Anmerkungen:
Tag der Verteidigung: 15.03.2022
Zusammenfassungen in deutscher und englischer Sprache
Beschreibung:
G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors in human physiology, comprising more than 800 different genes. They sense diverse extracellular signals and initiate intracellular signaling responses via the activation of specific G proteins. The downregulation of GPCR signaling is mediated by four ubiquitously expressed GPCR kinases (GRK2, 3, 5, and 6) and two β-arrestin isoforms (β-arrestin1 and 2). GRKs phosphorylate intracellular domains of active receptors to facilitate high-affinity β-arrestin-binding. Depending on the specific GPCR–β-arrestin interaction, β-arrestins undergo different conformational changes to mediate receptor desensitization, internalization, and mitogen-activated protein kinase (MAPK) signal-amplification. However, the impact of individual GRK isoforms on these processes has not been comprehensively assessed until now. Moreover, whether β-arrestin1 and 2 undergo different conformational changes upon binding to the same GPCR is still unknown. The first part of this thesis focusses on the elaboration of GRK isoform-specific aspects of GPCR signaling. For this, a panel of elven in-house created combinatorial HEK293 knockout cell clones, lacking GRK2/3/5/6 (including four single, two double, four triple, and a quadruple GRK knockout cell line), was extensively used. To investigate GRK isoform-specific β-arrestin recruitment, a NanoLuc–HaloTag-based BRET system was established and combined with the unique possibility to vary individual GRK expression levels using different ΔGRK knockout cell lines. Hence, the GRK-specificity of β-arrestin-binding was assessed with two different strategies: first, utilizing the triple GRK knockout cell lines, featuring the endogenous expression of only one GRK isoform (ΔGRK3/5/6, ΔGRK2/5/6, ΔGRK2/3/6, ΔGRK2/3/5), and additionally by re-introduction of a single GRK isoform in the quadruple GRK knockout cell line (ΔQ-GRK).