Regulation of vascular permeability by thrombin mediated signaling pathways

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The goal of this proposal is to understand signal integration of multiple G protein pathways downstream of a single receptor. We propose to build up multiple computable "signaling modules", which represent G protein signalingpathways downstream of a multiply coupled receptor, PAR1, the thrombin receptor. PAR1 is known to mediate its complex actions in endothelium via its ability to couple to multiple Gproteins including members of Gi, Gq and G12/13 families thus leading to activation of these signaling pathways to modulate endothelial barrier function. The mathematical ground work laid in modeling of G protein signaling pathways has not yet been applied to such a multiple pathway integration problem. Endothelial cells (EC) form a dynamicallyregulated barrier between blood and interstitial tissues; thrombin is the most potent regulator of barrierpermeability. Barrier dysfunctionleads to edema, a normal consequence of wounding and inflammation, but when uncontrolled is a devastating component of many diseases such as acute lung injury and sepsis. Our aims are to 1) Construct a mathematical and computational model that describes the multiple G protein signaling pathways downstream of PARs that will serve as a tool to systematically investigate the detailed molecular mechanisms that regulate endothelial permeability. 2) Determine the mechanisms by which individualG proteins contribute to the physiological response. The laboratory has developed innovative tools to uncouple one G protein pathway at a time and determine the effects on cellular responses. 3) Use the model to help to identify key sites that would be novel targets for therapeutic intervention in these disorders. Using an early iteration of the model, we showed that thrombin and the thrombin receptor activating peptide differentially regulate different classes of G protein signaling pathways. We will investigate the hypothesis that different conformations of PAR1 traffic to different G proteins, and differentially regulate barrier function. This would provide a therapeutic window to search for allosteric modulators of PARs that work at different sites than the tethered ligand. Throughout the grant, the modeling, simulations and experimentation will be conducted iteratively. The long term goals are to build toward production of a validated computational model of the complex signaling processes used in thrombin regulation of the endothelial barrier. The model will help to identify key sites that would be novel targets for therapeutic intervention in these disorders.