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.
Levin
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Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.
Reed
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RR
D000262:Adenylyl Cyclases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002460:Cell Line; D016384:Consensus Sequence; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017354:Point Mutation; D011493:Protein Kinase C / Q000378:metabolism; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D015183:Restriction Mapping; D013329:Structure-Activity Relationship; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D002147:Calmodulin; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D011493:Protein Kinase C; D000262:Adenylyl Cyclases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium