CONVERSION OF FORSKOLIN-INSENSITIVE TO FORSKOLIN-SENSITIVE (MOUSE-TYPE-IX) ADENYLYL-CYCLASE

Forskolin potently activates all cloned mammalian adenylyl cyclases ex cept 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 for skolin. The C-1/C-2 complex has only one forskolin, one ATP, and one b inding site for the alpha subunit of the G protein that stimulates ade nylyl cyclase (Gs(alpha)) and its structure may be modeled using the t hree-dimensional structure of (IIC2/forskolin)(2). The Ser942 mutation defines which forskolin in the (IIC2/forskolin)(2) structure exists i n 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 s ite. Mutation from Leu912 of IIC2 protein to tyrosine or alanine sever ely reduces G(s alpha) activation and completely prevents forskolin ac tivation. The corresponding residue of Leu912 is Tyr1082 at type IX is oform of adenylyl cyclase. Similar to recombinant type IX enzyme, solu ble adenylyl cyclase derived from mouse-type IX adenylyl cyclase is se nsitive to G(s alpha) activation but not to forskolin. Changing Tyr108 2 to leucine makes soluble type IX adenylyl cyclase forskolin-responsi ve.