Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.6.1.1 (adenylate cyclase)
19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Saccharomyces cerevisiae strains with a disrupted RAS1 gene and with an intact RAS2 gene (ras1- RAS2 strains) grew well on both fermentable and nonfermentable carbon sources. By constructing isogenic mutants having a disrupted RAS1 locus and a randomly mutagenized chromosomal RAS2 gene, we obtained yeast strains with specific growth defects. The strain TS1 was unable to grow on nonfermentable carbon sources and galactose at 37 degrees C, while it could grow on glucose at the same temperature. The mutated RAS2 gene in TS1 cells encoded a protein with the glycines at positions 82 and 84 replaced by serine and arginine respectively. Both mutations were necessary for temperature sensitivity. We also isolated a mutant yeast that was unable to grow on nonfermentable carbon sources both at 30 and 37 degrees C, while growing on glucose at both temperatures. This phenotype was caused by a single chromosomal mutation, leading to the replacement of aspartic acid 40 of the RAS2 protein by asparagine. A ras1- yeast strain with a chromosomal RAS2 gene harbouring the three mutations together did not grow at any temperature using non-fermentable carbon sources, but it was able to grow on glucose at 30 degrees C, and not at 37 degrees C. The mutated proteins were much less effective than the wild-type RAS2 protein in the stimulation of adenylate cyclase, but were efficiently expressed in vivo. The possible roles of residues 40, 82 and 84 of the RAS2 protein in the regulation of adenylate cyclase are discussed.
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PMID:Yeast mutants temperature-sensitive for growth after random mutagenesis of the chromosomal RAS2 gene and deletion of the RAS1 gene. 314 92

The light chain of type C2 toxin produced by Clostridium botulinum was isolated by high-performance liquid chromatography. The protein eluted as a single peak; as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, it had an apparent molecular weight of 51,000 daltons. The light chain was an enzyme that possessed ADP-ribosylating activity. In experiments with synthetic substrates (homo-poly-L-amino acids; alanine, arginine, asparagine, aspartic acid, histidine, leucine, lysine, methionine, phenylalanine, proline, serine and tryptophan), only poly-L-arginine was ADP-ribosylated by the enzyme. In experiments with endogenous substrates (50,000 X g pellet and 50,000 X g supernatant from homogenates of mouse brain, liver and lung), the enzyme ADP-ribosylated proteins or polypeptides in both the particulate and soluble fractions. ADP-ribosylation of the soluble substrate was antagonized by adenine (K1 approximately 2.1 X 10(-5) M) and by adenosine (K1 approximately 2.7 X 10(-4) M); the reaction was reversed by a large molar excess of nicotinamide (0.1 M). ADP-ribosylation of soluble substrate was diminished when the substrate had been pretreated with 1,2-cyclohexane-dione (0.1 M), a site reactive reagent that modified selectively arginine residues. Neither the light chain nor the heavy chain of the binary toxin possessed adenylate cyclase activity. Tissue fractions did possess endogenous adenylate cyclase activity, but the toxin did not stimulate this activity. The data indicate that the binary toxin produced by Clostridium botulinum resembles other protein toxins.
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PMID:Molecular basis for the pharmacological actions of Clostridium botulinum type C2 toxin. 623 95

The neurotoxic effect of glutamate in cultured mouse mesencephalic dopaminergic neurons was investigated. Neuron-rich cell cultures were prepared from 13-14-day-old fetal mouse ventral mesencephalic tissue. Cultures were exposed to glutamate for 10 min and evaluated for glutamate neurotoxicity (GNT) 18-24 hr later by tyrosine hydroxylase (TH) immunostaining, microtubule associated protein-2 (MAP2) immunostaining, and radiolabeled dopamine uptake assay. In glutamate-exposed cultures, the number of TH-positive neurons and the level of dopamine uptake were reduced to 40% (35-45%) and 50% (47-52%), respectively, of control cultures. The number of MAP2-positive neurons was also reduced to 47%, indicating that the GNT was not restricted or selective to dopaminergic neurons. It is concluded that GNT was mediated by the N-methyl-D-aspartic acid (NMDA) receptor from the following observations: 1) GNT was completely blocked by MK-801, an NMDA receptor antagonist; 2) NMDA itself was as toxic as glutamate; 3) 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an antagonist of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate (AMPA/KA) receptor, did not block GNT; 4) kainate did not show neurotoxicity at a low concentration; and 5) two modulators of the NMDA receptor, 7-chlorokynurenic acid and magnesium, were effective in blocking GNT. Protective effects of phorbol myristate acetate, a tumor promoter, and gangliosides (GM1 and GT1b) on GNT were also demonstrated. Possible interactions between GNT and several protein kinase cascades were also investigated. Forskolin, an activator of adenyl cyclase and protein kinase A, showed some protective effect on GNT. But okadaic acid, an inhibitor of phosphatases, and genistein, a tyrosine kinase inhibitor, did not show any protective effect. These results suggest that 1) glutamate is capable of causing neuronal death in the substantia nigra; 2) GNT on dopaminergic neurons is mainly mediated by the NMDA receptor under the conditions of our study; 3) protein kinase C translocation is a key mechanism of GNT; and 4) there is an interplay of a signal transduction system in the pathomechanism of GNT.
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PMID:Glutamate neurotoxicity in mesencephalic dopaminergic neurons in culture. 790 39

Edema toxin of Bacillus anthracis is composed of protective antigen (PA) and edema factor (EF), a calcium- and calmodulin-dependent adenylate cyclase. At least five different antigenic regions of EF were identified using a competitive-binding, enzyme-linked immunosorbent assay of paired monoclonal antibodies (mAbs). Two mAbs, 9F5 and 7G10, inhibited the binding of 125I-EF to cell-bound PA. However, only 9F5 inhibited the elongation response of Chinese hamster ovary cells in the presence of edema toxin. Cleavage of EF at the two aspartic acid-proline residues by acid hydrolysis resulted in three fragments: a C-terminal 17 kDa fragment, a central 53 kDa fragment, and an N-terminal 18 kDa fragment. Immunoblots of EF cleaved by formic acid mapped mAbs 9F5 and 7G10 to the N-terminal 18 kDa fragment, mAb 1E6 to the C-terminal 17 kDa fragment, and the remaining 7 mAbs to the central 53 kDa fragment. mAbs 7G10 and 9F5 defined an antigenic region within amino acids 1-156 of EF which is involved in interaction with PA in forming edema toxin.
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PMID:Structure-function analysis of Bacillus anthracis edema factor by using monoclonal antibodies. 813 9

Several glucagon analogs containing substitutions for serine have been synthesized to assess the role of the four serine residues in the hormone. The strategic importance of His1 has been confirmed, and we have previously identified an aspartic acid critical for activity at position 9. While these findings have led to a series of pure glucagon antagonists, the details of specific glucagon-receptor interactions that switch on the ensuing signaling events are still not readily apparent. The requirement for serine was tested by the chemical synthesis of a series of analogs containing substitutions for the hydrophilic hydroxyl group in each of the highly conserved serine residues at positions 2, 8, 11, and 16 of glucagon. The resulting analogs were analyzed in rat hepatocyte membranes for their receptor-binding affinities as well as their abilities to stimulate adenylate cyclase. Positions 2 and 8 were the most sensitive to modification, where both binding and activity were adversely affected. This is consistent with the notion that although the sequence responsible for transduction lies in the amino-terminal half of glucagon, some residues at that end also contribute to binding affinity. Modifications at position 11 generated high-binding-affinity derivatives that were full or moderate agonists. In contrast, position 16 replacement analogs maintained significant receptor binding affinities while the agonist properties were almost completely lost, thus separating binding and transduction functions. Therefore, Ser16 is a third critical residue that determines glucagon activity. It is postulated, but not proven, that a serine residue, together with His1 and Asp9, may participate in the putative active center of glucagon, which, upon initial recognition and binding to receptor, leads to transduction of the biological signal. A dependence of the glucagon action on a three-residue cooperative mechanism might be analogous to the charge-relay scheme of serine proteases. It is suggested that, after binding to its receptor, glucagon becomes activated and functions like a coenzyme in catalyzing the specific hydrolysis of a peptide bond in the receptor, generating new amino and carboxyl end groups, and that one of these exposed chains may contact the GTP-binding protein and activate it for further interaction with adenylate cyclase. This idea was supported by inhibition experiments with 4-amidinophenylmethanesulfonyl fluoride (APMSF), a specific and irreversible inhibitor of serine proteases, which at a concentration of 5 mM completely suppressed cAMP formation by glucagon in liver membranes. cAMP formation was not affected if either glucagon or membranes were separately pretreated with APMSF and then assayed.
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PMID:Identification of an essential serine residue in glucagon: implication for an active site triad. 829 May 48

Extensive evidence gathered from structure-activity relationship analysis has identified and confirmed specific positions in the glucagon sequence that are important either for binding to its receptor or for signal transduction. Fifteen glucagon analogues have been designed and synthesized by incorporating structural changes in the N-terminal region of glucagon, in particular histidine-1, phenylalanine-6, and aspartic acid-9. This investigation was conducted to study the role of phenylalanine at position 6 on the glucagon mechanism of action. These glucagon analogues have been made by either deleting or substituting hydrophobic groups, hydrophilic groups, aromatic amino acids, or a D-phenylalanine residue at this position. The structures of the new analogues are as follows: [des-His1, des-Phe6, Glu9]glucagon-NH2 (1); [des-His1,Ala6,Glu9]glucagon-NH2 (2); [des-His1,Tyr6,Glu9]glucagon-NH2 (3); [des-His1,Trp6,Glu9]-glucagon-NH2 (4); [des-His1,D-Phe6,Glu9]glucagon-NH2 (5); [des-His1,Nle6,Glu9]glucagon-NH2 (6); [des-His1,Asp6,Glu9]glucagon-NH2 (7); [des-His1,des-Gly4,Glu9]glucagon-NH2 (8); [desPhe6,-Glu9]glucagon-NH2 (9); [des-Phe6]glucagon-NH2 (10); [des-His1, des-Phe6]glucagon-NH2 (11); [des-His1, des-Phe6,Glu9]glucagon (12); [des-Phe6,Glu9]glucagon (13); [des-Phe6]glucagon (14); and [des-His1, des-Phe6]glucagon (15). The receptor binding potencies IC50 values are 48 (1), 126 (2), 40 (3), 19 (4), 100 (5), 48 (6), 2000 (7), 52 (8), 113 (9), 512 (10), 128 (11), 1000 (12), 2000 (13), 500 (14), and 200 nM (15). All analogues were found to be antagonists unable to activate the adenylate cyclase system even at concentrations as high as 10(-5) M except for analogues 6 and 8, which were found to be weak partial agonists/partial antagonists with maximum stimulation between 6-12%. In competitive inhibition experiments, all the analogues caused a right shift of the glucagon-stimulated adenylate cyclase dose-response curve. The pA2 values were 8.20 (1), 6.40 (2), 6.20 (3), 6.25 (4), 6.30 (5), 6.30 (7), 6.05 (8), 6.20 (9), 6.30 (10), 6.25 (11), 6.10 (12), 6.20 (13), 6.20 (14), and 6.35 (15).
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PMID:The role of phenylalanine at position 6 in glucagon's mechanism of biological action: multiple replacement analogues of glucagon. 925 62

This review examines the recent progress in the field of angiotensin receptors. Multiplicity of these receptors was demonstrated initially on the basis of pharmacologic differences and then confirmed by expression cloning. AT1 receptors are predominant in the adult. They are widely distributed and mediate all of the known biologic effects of angiotensin II (AngII) through a variety of signal transduction systems, including activation of phospholipases C and A2, inhibition of adenylate cyclase, opening of calcium channels, and activation of tyrosine kinases. AT2 receptors are predominant in the fetus, but also present in adult tissues such as the adrenals, ovaries, uterus, and brain. AngII via these receptors exerts effects often opposed to those mediated by the AT1 receptors. Signal transduction implicates protein tyrosine phosphatase stimulation. AT1 and AT2 receptor expressions are regulated differently, and regulation is also tissue-specific. AT1 and AT2 receptors have been demonstrated in endothelial cells. Activation of AT1 receptors results in production of vasodilatory agents, nitric oxide, and prostacyclin (PGI2), which counteract the direct vasoconstrictor effects of Ang II on the adjacent smooth muscle cells. AT1 receptors on mesangial cells, smooth muscle cells, and fibroblasts are involved in cell growth and fibrosis, the latter being due both to an increase in the synthesis and a decrease in the degradation of the main components of the extracellular matrix. These AT1 receptor-dependent effects are for the most part indirect and mediated by growth factors, cytokines, and other peptides, including endothelin, transforming growth factor-beta1, and platelet-derived growth factor. AngII is metabolized into active fragments by deletion of the terminal amino acids on both ends. AngIII and AngIV are formed by successive deletions of aspartic acid and arginine at the N terminus. AngII (1-7) is obtained by deletion of phenylalanine at the C terminus. AngIII shares the same receptors and exerts the same effects as AngII. AngIV and AngII (1-7) recognize the AT1 and AT2 receptors with a lesser affinity than AngII and, in addition, possess their own receptors that mediate effects often opposed to those of AngII.
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PMID:Angiotensin II receptors. 989 38

Proteins that bind to G protein-coupled receptors have recently been identified as regulators of receptor anchoring and signaling. In this study, actin-binding protein 280 (ABP-280), a widely expressed cytoskeleton-associated protein that plays an important role in regulating cell morphology and motility, was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. The specificity of this interaction was originally identified in a yeast two-hybrid screen and confirmed by protein binding. The functional significance of the D(2) receptor-ABP-280 association was evaluated in human melanoma cells lacking ABP-280. D(2) receptor agonists were less potent in inhibiting forskolin-stimulated cAMP production in these cells. Maximal inhibitory responses of D(2) receptor activation were also reduced. Further yeast two-hybrid experiments showed that ABP-280 association is critically dependent on the carboxyl domain of the D(2) receptor third cytoplasmic loop, where there is a potential serine phosphorylation site (S358). Serine 358 was replaced with aspartic acid to mimic the effects of receptor phosphorylation. This mutant (D(2)S358D) displayed compromised binding to ABP-280 and coupling to adenylate cyclase. PKC activation also generated D(2) receptor signaling attenuation, but only in ABP-containing cells, suggesting a PKC regulatory role in D(2)-ABP association. A mechanism for these results may be derived from a role of ABP-280 in the clustering of D(2) receptors, as determined by immunocytochemical analysis in ABP-deficient and replete cells. Our results suggest a new molecular mechanism of modulating D(2) receptor signaling by cytoskeletal protein interaction.
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PMID:Modulation of dopamine D(2) receptor signaling by actin-binding protein (ABP-280). 1069 83

We hypothesized that ethanol (EtOH) might act through the endocannabinoid system to inhibit luteinizing hormone-releasing hormone (LHRH) release. Therefore, we examined the mechanism by which EtOH and anandamide (AEA), an endogenous cannabinoid, inhibit LHRH release from incubated medial basal hypothalamic explants. In previous work, we demonstrated that EtOH inhibits the N-methyl-D-aspartic acid-stimulated release of LHRH by increasing the release of two neurotransmitters: beta-endorphin and gamma-aminobutyric acid (GABA). In the present work, bicuculline, a GABAergic antagonist, completely prevented the inhibition of AEA (10(-9)M) on N-methyl-D-aspartic acid-induced LHRH release, but naltrexone, a micro-opioid receptor antagonist, had no effect. AEA also significantly increased GABA release but had no effect on beta-endorphin release. Therefore, AEA could inhibit LHRH release by increasing GABA but not beta-endorphin release. Because EtOH and AEA acted similarly to inhibit LHRH release, we investigated whether both substances would affect the adenylate cyclase activity acting through the same GTP-coupled receptors, the cannabinoid receptors 1 (CB1-rs). AEA and EtOH (10(-1)M) reduced the forskolin-stimulated accumulation of cAMP, but AM251, a specific antagonist of CB1-r, significantly blocked that inhibition. Additionally we investigated whether CB1-r is involved in the inhibition of LHRH by EtOH and AEA. AEA and EtOH reduced forskolin-stimulated LHRH release, but AM251 significantly blocked that inhibition. Also, we demonstrated that EtOH did not act by increasing AEA synthase activity to inhibit LHRH release in our experimental conditions. Therefore, our results indicate that EtOH inhibits the release of LHRH acting through the endocannabinoid system.
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PMID:Alcohol inhibits luteinizing hormone-releasing hormone release by activating the endocannabinoid system. 1498 Dec 61

Efficient signaling requires accurate spatial and temporal compartmentalization of proteins. RACK1 is a scaffolding protein that fulfils this role through interaction of binding partners with one of its seven WD40 domains. We recently identified the kinase Fyn and the NR2B subunit of the N-methyl-D-Aspartate receptor (NMDAR) as binding partners of RACK1. Scaffolding of Fyn near its substrate NR2B by RACK1 inhibits Fyn phosphorylation of NR2B and thereby negatively regulates channel function. We found that Fyn and NR2B share the same binding site on RACK1; however, their binding to RACK1 is not mutually exclusive (Yaka, R., Thornton, C., Vagts, A. J., Phamluong, K., Bonci, A., and Ron, D. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5710-5715). We therefore tested the hypothesis that RACK1 forms a homodimer that allows the simultaneous binding of Fyn and NR2B. We found that RACK1 binds to itself both in vitro and in the brain. Deletion analyses identified a RACK1-RACK1 dimer-binding site within the 4th WD40 repeat, and application of the 4th WD40 repeat or a peptide derivative to hippocampal slices inhibited NMDAR activity. We further found that in hippocampal slices, both RACK1 and NR2B associated with another WD40 protein, the beta-subunit of G protein (Gbeta), previously shown to heterodimerize with RACK1 in vitro (Dell, E. J., Connor, J., Chen, S., Stebbins, E. G., Skiba, N. P., Mochly-Rosen, D., and Hamm, H. E. (2002) J. Biol. Chem. 277, 49888-49895). However, activation of the pituitary adenylate cyclase polypeptide (1-38) G protein-coupled receptor, previously found to induce the dissociation of RACK1 from the NMDAR complex (Yaka, R., He, D. Y., Phamluong, K., and Ron, D. (2003) J. Biol. Chem. 278, 9630-9638), attenuated the association of Gbeta with RACK1 and NR2B. Based on these results, we propose that WD40-mediated homo- and heterodimerization of RACK1 mediate the formation of a transient signaling complex that includes the NMDAR, a G protein and Fyn.
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PMID:Spatial and temporal regulation of RACK1 function and N-methyl-D-aspartate receptor activity through WD40 motif-mediated dimerization. 1514 Aug 93


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