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

Abstract

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.GPCR | G protein | preassembly | conformation dynamics | 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 β 2 -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).

A Significant Role of the Truncated Ghrelin Receptor GHS-R1b in Ghrelin-induced Signaling in Neurons

“…The ability of GHS-R1a homodimers to oligomerize with one or several (at least two) GHS-R1b protein molecules provides a frame for oligomerization to be involved in this fine-tuning, stoichiometry-dependent modulation of GHS-R1a function. Because GHS-R1a seems to be the minimal functional unit (19), one possible scenario is that one GHS-R1b molecule per one GHS-R1a homodimer facilitates trafficking but not a negative allosteric modulation of ghrelin, whereas two (or more) GHS-R1b molecules would not facilitate trafficking but would allosterically decrease ghrelin-mediated signaling.…”

“…In isolation, the GHS-R1a receptor has been shown to couple to G q proteins, resulting in activation of phospholipase C, inositol 1,4,5-trisphosphate, and Ca 2ϩ mobilization (5,19,20), but it has also been reported to produce signaling dependent on pertussis toxin-sensitive G i/o proteins (21,22). Thus, ghrelin seems to be able to activate different signaling pathways in a tissue-specific manner.…”

“…Primary cultures of striatal, hippocampal, and cortical neurons were obtained from fetal Sprague-Dawley rats (embryonic day 19). Cells were isolated as described in Ref.…”

Conformational Equilibrium of CDK/Cyclin Complexes by Molecular Dynamics with Excited Normal Modes

“…Recently, we proposed MD with excited NMs (MDeNM) (27) as an alternative method that uses the low-frequency NMs computed in vacuum as privileged directions in MD simulations with an explicit representation of the surrounding medium. In addition, we applied this method to simulate large protein/protein complexes in a membrane model (28).…”

Agonism, Antagonism, and Inverse Agonism Bias at the Ghrelin Receptor Signaling

“…Unlike the endogenous ligand that usually activates all the G protein and ␤-arrestin-dependent pathways, the synthetic ligands could thus selectively activate only some of them, for instance activation of ␤-arrestin with no effect on G proteins (16). Although molecular mechanisms responsible for ligand-directed functional selectivity are not fully understood, there is increasing evidence that biased activity results from selective stabilization of different receptor conformations that differ in their ability to couple to different downstream effectors (17)(18)(19)(20)(21). Importantly, in vivo studies demonstrated that side effects of classical drugs might be diminished by the use of biased molecules, suggesting this kind of molecules could have potential clinical application (16,22).…”