Investigation of Gai1 protein homodimerization in live cells using förster resonance energy transfer (FRET) and biomolecular fluorescence complementation assay (BIFC)

Atay, Özge
The classical GPCR signaling pathway, where a heterotrimeric G protein-GPCR interaction is sufficient to transmit the signal to effector proteins has been replaced by a heteromeric G protein-GPCR homo- or hetero-dimer interaction model over the past two decades. These studies demonstrate that GPCRs that interact with each other couple with a heteromeric G protein. In recent years, evidence suggests that dimer of GPCR dimers is required for some complex signal transductions. In these studies, it was proposed that this heteromeric tetramer formed by the dimerization of the dimers brought two G proteins close enough to each other for protein-protein interaction. It is not clear if GPCR tetramerization is required for G-protein dimerization or dimerization can occur independent of the GPCRs. On the other hand, studies on small G-proteins (Ras family), which are structural homologs of G alpha subunits of heteromeric G-proteins, shows that dimerization can be independent of the receptors and necessary for various signaling pathways.Within the scope of this study, G alpha protein homodimerizations were qualitatively and quantitatively investigated in live cells using Bimolecular Fluorescent Complementation Assay (BiFC) and Förster resonance energy transfer (FRET) method.To achieve this, Galphai1 protein was labeled from various positions, including (G60-Y61, L91-K92 and A121-E122), with Enhance Green Fluorescent Protein (EGFP) which was derived from Aequorea victoria and mCherry fluorescent protein which is a monomeric derivative of DsRed.In addition Gaphai1 gene was labeled with split EGFP parts which are N-terminus EGFP and C-terminus EGFP for the Bimolecular Fluorescence Complementation Assay. All labeled proteins were cotransfected into Mus musculus Neuroblastoma-2a (N2a) cells and interactions were imaged using spinning disc confocal microscope and analyzed.The findings of this study could help us understand the molecular mechanisms required for Galpha dimerization and the dynamics of these proteins.Also,GPCR interactions with various effectors during complex signal transductions and the requirement of these receptors during Galpha dimerizations can be studied with the techniques optimized in this study.


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...
Integration of a Glutamate Sensitive Genetically Encoded Sensor Protein into Photocrosslinkable Hydrogel Optrodes
Kahyaoğlu, Leyla Nesrin (2016-01-01)
Immobilization into 3D matrices stabilizes proteins in comparison to flat planar surfaces and facilitates the study of the biomolecular interactions as well as integration into optrodes for cell physiology. Photocrosslinkable hydrogels have received significant attention in recent years as they provide not only a highly hydrophilic 3D environment to promote protein stabilization and its interactions with analyte molecules, but enable optically addressable patterning for spatial control of protein localizati...
Analysis of ligand-receptor cross-linked fragments by mass spectrometry
Son, Çağdaş Devrim; Hurst, GB; Naider, F; Becker, JM (Wiley, 2005-03-01)
G-protein coupled receptors (GPCRs) are a class of integral membrane receptor proteins that are characterized by a signature seven-transmembrane (7-TM) configuration. The alpha-factor receptor (Ste2p) from Saccharomyces cerevisiae is a GPCR that, upon binding of a peptide ligand, transduces a signal to initiate a cascade of events leading to the mating of haploid yeast cells. This study summarizes the application of affinity purification and of matrix-assisted laser-desorption ionization time-of-flight (MAL...
GPCR-Gα protein precoupling: Interaction between Ste2p, a yeast GPCR, and Gpa1p, its Gα protein, is formed before ligand binding via the Ste2p C-terminal domain and the Gpa1p N-terminal domain
Cevheroğlu, Orkun; Becker, Jeffrey M.; Son, Çağdaş Devrim (Elsevier BV, 2017-12)
G protein coupled receptors bind ligands that initiate intracellular signaling cascades via heterotrimeric G proteins. In this study, involvement of the N-terminal residues of yeast G-alpha (Gpa1p) with the C-terminal residues of a full-length or C-terminally truncated Ste2p were investigated using bioluminescence resonance energy transfer (BRET), a non-radiative energy transfer phenomenon where protein-protein interactions can be quantified between a donor bioluminescent molecule and a suitable acceptor fl...
The yeast Ste2p G protein-coupled receptor dimerizes on the cell plasma membrane
Cevheroglu, Orkun; Kumas, Gozde; Hauser, Melinda; Becker, Jeffrey M.; Son, Çağdaş Devrim (2017-05-01)
Dimerization of G protein-coupled receptors (GPCR) may play an important role in maturation, internalization, signaling and/or pharmacology of these receptors. However, the location where dimerization occurs is still under debate. In our study, variants of Ste2p, a yeast mating pheromone GPCR, were tagged with split EGFP (enhanced green fluorescent protein) fragments inserted between transmembrane domain seven and the C-terminus or appended to the C-terminus. Bimolecular Fluorescence Complementation (BiFC) ...
Citation Formats
Ö. Atay, “Investigation of Gai1 protein homodimerization in live cells using förster resonance energy transfer (FRET) and biomolecular fluorescence complementation assay (BIFC),” Thesis (M.S.) -- Graduate School of Natural and Applied Sciences. Biochemistry., Middle East Technical University, 2019.