Thrombin receptor signaling to cytoskeleton requires Hsp90.
Thrombin is a serine protease that evokes various cellular responses involved in injury and repair of the nervous system through the activation of protease-activated receptor-1 (PAR-1). Signals that modulate cell morphology precede most PAR-1 effects, but the initial signal transduction molecules are not known. Using the yeast two-hybrid system, we identified Hsp90, a chaperone with known signaling properties, as a binding partner of PAR-1. The interaction was confirmed by glutathione S-transferase pull-down, overlay, and co-immunoprecipitation assays. Morphological assays in mouse astrocytes were carried out to evaluate the importance of Hsp90 during cytoskeletal signaling. Reducing Hsp90 levels by antisense treatment or disruption of the Hsp90.PAR-1 complex by the Hsp90-specific drug geldanamycin attenuated thrombin-mediated astrocyte shape changes. Furthermore, overexpression of the PAR-1 cytoplasmic tail abrogated thrombin-induced cytoskeletal changes in neuronal 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 together, these studies demonstrate that Hsp90 may be essential for PAR-1-mediated signaling to the cytoskeleton.
Pai
K S
KS
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, California 92697-4025, USA.
Mahajan
V B
VB
Lau
A
A
Cunningham
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DD
D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000502:physiology; D003001:Cloning, Molecular; D003599:Cytoskeleton / Q000502:physiology*; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009474:Neurons / Q000166:cytology / Q000502:physiology; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology*; D013917:Thrombin / Q000494:pharmacology*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D018841:HSP90 Heat-Shock Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D011993:Recombinant Fusion Proteins; D013917:Thrombin; D020742:rhoA GTP-Binding Protein