| Summary: | 碩士 === 國立清華大學 === 分子與細胞生物研究所 === 93 === Protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR), is activated by thrombin. Recently, caveolin-1 has been found to be involved in the regulation of cell signaling of several GPCRs. However, it is still unknown whether caveolin-1 plays any role in PAR1 signaling. To determine whether caveolin-1 is involved in PAR1 signaling, I have examined the effect of caveolin-1 knockdown on adenylyl cyclase activity and extracellular signal-regulated kinase (ERK) activation induced by PAR1. Because the expression of caveolin-1 is highly regulated, the half-life of caveolin-1 was examined by the addition of cycloheximide to inhibit proteins synthesis. I found that the half-life of caveolin-1 is about 48 hours and caveolin-1 could be almost degraded in 72 hours. Since the cell confluency has been reported to influence the expression of caveolin-1, the expression of caveolin-1 at different cell confluency was also determined after applying small interfering RNA (siRNA) to knock down caveolin-1. It was found that the cell conflunecy should be below 75% as the optimal condition for caveolin-1 knockdown. By applying siRNA-mediated knockdown of caveolin-1, I found that after PAR1 activation, caveolin-1 is not involved in Gi-dependent inhibition of adenylyl cyclase and that loss of caveolin-1 attenuates the duration of ERK activation induced by PAR1. The results indicate that caveolin-1 extends the duration of ERK activation but not regulates Gi-dependent inhibition of adenylyl cyclase induced by PAR1 in HEK 293 cells.
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