Analysis of interactions between BETA-2 adrenergic receptor & BETA arrestin-2 using förster resonance energy transfer (FRET) method in live cells

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2019

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Evci, Hüseyin

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Cells communicate with their environment and with other cells using receptors. Receptor takes signal from outside and transmits it into the cell. After transmitting the signal, receptors could be desensitized and/or endocytosed to stop signal transduction. Following receptor activation many receptors got phosphorylated which will trigger additional protein-protein interactions that could lead to receptor internalization. In the case of Beta-2 Adrenergic receptor (B2AR), key protein recognising the phosphorylation is Beta Arrestin-2. It is known that in the C terminus of the BAR, 4 amino acids get phosphorylated and play role in the interaction between B2AR and beta arrestin-2. In this thesis, we planned to further investigate the importance of each one of these 4 amino acids on the interaction by site directed mutagenesis. FRET technique will be used for the quantitative analysis of the interaction in live cells. For this purpose, mutations were done on B2AR. After mutations, both receptor and the B-arrestin 2 protein tagged with both mEGFP and mCherry. Receptor was tagged from 5 different places and ß-arrestin 2 was tagged from both N and C terminus. Receptor and B-arrestin 2 were cotransfected together to Mus musculus Neuroblastoma-2a (N2a) cells as various combinations. Images were taken via spinning disc confocal microscopy to analyze the possible interactions, and FRET efficiency was calculated by using pixFRET Plugin for ImageJ software. Single residue mutations were had a slightly increased FRET signal than wild-type receptor except S355A. One of the two residue mutations (S355A-S356G) was had no interaction with B-arrestin 2 while other one was had the same FRET signal with wild type receptor. Further mutations and experiments can be done to understand which residue is more important for the interaction between B2AR and B-arrestin 2.

Subject Keywords

Adrenaline, Adrenaline Receptors., Beta-2 Adrenergic Receptor, Beta-Arrestin 2, Receptor Internalization, FRET,Site-Directed Mutagenesis

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http://etd.lib.metu.edu.tr/upload/12623559/index.pdf
https://hdl.handle.net/11511/43784

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Graduate School of Natural and Applied Sciences, Thesis

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Citation Formats

H. Evci, “Analysis of interactions between BETA-2 adrenergic receptor & BETA arrestin-2 using förster resonance energy transfer (FRET) method in live cells,” Thesis (M.S.) -- Graduate School of Natural and Applied Sciences. Biochemistry., Middle East Technical University, 2019.