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.