Phosphoinositide-specific phospholipase C (PLC) hydrolyzes phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) to yield inositol 1,4,5-trisphosphate and diacylglycerol, two intracellular second messengers that are active in all eukaryotes. Operating through calcium and protein kinases, these signaling molecules trigger a broad range of bioligical responses that include hormone and neurotransmitter secretion, smooth muscle contraction and mitosis. Although the broad outlines of PLC regulation are known, the molecular basis for its activation is not. Contributing to this lack of progress is the misunderstood role of the membrane surface, which is both a barrier to substrate access and an actiation cofactor. The goals of this project are (1) to identify the regions of PLC that facilitate phospholipid binding and access to substrate, (2) to determine how calcium ions and GTP-binding proteins control catalysis and (3) to test the idea that penetration of the membrane surface activates the enzyme by an 'induced fit' mechanism. PLC-delta1 and beta2, isozymes with distinct modes of regulation, will be studies in parallel to gain insight into the activation process. A combination of genetic and biophysical approaches will be used. The modular regions found in the PLC isozymes (and a host of other signaling proteins), including the pleckstrin homology (PH), EF-hand and C-2 domains, will be expressed as isolated fusion proteins. Function in the intact enzyme will be further tested by subjecting critical residues to site-directed mutagenesis and by exchanging each region of PLC-delta1 with the corresponding module found in PLC-beta2. The binding properties and catalytic behaviors of these engineered proteins will be studied in membrane bilayers, monolayers, and detergent/phospholipid mixed micelles, and in solution with monomeric substrate analogs. High pressure fluorescence spectrosopy will be used to examine the relationships between emebrane penetration, catalysis and the enzyme's conformational state.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM043422-06A1
Application #
2022352
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1989-12-01
Project End
2001-04-30
Budget Start
1997-05-01
Budget End
1998-04-30
Support Year
6
Fiscal Year
1997
Total Cost
Indirect Cost
Name
State University New York Stony Brook
Department
Anesthesiology
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794