The regulation of phosphatidylinositol-specific phospholipase C (PIPLC) activity by hormones controls the evolution of two second messengers, IP3 and diacylglycerol, and their subsequent effects on intracellular free Ca2+ and the activity of protein kinase C. G proteins of the Gq subfamily have been identified as mediators of PLCbeta1 activity. We have extended this regulation to two other isotypes of PLCbeta, PLCbeta2 and PLCbeta3. All three isotypes of PIPLC can be activated to different extents by both alpha-q and beta-gamma subunits. This application is designed to extend these studies in three major directions. First, we propose to determine the structural determinants of the PLCbeta enzymes that are important for their .regulation by G proteins and action in general. This will be done through expression and characterization of mutant proteins with a Baculovirus expression system. Second, we plan to search for other PIPLC enzymes that respond to alpha-q and/or beta-gamma subunits. This will include experiments with crude preparations from tissues and cells as well as characterization of expressed PLCdelta isoforms. Finally, initial evidence suggests that the purified PLCbeta3 contains an inhibitory subunit. Plans are presented to verify this observation and to characterize the inhibitory protein. We also propose to determine if other putative inhibitory subunits exist for PLCbeta1 and PLCbeta2. This work will increase our understanding of G protein dependent regulation of intracellular free Ca2+ and protein phosphorylation by hormones. A better understanding of the mechanisms of PIPLC regulation will help in the development of better tools to manipulate their function. The identification of specific inhibitory subunits for PLCbeta isotypes would establish a potentially powerful tool for dissecting the roles of these individual enzymes in intracellular regulation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM049993-01
Application #
3309132
Study Section
Toxicology Subcommittee 2 (TOX)
Project Start
1993-08-01
Project End
1997-06-30
Budget Start
1993-08-01
Budget End
1994-07-31
Support Year
1
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Type
Schools of Medicine
DUNS #
City
Dallas
State
TX
Country
United States
Zip Code
75390