FUNCTIONAL DOMAINS OF SOLUBLE GUANYLYL CYCLASE

Soluble guanylyl cyclase is a heterodimer consisting of an alpha and b eta subunit and stimulation occurs upon binding of NO to a prosthetic group. Little is known about the localization of catalytic and regulat ory domains within the subunits of soluble guanylyl cyclase. We used d eletion mutagenesis to identify the regions of alpha(1) and beta(1) su bunits that are responsible for cGMP production or NO-heme-mediated ac tivation. The amino terminus of the beta(1) subunit was necessary for NO stimulation since deletion of the 64 NH2-terminal amino acids resul ted in a mutant with intact basal activity but complete loss of NO act ivation. The amino terminus of the alpha(1) subunit also appeared to b e essential for NO sensitivity since deletion of 131 NH2-terminal amin o acids of alpha(1) led to markedly reduced NO activation. These resul ts suggest that NH2-terminal regions of alpha(1) and beta(1) are invol ved in NO-heme-mediated signal transduction. The NH2 terminally trunca ted beta(1) subunit exerted a dominant negative effect exclusively on the NO-stimulated activity of the wild type enzyme, further underlinin g that the regulatory domain is located within the NH2 terminus of the enzyme, Aside from the structural implications, the mutant represents a powerful tool to investigate nitric oxide-sensitive signaling pathw ays. Coexpression of the COOH-terminal halves of alpha(1) and beta(1) were sufficient for basal cGMP production while either of the halves e xpressed alone was inactive. Therefore the COOH-terminal regions appea r to contain sufficient information for dimerization and basal enzymat ic activity. Thus, we provide the first evidence that the regulatory a nd catalytic properties of soluble guanylyl cyclase can be attributed to different regions of the subunits and that the catalytic domain can be functionally expressed separately from the NH2-terminal regulatory domain. Taken together with findings on the membrane bound enzyme for m, guanylyl cyclases, appear to resemble fusion proteins where differe nt regulatory domains have been joined with a common cGMP-forming segm ent.