Detection of homo/hetero-dimerizations between adenosine A2A, dopamine D2 and NMDA receptors by use of FRET and BiFC assays

Thesis Type: Postgraduate

Institution Of The Thesis: Orta Doğu Teknik Üniversitesi, Faculty of Arts and Sciences, Department of Biology, Turkey

Approval Date: 2014




Recent studies showed that GPCRs on the cell surface can exist as homo- or hetoro dimers and/or oligomers. To date, several G-protein coupled receptors have been revealed to interact either with other G-protein coupled receptors or other membrane proteins, including ion channels. Adenosine A 2A-Dopamine D2 and Dopamine D1-NMDA interactions are examples for well-studied heterodimerizations. All these receptors have critical functions in physiological processes. Adenosine A2A receptor modulates neurotransmission, cardiovascular system and immune response; while Dopamine D2 receptor controls the regulation of locomotion, food intake, learning, emotion and behavior. On the other hand, NMDAR mediates several signaling pathways necessary for synaptic plasticity, synaptic efficacy, neuronal morphology and memory formation by inducing long term potentiation. Dysfunctions in signaling mediated through these receptors have been shown in many neurological disorders, including Parkinson’s disease, schizophrenia and Alzheimer’s disease. The purpose of this study is detection of physical interactions between A2AR, D2R and NMDARs using fluorescent based detection methods, specifically Bimolecular Fluorescence Complementation (BiFC) and Fluorescence Resonance Energy Transfer (FRET) assays. For this purpose, receptors were labelled with split-EGFP fragments, and full length EGFP and mCherry proteins from their C-tail; and by co-transfecting the labelled receptors to N2a cells with various combinations, homo- and heterodimerizations were analyzed with and without agonist treatment using laser scanning confocal microscope. Finally, the observed dimerizations were quantified using PixFRET, an imageJ plugin. Moreover, further studies are planned to study oligomerization of these three receptors by combining BiFC and FRET techniques. Establishing this model will facilitate to understand the molecular mechanisms of these receptor interactions; and understanding these interactions in detail could help us design new treatment options. Additionally, the fluorescence based live cell model will be used to detect possible effects of potential drug candidates on the interactions of these receptors.