Verbatim): Thrombin cleavage of the thrombin receptors activates an extraordinarily diverse array of physiologic responses. These include platelet aggregation, cellular proliferation/apoptosis, cell-cell adhesion and inflammation, and potentially the invasive and tissue-reorganizing processes involved in cancer. Activation of platelet thrombin receptors is likely to play a major role in the initiation and maintenance of pathological arterial and venous thromboses and the development of atherosclerotic lesions. Four G protein-coupled, protease-activated receptors have been identified: PAR1, PAR2, PAR3, and PAR4. The prototypical protease-activated receptor, PAR1, has been shown to couple to Gq., Gi(betagamma), and G12/13 under a variety of in vitro conditions. The specifics of how PAR1 interacts with the G proteins and the relative importance and temporal ordering of differential G protein activation is still unknown. Far less is known about the identity of the G proteins that couple to the newly discovered PAR3 and PAR4 thrombin receptors. The goals of these studies are: 1) to investigate the mechanistic basis of differential G protein activation by PAR1 under in vivo conditions, 2) to understand the underlying differences between PAR4 and PAR1 G-protein dependent signaling and signal termination, and 3) to expand the repertoire of intracellular proteins that might interact with the class of protease-activated receptors. In the first specific aim we will exploit our newly discovered class of cell-penetrating pepducins. These reagents rapidly penetrate the plasma membrane of intact cells such as platelets and fibroblasts and cause specific activation and/or inhibition of receptor-dependent intracellular signal transduction under in vivo conditions. We have demonstrated that these pepducins exhibit properties that are normally attributable to activated PAR1. These include full platelet aggregation and shape change, stimulation of regulated Ca++ fluxes, activation of phospholipase C, and desensitization of thrombin receptor responses. We anticipate that the use of the pepducins to study intracellular signal transduction will open new avenues of experimental research in cells previously not amenable to molecular techniques. Ultimately, these may prove to be the first therapeutically useful agents that are targeted at receptor-G protein interfaces.
The second aim will explore the mechanistic basis of PAR4 versus PARI-activation of intracellular signaling pathways using a variety of genetic and biochemical techniques. We will expand on recent work done in our lab that demonstrated that PAR1 and PAR4 have distinct kinetics of Ca++ signaling in platelets. In the third aim, we will study coexpressed PAR1 and mammalian G proteins in yeast and will identify novel proteins which specifically interact with PAR1 and PAR4 intracellular domains.
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