Abstract

Forskolin potently activates all cloned mammalian adenylyl cyclases except type IX by interacting with two homologous cytoplasmic domains (C₁ and C₂) that form the catalytic core. A mutational analysis of the IIC₂ protein (C₂domain from type II adenylyl cyclase) and forskolin analogs suggests that Ser942 interacts with the 7-acetyl group of forskolin. The C₁/C₂ complex has only one forskolin, one ATP, and one binding site for the α subunit of the G protein that stimulates adenylyl cyclase (G_sα) 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)₂structure exists in C₁/C₂ complex. Thus, the forskolin-binding site is close to the G_sα-binding site but distal (15–20Å) from the catalytic site. Mutation from Leu912 of IIC₂ protein to tyrosine or alanine severely reduces G_sα 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_sα activation but not to forskolin. Changing Tyr1082 to leucine makes soluble type IX adenylyl cyclase forskolin-responsive.