Modeling of Gαs and Gαi Regulation of Human Type V and VI Adenylyl Cyclase

Abolanle N Lukan,Misty Chen-Goodspeed,Carmen W Dessauer

doi:10.1074/jbc.m409172200

Abolanle N Lukan, Misty Chen-Goodspeed + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m409172200

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Abstract

We examined the kinetics of Galpha(s) and Galpha(i) 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 Galpha(s) displayed classical Michaelis-Menten kinetics, whereas AC V activation by Galpha(s) 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 Galpha(i). Both enzymes showed greater inhibition by Galpha(i) at low Galpha(s) concentrations; however, human AC V was activated by Galpha(i) at high Galpha(s) concentrations. Neither regulator had an effect on the K(m) for Mg-ATP. Mutations made within the Galpha(s) binding pocket of AC V (N1090D) and VI (F1078S) displayed 6- and 14-fold greater EC(50) values for Galpha(s) activation but had no effect on Galpha(i) inhibition of basal activity or K(m) for Mg-ATP. Galpha(s)-stimulated AC VI-F1078S was not significantly inhibited by Galpha(i), despite normal inhibition by Galpha(i) upon forskolin stimulation. Mechanistic models for Galpha(s) and Galpha(i) 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 Galpha(i) in the presence of Galpha(s). For AC VI, Galpha(s) is required for inhibition but not binding by Galpha(i). For AC V, binding of two molecules of Galpha(s) and Galpha(i) 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.

Highlights

From the Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, Texas 77030
In an attempt to fully understand the regulation of full-length human adenylyl cyclase (AC) V and VI, we have modeled the kinetics of G␣s/G␣i regulation
The difference in the -fold activation lies in the low basal activity of AC VI [28]

Summary

Introduction

We examined the kinetics of G␣s and G␣i regulation of human type V and type VI adenylyl cyclase Fold greater EC50 values for G␣s activation but had no effect on G␣i inhibition of basal activity or Km for MgATP. For AC VI, G␣s is required for inhibition but not binding by G␣i. For AC V, binding of two molecules of G␣s and G␣i 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

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Conclusion