The recent determination of the crystal structure of adenylyl cyclase has elucidated many structural features that determine the regulatory properties of the enzyme. In addition, the characterization of adenylyl cyclase by mutagenic techniques and the identification of the binding site for P-site inhibitors have led to modeling studies that describe the ATP-binding site. Despite these advances, the catalytic mechanism of adenylyl cyclase remains uncertain, especially with respect to the role that magnesium ions may play in this process. We have identified four mutant mammalian adenylyl cyclases defective in their metal dependence, allowing us to further characterize the function of metal ions in the catalytic mechanism of this enzyme. The wild-type adenylyl cyclase shows a biphasic Mg 2؉ dose-response curve in which the high-affinity component displays cooperativity (Hill coefficient of 1.4). Two mutations (C441R and Y442H) reduce the affinity of the adenylyl cyclase for Mg 2؉ dramatically without affecting the binding of MgATP, suggesting that there is a metal requirement in addition to the ATP-bound Mg 2؉ . The results of this study thus demonstrate multiple metal requirements of adenylyl cyclase and support the existence of a Mg 2؉ ion essential for catalysis and distinct from the ATP-bound ion. We propose that adenylyl cyclase employs a catalytic mechanism analogous to that of DNA polymerase, in which two key magnesium ions facilitate the nucleophilic attack of the 3-hydroxyl group and the subsequent elimination of pyrophosphate.Intracellular levels of cAMP are primarily regulated at the level of its synthesis by adenylyl cyclase. cAMP, in turn, regulates a wide variety of cellular processes such as protein phosphorylation levels, gene expression, and ion channel conductance. Currently, nine isoforms of mammalian adenylyl cyclase have been identified by molecular cloning techniques (reviewed in Refs. 1-3), and they display a common deduced topology composed of a short cytoplasmic amino terminus followed by a region of six transmembrane domains (M1) and a large cytoplasmic loop (C1). This motif is then repeated with a second transmembrane region (M2) and a large cytoplasmic carboxyl terminus (C2). Sequence comparison has revealed that each cytoplasmic loop contains subdomains (denoted C1a and C2a) that are highly conserved among all adenylyl cyclase isoforms and that also display great homology to each other. The C1a and C2a domains can be expressed separately, and catalytic activity is reconstituted when they are mixed in vitro, although each domain by itself shows no activity (4, 5).Many of the structural motifs of adenylyl cyclase responsible for catalytic activity and for the recognition of regulatory molecules are presently being uncovered. A region in the C2 domain of type II adenylyl cyclase (residues 956 -982), for example, has been implicated in the binding of the G protein 1 ␤␥ subunits (6), whereas another region in the C1 domain of type I adenylyl cyclase (residues 495-522) appears to be involved ...

We describe the development of a genetic system allowing for the isolation of mutant mammalian adenylyl cyclases defective in their responses to G protein subunits, thus allowing for the identification of structural elements within the cyclase that are responsible for the recognition of these regulators. Expression of mammalian type V adenylyl cyclase in a cyclase-deleted yeast strain can conditionally complement the lethal phenotype of this strain. Type V adenylyl cyclase-expressing yeast grow only when the cyclase is activated by coexpression of G s␣ or addition of forskolin to the medium; however, growth arrest is observed in the presence of both activators or under basal conditions. Utilizing this genetic system, we have isolated 25 adenylyl cyclase mutants defective in their response to G s␣ . Sequence analysis and biochemical characterization of these mutants have identified residues in both cytoplasmic domains of the cyclase that are involved in the specific binding of and regulation by G s␣ .Regulation of intracellular cyclic AMP concentrations is principally controlled at the level of its synthesis, through the hormonal regulation of adenylyl cyclase, the enzyme responsible for the conversion of ATP into cyclic AMP. The adenylyl cyclase system comprises three components: seven transmembrane-spanning receptors for a variety of hormones and neurotransmitters, heterotrimeric G proteins, 1 and the catalytic entity itself. Currently, nine isoforms of membrane-bound adenylyl cyclases have been identified by molecular genetic approaches, and studies of these enzymes reveal both common and unique regulatory features (1, 2). All isoforms tested to date are activated by the GTP-bound form of G s␣ and by forskolin; for some of the isoforms, such as the type V, these stimulators synergistically activate the enzyme. All isoforms of adenylyl cyclase are further regulated by additional inputs in an isoform-specific pattern. For example, G i␣ inhibits the types I, V, and VI isoforms (3-5); ␤␥ subunits can activate (types II, IV, and VII) or inhibit (type I) adenylyl cyclase activity (6 -10). Increases in intracellular calcium concentrations will inhibit the types V and VI isoforms (11-13) while indirectly activating (via a calmodulin-dependent process) types I and VIII (14,15) and inhibiting (by calmodulin kinase) the type III isoform (16).Structural motifs responsible for the recognition of these regulatory molecules by the adenylyl cyclases are starting to be uncovered. A region of type II adenylyl cyclase (residues 956 -982 from the C 2 domain) containing a QXXER motif has been shown to be important for the regulation of this enzyme by G protein ␤␥ subunits (17). This site has been proposed to interact with the ␤␥ subunit of the G protein, within the aminoterminal 100 residues of ␤ (18, 19). Synthetic peptide and mutational approaches have identified sequences located within the first cytoplasmic (C 1a ) region of type I adenylyl cyclase (residues 495-522) important for calmodulin activation (20, 21); this sequence ...

We have implemented a yeast genetic selection developed previously by our laboratory to identify mutant mammalian type V adenylyl cyclases insensitive to inhibition by G(ialpha.) One mutation isolated was localized to the first cytoplasmic domain at a Phe residue (position 400), which is conserved in all nine isoforms of membrane-bound mammalian adenylyl cyclase. Biochemical characterization of the F400Y mutant revealed a dramatic conversion of the G(ialpha) response from inhibitory to stimulatory. This mutation results in additional activating effects. The mutant exhibits an enhanced sensitivity toward activation by either G(salpha) or forskolin. Synergism between G(salpha) and forskolin is not observed for the F400Y mutant, presumably because the mutant already is in the sensitized state. Additionally, an enhancement of the basal unstimulated activity was observed. This mutation, which is the first demonstration of an activating point in a mammalian adenylyl cyclase, mimics a sensitized conformation of the wild-type enzyme that underlies the synergism between stimulatory inputs, and additionally, removes the inhibitory regulatory input provided by G(ialpha). Because sensitizing adenylyl cyclase toward its stimulators can have profound biological implications, this raises the possibility that naturally occurring mutations resembling those at the Phe400 residue may be associated with human disease states.