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.
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