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

Download
2014
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.

Suggestions

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...
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 ...
Enzyme prediction with word embedding approach
Akın, Erkan; Atalay, M. Volkan.; Department of Computer Engineering (2019)
Information such as molecular function, biological process, and cellular localization can be inferred from the protein sequence. However, protein sequences vary in length. Therefore, the sequence itself cannot be used directly as a feature vector for pattern recognition and machine learning algorithms since these algorithms require fixed length feature vectors. We describe an approach based on the use of the Word2vec model, more specifically continuous skip-gram model to generate the vector representation o...
More than just a dimer: detection of G protein-coupled receptor oligomers using fluorescent protein reassembly of Ste2p, a yeast pheromone receptor
Cevheroğlu, Orkun; Son, Çağdaş Devrim; Akkaya, Mahinur S.; Department of Biotechnology (2015)
GPCRs are known to form homo- and hetero-dimers and this interaction could have important roles in internalization, maturation, function and/or pharmacology of these receptors. In the first part of the study bimolecular fluorescence complementation (BiFC) using split enhanced green fluorescent protein (EGFP) was used to determine the interaction and cellular location between various Ste2p constructs. Co-expression of two constructs, one with the N-terminus of EGFP inserted into the full-length receptor at t...
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.