The long-term goal of this project is to understand how prostanoid receptors mediate human physiological and pathological vascular and hemodynamic processes, including hemostasis thrombosis and stroke. Prostanoids, including thromboxane (TXA2) and prostaglandin I2 (PGI2) are a family of bioactive oxygenase metabolites of polyunsaturated fatty acids synthesized by vascular smooth muscle and endothelium. TXA2 is a potent stimulator of platelet aggregation and a construction of smooth vasodilator and inhibitor of platelet aggregation actions essentially opposite to those of TXA2. The functions of TXB2 and PGI2 are mediated by specific cell surface receptors. Eight types of human prostanoid receptors have been defined. They belong to the G protein-coupled receptor family, with seven transmembrane domains and are coupled to different signal transduction systems. However, the structure and function relationship of the TXA2 receptor (TP) and PGI2 receptor (IP) are poorly defined, with little structural information on how the prostanoids specifically recognize their receptors and how the specific signal transduction is achieved by the initial contact of the receptor with G protein. The opposite receptor-mediated actions of TXA2 and PGI2 led us to hypothesize that the two receptors have distinct 3D structures in their extracellular ligand recognition sites which determine ligand selectivity, and in their intracellular G protein-coupled sites which determine signal transduction specificity. We further hypothesize that the ligand-recognition sites are formed by multiple extracellular domains. Our initial focus will be on the human TP receptor.
The specific aims are to use recombinant receptor mutants, synthetic peptides, peptide antibodies, circular dichroism and 2D nuclear magnetic resonance spectroscopy, molecular modeling and functional assays to: 1) Identify the peptide segments and key residues making up the ligand recognition site(s) of the TP receptor; 2) Determine the 3D structures of the peptides mimicking the extracellular domains of the receptor, 3) Characterize the structures of the TP receptor ligand, and complexes of the ligand with the receptor fragments and thus build a 3D structural model of the receptor/ligand interaction; and 4) Localize the G protein-coupling site(s) and determine 3D structures of the segments comprising the intracellular domains of the receptor.. Results from the proposed studies with the TP receptor will guide later characterization of the IP receptor, will be important for understanding the structure/function relationships of the TP and IP receptors, and will also develop generally useful approaches to solving the 3D structures of these important domains in other prostanoid receptors, and in other G protein-coupled receptors.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Specialized Center (P50)
Project #
2P50NS023327-14
Application #
6112248
Study Section
Project Start
1999-02-01
Project End
2000-01-31
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
14
Fiscal Year
1999
Total Cost
Indirect Cost
City
Houston
State
TX
Country
United States
Zip Code
77225
Chen, Pei-Feng; Wu, Kenneth K (2009) Two synthetic peptides corresponding to the proximal heme-binding domain and CD1 domain of human endothelial nitric-oxide synthase inhibit the oxygenase activity by interacting with CaM. Arch Biochem Biophys 486:132-40
Wu, Jui-Sheng; Cheung, Wai-Mui; Tsai, Yau-Sheng et al. (2009) Ligand-activated peroxisome proliferator-activated receptor-gamma protects against ischemic cerebral infarction and neuronal apoptosis by 14-3-3 epsilon upregulation. Circulation 119:1124-34
Liou, Jun-Yang; Ellent, David P; Lee, Sang et al. (2007) Cyclooxygenase-2-derived prostaglandin e2 protects mouse embryonic stem cells from apoptosis. Stem Cells 25:1096-103
Liou, Jun-Yang; Lee, Sang; Ghelani, Dipak et al. (2006) Protection of endothelial survival by peroxisome proliferator-activated receptor-delta mediated 14-3-3 upregulation. Arterioscler Thromb Vasc Biol 26:1481-7
Cieslik, Katarzyna A; Deng, Wu-Guo; Wu, Kenneth K (2006) Essential role of C-Rel in nitric-oxide synthase-2 transcriptional activation: time-dependent control by salicylate. Mol Pharmacol 70:2004-14
Deng, Wu-Guo; Tang, Shao-Tzu; Tseng, Hui-Ping et al. (2006) Melatonin suppresses macrophage cyclooxygenase-2 and inducible nitric oxide synthase expression by inhibiting p52 acetylation and binding. Blood 108:518-24
Wu, Kenneth K (2006) Analysis of protein-DNA binding by streptavidin-agarose pulldown. Methods Mol Biol 338:281-90
Lin, Teng-Nan; Cheung, Wai-Mui; Wu, Jui-Sheng et al. (2006) 15d-prostaglandin J2 protects brain from ischemia-reperfusion injury. Arterioscler Thromb Vasc Biol 26:481-7
Wu, Kenneth K (2006) Transcription-based COX-2 inhibition: a therapeutic strategy. Thromb Haemost 96:417-22
Liou, Jun-Yang; Aleksic, Nena; Chen, Shu-Fen et al. (2005) Mitochondrial localization of cyclooxygenase-2 and calcium-independent phospholipase A2 in human cancer cells: implication in apoptosis resistance. Exp Cell Res 306:75-84

Showing the most recent 10 out of 63 publications