ABSTRACT Phospholipase C (PLC) enzymes, in particular PLCepsilon, are essential for normal cellular function in the cardiovascular system. There, they generate second messengers in response to extracellular signals that increase calcium levels and activate protein kinase C. Misregulation of PLCepsilon is highly deleterious and can lead to maladaptive changes in cardiac contractility, cell remodeling, and hypertrophy. Consequently, PLCepsilon is tightly regulated both in its activity and its subcellular localization. Under basal conditions, the enzyme resides in the cytoplasm and exhibits low activity. Following the activation of G protein-coupled receptors and/or receptor tyrosine kinases, the enzyme is allosterically activated and translocated to specific membranes through direct interactions with small molecular weight GTPases. The small GTPase Rap1A activates and translocates PLCepsilon to the perinuclear membrane where it stimulates protein kinase cascades involved in cardiac hypertrophy. However, very little is known about the molecular mechanisms that underlie allosteric activation of PLCepsilon and its localization by Rap1A. The goal of this proposal is to use an array of structural, functional, and cell-based studies to provide insights into the molecular mechanisms underlying the normal and pathological functions of PLCepsilon. In the future, our experiments will aid in the identification of selective chemical probes for this enzyme and facilitate the development of novel therapeutic approaches to treat cardiovascular disease, the leading cause of death in the United States and a rapidly growing problem worldwide.