Gαi ve gαs proteinlerinin fiziksel etkileşimlerinin canlı hücrelerde FRET Metodu ile araştırılması

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 appropriately. Studies with A1 and A2A heterotetramer and A2A – D2R heterotetramer showed that these interactions theoretically allow G protein α subunit dimer formation according to Navarro et al. and Ferré et al. This theoretical interaction has never been validated via an advanced fluorescent microscopy technique like FRET. This technique allows the detection of physical protein-protein interaction of fluorescently labeled proteins via resonance energy transfer between donor and acceptor fluorophores. In this study, Gαi and Gαs interaction was investigated by FRET technique. In addition, the effect of agonist treatment, CGS (CGS-21680), and quinpirole for A2A and D2R heterotetramer, respectively, was investigated. Furthermore, based on the G protein family member K-Ras studies, which has high structural homology with G protein α subunits, that forms a homodimer independent from its receptor (Dempke & Heinemann, 2009), we tested the effect of receptor binding on the physical interaction of G protein α subunit using G protein α subunit-specific minigenes.