Mechanisms Underlying Regulation of PLCbeta by heterotrimeric G proteins - Phospholipase C b (PLCb) enzymes increase intracellular calcium in response to diverse extracellular signals, regulating numerous processes including cell proliferation and survival. Dysregulation of their expression or activity contributes to pathophysiological conditions such as heart disease, cancer, and addiction. All four PLCb isoforms bind to the cytoplasmic leaflet of the plasma membrane where they hydrolyze phosphatidylinositol-4,5- bisphosphate (PIP2). They are activated via direct interactions with the heterotrimeric G proteins Gaq, and in most cases, by Gbg. All PLCb isozymes have two elements that profoundly autoinhibit PIP2 hydrolysis: a lid that blocks access to the active site, and a helix in the proximal C-terminal domain that binds to the catalytic core. G protein binding and recruitment of the lipase to a membrane surface has been proposed to displace all the autoinhibitory elements, resulting in activation. However, recent cryo-electron microscopy (cryo-EM) structures of G protein–PLCb3 complexes bound to model membranes reveal that this is model is insufficient. In these structures, the catalytic core fails to engage the membrane and the active site remains blocked. Thus, the mechanism of activation and the molecular basis of isoform-specific responses to G proteins remain unresolved. Furthermore, G proteins may also increase PLCb activity by altering their behavior on the membrane surface, an aspect that cannot be assessed via structures or cell-based assays alone. To address these gaps, we propose a synergistic and interdisciplinary combination of functional and structural studies, cell-based assays, and single molecule microscopy to investigate the structure and regulation of the four PLCb isoforms. In Aim 1, we use functional analyses to test hypotheses derived from new structures of PLCb complexes. We will also determine cryo-EM reconstructions of the four PLCb isoforms in solution, and a subset in complex with G proteins on model membranes known to promote activity. Aim 2 utilizes bioluminescence resonance energy transfer (BRET) and signaling assays to monitor the location and disposition of PLCb isoforms within a cell and measure the kinetics of G protein-dependent activation in response to receptor stimulation. In Aim 3, single molecule total internal reflection fluorescence (TIRF) microscopy will be used to dissect the contributions of the PLCb regulatory domains, PIP2, and G proteins to the kinetic behavior of individual lipase molecules on model membranes. Strong preliminary data is included in support of each aim. Our work will not only reveal new mechanistic insights in PLCb regulation but allow us to identify regulatory features unique to each isozyme that could be targeted by novel selective chemical probes.
