Inactivation of soluble guanylyl cyclase in living cells proceeds without loss of haem and involves heterodimer dissociation as a common step.

Background and Purpose: Nitric oxide (NO) activates soluble guanylyl cyclase (sGC) for cGMP production, but in disease, sGC becomes insensitive towards NO activation. What changes occur to sGC during its inactivation in cells is not clear.
Experimental Approach: We utilized HEK293 cells expressing sGC proteins to study the changes that occur regarding its haem content, heterodimer status and sGCβ protein partners when the cells were given the oxidant ODQ or the NO donor NOC12 to inactivate sGC. Haem content of sGCβ was monitored in live cells through use of a fluorescent-labelled sGCβ construct, whereas sGC heterodimer status and protein interactions were studied by Western blot analysis. Experiments with purified proteins were also performed.
Key Results: ODQ- or NOC12-driven inactivation of sGC in HEK293 cells was associated with haem oxidation (by ODQ), S-nitrosation of the sGCβ subunit (by NOC12), sGC heterodimer breakup and association of the freed sGCβ subunit with cell chaperone Hsp90. These changes occurred without detectable loss of haem from the sGCβ reporter construct. Treating a purified ferrous haem-containing sGCβ with ODQ or NOC12 caused it to bind with Hsp90 without showing any haem loss.
Conclusion and Implications: Oxidative (ODQ) or nitrosative (NOC12) inactivation of cell sGC involves sGC heterodimer dissociation and rearrangement of the sGCβ protein partners without any haem loss from sGCβ. Clarifying what changes do and do not occur to sGC during its inactivation in cells may direct strategies to preserve or recover NO-dependent cGMP signalling in health and disease.
Linked Articles: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.
(© 2021 The British Pharmacological Society.)