Thrombin receptor signaling to cytoskeleton requires Hsp90

Thrombin is a serine protease that evokes various cellular responses involv ed in injury and repair of the nervous system through the activation of pro tease-activated receptor-1 (PAR-1). Signals that modulate cell morphology p recede most PAR-1 effects, but the initial signal transduction molecules ar e not known. Using the yeast two-hybrid system, we identified Hsp90, a chap erone with known signaling properties, as a binding partner of PAR-1. The i nteraction was confirmed by glutathione S-transferase pull-down, overlay, a nd co-immunoprecipitation assays. Morphological assays in mouse astrocytes were carried out to evaluate the importance of Hsp90 during cytoskeletal si gnaling. Reducing Hsp90 levels by antisense treatment or disruption of the Hsp90-PAR-1 complex by the Hsp90-specific drug geldanamycin attenuated thro mbin-mediated astrocyte shape changes. Furthermore, overexpression of the P AR-1 cytoplasmic tail abrogated thrombin-induced cytoskeletal changes in ne uronal cells. Treatment with geldanamycin specifically inhibited activation of RhoA without affecting thrombin-mediated intracellular calcium release, revealing the regulation of a distinct signaling pathway by Hsp90. Taken t ogether, these studies demonstrate that Hsp90 may be essential for PAR-1-me diated signaling to the cytoskeleton.