Cyclase

About: Cyclase is a research topic. Over the lifetime, 10162 publications have been published within this topic receiving 388566 citations.

Site-directed mutagenesis of human beta-adrenergic receptors: substitution of aspartic acid-130 by asparagine produces a receptor with high-affinity agonist binding that is uncoupled from adenylate cyclase

Abstract: By using oligonucleotide-directed mutagenesis, we have produced a point mutation (guanine to adenine) at nucleotide 388 of the gene for human beta-adrenergic receptor (beta AR) that results in a substitution of asparagine for the highly conserved aspartic acid at position 130 in the putative third transmembrane domain of the human beta AR ([Asn130]beta AR). We have examined the functional significance of this mutation in B-82 cells continuously expressing the mutant [Asn130]beta AR. The mutant [Asn130]beta AR displayed normal antagonist binding but unusually high-affinity agonist binding (5- to 10-fold higher than wild-type beta AR), consistent with a single class of high-affinity binding sites. The mutant beta AR displayed guanine nucleotide-sensitive changes in agonist affinity (3- to 5-fold shift) implying an interaction between the beta AR and the stimulatory guanine nucleotide-binding regulatory protein; however, the ability of guanine nucleotides to alter agonist affinity was attenuated. Addition of saturating concentrations of isoproterenol to cell cultures expressing mutant [Asn130]-beta ARs had no effect on intracellular levels of cAMP, indicating that the mutant beta AR is unable to affect stimulation of adenylate cyclase. These results indicate that substitution of the aspartic acid with asparagine at residue 130 of the human beta AR dissociates the well-characterized guanine nucleotide effects on agonist affinity from those on activation of the stimulatory guanine nucleotide-binding regulatory protein and adenylate cyclase and suggests the existence of two distinct counterions for the amine portion of catecholamines that are associated with high- and low-affinity agonist binding states of beta AR.

The 5-hydroxytryptamine 5-HT1D receptor subtype is negatively coupled to adenylate cyclase in calf substantia nigra.

Abstract: 1) The possibility was explored that the recently defined 5-HT1D binding sites could be negatively coupled to adenylate cyclase in calf substantia nigra. 2) 5-HT inhibited forskolin-stimulated adenylate cyclase activity in a concentration-dependent manner (EC50 valu e = 24.0 nmol/l, Emax = 22.7% inhibition) in the presence of GTP (10 μmol/l), which was required for this inhibitory effect. 3) The following 5-HT receptor agonists inhibited adenylate cyclase activity (in decreasing order of potency): 5-carboxamidotryptamine > 5-HT > 5-methoxytryptamine > 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H indole (RU 24969) ≥ N1N-dipropyl-5-carboxamidotryptamine > 8-hydroxy-2(di-n-propylamino)-tetralin (8OH-DPAT) > buspirone > ipsapirone; the latter two compounds apparently behaved as partial agonists. 4) Other compounds displaying agonist activity in this system were: metergoline > methysergide ≥ rauwolscine ≥ cyanopindolol > yohimbine > (±)-4(3-tert-butyl-amino 2-hydroxypropoxy)-indol-2 carbonic acid isopropylester (21-009) > corynanthine. 5) Methiothepin, mianserin and spiperone displaced the concentration-effect curve of 5-HT to the right without depressing the E max value. The same held true for the partial agonists ipsapirone, buspirone and corynanthine. 6) The rank order of potency of agonists as well as of antagonists in this system was in full agreement with their affinities at 5-HT1D binding site. A highly significant correlation was found between both parameters (r = 0.94, P = 0.0001). 7) The results strongly support the contention that 5-HT1D binding sites are negatively coupled to adenylate cyclase in calf substantia nigra.

Insulin triggers cyclic AMP-dependent activation and phosphorylation of a plasma membrane cyclic AMP phosphodiesterase

Abstract: Regulation of blood glucose levels by the liver is primarily achieved by the action of two peptide hormones, insulin and glucagon, which bind to specific receptors associated with the hepatocyte plasma membrane. Whilst the molecular action of glucagon at the level of the cell plasma membrane in activating adenylate cyclase is relatively well understood, we know little, if anything, of the molecular consequences of insulin occupying its receptor. We demonstrate here that insulin, at physiologically relevant concentrations, can trigger the cyclic AMP-dependent activation and phosphorylation of a low Km cyclic AMP phosphodiesterase attached to the liver plasma membrane. Such an effect may in part explain the ability of insulin to inhibit the increase in cellular cyclic AMP content that glucagon alone produces by activation of adenylate cyclase. Our observation that basal, intracellular cyclic AMP levels are insufficient to allow insulin to activate the cyclic AMP phosphodiesterase, yet those cyclic AMP levels achieved after exposure of the cells to glucagon are sufficient, gives a molecular rationale to Butcher and Sutherland's proposal that it is necessary to first elevate cellular cyclic AMP levels before they can be depressed by insulin.