DIFFERENTIAL REGULATION OF ADENYLYL CYCLASES BY G-ALPHA(S)

Regulation of adenylyl cyclases 1, 2, and 6 by G alpha(s) was studied, All three mammalian adenylyl cyclases were expressed in insect (Sf9 o r Hi-5) cells by baculovirus infection. Membranes containing the diffe rent adenylyl cyclases were stimulated by varying concentrations of mu tant (Q227L) activated G alpha(s) expressed in reticulocyte lysates, G alpha(s) stimulation of AC1 involved a single site and had an apparen t K-act of 0.9 nM. G alpha(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 affini ty site appears to be sufficient for G beta gamma stimulation of AC2. G alpha(s) stimulation of AC6 was also best explained by a two-site mo del 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 inter actions between the two sites best fit the AC6 data, With 100 mu M for skolin, G alpha(s) stimulation of all three adenylyl cyclases showed v ery similar profiles, G alpha(s) stimulation in the presence of forsko lin involved a single site with apparent K-act of 0.1-0.4 nM. These ob servations indicate a conserved mechanism by which forskolin regulates G alpha(s) coupling to the different adenylyl cyclases, However, ther e are fundamental differences in the mechanism of G alpha(s) stimulati on 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.