Abstract
Despite the crowded nature of the cellular milieu, ligand–GPCR–G protein interactions are traditionally viewed as spatially and temporally isolated events. In contrast, recent reports suggest the spatial and temporal coupling of receptor–effector interactions, with the potential to diversify downstream responses. In this study, we combine protein engineering of GPCR–G protein interactions with affinity sequestration and photo-manipulation of the crucial Gα C terminus, to demonstrate the temporal coupling of cognate and non-cognate G protein interactions through priming of the GPCR conformation. We find that interactions of the Gαs and Gαq C termini with the β2-adrenergic receptor (β2-AR), targeted at the G-protein-binding site, enhance Gs activation and cyclic AMP levels. β2-AR–Gα C termini interactions alter receptor conformation, which persists for ~90 s following Gα C terminus dissociation. Non-cognate G-protein expression levels impact cognate signaling in cells. Our study demonstrates temporal allostery in GPCRs, with implications for the modulation of downstream responses through the canonical G-protein-binding interface.
Highlights
Despite the crowded nature of the cellular milieu, ligand–GPCR–G protein interactions are traditionally viewed as spatially and temporally isolated events
We have recently demonstrated that native peptides from the Gα C terminus are sufficient for GPCR priming[9]
We find that tethering of Qp or Sp to β2-adrenergic receptor (β2-AR) in live cells using a Systematic Protein Affinity Strength Modulation (SPASM) sensor enhanced potency (EC50) and Emax with isoproterenol, the β2-AR-selective ligand fenoterol[22], and the endogenous agonist epinephrine (Fig. 1h, i), recapitulating GPCR priming
Summary
Despite the crowded nature of the cellular milieu, ligand–GPCR–G protein interactions are traditionally viewed as spatially and temporally isolated events. The traditional model of GPCR signaling views the efficacy of effector activation by a GPCR as an isolated event that is spatially and temporally separated from the cellular milieu[2,3,4]. Emerging data suggest that GPCR–effector interactions can be spatially and temporally coupled, thereby enhancing the context-dependent multiplicity inherent in physiological signaling pathways. In addition to SPASM sensors, we employ affinity sequestration and UV irradiation of synthetic, photocleavable (PC) Gα C-terminal peptides to investigate the temporal regulation of GPCR–G protein interactions. Taken together, these approaches delineate the sequence of events underlying GPCR priming. We use a prototypical Gs-coupled receptor, β2-
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