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Ubiquitination of protease‐activated receptor‐1 reveals a new mode of signal regulation

Protease‐activated receptor‐1 (PAR1) is irreversibly proteolytically activated by thrombin. The regulatory mechanisms that control PAR1 signaling are critical for proper cellular responses. Here, we show that PAR1 is ubiquitinated after agonist stimulation. However, ubiquitin is not essential for PAR1 internalization and lysosomal degradation. This study seeks to identify the PAR1 ubiquitin modifying enzymes and the function of receptor ubiquitination. A candidate siRNA screen revealed a NEDD4 family E3 ligase to be a critical mediator of agonist‐stimulated PAR1 ubiquitination. NEDD4 formed a stable interaction with PAR1 after agonist stimulation. To determine if PAR1 signaling regulated NEDD4 recruitment we examined the role Ca2+. Treatment with the Ca2+ chelator BAPTA blocks PAR1‐ stimulated Ca2+ mobilization, NEDD4 recruitment and receptor ubiquitination. Furthermore, agonist induced PAR1 ubiquitination mediates the recruitment and activation of MAPK signaling. This study is the first example of GPCR stimulation, in this in case PAR1, regulating the activation and recruitment of NEDD4 E3 ligase to mediate itâ€™s own modification, which is critical for activation of MAPK signaling. Supported by NIH R01 HL073328