G protein-coupled receptor (GPCR) signaling is a dynamic process involving various conformational intermediates in addition to those captured in static three-dimensional structures. Here, we used newly developed G protein peptidomimetics to characterize the interactions of the ghrelin receptor (GHSR) with G proteins. Coupling to the G protein peptidomimetic not only affected the conformational features of the cytoplasmic regions of the receptor where the G protein binds but also allosterically affected the extracellular ligand-binding pocket. These conformational and allosteric changes increased the affinity of G protein-coupled GHSR for the endogenous agonist ghrelin. In addition, our data identified different complexes along the G protein activation pathway that differed in the engagement of the Gαq C-terminal helix. Given that this helix is the main link between the activated receptor and the Gα nucleotide-binding pocket, these findings suggested a stepwise process involving distinct states in GPCR-catalyzed G protein activation. Collectively, our results provide evidence for the dynamic behavior of GPCR-G protein signaling complexes, with such dynamics most likely contributing to signaling selectivity and/or efficacy.
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