Year: 2017
Type: dissertation
Abstract: Cells sense change in their external environment and react appropriately through the action of signaling pathways. This process is initiated by receptor proteins with high degrees of specificity for a particular stimulus. G protein-coupled receptors form the largest family of membrane protein receptors and their ability to sense a broad variety of ligands is unparalleled despite their common ancestral origin. How GPCRs evolved their unique specificities is unknown, although ligand binding affinity is often given a central role. The goal of this thesis was to assess the possible contributions of secondary mechanisms of specificity, namely ligand efficacy and downstream signaling regulation, to changes in ligand recognition. Through directed evolution, we generated a yeast pheromone receptor with an altered specificity in two steps. First, promiscuous receptors were obtained through either improved binding affinity or weaker molecular interaction with a negative regulator of signaling. Second, a ligand-discriminating receptor was obtained from a promiscuous variant solely by reducing the efficacy of the native pheromone. These findings demonstrate the importance of assessing GPCRsâ pharmacological profiles in their native context, where signaling trumps binding affinity in significance.
Source: TSpace (University of Toronto)
Author: RaphaĂŤl Brisset Di Roberto
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2892350899