RESUMEN
The γ-aminobutyric acid type B (GABAB) receptor is a prototypical family C G protein-coupled receptor (GPCR) that plays a key role in the regulation of synaptic transmission. Although growing evidence suggests that GPCR signaling in neurons might be highly organized in time and space, limited information is available about the mechanisms controlling the nanoscale organization of GABAB receptors and other GPCRs on the neuronal plasma membrane. Using a combination of biochemical assays in vitro, single-particle tracking, and super-resolution microscopy, we provide evidence that the spatial organization and diffusion of GABAB receptors on the plasma membrane are governed by dynamic interactions with filamin A, which tethers the receptors to sub-cortical actin filaments. We further show that GABAB receptors are located together with filamin A in small nanodomains in hippocampal neurons. These interactions are mediated by the first intracellular loop of the GABAB1 subunit and modulate the kinetics of Gαi protein activation in response to GABA stimulation.
Asunto(s)
Receptores de GABA-B , Receptores de GABA , Receptores de GABA/metabolismo , Filaminas , Receptores de GABA-B/metabolismo , Membrana Celular/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Mechanisms that control mobilization of cytosolic calcium [Ca2+]i are key for regulation of numerous eukaryotic cell functions. One such paradigmatic mechanism involves activation of phospholipase Cß (PLCß) enzymes by G protein ßγ subunits from activated Gαi-Gßγ heterotrimers. Here, we report identification of a master switch to enable this control for PLCß enzymes in living cells. We find that the Gαi-Gßγ-PLCß-Ca2+ signaling module is entirely dependent on the presence of active Gαq. If Gαq is pharmacologically inhibited or genetically ablated, Gßγ can bind to PLCß but does not elicit Ca2+ signals. Removal of an auto-inhibitory linker that occludes the active site of the enzyme is required and sufficient to empower "stand-alone control" of PLCß by Gßγ. This dependence of Gi-Gßγ-Ca2+ on Gαq places an entire signaling branch of G-protein-coupled receptors (GPCRs) under hierarchical control of Gq and changes our understanding of how Gi-GPCRs trigger [Ca2+]i via PLCß enzymes.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/genética , Proteínas de Unión al GTP Heterotriméricas/genética , Fosfolipasa C beta/genética , Calcio/metabolismo , Señalización del Calcio/genética , Citosol/metabolismo , Células HEK293 , Humanos , Unión Proteica/genética , Receptores Acoplados a Proteínas G/genética , Transducción de Señal/genéticaRESUMEN
µ-Opioid receptors (µ-ORs) play a critical role in the modulation of pain and mediate the effects of the most powerful analgesic drugs. Despite extensive efforts, it remains insufficiently understood how µ-ORs produce specific effects in living cells. We developed new fluorescent ligands based on the µ-OR antagonist E-p-nitrocinnamoylamino-dihydrocodeinone (CACO), that display high affinity, long residence time and pronounced selectivity. Using these ligands, we achieved single-molecule imaging of µ-ORs on the surface of living cells at physiological expression levels. Our results reveal a high heterogeneity in the diffusion of µ-ORs, with a relevant immobile fraction. Using a pair of fluorescent ligands of different color, we provide evidence that µ-ORs interact with each other to form short-lived homodimers on the plasma membrane. This approach provides a new strategy to investigate µ-OR pharmacology at single-molecule level.
