Thromboxane A2 (TxA2) is a potent platelet aggregating and vasoconstrictor eicosanoid which contributes to the pathogenesis kidney diseases. Its effects are mediated by activating specific cell surface receptors. Receptor activation depends not only on the extracellular concentration of TxA2 but also on receptor responsiveness and number. Recent studies suggest that TxA2 receptor activity is modulated by distinct biochemical processes. Exposure of TxA2 receptors to agonist leads to inactivation of the receptor on the cell surface followed by a loss of binding sites from membrane fractions of the cell. Central to these regulatory processes are protein kinases which appear to inhibit TxA2 receptor activity, perhaps by directly phosphorylating the receptor protein. The kinase pathways involved in TxA2 receptor regulation are not specifically characterized. Moreover, the role of receptor phosphorylation in modulating responses to TxA2 is not known. The purpose of this grant proposal is to investigate the role or protein kinases in regulating TxA2 receptor activity. To accomplish this goal, we have stably expressed the mouse TxA2 receptor gene in COS-l cells and obtained high levels of functional TxA2 receptors which are regulated like TxA2 receptors in the kidney. Using this cell system, we propose to investigate TxA2 regulation focusing on the following specific aims: 1. characterization of TxA2 receptor regulation by protein kinases 2. defining regulatory domains of the TxA2 receptor protein, and 3. examination of kinetics and patterns of TxA2 receptor phosphorylation. To investigate these specific aims we will use three complementary approaches. First, we will use pharmacological agents to modulate the activity of protein kinases and determine the effects of these agents on TxA2 receptor responsiveness. Secondly, mutant TxA2 receptors will be generated to study potential regulatory domains of the TxA2 receptor protein. Lastly, we will create epitope tagged TxA2 receptors to allow purification of TxA2 receptor proteins and assessment of their phosphorylation state. These studies should provide new, important insights into the mechanisms TxA2 receptor regulation. Because TxA2 is a central component in the pathogenesis of diverse kidney diseases, understanding regulation of its receptor may suggest novel approaches to the treatment of disease processes affecting the kidney.
