The experiments outlined in this proposal are designed to define mechanisms and structural features involved in regulation of phosphatidylinositol specific phospholipase C (PLC) beta and phosphoinositide 3-kinase (PI3K) by G protein beta-gamma subunits and phosphatidylinositol 4,5-bisphosphate (PIP2). The family of effectors regulated by the beta-gamma subunits of heterotrimeric GTP binding proteins (G proteins) continues to expand and includes a wide variety of enzymes and ion channels. An emerging concept in signal transduction is that PIP2 is a central molecule in the regulation of a variety of cellular processes by a mechanism not necessarily related to its role as a precursor for reactions catalyzed by PLC and PI3K. By analyzing specific interactions for two different G protein-beta-gamma First, the influence of specific lipids and beta-gamma subunits on interactions with lipid vesicles will be examined using fluorescence resonance energy transfer (FRET). These experiments will address the hypothesis that PLC or PI3K can be regulated by controlling interactions with membrane surfaces. The involvement of pleckstrin homology (PH) domains, present in the primary sequences from PLC/PI3K, in interactions with lipid bilayer interfaces and G protein beta-gamma subunits will be determined by deleting the domains and/or expressing the domains in isolation. The resulting purified proteins will be examined for their ability to interact with either PIP2 vesicles or beta-gamma subunits using FRET and enzymatic analysis. Additionally, roles for these domains in cellular localization will be examined. Proteins that contain PH domains could potentially interact with either PIP2 or beta-gamma subunits to regulate PLC/PI3K. This will be investigated specifically for pleckstrin, a major protein kinase C substrate in platelets. Covalent lipid modifications of G protein gamma subunits are thought to be important for the interaction of beta-gamma subunits with lipid bilayers and membranes. In this proposal, the importance of these modifications for direct interaction with PLC beta 2 will be investigated using FRET. Finally, oligomerization of PLC and the potential involvement of this process in regulating PLC activity will be investigated by FRET, sucrose density gradient centrifugation, and fluorescence polarization anisotropy.
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