Calmodulin-binding sites on adenylyl cyclase type VIII

Ca2+ stimulation of adenylyl cyclase type VIII (ACVIII) occurs through loos ely bound calmodulin. However, where calmodulin binds in ACVIII and how the binding activates this cyclase have not yet been investigated. We have loc ated two putative calmodulin-binding sites in ACVIII, One site is located a t the N terminus as revealed by overlay assays; the other is located at the C terminus, as indicated by mutagenesis studies. Both of these calmodulin- binding sites were confirmed by synthetic peptide studies. The N-terminal s ite has the typical motif of a Ca2+-dependent calmodulin-binding domain, wh ich is defined by a characteristic pattern of hydrophobic amino acids, basi c and aromatic amino acids, and a tendency to form amphipathic cu-helix str uctures. Functional, mutagenesis studies suggest that this binding makes a minor contribution to the Ca2+ stimulation of ACVIII activity, although it might be involved in calmodulin trapping by ACVIII. The primary structure o f the C-terminal site resembles another calmodulin-binding motif, the so-ca lled IQ motif, which is commonly Ca2+-independent. Mutagenesis and function al assays indicate that this latter site is a calcium-dependent calmodulin- binding site, which is largely responsible for the Ca2+ stimulation of ACVI II, Removal of this latter calmodulin-binding region from ACVIII results in a hyperactivated enzyme state and a loss of Ca2+ sensitivity. Thus, Ca2+/c almodulin regulation of ACVIII may be through a disinhibitory mechanism, as is the case for a number of other targets of Ca2+/calmodulin.