Visualization of interactions between fluorescently tagged g protein α11, α12/13 subtypes and adenosine 2a, dopamine 2 or homodimer adenosine 2a/2a receptors

Kostromin, İrmak Begüm
G-Protein-coupled receptors (GPCRs) belong to one of the largest family of cell surface receptors, which transmit extracellular signals to intracellular responses by interacting with G- proteins. The G proteins are known as molecular switches that regulates different pathways via control of secondary messengers and signaling proteins. Adenosine 2A (A2A) and Dopamine 2 (D2) receptors belong to G-Protein-coupled receptors (GPCRs) family and are located mostly in striatopallidal γ-aminobutyric acid (GABA) containing neurons. Therefore, problems related to these receptors are associated with physiological disorders such as schizophrenia, and Parkinson disease. A2A receptors regulate neurotransmission, cardiovascular system and immune response; On the other hand, D2 receptors modulate the regulation of locomotion, food intake, learning, emotion and behavior. Studies showed that, A2A and D2 receptors change the cAMP level by stimulating or inhibiting adenylyl cyclase via interacting with Gαs and Gαi proteins. When D1 and D2 receptors heterodimerize, Gαq11 signal pathway was preferred and activate PIP2 and DAG secondary messengers. However, interaction between Gαq11 protein and A2A, D2R or A2A/A2A homodimer is not reported. GPCRs could also signal through Gα12/13 which regulates actin cytoskeleton and cell migration through activation of Rho kinases. It is established that, Gα12/13 mutant cells show neuronal over migration and dysfunction at cerebral cortex. In addition, cell migration is effected positively by A2A receptor activation while it could be negatively regulated by D2R receptor activation. However, it is not clear if Gα12/13 proteins are interacting with A2A and D2 receptors triggering related pathways. The aim of this study is to analyze possible physical interaction between Gαq11-A2A, Gαq11-D2R, Gαq11-A2A/A2A; Gα12/13-A2A, Gα12/13 -D2R and Gα12/13 -A2A/A2A using Fluorescence Resonance Energy Transfer (FRET) method. For this purpose, G-protein α subunits (Gα11,G α12 and Gα13) were labeled with mCherry or EGFP fluorescent proteins from five different internal locations; receptors were labelled with split-EGFP fragments, and full length EGFP fluorophore, and then transfected to Mus musculus Neuroblastoma-2a (N2a) cells as various combinations. To analyze the possible interactions, images were taken via spinning disc confocal microscopy and FRET efficiency was calculated by using pixFRET Plugin for ImageJ software. Images obtained from Gα11+mCherry and D2R+EGFP transfected N2a cells; Gα13+mCherry and A2A+EGFP have high FRET signal mostly located on cell membrane. Gα13-D2R, Gα11A2A and Gα11-A2A/A2A pairs had lower signal than Gα11-D2R and Gα13-A2A pairs. Further experiments are necessary to prove an interaction between these pairs if it ever exists. On the othe hand Gα12-A2A and Gα13-A2A/A2A pairs had very low signal that was impossible to difrentiate from background signal indicating a lack of interaction. Protein interaction studies like the ones detailed in this study could help us understand signal transduction pathways much better, thus will be useful to design new treatment models and discover drug candidates in future.