nuclear magnetic resonance; NMR; GPCR; signalling; structure; dynamics; structural biology
Isogai Shin, Deupi Xavier, Opitz Christian, Heydenreich Franziska M, Tsai Ching-Ju, Brueckner Florian, Schertler Gebhard F X, Veprintsev Dmitry B, Grzesiek Stephan (2016), Backbone NMR reveals allosteric signal transduction networks in the β1-adrenergic receptor., in Nature
, 530(7589), 237-41.
Chakrabarti Kalyan S, Agafonov Roman V, Pontiggia Francesco, Otten Renee, Higgins Matthew K, Schertler Gebhard F X, Oprian Daniel D, Kern Dorothee (2016), Conformational Selection in a Protein-Protein Interaction Revealed by Dynamic Pathway Analysis., in Cell reports
, 14(1), 32-42.
Opitz Christian, Isogai Shin, Grzesiek Stephan (2015), An economic approach to efficient isotope labeling in insect cells using homemade 15N-, 13C- and 2H-labeled yeast extracts., in Journal of biomolecular NMR
, 62(3), 373-85.
Milić Dalibor, Veprintsev Dmitry B (2015), Large-scale production and protein engineering of G protein-coupled receptors for structural studies., in Frontiers in pharmacology
, 6, 66-66.
Heydenreich Franziska M, Vuckovic Ziva, Matkovic Milos, Veprintsev Dmitry B (2015), Stabilization of G protein-coupled receptors by point mutations., in Frontiers in pharmacology
, 6, 82-82.
G protein-coupled receptors (GPCRs) are integral membrane proteins that transmit extracellular neural, endocrine, olfactory and visual signals across the plasma membrane. When activated by light or a ligand GPCRs undergo a conformational change leading to activation of a G protein, arrestin and other signaling pathways. Over 30% of compounds used in medicine today modulate the activity of GPCRs. Some GPCRs can bind a range of ligands, some of which, instead of fully activating the receptor, rather bias the signal transduction cascade toward distinct intracellular pathways. The currently available X-ray structures of several GPCRs and of a ?2-adrenoreceptor/Gs signaling complex provide a frozen snapshot of a signal transduction event, but do not fully explain how the ligand selectivity is achieved, and how the ligand binding results in preferential binding of a specific signaling protein such as Gi, Gs or arrestin. Understanding the precise structural and dynamic nature of these phenomena is critical to knowing the mechanism of GPCR activation and will help further development of pharmaceuticals with desired pharmacological properties. We propose here to provide the missing dynamical information on GPCR function by Nuclear magnetic resonance spectroscopy (NMR), which shall determine the conformational changes and dynamics of the receptors and signaling complexes in solution. This should then a yield a comprehensive view of GPCR activation of and signal transduction. We will initially focus on rhodopsin and ?1-adrenoreceptor (?1AR), as well as their signaling complexes with G proteins and arrestin, and later apply developed expertise to the vasopressin receptor V2R and its signaling complexes.In particular, we propose: a) to label GPCRs for NMR analysis; b) to obtain a dynamic view of the conformational changes in GPCRs induced by a range of ligands with different activities including agonists, antagonists and biased ligands; c) to investigate the effect of G proteins and arrestin binding on the receptors; d) to identify the molecular basis for signaling selectivity via a comparative NMR and biophysical analysis of receptor binding to the various ligands, G proteins and arrestin.Due to the high sequence conservation among G protein-coupled receptor proteins our results are expected to have wide implications for signal transduction by GPCRs in general.