TY  - JOUR
A1  - Christian, Gentzsch
A1  - Seier, Kerstin
A1  - Drakopoulos, Antonios
A1  - Jobin, Marie-Lise
A1  - Lanoiselée, Yann
A1  - Koszegi, Zsombor
A1  - Maurel, Damien
A1  - Sounier, Rémy
A1  - Hübner, Harald
A1  - Gmeiner, Peter
A1  - Granier, Sébastien
A1  - Calebiro, Davide
A1  - Decker, Michael
T1  - Selective and Wash‐Resistant Fluorescent Dihydrocodeinone Derivatives Allow Single‐Molecule Imaging of μ‐Opioid Receptor Dimerization
JF  - Angewandte Chemie International Edition
N2  - μ‐Opioid receptors (μ‐ORs) play a critical role in the modulation of pain and mediate the effects of the most powerful analgesic drugs. Despite extensive efforts, it remains insufficiently understood how μ‐ORs produce specific effects in living cells. We developed new fluorescent ligands based on the μ‐OR antagonist E‐p‐nitrocinnamoylamino‐dihydrocodeinone (CACO), that display high affinity, long residence time and pronounced selectivity. Using these ligands, we achieved single‐molecule imaging of μ‐ORs on the surface of living cells at physiological expression levels. Our results reveal a high heterogeneity in the diffusion of μ‐ORs, with a relevant immobile fraction. Using a pair of fluorescent ligands of different color, we provide evidence that μ‐ORs interact with each other to form short‐lived homodimers on the plasma membrane. This approach provides a new strategy to investigate μ‐OR pharmacology at single‐molecule level.
KW  - single-molecule microscopy
KW  - fluorescent probes
KW  - G-protein coupled receptor
KW  - homodimerization
KW  - opioid ligands
Y1  - 2020
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-212398
VL  - 59
IS  - 15
ER  - 
TY  - JOUR
A1  - Weigand, Isabel
A1  - Ronchi, Cristina L.
A1  - Vanselow, Jens T.
A1  - Bathon, Kerstin
A1  - Lenz, Kerstin
A1  - Herterich, Sabine
A1  - Schlosser, Andreas
A1  - Kroiss, Matthias
A1  - Fassnacht, Martin
A1  - Calebiro, Davide
A1  - Sbiera, Silviu
T1  - PKA Cα subunit mutation triggers caspase-dependent RIIβ subunit degradation via Ser\(^{114}\) phosphorylation
JF  - Science Advances
N2  - Mutations in the PRKACA gene are the most frequent cause of cortisol-producing adrenocortical adenomas leading to Cushing’s syndrome. PRKACA encodes for the catalytic subunit α of protein kinase A (PKA). We already showed that PRKACA mutations lead to impairment of regulatory (R) subunit binding. Furthermore, PRKACA mutations are associated with reduced RIIβ protein levels; however, the mechanisms leading to reduced RIIβ levels are presently unknown. Here, we investigate the effects of the most frequent PRKACA mutation, L206R, on regulatory subunit stability. We find that Ser\(^{114}\) phosphorylation of RIIβ is required for its degradation, mediated by caspase 16. Last, we show that the resulting reduction in RIIβ protein levels leads to increased cortisol secretion in adrenocortical cells. These findings reveal the molecular mechanisms and pathophysiological relevance of the R subunit degradation caused by PRKACA mutations, adding another dimension to the deregulation of PKA signaling caused by PRKACA mutations in adrenal Cushing’s syndrome.
KW  - mutation triggers
KW  - phosphorylation
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-270445
VL  - 7
IS  - 8
ER  - 
TY  - JOUR
A1  - Godbole, Amod
A1  - Lyga, Sandra
A1  - Lohse, Martin J.
A1  - Calebiro, Davide
T1  - Internalized TSH receptors en route to the TGN induce local G\(_{S}\)-protein signaling and gene transcription
JF  - Nature Communications
N2  - 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.
KW  - G protein-coupled receptors
KW  - fluorescence imaging
KW  - hormone receptors
KW  - trans-Golgi network
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170375
VL  - 8
IS  - 443
ER  - 
TY  - JOUR
A1  - Weigand, Isabel
A1  - Ronchi, Cristina L.
A1  - Rizk-Rabin, Marthe
A1  - Dalmazi, Guido Di
A1  - Wild, Vanessa
A1  - Bathon, Kerstin
A1  - Rubin, Beatrice
A1  - Calebiro, Davide
A1  - Beuschlein, Felix
A1  - Bertherat, Jérôme
A1  - Fassnacht, Martin
A1  - Sbiera, Silviu
T1  - Differential expression of the protein kinase A subunits in normal adrenal glands and adrenocortical adenomas
JF  - Scientific Reports
N2  - Somatic mutations in protein kinase A catalytic α subunit (PRKACA) were found to be causative for 30-40% of cortisol-producing adenomas (CPA) of the adrenal gland, rendering PKA signalling constitutively active. In its resting state, PKA is a stable and inactive heterotetramer, consisting of two catalytic and two regulatory subunits with the latter inhibiting PKA activity. The human genome encodes three different PKA catalytic subunits and four different regulatory subunits that are preferentially expressed in different organs. In normal adrenal glands all regulatory subunits are expressed, while CPA exhibit reduced protein levels of the regulatory subunit IIβ. In this study, we linked for the first time the loss of RIIβ protein levels to the PRKACA mutation status and found the down-regulation of RIIβ to arise post-transcriptionally. We further found the PKA subunit expression pattern of different tumours is also present in the zones of the normal adrenal cortex and demonstrate that the different PKA subunits have a differential expression pattern in each zone of the normal adrenal gland, indicating potential specific roles of these subunits in the regulation of different hormones secretion.
KW  - kinases
KW  - immunohistochemistry
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-157952
VL  - 7
IS  - 49
ER  - 
TY  - JOUR
A1  - Maiellaro, Isabella
A1  - Lohse, Martin J.
A1  - Kitte, Robert J.
A1  - Calebiro, Davide
T1  - cAMP Signals in Drosophila Motor Neurons Are Confined to Single Synaptic Boutons
JF  - Cell Reports
N2  - The second messenger cyclic AMP (cAMP) plays an important role in synaptic plasticity. Although there is evidence for local control of synaptic transmission and plasticity, it is less clear whether a similar spatial confinement of cAMP signaling exists. Here, we suggest a possible biophysical basis for the site-specific regulation of synaptic plasticity by cAMP, a highly diffusible small molecule that transforms the physiology of synapses in a local and specific manner. By exploiting the octopaminergic system of Drosophila, which mediates structural synaptic plasticity via a cAMP-dependent pathway, we demonstrate the existence of local cAMP signaling compartments of micrometer dimensions within single motor neurons. In addition, we provide evidence that heterogeneous octopamine receptor localization, coupled with local differences in phosphodiesterase activity, underlies the observed differences in cAMP signaling in the axon, cell body, and boutons.
KW  - cAMP
KW  - synaptic plasticity
KW  - PDE
KW  - octopamine
KW  - FRET
KW  - active zone
KW  - dunce
KW  - GPCR
Y1  - 2016
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-162324
VL  - 17
IS  - 5
ER  - 
TY  - JOUR
A1  - Calebiro, Davide
A1  - Maiellaro, Isabella
T1  - cAMP signaling microdomains and their observation by optical methods
JF  - Frontiers in Cellular Neuroscience
N2  - The second messenger cyclic AMP (cAMP) is a major intracellular mediator of many hormones and neurotransmitters and regulates a myriad of cell functions, including synaptic plasticity in neurons. Whereas cAMP can freely diffuse in the cytosol, a growing body of evidence suggests the formation of cAMP gradients and microdomains near the sites of cAMP production, where cAMP signals remain apparently confined. The mechanisms responsible for the formation of such microdomains are subject of intensive investigation. The development of optical methods based on fluorescence resonance energy transfer (FRET), which allow a direct observation of cAMP signaling with high temporal and spatial resolution, is playing a fundamental role in elucidating the nature of such microdomains. Here, we will review the optical methods used for monitoring cAMP and protein kinase A (PKA) signaling in living cells, providing some examples of their application in neurons, and will discuss the major hypotheses on the formation of cAMP/PKA microdomains.
KW  - G protein-coupled receptor
KW  - cyclic AMP
KW  - signaling microdomain
KW  - fluorescence resonance energy transfer
KW  - neurons
Y1  - 2014
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-118252
SN  - 1662-5102
VL  - 8
ER  -