CONSTRUCTION OF A SOLUBLE ADENYLYL-CYCLASE ACTIVATED BY G(S)ALPHA ANDFORSKOLIN

A soluble adenylyl cyclase was constructed by linkage of portions of t he cytosolic domains of the mammalian type I and type II enzymes. The soluble enzyme was stimulated by both forskolin and the alpha subunit of the heterotrimeric guanine nucleotide-binding protein (G protein) G (s) (G(s) alpha). Expression of the construct complemented the catabol ic defect in a strain of Escherichia coli that is deficient in adenyly l cyclase activity. The active, approximately 60-kilodalton enzyme acc umulated in the cytoplasmic fraction of E. coli to yield activities in excess of 1 nanomole per minute per milligram of protein. The two set s of transmembrane helices of mammalian adenylyl cyclases are thus not necessary for the catalytic or the most characteristic regulatory act ivities of the enzyme. This system may be useful for both genetic and biochemical analysis of G protein-regulated adenylyl cyclases.