The present application proposes experiments which address three fundamental aspects of TXA2 receptor (TPR) signaling in platelets: 1.Cross-signaling between platelet TPRs and other GPCRs. Our hypothesis is that the process of GPCR-G protein (GP) complex formation is based on the principles of mass action, and that this principle defines and prioritizes platelet GPCR signaling and cross-signaling. This model predicts a dynamic state of platelet GPCR signaling, which can be perturbed by numerous factors including cardiovascular disease. We believe that this principle forms a molecular mechanism by which platelets can integrate their separate GPCR signaling pathways and by which platelet GPCR signaling preferences are established. Experiments are now proposed to further investigate the underlying principles of this dynamic process. 2. Map the TPR ligand-binding pocket. Our hypothesis is that ligands dock at C-terminal ED3 and interact with residues in TM5 and/or N-terminal ED4. We previously demonstrated that C183-D193in ED3 form a critical ligand binding domain of TPRs, and these results have recently been confirmed by NMR. We will now identify the residues in ED3, which participate in this ligand binding. Based on the effective coordination radius of TPR ligands, we will also examine the participation of neighboring residues (from TM5 and N-terminal ED4) in the docking/signal transduction process. Using site-directed mutagenesis and PG receptor chimeras, experiments will measure agonist/antagonist ligand affinity and functional responses as indices ligand docking and efficacy. In addition, a rabbit model using a new functional antibody will be employed to determine the contribution of TPR ED3 to platelet reactivity in vivo. 3. The molecular and functional consequences of cAMP-mediated G-alpha12/13 phosphorylation. Our hypothesis is that phosphorylation of the conformationally sensitive Switch I Region (SRI) of G-alpha12/13 plays an important role in modulating signaling through the G-alpha12/13 pathway. We recently demonstrated PKA-mediated phosphorylation within SW1 at Thr (203) of G13. We will now further define the molecular/functional consequences of such phosphorylation on platelet activation and inhibition. Site-directed peptides/mutagenesis and inducible minigenes will be used to define PKA modulation of G12/13-Rho signaling in resting/activated platelets. Subsequent experiments will identify G12/13-mediated platelet functional responses, and how these responses are modulated by G-alpha phosphorylation. Collectively, the proposed experiments should provide new and important information regarding TPR signal transduction. This information should, in turn, aid in the development of therapeutic approaches for controlling TXA2-mediated thromboembolism ? ?
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