Identification of functional domains of adenylyl cyclase using in vivo chimeras.

Adenylyl cyclase, the effector molecule of the cAMP signaling pathway, is composed of a family of isoforms that differ in their modes of regulation. Many of these modulatory interactions are dependent upon well characterized molecules from various second messenger pathways; however, very little is known about their mechanisms or sites of action on adenylyl cyclase. Chimeras were produced by a novel in vivo mechanism between two differentially modulated adenylyl cyclases to identify their regulatory domains. The basal activity of the type I adenylyl cyclase (AC1) is activated by calcium/calmodulin, inhibited by G protein beta gamma subunits, and insensitive to protein kinase C regulation. In contrast, type II adenylyl cyclase (AC2) is insensitive to calcium/calmodulin regulation and is activated by G protein beta gamma subunits as well as by activated protein kinase C. Expression and biochemical characterization of chimeras between AC1 and AC2 identified a single specific domain of AC1 responsible for calmodulin binding and a small, well defined region near the C terminus of AC2 required for protein kinase C activation.