Interaction of the two cytosolic domains of mammalian adenylyl cyclase.

Whisnant, Richard E.; Gilman, Alfred G.; Dessauer, Carmen W.

doi:10.1073/pnas.93.13.6621

Cited by 126 publications

(126 citation statements)

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“…The fact that the G s␣ binding site that we have determined by the mutational analysis comprises residues located in both C 1 and C 2 domains is consistent with the observation that G s␣ increases the affinity of the cytoplasmic domains for each other (37,38). That most of the G s␣ -insensitive mutants isolated in this study map to residues in the C 2 domain is consistent with the observation that recombinant soluble C 2 domain constructs can bind to G s␣ in vitro, whereas the C 1 domain cannot but can be covalently attached to G s␣ in the presence of chemical crosslinkers (39).…”

Section: Discussionsupporting

confidence: 88%

“…Mutation of this aspartate would disrupt its ability to hydrogen-bond to the backbone nitrogen of these residues, thereby decreasing the interaction of the two cytoplasmic domains. In light of the finding that both G s␣ and forskolin activate adenylyl cyclase by increasing the affinity of C 1 for C 2 (37,38), the predicted consequence of mutating Asp-424 would be precisely what was observed for the D424N mutant: reduced sensitivity to stimulation by either forskolin or G s␣ alone, and a retention of the ability to synergistically integrate these stimulatory inputs.…”

Section: Discussionmentioning

confidence: 90%

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Genetic Selection of Mammalian Adenylyl Cyclases Insensitive to Stimulation by Gsα

Zimmermann

Zhou

Taussig

1998

Journal of Biological Chemistry

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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 ...

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Section: Discussionsupporting

confidence: 88%

Section: Discussionmentioning

confidence: 90%

Genetic Selection of Mammalian Adenylyl Cyclases Insensitive to Stimulation by Gsα

Zimmermann

Zhou

Taussig

1998

Journal of Biological Chemistry

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“…1A). Recent studies from Gilman and coworkers have demonstrated that AC activity can be reconstituted by the expression of the two major cytosolic regions of type I and II enzyme that are either joined together by a linker (20) or expressed separately and then mixed together (21). Thus, the engineered soluble forms of AC not containing the membrane spanning regions can be utilized to investigate the regions on the molecule that are important for its regulation by various modulators.…”

mentioning

confidence: 99%

Characterization of soluble forms of nonchimeric type V adenylyl cyclases

Scholich

Barbier

Mullenix

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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Type V adenylyl cyclase (ACV) belongs to the family of Ca 2+ - inhibited cyclases. We have generated two soluble forms of the enzyme containing the C1 or C1a region (which lacks the C-terminal 112 amino acids) linked to the C2 domain and compared their regulation with the full-length ACV. All three forms of ACV were stimulated by the α subunit of the stimulatory G protein G s (G sα ) and forskolin. However, the synergistic stimulation by both these activators was markedly enhanced in the soluble enzymes. Moreover, the α subunit of the inhibitory G protein G i (G iα ) inhibited all forms of the enzyme, indicating that the regions for G sα and G iα interaction are preserved in the soluble forms. Ca 2+ inhibited forskolin-stimulated adenylyl cyclase (AC) activity of the full-length and C1-C2 forms of ACV but did not alter the activity of the C1a-C2 form. Maximal stimulation of AC activity by combination of G sα and forskolin obliterated the Ca 2+ -mediated inhibition of the full-length and C1-C2 forms of ACV. In 45 Ca 2+ overlay experiments, the C1-C2 but not the C1a-C2 soluble ACV bound Ca 2+ . Moreover, proteins corresponding to the C1a and C2 domains did not bind calcium. On the other hand, the proteins corresponding to C1 and its C-terminal 112 amino acids (C1b) bound 45 Ca 2+ . To our knowledge, this is the first report of nonchimeric soluble forms of AC in which regulation by G sα and G iα is preserved. Moreover, we demonstrate that the 112 amino acid C1b region of ACV is responsible for the binding of Ca 2+ and inhibition of enzyme activity.

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“…BW245C at 10 μM induced about 6-fold increase, while 300 nM forskolin induced about 11-fold increase in intracellular cAMP level. Forskolin is known to bind directly to the catalytic subunit of AC, therefore leading to abundant cAMP increase (26).…”

Section: Discussionmentioning

confidence: 99%

Relaxant Effect of Prostaglandin D2–Receptor DP Agonist on Liver Myofibroblast Contraction

et al. 2011

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Abstract. Increased intrahepatic resistance causes portal hypertension in cirrhosis. Liver myofibroblasts (MFs) are now regarded as the principle cells involved in sinusoidal blood flow regulation. Many other prostaglandin-receptor agonists have been reported to regulate liver MF contraction, but the role of the prostaglandin D 2 -receptor DP is unknown. In this study, we investigated the effect of a synthetic agonist of prostanoid DP receptor, BW245C, on contractile properties of primary rat liver MFs. Collagen gel contraction assay revealed that BW245C alone (1 and 10 μM) did not induce contraction but induced cell relaxation. Pretreatment with BW245C (10 μM, 30 min) attenuated bradykinin (100 nM)-induced liver MF contraction. Elevation of [Ca 2+ ] i induced by bradykinin (100 nM) was partially suppressed by BW245C pretreatment (10 μM, 3 min). BW245C (1 and 10 μM) significantly increased intracellular cAMP level in a dose-dependent manner. Pretreatment with forskolin (30 -300 nM, 30 min) and dibutyryl-cAMP (3 -30 μM, 30 min) significantly reduced bradykinin-induced contraction. Furthermore, a protein kinase A (PKA) inhibitor KT5720 (10 nM to 1 μM, 30 min) blocked the relaxant effect of BW245C. These results suggest that prostanoid DP receptor agonism inhibits bradykinin-induced [Ca 2+ ] i elevation and contraction through cAMP-PKA signal activation in rat liver MFs.

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Interaction of the two cytosolic domains of mammalian adenylyl cyclase.

Cited by 126 publications

References 10 publications

Genetic Selection of Mammalian Adenylyl Cyclases Insensitive to Stimulation by Gsα

Genetic Selection of Mammalian Adenylyl Cyclases Insensitive to Stimulation by Gsα

Characterization of soluble forms of nonchimeric type V adenylyl cyclases

Relaxant Effect of Prostaglandin D2–Receptor DP Agonist on Liver Myofibroblast Contraction

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