Ligands and signaling proteins govern the conformational landscape explored by a G protein-coupled receptor

Abstract

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.membrane protein | structural repertoire | biased ligands | constitutive activity M ajor progress has been made in past years in understanding the molecular bases of G protein-coupled receptor (GPCR)-mediated signaling. Spectacular developments in GPCR stabilization and crystallization have resulted in many different crystal structures of active and inactive receptors (1) and ultimately led to the structure of the agonist-activated β 2 -adrenergic receptor in complex with its cognate Gs protein (2). However, these structures still represent unique states, whereas the dynamic character of receptors is essential for their physiological functions. Indeed, many reports point at a model where GPCRs are highly dynamic proteins capable of adopting a large number of conformational states that signal with different efficacies to various pathways (3). The most detailed description of the conformational repertoire that a GPCR can adopt has been obtained in the case of the β 2 -adrenergic receptor with an experimental demonstration of a complex conformational landscape where ligands with distinct pharmacological properties shift the conformation of the receptor from one state to another (4-11). In the same way, we showed that ligands with different efficacies stabilize different conformations of the serotonin receptor (12). Despite this wealth of data, understanding of GPCR activation mechanisms, and in particular of the necessary influence of signaling proteins on the receptor conformationa...

Allosteric Modulation of a Chemogenetically Modified G Protein-Coupled Receptor

“…In agreement with our previous study (Canals et al, 2012), BQCA exhibited key hallmarks of allostery within a two-state system at the WT M 1 mAChR, including positive or negative modulation of orthosteric ligand activity depending on the nature of the orthosteric ligand (i.e., positive or inverse agonist) and different strengths of cooperativity depending on the intrinsic efficacy of the orthosteric ligand and the magnitude of stimulus-response coupling of the studied signal pathway. It can, of course, be questioned as to how an allosteric modulator can behave in a manner consistent with a two-state model at GPCRs given that these receptors adopt a larger spectrum of biologically active states, a prerequisite, in fact, for biased signaling (Vaidehi and Kenakin, 2010;Mary et al, 2012). This can be reconciled within a model whereby the modulator changes the abundance, but not the quality/nature, of different microstates that govern receptor activity; an overall change in abundance of active microstates in one direction relative to "inactive" microstates would still appear, at a macroscopic level, as a "two-state" system.…”

Advances in G Protein-Coupled Receptor Allostery: From Function to Structure

“…Early work by Gurevich et al (1997) found that a b-arrestin bound receptor showed a markedly higher affinity for agonists than the nonoccupied receptor, which is in accordance with the expectations of a ternary complex model. A more recent study using double-exponential fluorescent lifetime decay analysis showed distinct ghrelin receptor conformational changes upon addition of b-arrestin (or G q protein) in reconstituted lipid nanodiscs (Mary et al, 2012). The receptor activity modifying proteins (RAMPs) are another striking example of a single-transmembrane-spanning protein family that can associate with certain GPCRs (predominantly class B receptors) to create new receptor phenotypes or to bias signaling (McLatchie et al, 1998;Christopoulos et al, 1999Christopoulos et al, , 2003Hay et al, 2006).…”

The Effective Application of Biased Signaling to New Drug Discovery

“…Different cobinding ligands have been shown to affect the receptor affinity of ligands in accordance with this allosteric reciprocity (i.e., salvanorin affinity for k-opioid receptors with Ga 16 versus Ga i2 ; Yan et al, 2008 and changes in ghrelin receptors with addition of b-arrestin to nanodiscs, Mary et al, 2012). In view of the fact that functional response depends on the ternary complex of agonist/receptor/signaling protein (Onaran and Costa, 2012), it is conceivable that the affinity of the receptor-signaling complex shows variable affinity for agonists depending on the nature of the signaling protein interacting with the receptor.…”