Fluorescent techniques including bioluminescence and fluorescence resonance energy transfer (BRET and FRET) and bimolecular fluorescence complementation (BiFC) were used to test if the heptahelical beta2-adrenergic receptor (b2AR), heterotrimeric G protein subunits and adenylyl cyclase (AC) are all simultaneously part of the same signaling complex. FRET and BRET both revealed such oligomeric complexes in HEK cells transiently expressing fluorescent-protein-tagged receptor, G protein and adenylyl cyclase. Dominant negative inhibitors of anterograde protein trafficking, including dominant negative forms of Rab 1, Rab 2 and Sar 1, did not block the formation of these complexes even though they prevented them from being transported out of the endoplasmic reticulum to the cell surface. These data suggest that complex assembly begins during or shortly after protein synthesis. RNA interference techniques are being used to determine if the subunit composition of the Gbeta-gamma heterodimer plays a role with regard to the specific receptors and effectors that are incorporated into signaling complexes. HEK 293 cells express a large number of endogenous signaling proteins. Among them are at least 18 different receptors including the b2AR, and several effectors including AC. Mass spectrometer experiments indicate the presence of endogenous Galpha-s, Galpha-i1-3, Galpha-q, Galpha-o, Galpha-z, Galpha-12 and Galpha-13 and mRNAs for endogenous Gbeta-1-5, Ggamma-2, Ggamma-4, Ggamma-5, Ggamma-7, Ggamma-10, Ggamma-11, and Ggamma-12 have been identified. Preliminary results indicate that specifically knocking down Gbeta-1 or Ggamma-2 prevent the b2AR from reaching the cell surface while knock down of G2 was ineffective in this regard. The consequences of knocking down other G protein subunits remain to be determined, but the initial results suggest that Gbeta-gamma heterodimers with a specific subtype composition may play a role in the assembly of signaling complexs. AC is stimulated by receptors such as the b2AR which activates the stimulatory G protein Gs, and inhibited by receptors such as the D4.2 dopamine receptor (D4) which activates the inhibitory G protein Gi. BRET experiments have demonstrated that exogenously expressed b2AR and D4 are part of a signaling complex that includes Gs, Gi and AC. During signaling BRET revealed that conformational changes take place within the complex, but suggest that all of the components remain associated with the complex during signaling. The presence of both the b2AR and D4 in a complex was confirmed by co-immunoprecipitaton of the exogenously expressed proteins from HEK 293 cells. Several mammalian tissues including brain and heart contain both beta2-adrenergic and D4 dopamine receptors. The association of these receptors within a signaling complex would allow for the efficient bimodal regulation of adenylyl cyclase, and current investigations are aimed at determining if these receptors are part of the same complex in mouse brain and/or heart. Experiments are currently in progress to test for this.
|Rebois, R Victor; Maki, Karl; Meeks, Julie A et al. (2012) D2-like dopamine and ýý-adrenergic receptors form a signaling complex that integrates Gs- and Gi-mediated regulation of adenylyl cyclase. Cell Signal 24:2051-60|
|Dupré, Denis J; Robitaille, Mélanie; Rebois, R Victor et al. (2009) The role of Gbetagamma subunits in the organization, assembly, and function of GPCR signaling complexes. Annu Rev Pharmacol Toxicol 49:31-56|