The activation of the coagulation cascade is intimately linked to vascular cell signaling in development, inflammation and cancer biology. The TF initiation complex in primary endothelial cells activates PAR1 and PAR2, but it is currently poorly understood how these signaling events are distinct from the better characterized thrombin signaling. Our preliminary data show that activation of PAR2 specifically results in TF cytoplasmic domain Ser258 phosphorylation which serves as the major switch to regulate adaptor recruitment to the TF cytoplasmic domain. Furthermore, TF cytoplasmic domain deleted mice show that the intracellular domain has negative regulatory roles in angiogenesis in vivo. The primary objective of this project is to characterize the signaling cross-talk of TF with PAR2 by in vitro experiments and to validate significance of TF signaling by genetic approaches in vivo.
Aim 1 is to define how the PAR2 specificity in phosphorylation of the TF cytoplasmic domain is generated. We will test the hypothesis that the carboxyl-terminus of PAR2 recruits a specific kinase complex that phosphorylates TF. We will map the relevant region in PAR2, establish the involvement of the transmembrane domain of TF, characterize the role of arrestin and test whether Ser/Thr kinases of the MAP kinase pathway phosphorylate TF. These experiments will provide novel insight into how specificity is created in the co-signaling of protease-binding receptors with PARs.
Aim 2 is to test the hypothesis that PAR1 and PAR2 activation by the ternary initiation complex results in distinct trafficking and internalization routes. These experiments will characterize the role of PAR signaling in the regulation of the thrombogenic cascade.
Aim 3 is to determine whether deletion of the TF cytoplasmic domain results in deregulated PAR signaling in angiogenesis. We will analyze whether the gain of function phenotype of TF cytoplasmic domain deleted mice can be ablated by genetic crosses with PAR1 or PAR2 deficient mice. These experiments will generate genetic evidence that links the TF cytoplasmic domain to signaling through a specific PAR and validate the mechanistic studies in the previous aims in a relevant in vivo model. In a collaborative effort with Project 4, we will elucidate the structural basis of ligand binding specificity with recently identified adaptors that bind the TF cytoplasmic domain. The proposed studies thus characterize the biochemical basis, cell biology and in vivo relevance of the PAR2-dependent cell signaling specificity of the TF initiation complex. These experiments will provide novel insight into the endothelial cell biology of TF of relevance for development, cardiovascular disease and angiogenesis.
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