High Constitutive Activity Is an Intrinsic Feature of Ghrelin Receptor Protein

• Published in: The Journal of biological chemistry Vol. 287; no. 6; pp. 3630 - 3641
• Main Authors: Damian, Marjorie, Marie, Jacky, Leyris, Jean-Philippe, Fehrentz, Jean-Alain, Verdié, Pascal, Martinez, Jean, Banères, Jean-Louis, Mary, Sophie
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01-02-2012
• Subjects: 7-Helix Receptor; Arrestin; Constitutive Activity; Fluorescence; Ghrelin; Internalization; Membrane Proteins; Signal Transduction; Membrane Proteins; Signal Transduction; Ghrelin; Internalization; Fluorescence; Constitutive Activity; 7-Helix Receptor; Arrestin
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.288324

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 Gq 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.
Background: Constitutive activity is central to G protein-coupled receptor signaling but the mechanisms underlying it are still unknown.
Results: The ghrelin receptor monomer reconstituted in a lipid disc activates Gq without agonist and recruits arrestin in a ligand-dependent manner.
Conclusion: High constitutive activity is an intrinsic property of the ghrelin receptor.
Significance: This is the first demonstration that the ghrelin receptor has all the determinants for constitutive activity and ligand-regulated internalization.