CCTη, a novel soluble guanylyl cyclase-interacting protein

Abstract

Nitric oxide (NO) transduces most of its biological effects through activation of the heterodimeric enzyme, soluble guanylyl cyclase (sGC). Activation of sGC results in the production of cGMP from GTP. In this paper, we demonstrate a novel protein interaction between CCT (chaperonin containing t-complex polypeptide) subunit η and the α1β 1 isoform of sGC. CCTη was found to interact with the β1 subunit of sGC via a yeast-two-hybrid screen. This interaction was then confirmed in vitro with a co-immunoprecipitation from mouse brain. The interaction between, these two proteins was further supported by a co-localization of the proteins within rat brain. Using the yeast two-hybrid system, CCTη was found to bind to the N-terminal portion of sGC. In vitro assays with purified CCTη and Sf9 lysate expressing sGC resulted in a 30-50% inhibition, of diethylamine diazeniumdiolate-NO-stimulated sGC activity. The same assays were then performed using BAY41-2272, an NO-independent allosteric sGC activator, and CCTη had no effect on this activity. Furthermore, CCTη had no effect on basal or sodium nitroprusside-stimulated αβCys-105 sGC, a constitutively active mutant that only lacks the heme group. The N-terminal 94 amino acids of CCTη seem to be critical for the mediation of this inhibition. Lastly, a 45% inhibition of sGC activity by CCTη was seen in vivo in BE2 cells stably transfected with CCTη and treated with sodium nitroprusside. These data suggest that CCTη binds to sGC and, in cooperation with some other factor, inhibits its activity by modifying the binding of NO to the heme group or the subsequent cenformational changes.