Three discrete regions of mammalian adenylyl cyclase form a site for Gsalpha activation.

The interaction between the alpha subunit of G protein Gs (Gsalpha) and the two cytoplasmic domains of adenylyl cyclase (C1 and C2) is a key step in the stimulation of cAMP synthesis by hormones. Mutational analysis reveals that three discrete regions in the primary sequence of adenylyl cyclase affect the EC50 values for Gsalpha activation and thus are the affinity determinants of Gsalpha. Based on the three-dimensional structure of C2.forskolin dimer, these three regions (C2 alpha2, C2 alpha3/beta4, and C1 beta1) are close together and form a negatively charged and hydrophobic groove the width of an alpha helix that can accommodate the positively charged adenylyl cyclase binding region of Gsalpha. Two mutations in the C2 alpha3/beta4 region decrease the Vmax values of Gsalpha activation without an increase in the EC50 values. Since these three regions are distal to the catalytic site, the likely mechanism for Gsalpha activation is to modulate the structure of the active site by controlling the orientation of the C2 alpha2 and alpha3/beta4 structures.