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1 Vascular Biology Center, Medical College of Georgia, Augusta, Georgia, United States
2 Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia, United States
3 Vascular Biology Center, Medical College of Georgia, Augusta, Georgia, United States; Department of Pediatrics, Medical College of Georgia, Augusta, Georgia, United States
4 Vascular Biology Center, Medical College of Georgia, Augusta, Georgia, United States; Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia, United States
5 Carolina Cardiovascular Biology Center, University of North Carolina, Chapel Hill, North Carolina, United States
6 Dept of Pharmacy, University of Patras, Patras, Greece
* To whom correspondence should be addressed. E-mail: jcatrava{at}mcg.edu.
The nitric oxide receptor, soluble guanylyl cyclase (sGC) exists in multimeric protein complexes including hsp90 and eNOS. Inhibition of hsp90 by geldanamycin causes proteasomal degradation of sGC protein. In this study, we have investigated whether CHIP, a co-chaperone molecule involved in protein folding, but also a chaperone-dependent ubiquitin E3 ligase, could play a role in the process of degradation of sGC. Transient overexpression of CHIP in Cos-7 cells degraded heterologous sGC in a concentration-related manner; this down-regulation of sGC was abrogated by the proteasome inhibitor, MG 132. Transfection of TPR and U-box domain CHIP mutants attenuated sGC degradation suggesting that both domains are indispensable for CHIP function. Results from immunoprecipitation and indirect immunofluorescent microscopy experiments demonstrated that CHIP is associated with sGC, hsp90 and hsp70 in Cos-7 cells. Furthermore, CHIP increased the association of hsp70 with sGC. In in vitro ubiquitination assays using purified proteins and ubiquitin enzymes, E3 ligase CHIP directly ubiquitinated sGC; this ubiquitination was potentiated by geldanamycin in Cos-7 cells, followed by proteasomal degradation. In rat aortic smooth cells, endogenous sGC was also degraded by adenovirus-infected wild type CHIP, but not by the chaperone interaction deficient K30A CHIP, whereas CHIP, but not K30A attenuated sGC expression in, and NO donor-induced relaxation of rat aortic rings, suggesting that CHIP plays a regulatory role under physiological conditions. This study reveals a new mechanism for the regulation of sGC, an important mediator of cellular and vascular function.
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