Detecting g-protein coupled receptor interactions using enhanced green fluorescent protein reassembly

Kumaş, Gözde
The largest class of cell surface receptors in mammalian genomes is the superfamily of G protein-coupled receptors (GPCRs) which are activated by a wide range of extracellular responses such as hormones, pheromones, odorants, and neurotransmitters. Drugs which have therapeutic effects on a wide range of diseases are act on GPCRs. In contrast to traditional idea, it is recently getting accepted that G-protein coupled receptors can form homo- and hetero-dimers and this interaction could have important role on maturation, internalization, function or/and pharmacology. Bimolecular fluorescence complementation technique (BiFC); is an innovative approach based on the reassembly of protein fragments which directly report interactions. In our study we implemented this technique for detecting and visualizing the GPCR interactions in yeast cells. The enhanced green fluorescent protein (EGFP) fractionated into two fragments at genetic level which does not possess fluorescent function. The target proteins which are going to be tested in terms of interaction are modified with the non-functional fragments, to produce the fusion proteins. The interaction between two target proteins, in this study Ste2p receptors which are alpha pheromone receptors from Saccharomyces cerevisiae, enable the fragments to come in a close proximity and reassemble. After reassembly, EGFP regains its fluorescent function which provides a direct read-out for the detection of interaction. Further studies are required to determine subcellular localization of the interaction. Moreover, by using the fusion protein partners constructed in this study, effects of agonist/antagonist binding and post-translational modifications such as glycosylation and phosphorylation can be examined. Apart from all, optimized conditions for BiFC technique will guide for revealing new protein-protein interactions.


Identification of a contact region between the tridecapeptide alpha-factor mating pheromone of Saccharomyces cerevisiae and its G protein-coupled receptor by photoaffinity labeling.
Henry, Lk; Khare, S; Son, Çağdaş Devrim; Babu, Vv; Naider, F; Becker, Jm (2002-05-14)
Saccharomyces cerevisiae haploid cells communicate with their opposite mating type through peptide pheromones (alpha-factor and a-factor) that activate G protein-coupled receptors (GPCRs). S. cerevisiae was used as a model system for the study of peptide-responsive GPCRs. Here, we detail the synthesis and characterization of a number of a-factor (Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr) pheromone analogues containing the photo-cross-linkable group 4-benzoyl-L-phenylalanine (Bpa). Following chara...
Comparison of fluorescent protein labelled and wild type NMDA receptor distribution
Pirinçci, Şerife Şeyda; Son, Çağdaş Devrim; Department of Biology (2013)
NMDA (N-methyl D-aspartate) Receptor is a ligand and voltage gated ion channel and involved in many processes such as synaptic plasticity, memory formation, behavioral responses and cell survival. In the sense of functional activity, cellular localization of NMDAR is important since this receptor shows its activity on the membrane. Although NMDA receptor is intensely studied there are no satisfying study showing its localization with microscobic methods. Besides, the effect of florescent protein labelling o...
Balkan, Seyda Tuğçe; Son, Çağdaş Devrim; Küçük Baloğlu, Fatma; Department of Biochemistry (2021-8-11)
GPCR’s are seven-transmembrane receptors that transmit external signals to the intracellular environment via secondary messenger systems through heterotrimeric G proteins. Heterotrimeric G proteins consist of α and β-γ subunits. Until recent years, scientists thought that GPCR signal transduction occurs between one GPCR and one heterotrimeric G protein; however, recently, it has been shown that GPCR’s can make oligomers. Oligomerization of GPCR allows cells to tune the intensity of the signal and respond ap...
Characterization of extracellular beta-lactamases from penicillin G-resistant cells of Streptococcus thermophilus
Chirica, LC; Güray, Nülüfer Tülün; Gültekin, Güzin Candan; Bozoglu, F (International Association for Food Protection, 1998-07-01)
In this study, biochemical properties of two extracellular beta-lactamases produced by penicillin-resistant Streptococcus thermophilus cells were investigated. Both beta-lactamases showed specificity for penicillins but not for cephaloridins. The p-lactamases exhibited different affinities for penicillin G. The one with the higher molecular weight (F1) had a K(m) value of 3.44 mu M and a V(max), value of 8.33, mu mol/min/mg of protein, whereas the beta-lactamase with the lower molecular weight (FII) had a K...
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...
Citation Formats
G. Kumaş, “Detecting g-protein coupled receptor interactions using enhanced green fluorescent protein reassembly,” M.S. - Master of Science, Middle East Technical University, 2012.