BAY58-2667 activates different sGC species by distinct mechanisms that indicate its principal target in cells is the heme-free sGCβ-Hsp90 complex .

NO-unresponsive forms of soluble guanylyl cyclase (sGC) exist naturally and in disease can disable NO-sGC-cGMP signaling. Agonists like BAY58-2667 (BAY58) target these sGC forms but their mechanisms of action in living cells are unclear. We studied RFL-6 cells and human airway smooth muscle cells that naturally express sGC and HEK293 cells that we transfected to express sGC and variants. Cells were cultured to build up different forms of sGC, and we used fluorescence and FRET-based measures to monitor BAY58-driven cGMP production and any protein partner exchange or heme loss events that may occur for each sGC species. We found: (i) BAY58 activated cGMP production by the apo-sGCβ-Hsp90 species after a 5-8 min delay that was associated with apo-sGCβ exchanging its Hsp90 partner with an sGCα subunit. (ii) In cells containing an artificially constructed heme-free sGC heterodimer, BAY58 initiated an immediate and 3 times faster cGMP production. However, this behavior was not observed in cells expressing native sGC under any condition. (iii) BAY58 activated cGMP production by ferric heme sGC only after a 30 min delay, coincident with it initiating ferric heme loss from sGCβ. We conclude that the kinetics favor BAY58 activation of the apo-sGCβ-Hsp90 species over the ferric heme sGC species in living cells. The protein partner exchange events driven by BAY58 account for the initial delay in cGMP production and also limit the speed of subsequent cGMP production in the cells. Our findings clarify how agonists like BAY58 may activate sGC in health and disease. Significance Statement A class of agonists can activate cGMP synthesis by forms of sGC that do not respond to NO and accumulate in disease, but the mechanisms of action are unclear. This study clarifies what forms of sGC exist in living cells, which of these can be activated by the agonists, and the mechanisms and kinetics by which each form is activated. This information may help to hasten deployment of these agonists for pharmaceutical intervention and clinical therapy.