The production of lipid second messengers is stimulated by a variety of hormones and neurotransmitters that work through G protein coupled receptors (GPCR). One such lipid signaling system involves the hydrolysis of phosphatidylcholine, a major component of biological membranes, by phospholipase D (PLD) to produce phosphatidic acid (PA) and choline. Phosphatidic acid and its metabolic products, diacylglycerol and lyso-PA, have been postulated to modulate cell proliferation, vesicle transport membrane remodeling, long term goal of these studies is to achieve a better understanding of the molecular basis of PLD1 regulation. We decided to focus on the role of CDC42 in GPCR activation pathways because our preliminary findings suggests that Cdc42 modulates activation by other regulatory proteins and recent reports have shown that some heterotrimeric G proteins crosstalk to guanine nucleotide exchange factors specific to the Rho subfamily. The initial aim focuses on identifying sites on Cdc42 that bind and activate PLD1. We predict that several factors will be involved in heptahelical receptor activation of PLD1 and studies in the second aim will characterize two potentially novel modulators of PLD1 to determine their participation.
The third aim will determine whether a purinergic receptor stimulation of PLD1 is transduced through Cdc42. The role of Cdc42 has not previously been systematically investigated. The proposed studies combine pharmacological, biochemical, and molecular genetic approaches to elucidate the molecular mechanisms of PLD1 activation in native cell lines. These studies will utilize both intact cells and cell-free preparations that retain purinergic receptor-stimulated activation of PLD1.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058516-06
Application #
6711793
Study Section
Pharmacology A Study Section (PHRA)
Program Officer
Lograsso, Philip
Project Start
2000-03-01
Project End
2006-02-28
Budget Start
2004-02-29
Budget End
2006-02-28
Support Year
6
Fiscal Year
2004
Total Cost
$224,613
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Pharmacology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Garcia, Avalon; Zheng, Yang; Zhao, Chen et al. (2008) Honokiol suppresses survival signals mediated by Ras-dependent phospholipase D activity in human cancer cells. Clin Cancer Res 14:4267-74
Brown, H Alex; Henage, Lee G; Preininger, Anita M et al. (2007) Biochemical analysis of phospholipase D. Methods Enzymol 434:49-87
Milne, Stephen; Ivanova, Pavlina; Forrester, Jeffrey et al. (2006) Lipidomics: an analysis of cellular lipids by ESI-MS. Methods 39:92-103
Henage, Lee G; Exton, John H; Brown, H Alex (2006) Kinetic analysis of a mammalian phospholipase D: allosteric modulation by monomeric GTPases, protein kinase C, and polyphosphoinositides. J Biol Chem 281:3408-17
Milne, Stephen B; Ivanova, Pavlina T; DeCamp, Dianne et al. (2005) A targeted mass spectrometric analysis of phosphatidylinositol phosphate species. J Lipid Res 46:1796-802
Ivanova, Pavlina T; Milne, Stephen B; Forrester, Jeffrey S et al. (2004) LIPID arrays: new tools in the understanding of membrane dynamics and lipid signaling. Mol Interv 4:86-96
Forrester, Jeffrey S; Milne, Stephen B; Ivanova, Pavlina T et al. (2004) Computational lipidomics: a multiplexed analysis of dynamic changes in membrane lipid composition during signal transduction. Mol Pharmacol 65:813-21
Ge, Mingtao; Gidwani, Arun; Brown, H Alex et al. (2003) Ordered and disordered phases coexist in plasma membrane vesicles of RBL-2H3 mast cells. An ESR study. Biophys J 85:1278-88
Gidwani, Arun; Brown, H Alex; Holowka, David et al. (2003) Disruption of lipid order by short-chain ceramides correlates with inhibition of phospholipase D and downstream signaling by FcepsilonRI. J Cell Sci 116:3177-87
Fang, Yimin; Park, In-Hyun; Wu, Ai-Luen et al. (2003) PLD1 regulates mTOR signaling and mediates Cdc42 activation of S6K1. Curr Biol 13:2037-44

Showing the most recent 10 out of 18 publications