Küçük moleküllerle fosforilasyondan bağımsız βeta arrestin 2 ve βeta adrenerjik reseptör 2 nin aktivasyonu

The activities of neurohormones such as epinephrine are mediated by Adrenoceptors. In physiological processes, these messengers play an essential role; thus, Adrenoceptors are commonly found across the body. Agonist stimulated Adrenoceptor type 2 (β-2AR) works primarily by activating adenylyl cyclase through the stimulatory G protein (Gs), inducing an increase in the intracellular level of cAMP. This second messenger ultimately mediates downstream physiological effects. After few seconds of agonist stimulation, β-2ARs rapidly get phosphorylated by kinases (GRKs, PKA A, and C), resulting in uncoupling from Gs. High exposure to agonist, B2AR undergoes downregulation, mediated by a cytoplasmic adapter protein called Beta Arrestin. The magnitude of this interaction is determined by the receptor's phosphorylation level. This intensity of interaction influences the fate of the receptor, which can either be recycled back to the cell membrane or degraded in the cytoplasm, causing its downregulation. Mutations in the receptor at the amino vi acid residues that can be phosphorylated could prevent phosphorylation and the interaction with Arrestin. Therefore, the signal cannot be terminated. In this study, we try to facilitate phospho-deficient (P.D.) B2AR and wild-type βArrestin 2 interaction with the help of small molecules. Our aim is to find such compounds that promote the binding of β-Arrestin 2 to P.D.-β-2AR. We used Förster Resonance Energy Transfer (FRET) to monitor the interaction between P.D. or wild type β-2AR and Beta Arrestin 2. For co-localization and FRET studies, C-tail mCherry tagged β-2AR or P.D. β-2AR and 177th aa. position mEGFP tagged β–Arrestin 2 and GRK2 co-transfected in N2a cells were imaged using a spinning disc confocal microscope. Results showed that one of the tested chemicals increased FRET between P.D-β-2AR and β Arrestin 2, suggesting an interaction between these proteins. The kinetic of this chemical on β-2AR and Arrb2 interaction can be further investigated using the techniques optimized in this thesis study. Keywords Beta-2 Adrenoceptor, Βeta-Arrestin 2, Phospho-deficient Βeta-2- Adrenoceptor, FRET