCR/GIP complexes.