Abstract 1631: Interaction of Zyxin with Protease-activated Receptor-1: Role in Thrombin Signaling
INTRODUCTION Thrombin, generated at the surface of damaged endothelium, induces blood coagulation but also exerts a variety of functional effects on the endothelium. Thrombin induced micro-vascular dysfunction can have pathological consequences and contribute to organ reactions to inflammation and ischaemia. Protease-activated receptor1 (PAR-1) is the main receptor responsible for thrombin signaling in endothelial cells. Zyxin serves as a molecular adaptor that mediates the productive juxtaposition of protein partners. Zyxin participates in aspects of cytoskeletal assembly by recruiting components of actin assembly machinery. In human umbilical vein endothelial cells (HUVECs), thrombin stimulation induces zyxin redistribution along actin stress fibers. We hypothesized that zyxin participates in thrombin signaling via interaction with PAR-1.
METHODS Co-immunoprecipitation and GST fusion protein pull-down assay were performed to identify the domains of zyxin to interact with cytoplasmic tail of PAR-1 in COS-7 cells. Confocal immunofluorescence microscopy was utilized to observe the subcellular distribution of zyxin, PAR-1, and the actin was labeled with Alexa-594 phalloidin. Zyxin specific siRNA is generated to down-regulate 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 domain 1 and 2 of zyxin interacted with cytoplasmic tail of PAR-1. In HUVECs, Depletion of endogenous zyxin using siRNA technology inhibited thrombin-induced actin stress fiber formation and serum response element (SRE) dependent gene transcription. In addition, disruption of the interaction between zyxin and PAR-1 by overexpression of LIM domains of zyxin could also inhibit these thrombin responses. RhoA, a member of Ras superfamily, has been believed to be responsible for thrombin-induced stress fiber formation and SRE activity. In our study, downregulation of zyxin and disruption the zyxin-PAR-1 interaction could not inhibit thrombin-induced RhoA activation in HUVECs, suggesting that zyxin is involved in thrombin signaling in RhoA independent manner. These results suggest that zyxin is involved in thrombin signaling via direct interaction with PAR-1.