Two members of a widely expressed subfamily of hormone-stimulated adenylyl cyclases.

Abstract

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 ...

Characterization of soluble forms of nonchimeric type V adenylyl cyclases

“…4.6.1.1) is the enzyme that converts ATP to cAMP. To date, nine distinct isoforms of mammalian AC (designated I to IX) and two splice variants of the type VIII enzyme have been cloned and characterized (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Because of the diversity in their regulation, the various AC isoforms may be broadly divided into five groups.…”

“…The third group contains type II and type VII isoforms, which are phosphorylated and activated by protein kinase C (11,16). Type V and VI AC, which are inhibited by calcium (6)(7)(8)(9), form the fourth group. Finally, the recently characterized ACIX (originally named type X) is regulated by calcineurin (12), and because of this unique feature, it is classified in the fifth group.…”

Determination and cellular localization of adenylyl cyclase isozymes expressed in embryonic chick heart

“…As mammalian types V and VI adenylyl cyelase are inhibited by submicromolar Ca 2+ concentrations [7][8][9][10], and appear to be the maj or adenylyl cyclase isozymes expressed in mammalian heart [7,9,11], we postulated the embryonic chick myocytes express avian type V and/or type VI isozymes [14]. We now report that: (i) RT-PCR detects type V and VI messages in mRNA isolated from preparations of isolated myocytes, and (ii) RNase protection assays suggest that type V message in 'myocyte RNA' is 4-5 times that of type VI message.…”

“…One was chick AC V (2 clones) which had 97.2% identity with all current known mammalian type V isoforrns at the amino acid level [9][10][11]19], and 86.1% identity with all current known mammalian type VI isoforms [7,8,11,20]. The other was chick AC VI (15 clones), which had 91.7% identity with all current known mammalian type VI isoforms at the amino acid level [7,8,11,20] and had 84.7% identity with all current known mammalian type V isoforms [9][10][11]19]. The identity between the PCR amplified sequences of chick AC V and AC VI was 86.1% at the amino acid level and 75.1% at the nucleotide acid level.…”

“…Because of their tissue distributions, types I and VIII AC have been proposed to play important roles in development of long term potentiation in the hippocampus [3,5,6]. In contrast to type 1 and VIII adenylyl cyclase, types V and VI AC are inhibited by submicromolar concentrations of Ca 2+ [7 10]; Northern blot and PCR analyses suggest that these isozymes are the primary isozymes expressed in the heart [7,9,11]. Because of their abilities to be inhibited by submicromolar concentrations of Ca 2÷ and their tissue distributions, it is proposed that these forms of adenylyl cyclase may provide a key control point in the heart for regulating contractility [12,13].…”