Conversion of forskolin-insensitive to forskolin-sensitive (mouse-type IX) adenylyl cyclase

Forskolin potently activates all cloned mammalian adenylyl cyclases except type IX by interacting with two homologous cytoplasmic domains (C-1 and C-2) that form the catalytic core. A mutational analysis of the IIC2 protein (C-2 domain from type II adenylyl cyclase) and forskolin analogs suggests that Ser942 interacts with the 7-acetyl group of forskolin. The C-1/C-2 complex has only one forskolin, one ATP, and one binding site for the alpha subunit of the G protein that stimulates adenylyl cyclase (Gs(alpha)) and its structure may be modeled using the three-dimensional structure of (IIC2/forskolin)(2). The Ser942 mutation defines which forskolin in the (IIC2/forskolin)(2) structure exists in C-1/C-2 complex. Thus, the forskolin-binding site is close to the G(s alpha)-binding site but distal (15-20 Angstrom) from the catalytic site. Mutation from Leu912 of IIC2 protein to tyrosine or alanine severely reduces G(s alpha) activation and completely prevents forskolin activation. The corresponding residue of Leu912 is Tyr1082 at type IX isoform of adenylyl cyclase. Similar to recombinant type IX enzyme, soluble adenylyl cyclase derived from mouse-type IX adenylyl cyclase is sensitive to G(s alpha) activation but not to forskolin. Changing Tyr1082 to leucine makes soluble type IX adenylyl cyclase forskolin-responsive.