Regulation of Adenylyl Cyclases by a Region Outside the Minimally Functional Cytoplasmic Domains*

The highly conserved topological structure of G pro-tein-activated adenylyl cyclases seems unnecessary be-cause the soluble cytoplasmic domains retain regula-tory and catalytic properties. Yet, we previously isolated a constitutively active mutant of the Dictyoste-lium discoideum adenylyl cyclase harboring a single point mutation in the region linking the cytoplasmic and membrane domains (Leu-394). We show here that multiple amino acid substitutions at Leu-394 also dis-play constitutive activity. The constitutive activity of these mutants is not dependent on G proteins or cytoso-lic regulators, although some of the mutants can be ac-tivated to higher levels than wild type. Combining a constitutive mutation such as L394T with K482N, a point mutation that renders the enzyme insensitive to regula