Identification of an intramolecular interaction between small regions in type V adenylyl cyclase that influences stimulation of enzyme activity by G(sα)

Using the full-length and two engineered soluble forms (C1-C2 and Cla-C2) of type V adenylyl cyclase (ACV), we have investigated the role of an intramolecular interaction in ACV that modulates the ability of the α subunit of the stimulatory GTP-binding protein of AC (G(sα)) to stimulate enzyme activity. Concentration–response curves with G(sα) suggested the presence of high and low affinity sites on ACV, which interact with the G protein. Activation of enzyme by G(sα) interaction at these two sites was most apparent in the C1a-C2 form of ACV, which lacks the C1b region (K(572)–F(683)). Yeast two-hybrid data demonstrated that the C1b region interacted with the C2 region and its 64-aa subdomain, C2I. Using peptides corresponding to the C2I region of ACV, we investigated the role of the C1b/C2I interaction on G(sα)-mediated stimulation of C1-C2 and full-length ACV. Our data demonstrate that a 10-aa peptide corresponding to L(1042)–T(1051) alters the profile of the activation curves of full-length and C1-C2 forms of ACV by different G(sα) concentrations to mimic the activation profile observed with C1a-C2 ACV. The various peptides used in our studies did not alter forskolin-mediated stimulation of full-length and C1-C2 forms of ACV. We conclude that the C1b region of ACV interacts with the 10-aa region (L(1042)–T(1051)) in the C2 domain of the enzyme to modulate G(sα)-elicited stimulation of activity.