A wealth of experimental evidence accumulated over recent years suggests that G protein-coupled receptors (GPCRs) associate with each other in the plasma membrane, forming di-/oligomers. However, most studies have been inconclusive with regard to the variability across receptor subtypes, specificity of receptor-receptor interactions and, most importantly, the impact of GPCR oligomerization on signaling. These aspects need to be addressed to clarify the functional role of GPCR oligomers and, ultimately, devise new therapeutic strategies via specific effects on GPCR signaling. The objective of this research proposal is to provide breakthrough mechanistic insights into the spatio-temporal organization of the major opioid receptor (OR) subtypes, serving as prototypic GPCRs, in the cell membrane, and ultimately, its relation to function. To this end, we propose to use a synergistic, inter-disciplinary strategy integrating state-of-the-art molecular dynamics (MD) simulations, single molecule microscopy, and Frster resonance energy transfer (FRET) microscopy assays of GPCR signaling. This integration is timely and made ground-breaking by cutting-edge theoretical and experimental advances in the GPCR field, including new high- resolution crystal structures, recent technological developments for single-particle tracking of individual GPCR protomers in a living cell, implementation of efficient FRET assays for GPCR signaling, and high performance computational capabilities complemented by efficiently parallelized codes and multiscale system representations. Although we will use ORs as model systems, our proposed strategy can be applied to any complex of GPCRs or other membrane proteins. Specifically, we propose to study di-/oligomer formation of high- efficiency, covalently labeled OR subtypes with fluorescent dyes using single molecule total internal reflection fluorescence microscopy (TIR-FM) and FRET/BRET signals between GPCR protomers to characterize their possibly distinctive di-/oligomerization patterns. Based on an iterative computational-experimental approach, we will predict and then generate and investigate mutants to explore the interfaces forming the different OR dimers. Finally, we will investigate a possible relationship between dimer formation and signaling by assessing the effects of dimer-stabilizing or de-stabilizing mutations on signaling as measured with classic signaling assays and with newly developed real-time FRET sensors. We will move these studies from simple transfected cell lines to primary cells, and will also aim to move, in the future, from transfected fluorescently labeled receptors to endogenous receptors studied with fluorescently labeled ligands that we are developing in an independent collaboration. Thus, our studies will ultimately help to unravel general mechanisms of GPCR di/oligomerization and their effects on signaling and aim to identify new targets for GPCR-directed therapies.