G protein trafficking in cardiomyocytes and neurons

PROJECT SUMMARY/ABSTRACT G protein-coupled receptors (GPCRs) regulate virtually every aspect of excitable cell function and are targeted by approximately one-third of FDA-approved drugs. GPCRs regulate cardiomyocyte contractility, relaxation and hypertrophy, and neuronal excitability and neurotransmitter release, therefore understanding GPCR function in these excitable cells is essential to optimize treatment of cardiovascular and neurological diseases. Historically, GPCR signaling was thought to occur only at the plasma membrane, but abundant evidence has now shown that receptors can signal from endosomes, the Golgi apparatus, and other intracellular organelles. Intracellular GPCRs activate downstream signals that differ qualitatively or quantitatively from signals originating from the plasma membrane, therefore it is important to understand how receptors operate at different subcellular locations. While trafficking of GPCRs to different cellular compartments is well understood, it is not known how intracellular receptors are supplied with downstream transducers (G proteins) and signaling molecules (effectors). The aims of this project are to to fill critical gaps in our understanding of GPCR signaling from intracellular compartments of excitable cells by mapping the subcellular distribution of heterotrimeric G proteins using gene editing, advanced fluorescence microscopy, and bioluminescence resonance energy transfer (BRET) spectroscopy. The project will also determine how G proteins move or redistribute upon receptor activation, and whether or not G proteins are concentrated at specific plasma membrane subdomains (e.g. T-tubules and synapses) where important signaling events take place. A broader goal of the project is to develop BRET methods to enable similar studies of other signaling molecules, to ultimately produce a complete picture of GPCR signaling mechanisms throughout differentiated cells.