Dokument

Summary:
The signalling pathway from G-protein-coupled receptors to the second messenger cAMP is present in virtually all cells and of major physiological and pathophysiological importance. The membrane-spanning receptors can easily be targeted by pharmaceutical compounds and therapies to treat diseases like hypertension, asthma or pain affect cellular cAMP-levels. Biochemical studies have revealed a lot of important information about the interaction of the proteins participating in this signalling cascade, namely the receptors, the G-proteins and adenylyl cyclases. The development of new microscopic methods allowed to dynamically investigate protein/protein-interactions. While these techniques have already been widely used to investigate in vivo signalling dynamics of the receptors, G-proteins and second messengers, research on adenylyl cyclases (ACs) mainly relied on in vitro methods or steady-state interaction studies. An assay, based on Förster Resonance Energy Transfer (FRET), was developed within this study, to investigate the dynamic interaction between G-proteins and ACs in living cells, thereby providing a platform for biochemical analysis in vivo. Furthermore, a protocol was established to resolve Gi protein-mediated regulation of cAMP in single living cells by means of FRET. For the assays, a fluorescently labelled AC5 was cloned. Bioluminescence-based cAMP measurements proved the constructs wild-type-like functionality with respect to stimulation through forskolin and Gs-proteins. The new protocol for FRET-based cAMP measurements verified Gs- and Gi-protein signalling competence of the labelled AC5. The new assay was used to analyse agonist-dependent interaction between AC5 and Gαs-, Gαi1- and Gβ1γ2-subunits, respectively. Although no basal interaction between the G-protein subunits and AC5 was observed, all subunits showed an increase in FRET upon agonist-stimulation of the according receptors. Interestingly, the different combinations of AC5 and G-protein subunits showed distinct FRET-signals. The agonist-dependent Gβ1γ2/AC5-FRET increase and decrease followed an exponential course, closely resembling other FRET-signals observed for G-proteins. FRET between Gαs and AC5 was characterised by a transient peak in the onset of the signal. Contrastingly, Gαi1 and AC5 showed an additional transient increase in their FRET-signal upon agonist withdrawal. The signal-phenotypes observed between Gα-subunits and AC5 possibly indicate additional conformational changes within the G-protein/AC5 complex. The onset-kinetics of the interaction between AC5 and G-protein-subunits were fast and in the same range as previously observed G-protein activation-kinetics. The interaction between Gαi1 and AC5 was found to be especially sensitive and proved to be left-shifted in comparison to the activation of the Gi1-protein itself. Downstream events of Gi-dependent regulation of AC5 and Gi-protein activity further verified this difference on a functional level. Gi-dependent inhibition of AC5-regulated cAMP levels was determined with the newly established protocol for the FRET-based cAMP sensor Epac1-camps. In comparison to GIRK currents, which reflect Gi1 protein activity, the receptor-induced Gi-protein-mediated inhibition of stimulated AC5 activity was shifted by two orders of magnitude. This was in line with previous reports on higher sensitivity of cAMP-involving over Gi-protein activity-dependent pathways. After appropriate controls ruled out confounding mechanisms that could shift the apparent sensitivity of the assay, the interaction kinetics between AC5 and Gαi1 remained as a major contributing cause. Indeed the interaction of Gαi1-subunits with AC5 was prolonged in comparison to the deactivation of the Gi1-protein and could not be accelerated by RGS4. This indicates a slow dissociation of AC5 and Gαi1, which prevents the deactivation and reassembly of the Gi1 protein, thereby affecting the dynamics of the G-protein cycle. Presumably, the balance in the G protein cycle between inactive and active G-proteins is shifted towards a higher amount of AC5-bound active G-proteins, providing the putative molecular mechanism for the high sensitivity observed in the interaction studies.

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