GPCRs are highly mobile proteins and diffuse laterally in the cell membrane displaying various diffusion patterns. Using single-particle tracking and superresolution microscopy methods we have recently shown that agonist binding to GPCRs can change their diffusion to match the mobility of G proteins facilitating productive GPCR/G-protein coupling in specific cell-membrane compartments, so-called ‘hot spots’. However, it is essentially unknown how specific membrane environments, e.g. local membrane structure and composition, control specific membrane localization(s) of GPCRs and downstream signaling. In this funding period, we will use biosensor design, single-particle tracking and various fluorescence microscopy methods to study the molecular mechanisms and functional consequences of GPCR localization in cell membrane ‘hot spots’. Specifically, we will investigate the impact of membrane curvature and geometry (e.g. using t-tubuli and cilia as examples) and protein-protein interactions to elucidate the molecular basis of receptor localization. A combination of novel single-color GPCR biosensors (previously developed by C05) and designed biosensors to map localized GPCR signaling will reveal the molecular basis how receptor hot spots orchestrate downstream GPCR signaling.