Protein kinase C beta regulates heterologous desensitization of thrombin receptor (PAR-1) in endothelial cells.

We studied the effects of protein kinase C (PKC) activation on endothelial cell surface expression and function of the proteolytically activated thrombin receptor 1 (PAR-1). Cell surface PAR-1 expression was assessed by immunofluorescence (using anti-PAR-1 monoclonal antibody), and receptor activation was assessed by measuring increases in cytosolic Ca2+ concentration in human dermal microvascular endothelial cells (HMEC) exposed to alpha-thrombin or phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Immunofluorescence showed that thrombin and TPA reduced the cell surface expression of PAR-1. Prior exposure of HMEC to thrombin for 5 min desensitized the cells to thrombin, indicating homologous PAR-1 desensitization. In contrast, prior activation of PKC with TPA produced desensitization to thrombin and histamine, indicating heterologous PAR-1 desensitization. Treatment of cells with staurosporine, a PKC inhibitor, fully prevented heterologous desensitization, whereas thrombin-induced homologous desensitization persisted. Depletion of PKC beta isozymes (PKC beta I and PKC beta II) by transducing cells with antisense cDNA of PKC beta I prevented the TPA-induced decrease in cell surface PAR-1 expression and restored approximately 60% of the cytosolic Ca2+ signal in response to thrombin. In contrast, depletion of PKC beta isozymes did not affect the loss of cell surface PAR-1 and induction of homologous PAR-1 desensitization by thrombin. Therefore, homologous PAR-1 desensitization by thrombin occurs independently of PKC beta isozymes, whereas the PKC beta-activated pathway is important in signaling heterologous PAR-1 desensitization in endothelial cells.