We have examined the mechanism of inhibition of adenylate cyclase using the purified a and βγ subunits of bovine brain inhibitory guanine nucleotide regulatory protein (Ni) (i.e., ai and βγN) and bovine retinal transducin (aT and βγT) in reconstituted phospholipid vesicle systems. The addition of 07N or βγT to lipid vesicles containing the pure stimulatory guanine nucleotide regulatory protein (Ns) from human erythrocytes as well as a resolved preparation of the catalytic moiety (C) of bovine caudate adenylate cyclase results in significant inhibition of guanine nucleotide stimulated cyclase activity (80-90%). The inhibition by these βγ subunit complexes appears to fully account for the inhibitory effects observed with holo-Nior holotransducin. A variety of structure-function comparisons of the βγN and βγT complexes were performed in order to further probe the molecular mechanisms involved in the inhibitory pathway. Whereas the β subunits of βγN and βγT appear to be very similar, if not identical, on the basis of comparisons of their gel electrophoretic mobility and immunological cross-reactivity, clear differences exist in the apparent structures of γN and γT. Interestingly, functional differences are observed in the effectiveness of these two βγ complexes to inhibit adenylate cyclase activity. Specifically, while both βγN and βγT are capable of effecting significant levels of inhibition of the guanine nucleotide stimulated activities, the βγNcomplex is consistently more potent than βγT in inhibiting these activities. The difference in the effectiveness of these two complexes is most apparent when the inhibition of isoproterenol-stimulated adenylate cyclase is measured in vesicles containing the pure β-adrenergic receptor together with Ns and C. The functional differences between the βγ subunits of brain Ni and retinal transducin may reflect the clear structural differences in their 7 subunits, thus suggesting a possible role for 7 in the inhibition of adenylate cyclase by Ni. Overall, these results indicate that the βγ complexes, as well as the a subunits, may have an important role in imparting the functional specificity necessary for nucleotide regulatory protein mediated signal transduction.
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