Optimization of FRET method to detect dimerization of dopamine D2 and adenosine A2A receptors in live cells

Ünlü, Gökhan
Recent studies demonstrate that there are several G-protein coupled receptors (GPCRs) that dimerize with other GPCRs and form heterodimers. Adenosine A2A-Dopamine D2 receptor interaction is one of the examples for GPCR heterodimerization. Both receptors bear critical roles in physiological processes. Adenosine A2A receptor has functions in neurotransmission, cardiovascular system and immune response. On the other hand, dopamine receptors are the key point of dopaminergic system, which controls the regulation of memory, attention, food intake, endocrine regulation, psychomotor activity and positive reinforcement. Deregulation in dopamine signaling could cause neurological disorders such as Parkinson’s disease and schizophrenia. Dopamine D2R and adenosine A2AR have been shown to interact in striatum and modulate dopaminergic activity. The purpose of this study is to optimize Fluorescence Resonance Energy Transfer (FRET) method to detect dimerization of D2R and A2AR by tagging them with EGFP (enhanced green fluorescent protein) and mCherry (a red fluorescent protein) in live N2a cell line using laser scanning confocal microscope. Establishing this model will pave the ways for understanding mechanisms of interaction between dopamine and adenosine signaling, thereby, contributing to the understanding molecular mechanisms of some neurophysiological events and disorders. Moreover, the fluorescence based live cell model will be used to detect effects of potential anti-psychotic drugs on the interaction of these two receptors. Indeed, follow-up studies are necessary to extend the limits of this project. Further imaging analyses and drug-receptor interaction studies can be readily applied to extract more information on dopamine-adenosine signaling by using the system developed with this thesis study.


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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 emotio...
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This study aimed to compare two different approaches for the purification of enterocin B from Enterococcus faecium strain W3 based on the observation that the bacteriocin was found both in cell associated form and in culture supernatant. The first approach employed ammonium sulfate precipitation, cation-exchange chromatography, and sequential reverse-phase high-performance liquid chromatography. The latter approach exploited a pH-mediated cell adsorption-desorption method to extract cell-bound bacteriocin, ...
Citation Formats
G. Ünlü, “Optimization of FRET method to detect dimerization of dopamine D2 and adenosine A2A receptors in live cells,” M.S. - Master of Science, Middle East Technical University, 2011.