TY  - JOUR
A1  - Schönegge, Anne-Marie
A1  - Gallion, Jonathan
A1  - Picard, Louis-Philippe
A1  - Wilkins, Angela D.
A1  - Le Gouill, Christian
A1  - Audet, Martin
A1  - Stallaert, Wayne
A1  - Lohse, Martin J.
A1  - Kimmel, Marek
A1  - Lichtarge, Olivier
A1  - Bouvier, Michel
T1  - Evolutionary action and structural basis of the allosteric switch controlling β\(_2\)AR functional selectivity
JF  - Nature Communications
N2  - Functional selectivity of G-protein-coupled receptors is believed to originate from ligand-specific conformations that activate only subsets of signaling effectors. In this study, to identify molecular motifs playing important roles in transducing ligand binding into distinct signaling responses, we combined in silico evolutionary lineage analysis and structure-guided site-directed mutagenesis with large-scale functional signaling characterization and non-negative matrix factorization clustering of signaling profiles. Clustering based on the signaling profiles of 28 variants of the β\(_2\)-adrenergic receptor reveals three clearly distinct phenotypical clusters, showing selective impairments of either the Gi or βarrestin/endocytosis pathways with no effect on Gs activation. Robustness of the results is confirmed using simulation-based error propagation. The structural changes resulting from functionally biasing mutations centered around the DRY, NPxxY, and PIF motifs, selectively linking these micro-switches to unique signaling profiles. Our data identify different receptor regions that are important for the stabilization of distinct conformations underlying functional selectivity.
KW  - toxicology
KW  - functional clustering
KW  - molecular modelling
KW  - protein design
KW  - receptor pharmacology
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-172268
VL  - 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  - Lohse, Christian
A1  - Bock, Andreas
A1  - Maiellaro, Isabella
A1  - Hannawacker, Annette
A1  - Schad, Lothar R.
A1  - Lohse, Martin J.
A1  - Bauer, Wolfgang R.
T1  - Experimental and mathematical analysis of cAMP nanodomains
JF  - PLoS ONE
N2  - In their role as second messengers, cyclic nucleotides such as cAMP have a variety of intracellular effects. These complex tasks demand a highly organized orchestration of spatially and temporally confined cAMP action which should be best achieved by compartmentalization of the latter. A great body of evidence suggests that cAMP compartments may be established and maintained by cAMP degrading enzymes, e.g. phosphodiesterases (PDEs). However, the molecular and biophysical details of how PDEs can orchestrate cAMP gradients are entirely unclear. In this paper, using fusion proteins of cAMP FRET-sensors and PDEs in living cells, we provide direct experimental evidence that the cAMP concentration in the vicinity of an individual PDE molecule is below the detection limit of our FRET sensors (<100nM). This cAMP gradient persists in crude cytosol preparations. We developed mathematical models based on diffusion-reaction equations which describe the creation of nanocompartments around a single PDE molecule and more complex spatial PDE arrangements. The analytically solvable equations derived here explicitly determine how the capability of a single PDE, or PDE complexes, to create a nanocompartment depend on the cAMP degradation rate, the diffusive mobility of cAMP, and geometrical and topological parameters. We apply these generic models to our experimental data and determine the diffusive mobility and degradation rate of cAMP. The results obtained for these parameters differ by far from data in literature for free soluble cAMP interacting with PDE. Hence, restricted cAMP diffusion in the vincinity of PDE is necessary to create cAMP nanocompartments in cells.
KW  - fluorescence resonance energy transfer
KW  - yellow fluorescent protein
KW  - radii
KW  - adenylyl cyclase signaling cascade
KW  - cell fusion
KW  - cytosol
KW  - isoproterenol
KW  - absorption
KW  - cyclic nucleotides such as cyclic adenosine monophosphate
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170972
VL  - 12
IS  - 4
ER  -