In Vivo detection of GPCR dimerizations in saccharomyces cerevisiae using FRET and BIFC

Üstünkaya, Beren
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.


In vivo detection of yeast alpha mating pheromone receptor ste2p homodimerization by FRET
Bulut, Giray; Son, Çağdaş Devrim; Department of Biology (2014)
Ste2p is an alpha type pheromone sensing receptor of ‘a’ type Saccharomyces cerevisiae cells. Yeast life cycle could be haploid or diploid due to the signal sensed by Ste2p. This receptor is a G protein coupled receptor (GPCR). GPCRs are one of the most important drug targets because they are playing key roles in cell signaling. They have seven transmembrane domains and linked with a G protein in the cytosol. FRET is a method that is used for detecting protein-protein interactions by using the resonance ene...
Optimization of internal tagging of inhibitory G-proteins for investigating their interactions with dopamine receptor D2 via fret method
Özcan, Gizem; Son, Çağdaş Devrim; Özçubukçu, Salih; Department of Biochemistry (2016)
G-Protein Coupled Receptors (GPCRs) constitute a large family of receptors which act by sensing the molecules outside the cell and start a signal transduction inside the cell through interacting with their associated G-proteins. This interaction results in activation or repression of related signaling pathways via associated secondary messengers. Dopamine receptor D2 (D2R) is a member of D2-like Dopamine Receptor group, which also belongs to the GPCR family. It is known that D2R has critical roles in emotio...
Visualization of interactions between fluorescently tagged g protein α11, α12/13 subtypes and adenosine 2a, dopamine 2 or homodimer adenosine 2a/2a receptors
Kostromin, İrmak Begüm; Son, Çağdaş Devrim; Department of Biology (2018)
G-Protein-coupled receptors (GPCRs) belong to one of the largest family of cell surface receptors, which transmit extracellular signals to intracellular responses by interacting with G- proteins. The G proteins are known as molecular switches that regulates different pathways via control of secondary messengers and signaling proteins. Adenosine 2A (A2A) and Dopamine 2 (D2) receptors belong to G-Protein-coupled receptors (GPCRs) family and are located mostly in striatopallidal γ-aminobutyric acid (GABA) cont...
Investigation of the effect of GPCR oligomerization on the GNAi1 protein homodimerization in live cells using FRET
Nalli, Enise; Son, Çağdaş Devrim; Küçük Baloğlu, Fatma; Department of Biotechnology (2022-1-26)
G-Protein Coupled Receptors (GPCR) are membrane proteins that pass the cell membrane seven times. In classical GPCR signaling pathways, one GPCR-one heterotrimeric G-protein interaction model is enough to transmit the signal to effector proteins. Studies since 2000 showed that one GPCR dimer-one heterotrimeric G-protein interaction model is more likely, and GPCRs having homo- /hetero- dimers interact with a single G⍺-protein. Recently, studies on GPCRs indicated that more than two receptors interact to ...
Identification of interaction sites of G protein-coupled receptors using machine learning techniques
Şahin, Mehmet Emre; Can, Tolga; Department of Computer Engineering (2014)
G protein-coupled receptors (GPCRs), which play a crucial role in a host of pathophysiological pathways, form the largest and most divergent receptor family. Typically, they transmit outer signals to the inner cell by interacting with G-proteins. The emerging concept of GPCR dimerization has unsettled the classical idea that GPCRs function as monomeric units. Prediction of the interface residues of GPCR dimers is a challenging topic. The method proposed in this thesis trains itself with known interfaces fro...
Citation Formats
B. Üstünkaya, “In Vivo detection of GPCR dimerizations in saccharomyces cerevisiae using FRET and BIFC,” M.S. - Master of Science, Middle East Technical University, 2014.