ABSTRACTcDNA encoding a hormone-and guanine nu- Hormone-stimulated adenylyl cyclase [ATP pyrophosphatelyase (cyclizing), EC 4.6.1.1] is one of the best-studied guanine nucleotide-binding protein (G protein)-regulated signaling pathways. Detailed molecular information on the multiplicity of receptors and G proteins has been available for some time, but much less has been known about the effector until quite recently. Major progress was achieved with the development of affinity purification of adenylyl cyclase on forskolin-agarose (1) and the cloning of a calmodulinstimulated bovine brain adenylyl cyclase (2). This work has been followed by the recent cloning and characterization of three additional mammalian adenylyl cyclases (3-5). Hormone-regulated adenylyl cyclases have been functionally divided into two categories, calmodulin-sensitive and calmodulin-insensitive (6). Calmodulin-sensitive adenylyl cyclase activity is found predominantly in the brain, whereas liver and most other peripheral tissues contain only calmodulin-insensitive activity (6). We have had a long-standing interest in the glucagon-stimulated liver adenylyl cyclase system, and we set out to isolate the cDNA encoding the liver adenylyl cyclase. In this article we report on the isolation of cDNA clones encoding two distinct adenylyl cyclase enzymes that are highly related and widely expressed.* MATERIALS AND METHODSMaterials. Size-selected bovine cortex cDNA libraries in AZAP 11 (7), rat liver cDNA libraries in AZAP II (Stratagene) and in Agtll (Clonetech), and rat kidney libraries in AZAP II (Stratagene) and AgtlO (8) were used.Cloning. A 900-base-pair (bp) fragment encoding the final 300 amino acids of type 1 adenylyl cyclase (2) was amplified by PCR using specific primers from the published sequence, from 107 phage (9) of a >4.4-kilobase (kb) size-selected bovine cerebral cortex cDNA library in AZAP 11 (7). The product was subcloned in pBlueScript, sequenced, and used to screen a rat liver cDNA library in AZAP II by using standard methods (10). Positive inserts were recovered from the AZAP II vector by phagemid excision (11), and DNA sequences from double-stranded inserts in pBlueScript were determined by dideoxynucleotide chain-termination technique with specific oligonucleotide primers and T7 DNA polymerase (10). Ambiguous sequences were resequenced with 7-deaza-dGTP or dITP.Extreme 5' end clones were obtained by PCR screening of several rat liver and kidney cDNA libraries in various A phage vectors (9, 12). For each screening step, two nested antisense primers were prepared to known adenylyl cyclase sequences and used in sequential PCR reactions. In the first reactions, an aliquot of a cDNA library (107 phage) was amplified by using an antisense clone primer (21 bp) and one of a pair of primers that flank the virus multiple cloning site (i.e., T3 and T7 17-mer sequencing primers for AZAP II). Amplifications were performed for 35 cycles at 95TC for 1 min, 500C for 1 min, and 720C for 3 min. One microliter of the first reactions was used as ...

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