Abstract 492: CHIP (Carboxyl terminus of Hsp70-Interacting Protein) Promotes Basal and Geldanamycin-Induced Degradation of Soluble Guanylyl Cyclase-α1 Subunit.
The NO receptor, soluble guanylyl cyclase (sGC), exists in multimeric complexes that include NO synthases and heat shock protein 90 (hsp90), in both endothelial and smooth muscle cells. By acting as a E3 ligase, CHIP promotes hsp client protein ubiquitylation and proteasomal degradation. We tested the hypothesis that CHIP similarly regulates sGC protein turnover. Confocal imaging and co-immunoprecipitation studies demonstrated that sGCα1 is associated with CHIP. Exogenously over-expressed sGCα1 in COS7 cells was ubiquitylated and degraded by co-transfected CHIP. The degradation was partially blocked by MG132, a selective proteasome inhibitor, indicating that the proteasome pathway is involved in the degradation of the CHIP-ubiquitylated sGCα1. When the tetratricopeptide repeat (TPR) domain of CHIP (necessary for hsp binding) was mutated, the mutant CHIP lost its down-regulating effect on sGCα1, suggesting that hsp90/CHIP interaction is necessary for ubiquitylation of sGCα1. Conversely, the CHIP U-box mutant (H260Q) exhibited only partial loss of its degrading effect on sGCα1, compared to the wild-type CHIP. Exogenous expressed sGCα1 was also degraded by short-term (0.5–1h) exposure to the hsp90 binding inhibitor, geldanamycin (GA, 1μg/ml) in a time dependent manner; this effect was enhanced by co-expressed CHIP, suggesting that GA and CHIP act synergistically on sGCα1 down-regulation. These results suggest that sGCα1 is a target of CHIP ubiquitin ligase activity and present a novel mechanism for sGC regulation. (Supported by HL70412).