PROJECT SUMMARY Ghrelin is secreted by the stomach and act on hypothalamic AGRP neurons to promote food intake. Due to the potent orexigenic action of ghrelin, the ghrelin receptor GHSR1a is considered a promising target for the treatment of obesity. Our discoveries made during the first period of this project revealed that, in vivo, GHSR1a is in complex with the accessory protein MRAP2 and that, in this physiologically relevant environment, the pharmacology of GHSR1a is vastly different than what was depicted in heterologous systems. Indeed, in the presence of MRAP2, GHSR1a does not display its well documented elevated constitutive activity, has an enhanced response to ghrelin and only weekly interacts with b-arrestins. Those findings challenge our understanding of ghrelin action and GHSR1a signaling in vivo and, thus, may warrant a reevaluation of the drug development strategies targeting GHSR1a for the treatment of obesity. The goal of this proposal is to accurately elucidate the physiological relevance of each known signaling component of GHSR1a in vivo and their regulation by MRAP2 by 1) determining the contribution of MRAP2-mediated inhibition of b-arrestin recruitment to GHSR1a for ghrelin action in AGRP neurons; 2) Investigate the importance of MRAP2-mediated abrogation of GHSR1a basal activity on ghrelin action in AGRP neurons and 3) Assessing the requirement of MRAP2-mediated enhancement of ghrelin-stimulated Gaq/11 signaling in AGRP neurons. Using new tools developed in our lab, and state of the art technics for targeted gene silencing in specific neurons and feeding studies, we will accurately determine, in vivo, the role of each of those aspects of GHSR1a signaling for ghrelin function. We will also comprehensively investigate the relative importance of each of MRAP2 actions on GHSR1a signaling for the orexigenic action of ghrelin. Completion of this project will provide a clear understanding of GHSR1a signaling and regulation in its endogenous environment and establish the functional importance of the modulation of G- protein and b-arrestin dependent signaling by MRAP2. These results will have tremendous implications in informing future efforts to modulate GHSR1a signaling to promote weight loss and demonstrate the importance of studying GPCRs in their endogenous context and in the presence of their modulatory accessory proteins.