Thrombin mediated PAR1 stimulation results in sustained activation of Rap1 and downstream responses in human 1321N1 astroglioma cells

Abstract

G‐protein coupled receptors that activate RhoA can lead to aberrant cell growth and have been linked to human diseases including cancer. We have shown that in 1321N1 astroglioma cells, stimulation of the PAR1 receptor with thrombin activates RhoA and downstream signals required for mitogenesis. One mediator of thrombin induced mitogenesis is the matricellular protein CCN1 which signals through integrins (Walsh, FASEB J, 2008) and another is phospholipase C epsilon (Citro et al PNAS, 2007). Integrin signaling requires the low molecular weight GTPase Rap1, and PLC epsilon serves as an activator or Rap1. Accordingly we asked whether stimulation of the G12/13 and RhoA coupled PAR1 receptor in 1321N1 cells was coupled to activation of Rap1. Using a pull down assay to assess GTP liganded Rap1, we find that thrombin elicits a robust and sustained increase in Rap1 activation, increasing to 3 fold basal levels and maintained for at least 6 hours. Thrombin stimulation also elicits 4–5 fold increases ERK1/2 and PKD activation which are sustained for at least 6 hours. Our results suggest that thrombin mediated Rap1 activation is associated with sustained ERK1/2 and PKD activation. We are currently exploring the possibility of involvement of RhoA and PLC epsilon in thrombin induced Rap1 activation and the role of Rap1 in sustained ERK and PKD activation induced by thrombin in 1321N1 astroglioma cells.Supported by NIH grant, GM 36927