Abstract 970: A Novel Mechanism: Activation of (Pro)renin receptor requires its ectodomain shedding.
Background: Recent discovery, (pro)renin receptor (PRR) plays a pivotal effect of activation of renin angiotensin system through nonproteolytic activation of prorenin. It still remains, however, unclear about detail mechanism of activation of prorenin. To uncover this aspect, we performed following experiments.
Methods and results: Using COS-7 cells transfected with enhanced green fluorescent protein (EGFP)-tagged PRR, western blot analysis of eluted sample from these cells showed dual bands detected by anti-EGFP antibody at 74kd and 36kd respectively. To avoid toxicity characteristics of EGFP-tag to mammalian cells, myc6-tagged PRR was tarnsfected in COS-7 cells. Dual bands were detected by anti-myc antibody at 52kd and 28kd respectively in western blot analysis. After transfection with myc6-tagged trancated PRR (Delta 208 –307 or 108 –307) to COS-7 cells, 44kd and 28kd, or 38kd and 30kd bands were detected in western blot analysis (Figure⇓). These results suggested that 74kd band may indicate full-length PRR, and 32kd band may indicate cytoplasmic PRR shed at extra cellular region, since EGFP- or myc6 tag was fused into PRR at carboxyl terminus. To elucidate mechanism of ectodomain shedding of PRR, protease inhibitors such as DAPT, GM6001, furin I or furin II were pretreated in COS-7 cells transfected with myc6-tagged PRR. However, no protease inhibitor inhibited shedding of PRR. The medium of COS-7 cells transfected with myc-tagged PRR activated prorenin.
Conclusion: we found a novel mechanism of PRR for activation of prorenin. Ectodomain shedding of PRR may be required for activation of prorenin. This shedding may be not regulated but activated constitutively.