Mutations of the adenylyl cyclase gene that block RAS function in Saccharomyces cerevisiae

The interaction between RAS proteins and adenylyl cyclase was studied by using dominant interfering mutations of adenylyl cyclase from the yeast Saccharomyces cerevisiae. RAS proteins activate adenylyl cyclase in this organism. A plasmid expressing a catalytically inactive adenylyl cyclase was found to interfere dominantly with this activation. The interfering region mapped to the leucine-rich repeat region of adenylyl cyclase, which is homologous to domains present in several other proteins and is thought to participate in protein-protein interactions.     

The protein kinase domain of the ANP receptor is required for signaling

A plasma membrane form of guanylate cyclase is a cell surface receptor for atrial natriuretic peptide (ANP). In response to ANP binding, the receptor-enzyme produces increased amounts of the second messenger, guanosine 3',5'-monophosphate. Maximal activation of the cyclase requires the presence of adenosine 5'-triphosphate (ATP) or nonhydrolyzable ATP analogs. The intracellular region of the receptor contains at least two domains with homology to other proteins, one possessing sequence similarity to protein kinase catalytic domains, the other to regions of unknown function in a cytoplasmic form of guanylate cyclase and in adenylate cyclase. It is now shown that the protein kinase-like domain functions as a regulatory element and that the second domain possesses catalytic activity. When the kinase-like domain was removed by deletion mutagenesis, the resulting ANP receptor retained guanylate cyclase activity, but this activity was independent of ANP and its stimulation by ATP was markedly reduced. A model for signal transduction is suggested in which binding of ANP to the extracellular domain of its receptor initiates a conformational change in the protein kinase-like domain, resulting in derepression of guanylate cyclase activity.     

An M2 muscarinic receptor subtype coupled to both adenylyl cyclase and phosphoinositide turnover

To investigate whether a particular receptor subtype can be coupled to multiple effector systems, recombinant M2 muscarinic receptors were expressed in cells lacking endogenous receptor. The muscarinic agonist carbachol both inhibited adenylyl cyclase and stimulated phosphoinositide hydrolysis. The stimulation of phosphoinositide hydrolysis was significantly less efficient and more dependent on receptor levels than the inhibition of adenylyl cyclase. Both responses were mediated by guanine nucleotide binding proteins, as evidenced by their inhibition by pertussis toxin; the more efficiently coupled adenylyl cyclase response was significantly more sensitive. Thus, individual subtypes of a given receptor are capable of regulating multiple effector pathways.     

Effect of forskolin on voltage-gated K+ channels is independent of adenylate cyclase activation

Forskolin is commonly used to stimulate adenylate cyclase in the study of modulation of ion channels and other proteins by adenosine 3',5'-monophosphate (cAMP)-dependent second messenger systems. In addition to its action on adenylate cyclase, forskolin directly alters the gating of a single class of voltage-dependent potassium channels from a clonal pheochromocytoma (PC12) cell line. This alteration occurred in isolated cell-free patches independent of soluble cytoplasmic enzymes. The effect of forskolin was distinct from those of other agents that raise intracellular cAMP levels. The 1,9-dideoxy derivative of forskolin, which is unable to activate the cyclase, was also effective in altering the potassium channel activity. This direct action of forskolin can lead to misinterpretation of results in experiments in which forskolin is assumed to selectively activate adenylate cyclase.     

Adenylate cyclase activation shifts the phase of a circadian pacemaker

Forskolin, a highly specific activator of adenylate cyclase, produced both delay and advance phase shifts of the circadian rhythm recorded from the isolated eye of the marine mollusk Aplysia. The phase dependence of the response to forskolin was identical to that with serotonin, which also stimulates adenylate cyclase in the eye. The ability of agents to activate adenylate cyclase in homogenates was correlated with their ability to shift the phase of the circadian oscillator. These results along with earlier findings show that adenosine 3',5'-monophosphate mediates the effect of serotonin on the rhythm and regulates the phase of the circadian pacemaker in the eye of Aplysia.     

Type-specific regulation of adenylyl cyclase by G protein beta gamma subunits

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) dissociate into guanosine triphosphate (GTP)-bound alpha subunits and a complex of beta and gamma subunits after interaction with receptors. The GTP-alpha subunit complex activates appropriate effectors, such as adenylyl cyclase, retinal phosphodiesterase, phospholipase C, and ion channels. G protein beta gamma subunits have been found to have regulatory effects on certain types of adenylyl cyclase. In the presence of Gs alpha, the alpha subunit of the G protein that activates adenylyl cyclase, one form of adenylyl cyclase was inhibited by beta gamma, some forms were activated by beta gamma, and some forms were not affected by beta gamma. These interactions suggest mechanisms for communication between distinct signal-transducing pathways.     

Carboxyl terminal domain of Gs alpha specifies coupling of receptors to stimulation of adenylyl cyclase

The alpha subunits of Gs and Gi link different sets of hormone receptors to stimulation and inhibition, respectively, of adenylyl cyclase. A chimeric alpha i/alpha s cDNA was constructed that encodes a polypeptide composed of the amino terminal 60% of an alpha i chain and the carboxyl terminal 40% of alpha s. The cDNA was introduced via a retroviral vector into S49 cyc- cells, which lack endogenous alpha s. Although less than half of the hybrid alpha chain is derived from alpha s, its ability to mediate beta-adrenoceptor stimulation of adenylyl cyclase matched that of the normal alpha s polypeptide expressed from the same retroviral vector in cyc- cells. This result indicates that carboxyl terminal amino acid sequences of alpha s contain the structural features that are required for specificity of interactions with the effector enzyme, adenylyl cyclase, as well as with the hormone receptor.     

Digitonin effects on photoreceptor adenylate cyclase

Adenylate cyclase is described in a number of photoreceptor membranes. Vertebrate rod outer segments contain light-regulated cyclase, and light regulation is abolished by digitonin. Disruption of microvilli in cone and rhabdomphotoreceptors is also associated with loss of light regulation and retention of full enzymic activity. The data suggest that inhibitory constraint provides regulation in cyclase systems and that disruption of membrane structure uncouples catalytic and regulatory elements.     

Catecholamine-induced alteration in sedimentation behavior of membrane bound beta-adrenergic receptors

Incubation of astrocytoma cells with catecholamines results in a decrease in catecholamine-stimulated adenylate cyclase activity and a concomitant alteration in the sedimentation properties of particulate beta-adrenergic receptors. The altered receptors exhibit agonist binding properties similar to those of receptors that are "uncoupled" from adenylate cyclase.     

Glucagon-sensitive adenyl cylase in plasma membrane of hepatic parenchymal cells

The plasma membrane of hepatic parenchymal cells contains an adenyl cyclase system that is stimulated by glucagon. Adrenocorticotropin and epinephrine do not stimulate this adenyl cyclase, and very little cyclic phospho-diesterase activity is present in the membrane. These findings support the concept that glucagon exerts its regulatory action in the liver by stimulating adenyl cyclase activity in the plasma membrane.     

Hormone-sensitive adenyl cyclase: cytochemical localization in rat liver

An electron microscopic procedure has been developed, using rat liver, for the localization of hormone-sensitive adenyl cyclase. Isoproterenol-sensitive adenyl cyclase is located almost exclusively in the parenchymal cells. In contrast, glucagon-sensitive adenyl cyclase is located primarily in the reticulo-endothelial cells but is also present in parenchymal cells. Sodium fluoride-sensitive adenyl cyclase is found in both cell types.     

Facilitation of signal onset and termination by adenylyl cyclase

The alpha subunit (Gsalpha) of the stimulatory heterotrimeric guanosine triphosphate binding protein (G protein) Gs activates all isoforms of mammalian adenylyl cyclase. Adenylyl cyclase (Type V) and its subdomains, which interact with Gsalpha, promoted inactivation of the G protein by increasing its guanosine triphosphatase (GTPase) activity. Adenylyl cyclase and its subdomains also augmented the receptor-mediated activation of heterotrimeric Gs and thereby facilitated the rapid onset of signaling. These findings demonstrate that adenylyl cyclase functions as a GTPase activating protein (GAP) for the monomeric Gsalpha and enhances the GTP/GDP exchange factor (GEF) activity of receptors.     

Biotin enhances guanylate cyclase activity

Biotin and its analog, (+)-biotin-p-nitrophenyl ester enhanced guanylate cyclase activity two- to threefold in rat liver, kidney, colon, cerebellum, and heart. Dose-response relationships revealed that at concentrations as low as 1 micromolar, both biotin and its analog caused maximal augmentation of guanylate cyclase activity. These data suggest a role for the activation of guanylate cyclase in the mechanism of action of this vitamin.     

Thymus adenylate cyclase activity during murine leukemogenesis

Activity of thymus adenylate cyclase was more than three times higher in leukemic AKR mice than in nonleukemic AKR mice and CBA mice. Preleukemic AKR mice that had no evidence of leukemia but were expected to soon develop the disease exhibited similarly elevated activities of thymus adenylate cyclase.     

Message transmission: receptor controlled adenylate cyclase system

The adenylate cyclase system is composed of an activating hormone or neurotransmitter (H), its receptor (R), the guanosine triphosphate (GTP) binding protein (Gs), and the catalytic unit (C). The activation of the receptor R involves a transient change in conformation, from a loose binding of the neurotransmitter H to an extremely tight interaction, termed locking. The system is regulated in the activation steps and also by three deactivation processes. A guanosine triphosphatase activity is built into the Gs protein so that the active GsGTP has only a limited lifetime during which it is able to activate C. In addition, the continued occupation of R by H causes desensitization of R. Finally, there are inhibitory receptors, such as alpha-adrenergic and opiate receptors, which inhibit the adenylate cyclase by way of a specific GTP binding protein (Gi). Yet to be determined are the conformational transformations of pure R on binding of an agonist or a partial agonist; the genes that code for the many different receptors that activate the adenylate cyclase, and the possibility that the G components interact with systems in the cell other than the adenylate cyclase.     

Cyclic adenosine monophosphate: selective increase in caudate nucleus after administration of L-dopa

Treatment with the dopamine precursor L-dopa produced a significant accumulation of adenosine 3',5'-monophosphate (cyclic AMP) in the caudate nucleus of the rat. In contrast, there was no change in the amount of cyclic AMP in the cerebellum. Accumulation of cyclic AMP in the caudate nucleus after administration of L-dopa was prevented by prior treatment with the decarboxylase inhibitor RO 4-4602. These observations and those in other laboratories support the assumption that dopamine formed from L-dopa selectively activates striatal adenylate cyclase. The in vivo activation of adenylate cyclase after treatment with L-dopa may be a useful model for studying neurological and psychiatric disorders that are thought to involve the dopaminergic system of the brain.     

Identification of a specialized adenylyl cyclase that may mediate odorant detection

The mammalian olfactory system may transduce odorant information via a G protein-mediated adenosine 3',5'-monophosphate (cAMP) cascade. A newly discovered adenylyl cyclase, termed type III, has been cloned, and its expression was localized to olfactory neurons. The type III protein resides in the sensory neuronal cilia, which project into the nasal lumen and are accessible to airborne odorants. The enzymatic activity of the type III adenylyl cyclase appears to differ from nonsensory cyclases. The large difference seen between basal and stimulated activity for the type III enzyme could allow considerable modulation of the intracellular cAMP concentration. This property may represent one mechanism of achieving sensitivity in odorant perception.     

8-Arginine]-vasopressinoic acid: an inhibitor of rabbit kidney adenyl cyclase

Neurohypophyseal hormnones and several synthetic analogs stimulate adenyl cyclase prepared from rabbit kidney medullary tissue. [8-Arginine]-vasopressinoic acid inhibits the stimulation of medullary adenyl cyclase by neurohypophyseal peptides but does not influence the action of parathyroid hormone on adenyl cyclase from kidiney cortex.     

Adenylate cyclases of Trypanosoma brucei inhibit the innate immune response of the host

The parasite Trypanosoma brucei possesses a large family of transmembrane receptor-like adenylate cyclases. Activation of these enzymes requires the dimerization of the catalytic domain and typically occurs under stress. Using a dominant-negative strategy, we found that reducing adenylate cyclase activity by about 50% allowed trypanosome growth but reduced the parasite's ability to control the early innate immune defense of the host. Specifically, activation of trypanosome adenylate cyclase resulting from parasite phagocytosis by liver myeloid cells inhibited the synthesis of the trypanosome-controlling cytokine tumor necrosis factor-α through activation of protein kinase A in these cells. Thus, adenylate cyclase activity of lyzed trypanosomes favors early host colonization by live parasites. The role of adenylate cyclases at the host-parasite interface could explain the expansion and polymorphism of this gene family.     

CDC25: a component of the RAS-adenylate cyclase pathway in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae contains two functional homologues of the ras oncogene family, RAS1 and RAS2. These genes are required for growth, and all evidence indicates that this essential function is the activation of adenylate cyclase. In contrast, ras in mammalian cells does not appear to influence adenylate cyclase activity. To clarify the relation between ras function in yeast and in higher eukaryotes, and the role played by yeast RAS in growth control, it is necessary to identify functions acting upstream of RAS in the adenylate cyclase pathway. The evidence presented here indicates that CDC25, identified by conditional cell cycle arrest mutations, encodes such an upstream function.