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

Thesis Type: Postgraduate

Institution Of The Thesis: Middle East Technical University, Turkey

Approval Date: 2019

Student: Özge Atay



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where a heterotrimeric G protein-GPCR interaction is sufficient to transmit the signal to effector proteins has been replaced by a heteromeric G protein-GPCR homo- or hetero-dimer interaction model over the past two decades.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.