Differential Regulation of Adenylyl Cyclases by Gα_s*

Regulation of adenylyl cyclases 1, 2, and 6 by Gα_s was studied. All three mammalian adenylyl cyclases were expressed in insect (Sf9 or Hi-5) cells by baculovirus infection. Membranes containing the different adenylyl cyclases were stimulated by varying concentrations of mutant (Q227L) activated Gα_s expressed in reticulocyte lysates. Gα_sstimulation of AC1 involved a single site and had an apparentK _act of 0.9 nm. Gα_sstimulation of AC2 was best explained by a non-interactive two site model with a “high affinity” site at 0.9 nm and a “low affinity” site at 15 nm. Occupancy of the high affinity site appears to be sufficient for Gβγ stimulation of AC2. Gα_s stimulation of AC6 was also best explained by a two-site model with a high affinity site at 0.6–0.8 nm and a low affinity site at 8–22 nm; however, in contrast to AC2, only a model that assumed interactions between the two sites best fit the AC6 data. With 100 μm forskolin, Gα_s stimulation of all three adenylyl cyclases showed very similar profiles. Gα_s stimulation in the presence of forskolin involved a single site with apparentK _act of 0.1–0.4 nm. These observations indicate a conserved mechanism by which forskolin regulates Gα_s coupling to the different adenylyl cyclases. However, there are fundamental differences in the mechanism of Gα_s stimulation of the different adenylyl cyclases with AC2 and AC6 having multiple interconvertible sites. These mechanistic differences may provide an explanation for the varied responses by different cells and tissues to hormones that elevate cAMP levels.