Modeling of Gαs and Gαi regulation of human type V and VI adenylyl cyclase

Abstract

We examined the kinetics of Gαs and Gαi regulation of human type V and type VI adenylyl cyclase (AC V and AC VI) in order to better model interactions between AC and its regulators. Activation of AC VI by Gαs displayed classical Michaelis-Menten kinetics, whereas AC V activation by Gαs was cooperative with a Hill coefficient of 1.4. The basal activity of human AC V, but not that of AC VI, was inhibited by Gαi. Both enzymes showed greater inhibition by Gαi at low Gαs concentrations; however, human AC V was activated by Gαi at high Gαs concentrations. Neither regulator had an effect on the Km for Mg-ATP. Mutations made within the Gαs binding pocket of AC V (N1090D) and VT (F1078S) displayed 6- and 14-fold greater EC50 values for Gαs activation but had no effect on Gαi inhibition of basal activity or Km for Mg-ATP. Gαs- stimulated AC VI-F1078S was not significantly inhibited by Gαi, despite normal inhibition by Gαi upon forskolin stimulation. Mechanistic models for Gαs and Gαi regulation of AC V and VI were derived to describe these results. Our models are consistent with previous studies, predicting a decrease in affinity of Gαi in the presence of Gαs. For AC VI, Gαs is required for inhibition but not binding by Gαi. For AC V, binding of two molecules of Gαs and Gαi to an AC dimer are required to fully describe the data. These models highlight the differences between AC V and VI and the complex interactions with two important regulators.