Abstract
Protease-activated receptor 1 (PAR1) is a G protein-coupled receptor for thrombin. Src has been reported to be a signaling transducer of PAR1-mediated signal pathways. However, the regulation of Src kinase activity in these signal pathways is still unclear. The mechanisms of PAR1-mediated activation, de-activation, degradation, and re-expression of Src in HEK 293 cells were investigated. PAR1-mediated rapid activation of Src was increased with overexpression of □-arrestin1 or depletion of □-arrestin2. This activation of Src was decreased or eliminated with depletion of □-arrestin1 or overexpression of □-arrestin2. Of interest, stimulation of PAR1 induced lysosomal degradation of Src, and this degradation was blocked by depletion of □-arrestin2. Furthermore, long-term stimulation of PAR1 caused the re-expression of Src and PAR1 as well as the synthesis and secretion of vascular endothelial growth factor (VEGF). The re-expression of Src enhanced its kinase activity and was abolished by inhibiting its kinase activity. The synthesis of VEGF was also blocked by the inhibition of Src activity. Neutralization of secreted VEGF by anti-VEGF antibody reduced PAR1-mediated enhancement of cell viability. Collectively, these results indicate that Src is activated and de-activated by PAR1 through □-arrestin1- and □-arrestin2-dependent mechanisms, respectively. □-arrestin2 also appears to promote PAR1-induced lysosomal degradation of Src. Kinase activity of Src is involved in PAR1-mediated re-expression of Src and synthesis of VEGF. Synthesized VEGF is then secreted and participates in PAR1-mediated cell proliferation. Therefore, degradation and re-expression of Src provide a possible mechanism for terminating and re-activating PAR1 signaling. This work demonstrates that after stimulation of PAR1, Src was activated rapidly, then deactivated with degradation mechanism, and re-activated by re-expression of itself. The modulation of Src activity implies that PAR1 may manipulate Src activity to mediate thrombin-induced blood coagulation and angiogenesis.