In vivo detection of GPCR dimerizations in Saccharomyces cerevisiae using FRET and BiFC


Thesis Type: Postgraduate

Institution Of The Thesis: Orta Doğu Teknik Üniversitesi, Faculty of Arts and Sciences, Department of Biology, Turkey

Approval Date: 2014

Student: BEREN ÜSTÜNKAYA

Consultant: ÇAĞDAŞ DEVRİM SON

Abstract:

G protein-coupled receptors (GPCRs) are a class of membrane proteins that are composed of seven transmembrane domain and mediate physiological response to a diverse array of stimuli. In eukaryotic microorganisms, GPCRs regulate cell growth, development, morphogenesis, motility, and life span. In higher eukaryotic organisms as humans, they mediate the action of hundreds of peptide hormones, sensory stimuli, odorants, neurotransmitters, and chemokine. Due to their wide spectrum of action mechanisms, GPCRs are also targets for ~40-50% of current pharmaceuticals in the market. For a long time, these receptors have been thought to locate and function as monomeric units which activate a related heterotrimeric G protein to transmit the extracellular signal to inside of the cell. However, recent studies in last two decades have suggested that GPCRs form dimers (or higher order oligomers) for proper trafficking and/or functioning. After these early findings, many studies have been conducted to reveal, examine and understand dimerization of single type or different type of the receptors. So far, the dimers/oligomers have been reported to play important roles in regulation of receptor expression, ligand binding and second messenger activation whereas there is still limited information on how and where GPCR dimerization occurs, which type of them interact with each other, and where these dimers are located in the cells. In this thesis, we studied detection of dimer formation between nutrient sensing GPCR in Saccharomyces cerevisiae, Gpr1 proteins with its own type, and also with Ste2 protein, which is expressed as mating pheromone receptor by MATa type of haploid yeast cells. In vivo imaging after application of Förster Resonance Energy Transfer (FRET) and Bimolecular Fluorescence Complementation (BiFC) methods successfully showed that both dimer groups are located on plasma membrane and in intracellular compartments.