Abstract by Bjørn Behrens Sivertsen

G protein-coupled receptor (GPCRs) are one of the most important drug target families. They are involved in a large part of the hormonal- and paracrine signaling processes. They exert their function from the cell membrane where they sense extracellular signaling molecules and initiates a wide variety of signaling cascades. These signaling cascades were initially believed to be simple, in an “on/off” manner of a limited magnitude. It is now assumed that almost all GPCRs can initiate a vast network of intracellular responses in a very fine-tuned manner if given a particular stimulus. The signal outcome of GPCR activation is believed to be governed by direct interactions with GTP binding proteins, β-arrestins, and GRKs. The expression level of only a subset of these interaction partners can hugely shift the amplitude of the signaling cascades. The expression pattern of the GPCRs and interaction partners varies immensely depending on tissues, especially between the brain and the peripheral tissues. In this thesis we studied the ghrelin receptor, which is crucial in many energy-homeostasis and behavioral processes, however it is especially known for its role in appetite stimulation. The ghrelin receptor is highly expressed in the brain, where it is believed to exert its effects on energy homeostasis. The endogenous ligands for the ghrelin receptor are: an agonist, ghrelin, a peptide with an acylation on serine residue number 3 and a recently discovered antagonist LEAP-2. We studied the molecular pharmacology of the activation of the ghrelin receptor by mutation, fluorescent probes and measuring how the signaling for the receptor is conveyed by the G proteins, GRKs and β-arrestins. We found that the ghrelin receptor activates a plethora of intracellular signaling molecules and interacts with the subfamily of G proteins called Gα_12/13 to activate the Rho/ROCK family of second messengers. We also demonstrate that different ligand classes induce different conformations of the receptor by environmentally sensitive probes. We established a novel approach with less perturbation by the probes themselves compared to other commonly used probes. We lastly find that the ghrelin receptor is constitutively phosphorylated and that this phosphorylation increases β-arrestin recruitment to the receptor. We also find that without the kinase ability, GRK2 still hampers the constitutive and agonist-stimulated signaling of the receptor. Our findings are important to the early part of drug discovery; new interaction partners for the ghrelin receptor are verified and new tools to study the receptor are developed. Our findings also indicate that selecting of the most suitable assays in relation to the desired in vivo effect is important.