PROJECT SUMMARY The opioid crisis, cardiovascular disease, and cancer are the most pressing healthcare crises in the United States. PLCβ enzymes are essential for normal analgesic, cardiovascular, and proliferative signaling. Further, disruption of interactions between PLCβ and its G protein activators have been shown to improve outcomes in preclinical models of opioid induced analgesia, cardiac hypertrophy, and cancer. However, targeting these interactions specifically is nearly impossible in the absence of structural data that informs on the conformational differences between inactive, active, and G protein bound PLCβ states. The goal of this project is to understand mechanisms of PLCβ activation by Gβγ and/or the membrane through structural and functional studies. The Gβγ binding site on PLCβ has yet to be defined, and overall, Gβγ- dependent regulation of effector enzymes is a poorly understood process. This project would not only increase our overall understanding of PLCβ signaling at the membrane and in conjunction with Gβγ but would also provide insights into the regulation of other enzymes that act at the membrane interface. The completion of this study will synergistically increase our understanding of PLCβ activation mechanisms, providing insights that can be leveraged for the development of probes, and ultimately potential therapeutics, targeting G protein–PLCβ interactions.