Abstract
Despite its central role in signaling and the potential therapeutic applications of inverse agonists, the molecular mechanisms underlying G protein-coupled receptor (GPCR) constitutive activity remain largely to be explored. In this context, ghrelin receptor GHS-R1a is a peculiar receptor in the sense that it displays a strikingly high, physiologically relevant, constitutive activity. To identify the molecular mechanisms responsible for this high constitutive activity, we have reconstituted a purified GHS-R1a monomer in a lipid disc. Using this reconstituted system, we show that the isolated ghrelin receptor per se activates G(q) in the absence of agonist, as assessed through guanosine 5'-O-(thiotriphosphate) binding experiments. The measured constitutive activity is similar in its extent to that observed in heterologous systems and in vivo. This is the first direct evidence for the high constitutive activity of the ghrelin receptor being an intrinsic property of the protein rather than the result of influence of its cellular environment. Moreover, we show that the isolated receptor in lipid discs recruits arrestin-2 in an agonist-dependent manner, whereas it interacts with μ-AP2 in the absence of ligand or in the presence of ghrelin. Of importance, these differences are linked to ligand-specific GHS-R1a conformations, as assessed by intrinsic fluorescence measurements. The distinct ligand requirements for the interaction of purified GHS-R1a with arrestin and AP2 provide a new rationale to the differences in basal and agonist-induced internalization observed in cells.
Highlights
Constitutive activity is central to G protein-coupled receptor signaling but the mechanisms underlying it are still unknown
From GHS-R1a Expression in Inclusion Bodies to Assembly into Lipid Discs—To achieve a high-level production of the functional ghrelin receptor in lipid discs, we adopted a strategy that consists of accumulating the protein in E. coli inclusion bodies, folding it back to its native state, and assembling it into the lipid particles
The second peak corresponds to a homogeneous fraction with an apparent Stokes diameter in the 12-nm range, in agreement with the expected value for a lipid disc formed with MSP1E3 [49]
Summary
Constitutive activity is central to G protein-coupled receptor signaling but the mechanisms underlying it are still unknown. Despite its central role in signaling and the potential therapeutic applications of inverse agonists, the molecular mechanisms underlying G protein-coupled receptor (GPCR) constitutive activity remain largely to be explored In this context, ghrelin receptor GHS-R1a is a peculiar receptor in the sense that it displays a strikingly high, physiologically relevant, constitutive activity. The ability for a vast majority of receptors to display spontaneous constitutive activity has extended our insights into G protein-coupled receptor (GPCR)4-mediated signaling and provided new criteria for drug design and therapeutic strategies [1] Along this line, the behavior of very specific modulators of this basal activity shed light on inverse agonism [2, 3]. We directly demonstrate that the basal active state is an intrinsic feature of the monomeric GHS-R1a receptor
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