REGULATION OF FORSKOLIN INTERACTIONS WITH TYPE-I, TYPE-II, TYPE-V, AND TYPE-VI ADENYLYL CYCLASES BY G(S-ALPHA)

Several forms of adenylyl cyclase (types I, II, V, and VI) have been e xpressed using the recombinant baculovirus expression system in Sf9 ce lls. The activation of type I adenylyl cyclase by forskolin and G(s al pha) was not greater than additive. In contrast, there was synergistic activation of type II, V, and VI adenylyl cyclases by G(s alpha) and forskolin. G(s alpha) potentiated the effect of forskolin on type II a denylyl cyclase to the greatest extent. Type I and II adenylyl cyclase s were photolabeled specifically by an iodinated photoaffinity derivat ive of forskolin ([I-125]-6-AIPP-Fsk). Type I adenylyl cylcase was pho tolabeled efficiently in the absence of G(s alpha), and the addition o f G(s alpha) only slightly increased the labeling efficiency. In contr ast, type II adenylyl cyclase was not photolabeled efficiently in the absence of G(s alpha), and the addition of G(s alpha) greatly enhanced the labeling efficiency. Potolabeling of type V and VI adenylyl cycla ses was detected only in the presence of G(s alpha). Neither carcium/c almodulin nor G protein beta gamma subunits modulated the photolabelin g of type I or II adenylyl cyclases. Another iodinated derivative of f orskolin, [I-125]-6-IHPP-Fsk, bound to Sf9 cell membranes expressing t ype I adenylyl cyclase with high affinity in a filtration binding assa y, and the specific binding was not enhanced by the addition of G(s al pha). In contrast, specific binding of [I-125] -6-IHPP-Fsk to membrane s expressing type II adenylyl cyclase was detected only in the presenc e of G(s alpha). [I-125]-6- IHPP-Fsk bound to membranes expressing typ e I adenylyl cyclase with a K-d of 8 nM and a B-max of 6.4 pmol/mg pro tein. The K-d for binding of [(125)]-6-IHPP-Fsk to membranes expressin g type II adenylyl cyclase in the presence of G(s alpha) was 134 nM, a nd the B-max was 11.2 pmol/mg protein. Deoxy and desacetyl derivatives of forskolin displaced the binding of [I-125]-6-IHPP-Fsk to membranes expressing type I and II adenylyl cyclases with a similar rank order of potency. However, the affinity of these derivatives for the type I enzyme was between 6- and 20-fold higher than that for the type II enz yme.