Functional Differences in the βγ Complexes of Transducin and the Inhibitory Guanine Nucleotide Regulatory Protein

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 (N_i) (i.e., a_i and βγ_N) and bovine retinal transducin (a_T and βγ_T) in reconstituted phospholipid vesicle systems. The addition of 07_N or βγ_T to lipid vesicles containing the pure stimulatory guanine nucleotide regulatory protein (N_s) 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-N_ior holotransducin. A variety of structure-function comparisons of the βγ_N and βγ_T complexes were performed in order to further probe the molecular mechanisms involved in the inhibitory pathway. Whereas the β subunits of βγ_N and βγ_T 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 γ_N and γ_T. Interestingly, functional differences are observed in the effectiveness of these two βγ complexes to inhibit adenylate cyclase activity. Specifically, while both βγ_N and βγ_T are capable of effecting significant levels of inhibition of the guanine nucleotide stimulated activities, the βγ_Ncomplex is consistently more potent than βγ_T 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 N_s 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.