Definition of phosphorylation acceptor sites in the ghrelin receptor (GHSR1a)key elements determining functionality

  1. Bouzo Lorenzo, Mónica
Supervised by:
  1. Felipe Casanueva Freijó Director
  2. Jesús Pérez Camiña Co-director

Defence university: Universidade de Santiago de Compostela

Fecha de defensa: 03 February 2016

Committee:
  1. Adrian Butscher Chair
  2. Rubén Nogueiras Pozo Secretary
  3. Vicente Mouly Committee member
  4. María Magdalena Cid Fernández Committee member
  5. Rosalía Gallego Gómez Committee member

Type: Thesis

Teseo: 409648 DIALNET

Abstract

Ghrelin is a hormone peptide of 28 amino acids which was isolated from the stomach in 1999 and identified as the endogenous ligand for the growth hormone secretagogue receptor (GHSR1a). Ghrelin is capable of induce growth hormone (GH) secretion from the anterior pituitary gland, but it also plays a key role regulating metabolism and energy homeostasis. The GHSR1a regulates both, central and peripheral actions of ghrelin. GHSR1aclasically exerts intracellular effects through G-protein activation, mainly via Gq/11 and Gi/o. Nonetheless, more recently was demonstrated the role of β-arrestins as mediators of GHSR1a signaling pathways in a manner independent of G proteins. Therefore, in addition to its initial function as carrier proteins in the endocytosis of the receptor, the β-arrestins are key elements in the regulation of GHSR1a intracellular routes and thus in its associated functionality. However, the factors which regulate the binding of GHSR1a with β-arrestin, determining the signaling cascades, or the contribution of G-proteins to β-arrestin signaling are not clear. Studies of other G protein-coupled receptors (GPCR) has showed that the phosphorylation of these receptors play a key role in the internalization and activation of signaling pathways dependent on β-arrestins. Even was described that the phosphorylation pattern of a GPCR may act as a bar code that determines at least in part their intracellular signaling. For these reasons, in this work was studied the GHSR1a pattern of phosphorylation as a key element of its activation in order to define specifically its signaling mechanisms and their biological functionality. In conjunction with this we have also identified phosphorylation dependent signaling outcomes of the GHSR1a receptor. We also established the roles of G-protein coupled receptor kinases (GRKs), specifically the involvement of GRK 2 and 6, in the phosphorylation of GHSR1a and its relationship with G-proteins through protein kinase C-α (PKCα). This complex system determines the functionality of the receptor associated to the expression in the target tissue. Finally, we attempted to identify the bioactive centre of ghrelin to isolate the structural element of ghrelin which interacts with GHSR1a to produce a functional response. To investigate this, various truncations of ghrelin were studied in functional assays to determine the structure/activity relationship.