Hydrolysis of phosphatidylinositol 4,5 bisphosphate by phosphatidylinositol-specific phospholipase C (PLC) is a central process in hormone regulation of cellular physiology in virtually all mammalian tissues. G protein coupled receptors regulate PLC by stimulating the activation of either G protein alpha or beta-gamma subunits that modulate PLC activity through direct protein-protein interactions. Because there is no three dimensional crystal structure of the complex between G protein subunits and these molecules much needs to be elucidated about the nature of the interactions between G protein subunits and PLC beta that result in regulation of enzymatic activity. Experiments proposed in this application will further define the nature of the interactions between G proteins and phospholipase C isoforms and will lead to an understanding of the mechanism for regulation of these enzymes. More specifically: I. Site directed mutagenesis will be used to test the importance of an alpha-helix in the catalytic domain of PLC beta2 that may be a central transducing domain responsible for integrating signals from both beta-gamma and alpha subunits. A working hypothesis is that beta-gamma or alpha subunits either directly or indirectly alter this helix leading to alteration in the position of specific amino acids in the active site to change the rate of PIP2 hydrolysis. II. Chemical crosslinking of peptides derived from PLC beta2 to beta subunits and site directed mutagenesis of beta subunits will be used to confirm the involvement of the amino terminal coiled-coil of beta-gamma subunits in interactions with PLC beta. Additionally, chimeras between G betal and G beta5 isoforms will be constructed and tested to identify regions of the beta subunit involved in selective interactions with effectors. III. The mechanism for activation of a newly discovered phospholipase C isoform by the low molecular weight GTP binding protein, Ras, beta-gamma subunits and alpha subunits will be investigated. Given the well known involvement of Ras in mitogenesis and cancer, investigation of this mechanism could yield information about PLC involvement in cancer.
Showing the most recent 10 out of 29 publications
