The thirteen different mammalian phospholipase C (PLC) isozymes integrate signals from multiple upstream regulators acting as highly organized but poorly understood signaling nodes central to the action of many hormones, neurotransmitters, growth factors, and other extracellular stimuli. Our recent structures of PLC-? isozymes in the absence and presence of G protein activators led to the discovery that these signaling proteins are basally autoinhibited by the X/Y linker region of the catalytic TIM barrel. Results from our biochemical studies also are consistent with the hypothesis that activation by GTP-binding proteins and likely other modulators occurs by membrane-mediated removal of this autoinhibition by activator-promoted recruitment and orientation of the lipase active site at the membrane surface. We recently determined the structure of PLC-b bound to Gaq and used this information to propose a """"""""catch- and-release"""""""" fly-casting mechanism to interpret the rapid cycling of this complex within functional scaffolds that include G protein-coupled receptors. Here (Specific Aim 1) we propose to use a combination of innovative approaches, including molecular dynamics simulations, peptidomimetics, and biosensors to further understand this scaffolding and its biological ramifications. PLC-b isozymes also are directly activated by release of Gbg dimers from heterotrimeric G proteins. The mechanism whereby this occurs is unknown, and the goal of Specific Aim 2 will be to determine the structure of a PLC-b isozyme bound to Gbg.

Public Health Relevance

Phospholipase C-mediated activation of inositol lipid signaling pathways is obligatory in the physiological effects of many hormones, neurotransmitters, growth factors, and sensory stimuli, and this signaling protein is widely implicated in a broad range of diseases ranging from cancer to neurodegenerative disorders. The molecular mechanisms whereby activation/deactivation of phospholipase C signaling nodes occurs are poorly understood, and our research plan focuses on providing unambiguous molecular description of these critical signaling nodes. This information will be highly relevant to eventual identification of drugs that interdict phospholipase C-dependent signaling.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM057391-14
Application #
8297359
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Dunsmore, Sarah
Project Start
1998-05-01
Project End
2016-03-31
Budget Start
2012-05-01
Budget End
2013-03-31
Support Year
14
Fiscal Year
2012
Total Cost
$407,000
Indirect Cost
$132,000
Name
University of North Carolina Chapel Hill
Department
Pharmacology
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Charpentier, Thomas H; Waldo, Gary L; Barrett, Matthew O et al. (2014) Membrane-induced allosteric control of phospholipase C-β isozymes. J Biol Chem 289:29545-57
Schmitz, Anna-Lena; Schrage, Ramona; Gaffal, Evelyn et al. (2014) A cell-permeable inhibitor to trap Gαq proteins in the empty pocket conformation. Chem Biol 21:890-902
Huang, Weigang; Barrett, Matthew; Hajicek, Nicole et al. (2013) Small molecule inhibitors of phospholipase C from a novel high-throughput screen. J Biol Chem 288:5840-8
Hajicek, Nicole; Charpentier, Thomas H; Rush, Jeremy R et al. (2013) Autoinhibition and phosphorylation-induced activation of phospholipase C-γ isozymes. Biochemistry 52:4810-9
Dbouk, Hashem A; Vadas, Oscar; Shymanets, Aliaksei et al. (2012) G protein-coupled receptor-mediated activation of p110β by Gβγ is required for cellular transformation and invasiveness. Sci Signal 5:ra89
Gresset, Aurelie; Sondek, John; Harden, T Kendall (2012) The phospholipase C isozymes and their regulation. Subcell Biochem 58:61-94
Cherkis, Karen A; Temple, Brenda R S; Chung, Eui-Hwan et al. (2012) AvrRpm1 missense mutations weakly activate RPS2-mediated immune response in Arabidopsis thaliana. PLoS One 7:e42633
Wang, Xiaoyang; Barrett, Matthew; Sondek, John et al. (2012) Fluorescent phosphatidylinositol 4,5-bisphosphate derivatives with modified 6-hydroxy group as novel substrates for phospholipase C. Biochemistry 51:5300-6
Harden, T Kendall; Waldo, Gary L; Hicks, Stephanie N et al. (2011) Mechanism of activation and inactivation of Gq/phospholipase C-β signaling nodes. Chem Rev 111:6120-9
Huang, Weigang; Hicks, Stephanie N; Sondek, John et al. (2011) A fluorogenic, small molecule reporter for mammalian phospholipase C isozymes. ACS Chem Biol 6:223-8

Showing the most recent 10 out of 19 publications