Sub-cellular propagation of GPCR signaling; functional imaging in space and time

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G protein-coupled receptors (GPCR) form the largest family of proteins involved in the transfer of information across biological membranes. They are involved in the control of cellular homeostasis and in orchestrating a large number of physiological responses. In their classical representation, GPCR detect signals at the cell surface and transmit the information across the plasma membrane through conformational rearrangements leading to the engagement and activation of heterotrimeric G proteins attached to the inner face of the plasma membrane. The G proteins then interact and regulate the activity of effectors such as enzymes and channels. Following activation, phosphorylation of the receptors promotes the recruitment of the accessory protein βarrestin that leads to signal arrest and initiates receptor endocytosis. This traditional view is however rapidly evolving and ligand-promoted sub-cellular targeting of signaling complexes is emerging as an important determinant of GPCR signaling activities. We and others showed that βarrestin promotes G protein-independent signaling and may play a role in sustained endosomal G protein signaling. Nuclear translocation of GPCR and βarrestin has also been reported and linked to gene transcription regulation. Yet, most of the evidence for organelle-specific GPCR signaling are indirect and, in some cases, controversial. Directly monitoring organelle-specific signal propagation and its dynamic regulation remains a daunting challenge. We therefore propose a research program aimed at addressing this challenge by directly studying the dynamics of GPCR signal propagation in specific subcellular compartments. For this purpose, we will: 1) Develop new BRET-based biosensors detecting organelle-specific GPCR signalling activity, 2) Use high-resolution and time-resolved microscopy imaging to visualize the compartmentalized signalling activity. Using biosensors that directly monitor the real-time activation of the receptors' downstream effectors, we propose to monitor the organelle-specific signaling activity by targeting BRET probes engineered to restrict their localization to individual compartments. In addition to generate new fundamental knowledge about the dynamics and role of compartmentalized signaling of one of the most important class of signal transducers, the program will lead to the development of biophysical and imaging tools that will be useful for the study of other organelle-specific biological processes.