The dynamic character of G protein-coupled receptors is essential to their function. However, the details of how ligands stabilize a particular conformation to selectively activate a signaling pathway and how signaling proteins affect this conformational repertoire remain unclear. Using a prototypical peptide-activated class A G protein-coupled receptor (GPCR), the ghrelin receptor, reconstituted as a monomer into lipid discs and labeled with a fluorescent conformational reporter, we demonstrate that ligand efficacy and functional selectivity are directly related to different receptor conformations. Of importance, our data bring direct evidence that distinct effector proteins affect the conformational landscape of the ghrelin receptor in different ways. Whereas G proteins affect the balance between active and inactive receptor substates in favor of the active state, agonist-induced arrestin recruitment is accompanied by a marked change in the structural features of the receptor that adopt a conformation different from that observed in the absence of arrestin. In contrast to G proteins and arrestins, μ-AP2 has no significant effect on the organization of the transmembrane core of the receptor. Such a modulation of a GPCR conformational landscape by pharmacologically distinct ligands and effectors provides insights into the structural bases that decisively affect ligand efficacy and subsequent biological responses. This is also likely to have major implications for the design of drugs activating specific GPCR-associated signaling pathways.

β-arrestins are key molecular partners of G Protein-Coupled Receptors (GPCRs), triggering not only their desensitization but also intracellular signaling. Existing structural data point to high conformational plasticity of GPCR:β-arrestin interaction, with two completely different orientations between receptor and β-arrestin. Combining molecular dynamics simulations and fluorescence spectroscopy, we show that β-arrestin 1 interacts with membranes even in the absence of a receptor, an interaction that is enhanced by PI(4,5)P2, presumably holding the β-arrestin 1 C-edge loop into the lipid bilayer. This key interaction helps β-arrestin 1 to adopt a “receptor-ready” orientation and consequently favors its coupling to the ghrelin receptor (GHSR). In addition, we show that the GHSR:β-arrestin 1 assembly is a dynamic complex where β-arrestin can adopt several orientations. PI(4,5)P2 decreases the dynamics of the complex and shifts the equilibrium between the different arrangements, favoring one of them. Taken together, our results highlight how PI(4,5)P2 plays a true third-player role in the GPCR:β-arrestin interaction, not only by preparing β-arrestin for its further interaction with receptors but also by modulating its orientation once the protein:protein complex is formed.

Through their coupling to G proteins, G Protein-Coupled Receptors (GPCRs) trigger cellular responses to various signals. Some recent experiments have interestingly demonstrated that the G protein can also act on the receptor by favoring a closed conformation of its orthosteric site, even in the absence of a bound agonist. In this work, we explored such an allosteric modulation by performing extensive molecular dynamics simulations on the adenosine A2 receptor (A 2 aR) coupled to the Mini-Gs protein. In the presence of the Mini-Gs, we confirmed a restriction of the receptor’s agonist binding site that can be explained by a modulation of the intrinsic network of contacts of the receptor. Of interest, we observed similar effects with the C-terminal helix of the Mini-Gs, showing that the observed effect on the binding pocket results from direct local contacts with the bound protein partner that cause a rewiring of the whole receptor’s interaction network.