The rationale for the widespread use of aspirin in preventing coronary or cerebrovascular thrombosis is based on its ability to block the synthesis of TXA2. Thus, TXA2 signal transduction plays a critical role in the sequelae of events leading to certain occlusive vascular disorders. However, in spite of the clear biological importance of TXA2, significant questions remain concerning its signaling mechanisms. In the present application, we propose experiments which will address three fundamental aspects of TXA2-mediated signal transduction.
Specific Aim I. will Investigate Signaling Between Platelet Seven-Transmembrane Receptors.
This aim derives from our hypothesis that G-proteins serve as communication vectors within the platelet receptor pool. We believe this is a significant finding because it provides a molecular mechanism for the integration of the separate receptor signaling pathways in platelets (or other cells). This signaling process will now be further defined relative to its existence between different platelet/endothelial receptors, its effects on ligand binding affinities, and its molecular mechanism of action.
Specific Aim 2. will Investigate the Platelet TXA2 Receptor Ligand Binding Domain(s).
This aim i s directed at testing our hypothesis that the ligand binding domain(s) of the TXA2 receptor protein resides in the extracellular region of the protein. Recently, we have succeeded in developing a new class of biotinylated photoaffinity receptor probes, and are currently applying these probes to investigate the receptor binding pocket. Two complementary approaches to this Specific Aim will also be taken: a. site- directed mutagenesis of the receptor; and b. site-specific antibodies against the receptor. It is believed that the combined information obtained from each approach will provide more definitive information than could be obtained by the utilization of either photolabeling, mutagenesis or antibody targeting alone. Finally, Specific Aim 3. will Investigate cAMP-Mediated Phosphorylation of TXA2 receptor-associated G-alpha13.
This aim will test our hypothesis that TXA2 receptor signaling through G- alpha13 is coupled to IP3-independent Ca2+ fluxes in platelets; and that phosphorylation of G-alpha13 is associated with the peculiar sensitivity of TXA2 signaling to cAMP levels. Recently, we have demonstrated that the G-alpha13 subunit is associated with endogenous platelet TXA2 receptors, and that this alpha-subunit is phosphorylated by cAMP. Experiments are now proposed to study the function of G-alpha13 (i.e. Ca2+ fluxes), its localization in platelets (surface or internal membrane), and the biochemical consequences of its phosphorylation on the stability of the TXA2 receptor-G-alpha13 complex and the stability of the G-protein heterotrimer complex. In summary, the results obtained from the proposed experiments should provide new and important information regarding signal transduction through the TXA2 receptor pathway. This information will, in turn, be of significant benefit to the development of more specific and effective pharmacological approaches to the control of TXA2-mediated thromboembolism.
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