Homodimerization of soluble guanylyl cyclase subunits - Dimerization analysis using a glutathione S-transferase affinity tag

Soluble guanylyl cyclase (sGC) is an alpha/beta-heterodimeric hemoprotein t hat, upon interaction with the intercellular messenger molecule NO, generat es cGMP. Although the related family of particulate guanylyl cyclases (pGCs ) forms active homodimeric complexes, it is not known whether homodimerizat ion of sGC subunits occurs. We report here the expression in Sf9 cells of g lutathione S-transferase-tagged recombinant human sGC alpha 1 and pi subuni ts, applying a novel and rapid purification method based on GSH-Sepharose a ffinity chromatography, Surprisingly, in intact Sf9 cells, both homodimeric GST alpha/alpha: and GST beta/beta complexes were formed that were catalyt ically inactive, Upon coexpression of the respective complementary subunits , GST alpha/beta or GST beta/alpha heterodimers were preferentially formed, whereas homodimers were still detectable, When subunits were mixed after e xpression, e,g, GST beta and beta or;GST alpha and beta, no dimerization wa s observed, In conclusion, our data suggest the previously unrecognized pos sibility of a physiological equilibrium between homo- and heterodimeric sGC complexes.