@article{GodboleLygaLohseetal.2017,
  author    = {Godbole, Amod and Lyga, Sandra and Lohse, Martin J. and Calebiro, Davide},
  title     = {Internalized TSH receptors en route to the TGN induce local G\(_{S}\)-protein signaling and gene transcription},
  series = {Nature Communications},
  volume    = {8},
  journal   = {Nature Communications},
  number    = {443},
  doi       = {10.1038/s41467-017-00357-2},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170375},
  year      = {2017},
  abstract  = {A new paradigm of G-protein-coupled receptor (GPCR) signaling at intracellular sites has recently emerged, but the underlying mechanisms and functional consequences are insufficiently understood. Here, we show that upon internalization in thyroid cells, endogenous TSH receptors traffic retrogradely to the trans-Golgi network (TGN) and activate endogenous Gs-proteins in the retromer-coated compartment that brings them to the TGN. Receptor internalization is associated with a late cAMP/protein kinase A (PKA) response at the Golgi/TGN. Blocking receptor internalization, inhibiting PKA II/interfering with its Golgi/TGN localization, silencing retromer or disrupting Golgi/TGN organization all impair efficient TSH-dependent cAMP response element binding protein (CREB) phosphorylation. These results suggest that retrograde trafficking to the TGN induces local G\(_{S}\)-protein activation and cAMP/PKA signaling at a critical position near the nucleus, which appears required for efficient CREB phosphorylation and gene transcription. This provides a new mechanism to explain the functional consequences of GPCR signaling at intracellular sites and reveals a critical role for the TGN in GPCR signaling.},
  language  = {en}
}
@phdthesis{PerpinaViciano2020,
  author    = {Perpi{\~n}{\´a} Viciano, Cristina},
  title     = {Study of the activation mechanisms of the CXC chemokine receptor 4 (CXCR4) and the atypical chemokine receptor 3 (ACKR3)},
  doi       = {10.25972/OPUS-19237},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192371},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {The CXC chemokine receptor 4 (CXCR4) and the atypical chemokine receptor 3 (ACKR3) are seven transmembrane receptors that are involved in numerous pathologies, including several types of cancers. Both receptors bind the same chemokine, CXCL12, leading to significantly different outcomes. While CXCR4 activation generally leads to canonical GPCR signaling, involving Gi proteins and β-arrestins, ACKR3, which is predominantly found in intracellular vesicles, has been shown to signal via β-arrestin-dependent signaling pathways. Understanding the dynamics and kinetics of their activation in response to their ligands is of importance to understand how signaling proceeds via these two receptors. In this thesis, different F{\"o}rster resonance energy transfer (FRET)-based approaches have been combined to individually investigate the early events of their signaling cascades. In order to investigate receptor activation, intramolecular FRET sensors for CXCR4 and ACKR3 were developed by using the pair of fluorophores cyan fluorescence protein and fluorescence arsenical hairpin binder. The sensors, which exhibited similar functional properties to their wild-type counterparts, allowed to monitor their ligand-induced conformational changes and represent the first RET-based receptor sensors in the field of chemokine receptors. Additional FRET-based settings were also established to investigate the coupling of receptors with G proteins, rearrangements within dimers, as well as G protein activation. On one hand, CXCR4 showed a complex activation mechanism in response to CXCL12 that involved rearrangements in the transmembrane domain of the receptor followed by rearrangements between the receptor and the G protein as well as rearrangements between CXCR4 protomers, suggesting a role of homodimers in the activation course of this receptor. This was followed by a prolonged activation of Gi proteins, but not Gq activation, via the axis CXCL12/CXCR4. In contrast, the structural rearrangements at each step of the signaling cascade in response to macrophage migration inhibitory factor (MIF) were dynamically and kinetically different and no Gi protein activation via this axis was detected. These findings suggest distinct mechanisms of action of CXCL12 and MIF on CXCR4 and provide evidence for a new type of sequential signaling events of a GPCR. Importantly, evidence in this work revealed that CXCR4 exhibits some degree of constitutive activity, a potentially important feature for drug development. On the other hand, by cotransfecting the ACKR3 sensor with K44A dynamin, it was possible to increase its presence in the plasma membrane and measure the ligand-induced activation of this receptor. Different kinetics of ACKR3 activation were observed in response to CXCL12 and three other agonists by means of using the receptor sensor developed in this thesis, showing that it is a valuable tool to study the activation of this atypical receptor and pharmacologically characterize ligands. No CXCL12-induced G protein activation via ACKR3 was observed even when the receptor was re-localized to the plasma membrane by means of using the mutant dynamin. Altogether, this thesis work provides the temporal resolution of signaling patterns of two chemokine receptors for the first time as well as valuable tools that can be applied to characterize their activation in response to pharmacologically relevant ligands.},
  subject      = {G protein-coupled receptors},
  language  = {en}
}
@article{SchihadaVandenabeeleZabeletal.2018,
  author    = {Schihada, Hannes and Vandenabeele, Sylvie and Zabel, Ulrike and Frank, Monika and Lohse, Martin J. and Maiellaro, Isabella},
  title     = {A universal bioluminescence resonance energy transfer sensor design enables high-sensitivity screening of GPCR activation dynamics},
  series = {Communications Biology},
  volume    = {1},
  journal   = {Communications Biology},
  number    = {105},
  doi       = {10.1038/s42003-018-0072-0},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-228592},
  pages     = {1-8},
  year      = {2018},
  abstract  = {G-protein-coupled receptors (GPCRs) represent one of the most important classes of drug targets. The discovery of new GCPR therapeutics would greatly benefit from the development of a generalizable high-throughput assay to directly monitor their activation or de-activation. Here we screened a variety of labels inserted into the third intracellular loop and the C-terminus of the alpha(2 Lambda)-adrenergic receptor and used fluorescence (FRET) and bioluminescence resonance energy transfer (BRET) to monitor ligand-binding and activation dynamics. We then developed a universal intramolecular BRET receptor sensor design to quantify efficacy and potency of GPCR ligands in intact cells and real time. We demonstrate the transferability of the sensor design by cloning beta(2)-adrenergic and PTH1-receptor BRET sensors and monitored their efficacy and potency. For all biosensors, the Z factors were well above 0.5 showing the suitability of such design for microtiter plate assays. This technology will aid the identification of novel types of GPCR ligands.},
  language  = {en}
}