Ghrelin receptor conformational dynamics regulate the transition from a preassembled to an active receptor:Gq complex

How G protein-coupled receptor conformational dynamics control G protein coupling to trigger signaling is a key but still open question. We addressed this question with a model system composed of the purified ghrelin receptor assembled into lipid discs. Combining receptor labeling through genetic incorporation of unnatural amino acids, lanthanide resonance energy transfer, and normal mode analyses, we directly demonstrate the occurrence of two distinct receptor:Gq assemblies with different geometries whose relative populations parallel the activation state of the receptor. The first of these assemblies is a preassembled complex with the receptor in its basal conformation. This complex is specific of Gq and is not observed with Gi. The second one is an active assembly in which the receptor in its active conformation triggers G protein activation. The active complex is present even in the absence of agonist, in a direct relationship with the high constitutive activity of the ghrelin receptor. These data provide direct evidence of a mechanism for ghrelin receptor-mediated Gq signaling in which transition of the receptor from an inactive to an active conformation is accompanied by a rearrangement of a preassembled receptor:G protein complex, ultimately leading to G protein activation and signaling.G protein-coupled receptors (GPCRs), one of the largest cell surface receptor families, are involved in many cellular signaling processes (1). Based on this property, as well as their importance as drug targets, the molecular aspects of GPCR functioning have been extensively investigated. In particular, coupling to heterotrimeric G proteins has been the focus of numerous studies. Indeed, delineating the molecular mechanisms responsible for receptor:G protein interaction is absolutely required to better understand how signaling is controlled. Recent years have seen spectacular advances that have culminated in elucidation of the 3D structure of the β₂-adrenergic receptor:Gs complex (2). Nevertheless, the need for further progress remains, in particular to fully understand the dynamics of this interaction. This is a crucial question, given that how the receptor interacts with its G protein partner governs signaling, and thus biological and pathophysiological responses.To date, two different models for GPCR:G protein interaction have been proposed: collision coupling and preassembly. Originally, it was proposed that receptors and G proteins couple by collision (3, 4). One of the main features of this model is that only activated receptors interact with G proteins. Since then, alternative models of signaling have been developed. One of these, the preassembly model, proposes that the receptor and the G protein make a complex even in the absence of agonist (5–8). Discriminating between the two models is crucial. Indeed, signaling outputs, such as the kinetics of G protein activation, will be significantly different depending on whether the ligand-free receptor is always in complex with its G protein or must first be activated by the agonist to recruit the G protein and trigger signaling. Moreover, it has been shown that GPCR conformational dynamics (9–11) and signaling in the absence of ligand are key features of GPCR functioning (12). How receptor constitutive activity and conformational dynamics relate to their coupling to the G protein remains an open question.Here we used the purified ghrelin receptor GHS-R1a to analyze the way in which this GPCR interacts with its G protein partners. Ghrelin is a neuroendocrine peptide hormone that acts through its cognate GPCR to control important biological processes, such as growth hormone secretion, food intake, and reward-seeking behaviors (13). Among the GPCRs, GHS-R1a has been shown to have one of the highest basal Gq activation levels both in vitro (10, 14) and in vivo (15, 16). The physiological relevance of GHS-R1a basal activity is substantiated by the occurrence of a natural human mutation in the GHS-R1a gene (A²⁰⁴E substitution in the second extracellular loop of the receptor) that dramatically decreases constitutive activity and is associated with a short-stature phenotype (17). Along with its importance in drug design, GHS-R1a is a prototype for peptide-activated class A GPCRs.To delineate the way in which the ghrelin receptor interacts with G proteins, we used monomeric GHS-R1a reconstituted in a membrane-mimicking environment, lipid discs, and a combination of innovative biochemical [labeling with unnatural amino acid (UAA)] and biophysical [lanthanide resonance energy transfer (LRET) and normal mode (NM) analyses] approaches. By doing so, we provide the first direct evidence that ghrelin-mediated signaling involves a complex dialogue between the conformational dynamics of the receptor and its ability to interact with the different G protein subtypes to which it is coupled.