Isolation and characterization of constitutively active mutants of mammalian adenylyl cyclase

A genetic screen in Saccharomyces cerevisiae identified mutations in mammalian adenylyl cyclase that activate the enzyme in the absence of G_sα. Thirteen of these mutant proteins were characterized biochemically in an assay system that depends on a mixture of the two cytosolic domains (C₁ and C₂) of mammalian adenylyl cyclases. Three mutations, I1010M, K1014N, and P1015Q located in the β4-β5 loop of the C₂ domain of type II adenylyl cyclase, increase enzymatic activity in the absence of activators. The K1014N mutation displays both increased maximal activity and apparent affinity for the C₁ domain of type V adenylyl cyclase in the absence of activators of the enzyme. The increased affinity of the mutant C₂ domain of adenylyl cyclase for the wild type C₁ domain was exploited to isolate a complex containing VC₁, IIC₂, and G_sα-guanosine 5'-3-0-(thio)triphosphate (GTPγS) in the absence of forskolin and a complex of VC₁, IIC₂, forskolin, and P-site inhibitor in the absence of G_sα-GTPγS. The isolation of these complexes should facilitate solution of crystal structures of low activity states of adenylyl cyclase and thus determination of the mechanism of activation of the enzyme by forskolin and G_sα.