Characterization of two unusual guanylyl cyclases from Dictyostelium

Guanylyl cyclase A (GCA) and soluble guanylyl cyclase (sGC) encode GCs in Dictyostelium and have a topology similar to 12-transmembrane and soluble adenylyl cyclase, respectively. We demonstrate that all detectable GC activity is lost in a cell line in which both genes have been inactivated. Cell lines with one gene inactivated were used to characterize the other guanylyl cyclase (i.e. GCA in sgc(-) null cells and sGC in gea(-)null cells). Despite the different topologies, the enzymes have many properties in common. In vivo, extracellular cAMP activates both enzymes via a G-protein-coupled receptor. In vitro, both enzymes are activated by GTPgammaS (K-a = 11 and 8 muM for GCA and sGC, respectively). The addition of GTPgammaS leads to a 1.5-fold increase of V-max and a 3.5-fold increase of the affinity for GTP. Ca2+ inhibits both GCA and sGC with K-i of about 50 and 200 nM, respectively. Other biochemical properties are very different; GCA is expressed mainly during growth and multicellular development, whereas sGC is expressed mainly during cell aggregation. Folic acid and cAMP activate GCA maximally about 2.5-fold, whereas sGC is activated about 8-fold. Osmotic stress strongly stimulates sGC but has no effect on GCA activity. Finally, GCA is exclusively membrane-bound and is active mainly with Mg2+, whereas sGC is predominantly soluble and more active with Mn2+.