A large class of neurotransmitters and hormones activate phospholipase C beta-isozymes (PLC-betas) through G protein-linked signaling cascades. In response to activated G proteins, PLC-beta isozymes accelerate the hydrolysis of phosphatidylinositol-4,5 bisphosphate (PtdIns(4,5)P2) to the second messengers sn-1,2-diacylglycerol (DAG) and D-myo-inositol- 1,4,5-trisphosphate (Ins(1,4,5)P3). Production of soluble Ins (1,4,5)P3 leads to the release of intracellular calcium while DAG activates protein kinase C isozymes, and these events ultimately control a diverse array of cellular functions including proliferation, excitation, secretion, and contraction. The mechanism(s) of regulation of PLC-beta isozymes by G proteins, phospholipids, Ca2+, and other potential regulators is poorly understood at the molecular level. However, with the recent ability to purify multiple milligrams of functional PLC-beta isozymes, we are now in a favorable position to define clearly the modes of PLC-beta regulation. A series of studies will be carried out to optimize the production of homogeneous and monodisperse PLC-beta holoenzymes and domain fragments. These characterized proteins will then be utilized in a dual approach of quantitative binding analysis and crystallography to understand PLC-beta regulation. Particular attention will be given to delineating the role of the N-terminal PH domains of PLC-betas in binding phosphoinositides and possibly Gbetagamma subunits. Quantitative measurements of phosphoinositide and Gbetagamma binding to the PH domains of PLC-betas will be obtained from surface plasmon resonance measurements and microcalorimetry. Since different PLC-beta isozymes exhibit distinct G protein regulation, binding data for equivalent PH domains from different isozymes may delimit modes of isozyme-specific G protein activation. X-ray crystallography also will be used to determine atomic resolution structures of PLC-beta domains or full-length proteins. These structures will provide key information on interdomain interactions, relative domain orientations, and specific binding sites for small molecules. Overall, the combination of binding data and structural information is intended to provide an atomic- resolution framework for understanding and possibly manipulating the regulation of interfacial catalysis by PLC-beta isozymes. This work also will provide the foundation for understanding in greater detail the regulation of PLC-beta isozymes by other activated Galpha subunits and Gbetagamma dimers.
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