A Pre‐Assembled GPCR‐Signalosome Reveals Unappreciated Ligand Sensitivity and Mediates Unique Whole Cell Responses

Cells endogenously express many different receptors that activate the same signaling pathways but with diverse physiological outcomes. This suggests considerable organization and regulation of intracellular signaling, achieved by the assembly of multimeric protein complexes with diverse functions. The use of high‐resolution technologies has opened a new window of sensitivity to measure sub‐cellular signal organization by G protein‐coupled receptors (GPCRs). We previously discovered a novel signaling complex (signalosome) organized around the relaxin receptor RXFP1 that facilitates responses to femtomolar (fM) relaxin (Halls & Cooper 2010 EMBO J ). We now report that this is a previously undetected phenomenon common to many GPCRs including the prototypical β 2 ‐adrenoceptor. We show that constitutive assembly of the β 2 ‐signalosome is dependent on C‐terminal helix 8 and involves multiple signaling and scaffolding proteins. Activation by fM concentrations of ligand causes a sustained increase in cAMP, which is abolished following mutation of the orthosteric binding site. β 2 ‐signalosome activation is dependent on constitutive receptor recycling, and prolonged stimulation increases receptors at the cell membrane and decreases trafficking to lysosomes, suggesting a role in sensing the extracellular environment. In addition to sustained cAMP, activation of the β 2 ‐signalosome generates sustained nuclear ERK, increases global gene transcription and has unique effects at the proteomic level. The discovery of GPCR‐signalosomes that respond to very low concentrations of ligand has immediate relevance to our current understanding of GPCR function, and opens a new window for the therapeutic targeting of GPCRs.