Introduction Thrombin-induced increase in endothelial monolayer permeability depends on reorganization of cytoskeletal structures. Protease-activated receptor 1 (PAR-1) is the main receptor responsible for thrombin signaling in endothelial cells. Zyxin, a low-abundance phosphoprotein, serves as a molecular adaptor that mediates the productive juxtaposition of protein partners. Zyxin may participate in aspects of cytoskeletal assembly and dynamics by recruiting components of actin assembly machinery to specific sites in the cells. The subcellular distribution of zyxin is critical to its functions. In human umbilical vein endothelial cells (HUVECs), thrombin stimulation induces zyxin targeting to actin stress fibers. We proposed that zyxin participates in thrombin induced cytoskeletal reorganization by interaction with PAR-1.
Methods Coimmunoprecipitation and GST fusion protein pull-down assay were performed to identify the domains of zyxin to interact with carboxyl-terminal PAR-1 in COS-7 cells. Confocal immunofluorescence microscopy was utilized to observe the subcellular distribution of zyxin, PAR-1, actin stained with appropriate antibodies against these proteins. Zyxin-specific siRNA duplexes are generated to knock down the expression of endogenous zyxin.
Results We showed that PAR-1 interacted with zyxin both in vitro and in vivo. We have shown that LIM domains 1 and 2 of zyxin interacted with C-terminal domain of PAR-1. In human umbilical vein endothelial cells (HUVECs), thrombin promotes zyxin targeting to actin stress fibers. Down-regulation of zyxin using siRNA and disruption of the interaction between zyxin and PAR-1 resulted in reduced cellular responses to thrombin stimulation: actin stress fiber formation and serum response element (SRE)-dependent gene transcription. Zyxin-specific siRNA and C terminus of zyxin overexpression did not inhibit thrombin-induced RhoA activity, which plays a key role in thrombin-induced stress fiber formation and SRE activity and knockdown of endogenous zyxin could still attenuate the active RhoA-induced SRE activity, suggesting that zyxin may regulate thrombin-induced actin reorganization and SRE activity in an RhoA-independent manner. These results suggest that zyxin is involved in thrombin signaling by direct interaction with PAR-1.