IDENTIFICATION AND CHARACTERIZATION OF A WIDELY EXPRESSED FORM OF ADENYLYL-CYCLASE

A novel mammalian adenylyl cyclase was identified by reverse transcrip tion-polymerase chain reaction amplification using degenerate primers based on a conserved region of previously described adenylyl cyclases (Premont, R. T. (1994) Methods Enzymol, 238, 116-127), The fall-length cDNA sequence obtained from mouse brain predicts a 1353-amino acid pr otein possessing a 12-membrane spans topology, and containing two regi ons of high similarity with the catalytic domains of adenylyl cyclases , Comparison of this novel adenylyl cyclase with the eight previously described mammalian enzymes indicates that this type 9 adenylyl cyclas e sequence is the most divergent, defining a sixth distinct subclass o f mammalian adenylyl cyclases, The AC9 gene has been localized to huma n chromosome band 16p13.3-13.2. The 8.5-kb mRNA encoding the type 9 ad enylyl cyclase is widely distributed, being readily detected in all ti ssues tested, and is found at very high levels in skeletal muscle and brain, AC9 mRNA is found throughout rat brain but is particularly abun dant in hippocampus, cerebellum, and neocortex, An antiserum directed against the carboxyl terminus of the type 9 adenylyl cyclase detects n ative and expressed recombinant AC9 protein in tissue and cell membran es. Levels of the AC9 protein are highest in mouse brain membranes. Ch aracterization of expressed recombinant AC9 reveals that the protein i s a functional adenylyl cyclase that is stimulated by Mg2+, forskolin, and mutationally activated G(s) alpha. AC9 activity is not affected b y Ca2+/calmodulim or by G protein beta gamma-subunits. Thus AC9 repres ents a functional G protein-regulated adenylyl cyclase found in brain and in most somatic tissues.