An important functional role of the N terminus domain of type VI adenylyl cyclase in Galphai-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-DeltaA87) to be more greatly inhibited by a Galpha(i)-coupled receptor or activated Galpha(i) protein. Moreover, in vitro binding of the full-length N and C1a domain (designated C1a), which interacts with Galpha(i), 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 Galpha(i) 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 Galpha(i)-mediated inhibition of ACVI-DeltaA87. Further biochemical analyses of the effect of internal mutations of Leu-472/Val-476 on Galpha(i)-mediated inhibition of wild-type ACVI and ACVI-DeltaA87 suggested that N modulates the Galpha(i)-mediated inhibition of ACVI via binding to C1a when the level of Galpha(i) is low (i.e. around the IC(50) value) and that a more complicated interfering mode results when the level of Galpha(i) is high (i.e. approximately 10- to 20-fold of the IC(50) value). Collectively, data presented herein suggest a novel function of the N terminus of ACVI in Galpha(i)-mediated regulation.