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  - Lotz, C.
A1  - Kiesewetter, L.
A1  - Schmid, F. F.
A1  - Hansmann, J.
A1  - Walles, H.
A1  - Groeber-Becker, F.
T1  - Replacing the Draize eye test: impedance spectroscopy as a 3R method to discriminate between all GHS categories for eye irritation
JF  - Scientific Reports
N2  - Highly invasive animal based test procedures for risk assessment such as the Draize eye test are under increasing criticism due to poor transferability for the human organism and animal-welfare concerns. However, besides all efforts, the Draize eye test is still not completely replaced by alternative animal-free methods. To develop an in vitro test to identify all categories of eye irritation, we combined organotypic cornea models based on primary human cells with an electrical readout system that measures the impedance of the test models. First, we showed that employing a primary human cornea epithelial cell based model is advantageous in native marker expression to the primary human epidermal keratinocytes derived models. Secondly, by employing a non-destructive measuring system based on impedance spectroscopy, we could increase the sensitivity of the test system. Thereby, all globally harmonized systems categories of eye irritation could be identified by repeated measurements over a period of 7 days. Based on a novel prediction model we achieved an accuracy of 78% with a reproducibility of 88.9% to determine all three categories of eye irritation in one single test. This could pave the way according to the 3R principle to replace the Draize eye test.
KW  - biological models
KW  - electrical and electronic engineering
KW  - regenerative medicine
KW  - tissue engineering
KW  - toxicology
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-177492
VL  - 8
IS  - 15049
ER  -