An important functional role of the N terminus domain of type VI adenylyl cyclase in Gαi-mediated inhibition

Abstract

We show herein that removal of the first 86 amino acids (aa) of the N terminus (designated N) of type VI adenylyl cyclase (ACVI) caused the resultant ACVI mutant (ACVI-ΔA87) to be more greatly inhibited by a Gαi-coupled receptor or activated Gαi protein. Moreover, in vitro binding of the full-length N and C1a domain (designated C1a), which interacts with Gαi, was detected. A truncated N terminus (aa 1-86) also interacted with C1a, suggesting that the C1a-interacting region is located within aa 1-86. Mutation analyses further revealed that N might interact with C1a in the region (aa 434-505) where Gαi is bound. Mutations of two residues (Leu-472 and Val-476) located in this N-binding region of C1a suppressed the interaction between recombinant N and C1a and markedly reduced Gαi-mediated inhibition of ACVI-ΔA87. Further biochemical analyses of the effect of internal mutations of Leu-472/Val-476 on Gαi-mediated inhibition of wild-type ACVI and ACVI-ΔA87 suggested that N modulates the Gαi-mediated inhibition of ACVI via binding to C1a when the level of Gαi is low (i.e. around the IC50 value) and that a more complicated interfering mode results when the level of Gαi is high (i.e. ∼10- to 20-fold of the IC50 value). Collectively, data presented herein suggest a novel function of the N terminus of ACVI in Gαi-mediated regulation.