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

Evci, Hüseyin
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.


Balkan, Seyda Tuğçe; Son, Çağdaş Devrim; Küçük Baloğlu, Fatma; Department of Biochemistry (2021-8-11)
GPCR’s are seven-transmembrane receptors that transmit external signals to the intracellular environment via secondary messenger systems through heterotrimeric G proteins. Heterotrimeric G proteins consist of α and β-γ subunits. Until recent years, scientists thought that GPCR signal transduction occurs between one GPCR and one heterotrimeric G protein; however, recently, it has been shown that GPCR’s can make oligomers. Oligomerization of GPCR allows cells to tune the intensity of the signal and respond ap...
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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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Cell signaling is a list of chemical reactions that are used for intercellular and intracellular communication. Signaling pathways denote these chemical reactions in a systematic manner. Today, many signaling pathways are constructed by several experimental methods. However there are still many communication skills of cells that are needed to be discovered. RNAi system allows us to see the phenotypes when some genes are removed from living cells. By observing these phenotypes, we can build signaling pathway...
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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) cont...
Investigation of the effect of GPCR oligomerization on the GNAi1 protein homodimerization in live cells using FRET
Nalli, Enise; Son, Çağdaş Devrim; Küçük Baloğlu, Fatma; Department of Biotechnology (2022-1-26)
G-Protein Coupled Receptors (GPCR) are membrane proteins that pass the cell membrane seven times. In classical GPCR signaling pathways, one GPCR-one heterotrimeric G-protein interaction model is enough to transmit the signal to effector proteins. Studies since 2000 showed that one GPCR dimer-one heterotrimeric G-protein interaction model is more likely, and GPCRs having homo- /hetero- dimers interact with a single G⍺-protein. Recently, studies on GPCRs indicated that more than two receptors interact to ...
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.