Differential regulation of adenylyl cyclases by Gα(s)

Abstract

Regulation of adenylyl cyclases 1,2, and 6 Gα(s) was studied. All three mammalian adenylyl cyclases were expressed in insect (Sf9 of Hi-5) cells by baculovirus infection. Membranes containing the different adenylyl cyclases were stimulated by varying concentration of mutant (Q227L) activated Gα(s) expressed in reticulocyte lysates. Gα(s) stimulation of AC1 involved a single site and had an apparent K(act) of 0.9 nM. Gα(s) stimulation 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 GβΥ stimulation of Gα(s) 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 apparent K(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 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.