Electrostatic Regulation of Phospholipase C Beta Enzymes

Phospholipase C (PLC) proteins cleave phosphatidylinositol lipids from cellular membranes to produce the second messengers inositol triphosphate (IP3) and diacylglycerol (DAG). The PLC subfamily is among the best characterized of the PLC enzymes and is crucial for normal cardiovascular function. Under basal conditions, PLC has low activity and is partitioned between the plasma membrane and the cytoplasm. However, it is unclear how this low activity state is maintained from a molecular standpoint. One known autoinhibitory element is the X-Y linker, a loop within the catalytic domain that forms a lid over the active site and which is displaced through interfacial activation. Autoinhibition by the X-Y linker is thought to require a highly conserved acidic stretch of residues, as deletions encompassing this acidic region, or the entire linker, result in increased basal activity, decreased efficacy of G protein activation, and decreased thermal stability. These results, together with previous structural studies, suggest that the acidic stretch may regulate basal activity in part through interactions with conserved basic residues on the surface catalytic domain. I have used site-directed mutagenesis and functional assays to begin establishing the contribution of intramolecular electrostatic interactions to the regulation of PLC