Lipids are important regulators of the activity of many proteins including those involved in cardiac regulation, yet little is known about the molecular mechanisms mediating these effects. The overall aim of this proposal is to investigate the structural determinants of protein-lipid interactions, and to define how these interactions contribute to signal transduction in the cardiovascular system. Recent work has identified the pleckstrin homology domain as a lipid binding allosteric regulatory domain. This application to continue the molecular work describing the molecular mechanisms by which an isozyme of inositol phospholipid-specific phospholipase C (PI-PLC), PLC delta1 is regulated, and extend the studies to understand how lipids and PI-PLC isoforms mediate cellular effects in vivo. Specific Goals for the proposal include the following: 1. To investigate the modular nature of PH domains. A series of PLC delta1 chimeras containing PH domains from other molecules such as GRK2 will be constructed and characterized. A wide range of ligands and the generality of PH domain function will be investigated. 2. To characterize using molecular, biological and biochemical techniques high affinity non-substrate (PS) binding to PLC delta1. We hypothesize that the C2 domain of PLC delta1 is important in Ca2+ binding, phospholipid (PS) binding and activation. Mutagenesis will be performed to delineate the binding site. To determine the mechanisms by which PS activates PLC delta1. Contributions of enzyme anchoring, processivity, and catalysis to activation by lipids will be assessed. 4. To determine the function of phospholipase C delta 1 in vivo. Our philosophy will be to target mutant enzymes into the genome of mice. It will be determined if PLC delta 1 plays an important role in the pathophysiology of cardiovascular disease. Both transgenic and targeted replacement mice will be generated and assessed for the cardiovascular function. These studies should not only increase understanding of basic cardiovascular regulation, but also have the potential to promote the development of novel post-receptor therapeutics and new diagnostic tools.
