Understanding the Molecular Mechanism of PLCε Regulation and G Protein‐Mediated Activation

Abstract

Cardiovascular disease is the leading cause of death in the world. The phospholipase C (PLC) family of enzymes, in particular the PLCε subfamily, are essential for normal cardiovascular function. PLCε hydrolyzes phosphatidylinositol phosphates at cellular membranes, producing inositol phosphates (IPx) and diacylglycerol (DAG). These crucial secondary messengers activate multiple downstream pathways, including cardiac contractility and the expression of hypertrophic genes. In the cardiovascular system, PLCε is regulated through direct interactions with the RhoA and Rap1A small GTPases, which in turn are activated downstream of G protein‐coupled receptors (GPCRs). RhoA is reported to activate PLCε at the plasma membrane, whereas Rap1A translocates and activates PLCε at the perinuclear membrane. However, the elements within PLCε that regulate basal activity and membrane association have not been fully identified. Similarly, the domains involved in Rap1A versus RhoA binding, activation and translocation to different subcellular membranes have not been mapped. In this work, we use a structure‐guided approach, together with cell‐based activity assays, epifluorescence, and confocal microscopy to identify the roles of PLCε regulatory elements and domains in basal activity, subcellular localization, and regulation by RhoA and Rap1A GTPases. Functional studies show N‐ and C‐terminal regulatory domains of PLCε dictate its location within the cell, and contribute differently to basal and G protein‐dependent activity. We also show that regulatory insertions within the catalytic TIM barrel, including the X–Y linker and Y‐box, aid in interfacial activation and membrane association. These studies provide much needed insights into the molecular determinants of PLCε that regulate its localization and activity in cells, which is critical for elucidating its roles in cardiovascular function.