Understanding molecular mechanism of PLCe regulation by small G‐proteins

Abstract

Cardiovascular disease is the leading cause of death in the United States. The phospholipase C (PLC) family of enzymes catalyzes the hydrolysis of the inner membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2) to inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 and DAG are crucial secondary messengers that activate multiple signaling pathways and modulate gene expression to control cellular function and behavior. The PLCe subfamily is required for normal cardiovascular function, where it is regulated through direct interactions with the RhoA and Rap1A small GTPases, which are activated in response to G protein-coupled receptor (GPCR stimulation). RhoA activates PLCe at the plasma membrane, whereas Rap1A translocates and activates PLCe at the perinuclear membrane. However, the domains of PLCe involved in G protein binding and activation, and translocation to different subcellular membranes is largely unknown. In this work, we use cell-based activity assays, epifluorescence, and confocal microscopy to identify the domains of PLCe involved in basal activity, subcellular localization, and regulation by RhoA and Rap1A GTPases. Our preliminary studies demonstrate that the unique N- and C-terminal regulatory domains of PLCe dictate its location within the cell, and contribute differently to basal and G protein-dependent activity. These studies will provide needed insights into the regulation and localization of PLCe in cells, which is critical for its roles in cardiovascular function.