@phdthesis{Grushevskyi2022,
  author    = {Grushevskyi, Yevgenii},
  title     = {Activation kinetics of G-protein-coupled receptors},
  doi       = {10.25972/OPUS-20721},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-207215},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {G-protein-coupled receptors (GPCRs) are key biological switches that transmit both internal and external stimuli into the cell interior. Among the GPCRs, the "light receptor" rhodopsin has been shown to activate with a re-arrangement of the transmembrane helix bundle within ≈1 ms, while all other receptors are thought to become activated in subsecond range at saturating concentrations. Here we investigate activation kinetics of a dimeric GPCR, the metabotropic glutamate receptor-1 (mGluR1), and several class A GPCRs, as muscarinic receptor 3 (M3R), adrenergic (α2aAR and β1R) and opioid (µOR) receptors. We first used UV-light-triggered uncaging of glutamate in intact cells. Sub-millisecond F{\"o}rster resonance energy transfer recordings between labels at intracellular receptor sites were used to record conformational changes in the mGluR1. At millimolar ligand concentrations the initial rearrangement between the mGluR1 subunits occurs at a speed of τ1≈1-2 ms. These rapid changes were followed by significantly slower conformational changes in the transmembrane domain (τ2≈20 ms). We further characterized novel photoswitchable negative allosteric modulators for mGluR1, which bind to its transmembrane core and block the conformational change as well as the downstream signaling. Effects of the compounds were quantified in pharmacological cell assays in the dark and using UV and green light illumination. We finally develop a framework for image-based kinetic analysis of GPCRs which allowed us to measure activation kinetics of several prototypical class A GPCRs and to discover membrane heterogeneities of GPCR activation. It appears that GPCR activation signal is not only dependent on the amount of activated receptors, but also has some level of correlation with the local density of activated receptors.},
  subject      = {G-Protein gekoppelter Rezeptor},
  language  = {en}
}