Optimization of internal tagging of inhibitory G-proteins for investigating their interactions with dopamine receptor D2 via fret method

Özcan, Gizem
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 emotion and behavior related pathways. G-proteins take their name from their ability to bind to guanine nucleotide. They are key molecules for activation or deactivation for their related signaling pathways. D2R acts through inhibitory G-proteins which in turn reduces adenylyl cyclase activity. Deregulation of the dopaminergic signaling is shown to be related to many neurologic diseases including schizophrenia and Parkinson’s disease. Förster resonance energy transfer (FRET) technique is used for investigating distances between molecules. Energy transfer between two molecules is possible only when they are very close to each other, making it possible to conclude that these molecules are actually interacting when this transfer occurs. The purpose of the present study was to optimize labeling of inhibitory G-protein α subunits GNAO1 (Go1) and GNAI3 (Gi3) with fluorophores for the first time in the literature to be able to investigate their interactions with D2R via FRET method. To achieve this, D2R was labeled with Enhanced Green Fluorescent Protein (EGFP) from its C-terminus and G proteins were labeled with mCherry from three different internal locations. Co-transfections of all of the labeled proteins to Mus musculus Neuroblastoma-2a (N2a) cells were done and interactions were investigated via spinning disc confocal microscopy. Selected constructs were expressed in embyronic kidney (HEK293) cells to compare different cellular environment’s effects on their interaction. Results presented in this study are highlighting a possible starting point to investigate these subunits’ interactions with their receptors and other downstream molecules via FRET which adds a value to the GPCR targeted study grounds.