The thirteen different mammalian phospholipase C (PLC) isozymes integrate signals from multiple upstream regulators acting as highly organized but poorly understood signaling nodes central to the action of many hormones, neurotransmitters, growth factors, and other extracellular stimuli. Our recent structures of PLC-? isozymes in the absence and presence of G protein activators led to the discovery that these signaling proteins are basally autoinhibited by the X/Y linker region of the catalytic TIM barrel. Results from our biochemical studies also are consistent with the hypothesis that activation by GTP-binding proteins and likely other modulators occurs by membrane-mediated removal of this autoinhibition by activator-promoted recruitment and orientation of the lipase active site at the membrane surface. We recently determined the structure of PLC-b bound to Gaq and used this information to propose a "catch- and-release" fly-casting mechanism to interpret the rapid cycling of this complex within functional scaffolds that include G protein-coupled receptors. Here (Specific Aim 1) we propose to use a combination of innovative approaches, including molecular dynamics simulations, peptidomimetics, and biosensors to further understand this scaffolding and its biological ramifications. PLC-b isozymes also are directly activated by release of Gbg dimers from heterotrimeric G proteins. The mechanism whereby this occurs is unknown, and the goal of Specific Aim 2 will be to determine the structure of a PLC-b isozyme bound to Gbg.
Phospholipase C-mediated activation of inositol lipid signaling pathways is obligatory in the physiological effects of many hormones, neurotransmitters, growth factors, and sensory stimuli, and this signaling protein is widely implicated in a broad range of diseases ranging from cancer to neurodegenerative disorders. The molecular mechanisms whereby activation/deactivation of phospholipase C signaling nodes occurs are poorly understood, and our research plan focuses on providing unambiguous molecular description of these critical signaling nodes. This information will be highly relevant to eventual identification of drugs that interdict phospholipase C-dependent signaling.
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