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)

The albumen gland is a compound tubular exocrine gland found in the female reproductive tract of freshwater pulmonate snails such as Helisoma duryi. It secretes a perivitelline fluid, composed of protein and polysaccharide complexes, and coats each fertilized egg. A 288-kDa native glycoprotein, composed of several 66-kDa subunits, was identified in soluble extracts of albumen gland. Forskolin stimulates the release of secretory granules, containing both proteins and polysaccharides, from the cytoplasm of the glandular cells. An acid extract of the central nervous system or the adenosine-3', 5'-cyclic monophosphate (cAMP) analogue 8-bromo cAMP, stimulates protein secretion from the gland. Pretreatment of the albumen gland with cAMP antagonist (Rp isomer of cAMP) inhibits the stimulatory effect of a brain extract. Digestion of brain extract with proteolytic enzymes abolishes its activity, suggesting the factor from the brain is peptidergic. The neuroactive agents serotonin, Phe-Met-Arg-Phe-amide, Tyr-Gly-Gly-Phe-Met-Arg-Phe-amide, small cardioactive peptide B, and caudodorsal cell hormone were also tested for potential secretion-promoting ability. Brain extracts were partially purified with a Sep-Pak C18 reverse-phase cartridge and indicate the peptide is relatively hydrophobic. These results suggest that a brain peptide promotes the secretion of perivitelline fluid, and this is mediated by the adenylate cyclase/cAMP signal transduction pathway.
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PMID:Release of proteins and polysaccharides from the albumen gland of the freshwater snail Helisoma duryi: effect of cAMP and brain extracts. 963 57

The human beta-adrenoceptor is a member of the seven-transmembrane family of receptors, encoded by a gene on chromosome 5. beta-Adrenoceptors have been classified into beta1, beta2, and beta3 subgroups, with beta2-receptors being widely distributed in the respiratory tract, particularly in airway smooth muscle. Intracellular signaling following beta2-adrenoceptor activation is largely affected through a trimeric Gs protein coupled to adenylate cyclase. Cyclic AMP (cAMP) induces airway relaxation through phosphorylation of muscle regulatory proteins and attenuation of cellular Ca2+ concentrations. Alternative cAMP-independent pathways involving activation of membrane maxi-K+ channels and coupling through Gi to the MAP kinase system have also been described. Site-directed mutagenesis has identified Asp 113 and Ser 204/207 within the third and fourth membrane domains as the active site of the beta2-receptor, critical for beta2-agonist binding and activity. beta2-Agonists have been characterized as those that directly activate the receptor (albuterol), those that are taken up into a membrane depot (formoterol), and those that interact with a receptor-specific auxiliary binding site (salmeterol). These differences in mechanism of action are reflected in the kinetics of airway smooth muscle relaxation and bronchodilation in patients with asthma. beta-Adrenoceptor desensitization associated with beta2-agonist activation is a consequence of phosphorylation by beta-ARK and uncoupling of the receptor from Gs following beta-arrestin binding, of internalization and recycling of the receptor through processes of sequestration and resensitization and downregulation, modulated by an effect on receptor gene expression. The degree of receptor desensitization appears to differ, depending on the cell or tissue type, and is reflected in the different profiles of clinical tolerance to chronic beta2-agonist therapy. A number of polymorphisms of the beta2-receptor have been described that appear to alter the behavior of the receptor following agonist exposure. These include Arg-Gly 16, Glu-Gln 27, and Thr-lle 164. The Gly 16 receptor downregulates to a greater extent and is associated with increased airway hyperreactivity, nocturnal symptoms, and more severe asthma. The Glu 27 form appears to protect against downregulation and is associated with less reactive airways. An individual can be homozygous or heterozygous for given polymorphisms, and large populations will have to be studied to determine their importance to the asthma phenotype.
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PMID:The beta-adrenoceptor. 981 38

A recent study showed that the novel pituitary adenylate cyclase-activating peptide (PACAP)-27 analogue [Arg15,20,21,Leu17]-PACAP-27-Gly-Lys-Arg-NH2 causes sustained airway smooth muscle relaxation in vitro. This study examined whether this analogue also has bronchoprotective effects, by inhibiting muscarinic airway responsiveness in vivo. Total lung resistance was measured in anaesthetized, tracheostomized and ventilated guinea-pigs. Increasing doses of acetylcholine were given i.v. once before and thereafter repeatedly each hour after intratracheal instillation of either the PACAP-27 analogue or the clinical beta2-agonist bronchodilator salbutamol. Mean arterial blood pressure (MAP) was monitored to detect cardiovascular side-effects. Both the PACAP-27 analogue and salbutamol significantly attenuated the airway responsiveness to acetylcholine. The total inhibitory effect of the PACAP-27 analogue (350 nmol) corresponded to that of salbutamol (35 nmol). The inhibitory effect of salbutamol (35 nmol) peaked during the second hour and disappeared prior to 5 h after administration. In contrast, the corresponding effect of the analogue (350 nmol) gradually increased and peaked during the fifth hour after administration, whereas it did not fade during the observation period. Both the PACAP-27 analogue (350 nmol) and salbutamol (35 nmol) produced a transient decrease in MAP within 6 min after administration. In conclusion, the novel pituitary adenylate cyclase-activating peptide-27 analogue has bronchoprotective properties, by decreasing muscarinic airway responsiveness in guinea pigs in vivo. The time course of its effect is compatible with a more sustained duration of action compared with salbutamol.
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PMID:Effect of a novel PACAP-27 analogue on muscarinic airway responsiveness in guinea-pigs in vivo. 986 97

In the present study, the functional significance of the intracellular C-terminal loop of the mu-opioid receptor in activating Gi proteins was determined by constructing a C-terminal deletion mutant mu(C delta 45) receptor, which lacks the carboxyl 45 amino acids. When the truncated mu(C delta 45) receptor was stably expressed in human embryonic kidney (HEK) 293 cells, the efficacy and the potency of [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO), a specific mu-opioid receptor agonist, to inhibit forskolin-stimulated adenylate cyclase activity were not significantly affected. Similar to other G-coupled receptors, the third cytoplasmic loop of the mu-opioid receptor contains conserved basic residues (R276/R277/R280) at the C-terminal segment. Mutating these basic residues to neutral amino acids (L276/M277/L280) greatly impaired the ability of DAMGO to inhibit forskolin-stimulated cyclic AMP formation. Replacing R276/R277 with L276/M277 did not affect the efficacy and potency by which DAMGO inhibits the adenylate cyclase activity. In HEK 293 cells stably expressing mutant (R280L) mu-opioid receptors, the ability of DAMGO to inhibit forskolin-stimulated cyclic AMP production was greatly reduced. These results suggest that the intracellular carboxyl tail of the mu-opioid receptor does not play a significant role in activating Gi proteins and that the arginine residue (R280) at the distal third cytoplasmic loop is required for Gi activation by the mu-opioid receptor.
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PMID:A conserved arginine in the distal third intracellular loop of the mu-opioid receptor is required for G protein activation. 1003 4

Differences in the specificity of coupling of delta-opioid receptor with G-protein have been reported in the literature. We have observed a differential desensitization of delta-opioid receptors, endogenously expressed in the neuroblastoma cell line SK-N-BE, induced by peptide and alkaloid agonists. By combining photoaffinity labelling of receptor-activated G-proteins with [alpha-(32)P]azidoanilide-GTP and an anti-sense oligodeoxynucleotide strategy, we examined whether the chemical nature of opioid agonists, alkaloid or peptide, has a critical role in determining a G(i)alpha/G(o)alpha-protein-selective activation by the human delta-opioid receptors. Etorphine, a non-selective alkaloid agonist, was shown to stimulate the incorporation of [alpha-(32)P]azidoanilide-GTP into G(i)alpha1, G(i)alpha2, G(i)alpha3 and pertussis-toxin-insensitive Galpha subunits. In contrast, [d-Pen(2),d-Pen(5)]enkephalin (DPDPE; Pen is penicillamine) and Tyr-d-Ala-Phe-Asp-Val-Val-Gly-NH(2) (deltorphin I), selective peptide agonists, mainly activated G(i)alpha2 and G(o)alpha2 subunits. The 'knock-down' of G(o)alpha2 subunits by anti-sense oligodeoxynucleotides selectively decreased the inhibition of adenylate cyclase induced by DPDPE and deltorphin I, whereas anti-sense oligodeoxynucleotides directed against G(i)alpha2 subunits only decreased the potency of etorphine in inhibiting cAMP accumulation. These results suggest that the nature of the agonist, peptide or alkaloid is critical in determining the interaction between human delta-opioid receptors and Galpha subunits.
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PMID:Differential G-protein activation by alkaloid and peptide opioid agonists in the human neuroblastoma cell line SK-N-BE. 1043 2

Prior exposure (15 min at 37 degrees C) of several cell types (Vero, SH-SY5Y neuroblastoma, human intestinal epithelial T84) to 3 mm N-benzoyloxycarbonyl-Gly-Phe-amide (Cbz-Gly-Phe-NH(2)), a competitive substrate for metalloendoproteases, completely suppressed cholera toxin (CT)-induced intracellular cAMP accumulation. The specificity of the inhibitory effect was demonstrated by the complete lack of effect of the dipeptide Cbz-Gly-Gly-NH(2), an inactive analogue of Cbz-Gly-Phe-NH(2). The effect was reversible and dose- (IC(50) as low as 0.2 mm depending on the cell type) and time-dependent. Adding Cbz-Gly-Phe-NH(2) during the lag phase caused a diminution of its inhibitory effect similar to that observed with brefeldin A (BFA). Whereas the dipeptide completely suppressed the CT-induced adenylate cyclase (AC) activity, a direct effect on AC is unlikely since the elevation of intracellular cAMP by forskolin was only slightly reduced. The A(1) peptide of CT and NAD(+) activated the AC to the same extent in membranes from control and Cbz-Gly-Phe-NH(2)-treated cells or when Cbz-Gly-Phe-NH(2) was added directly to the assay. The inhibitory effects of suboptimal amounts of Cbz-Gly-Phe-NH(2) and BFA were not additive pointing to a similar mode of action of the two substances. However, Madin-Darby canine kidney cells of which the Golgi structure is BFA-resistant were not resistant to the inhibitory action of Cbz-Gly-Phe-NH(2) on CT cytotoxicity. Several lines of evidence indicate that a perturbation of intracellular Ca(2+) homeostasis by Cbz-Gly-Phe-NH(2) is not responsible for the inhibitory effect of the dipeptide. The dipeptide had also no effect on the binding of (125)I-CT to cells and even increased its intracellular internalization. In contrast with BFA, Cbz-Gly-Phe-NH(2) did not completely suppress the formation of the catalytically active A(1) fragment from bound CT. The data are compatible with a role of metalloendoprotease activity in the intracellular trafficking and processing of CT, although other mechanisms of action of Cbz-Gly-Phe-NH(2) cannot be excluded.
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PMID:A dipeptide metalloendoprotease substrate completely blocks the response of cells in culture to cholera toxin. 1083 1

The human VPAC(1) receptor for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide belongs to the class II family of G-protein-coupled receptors with seven transmembrane segments. Like for all class II receptors, the extracellular N-terminal domain of the human VPAC(1) receptor plays a predominant role in peptide ligand recognition. To determine the three-dimensional structure of this N-terminal domain (residues 1-144), the Protein Data Bank (PDB) was screened for a homologous protein. A subdomain of yeast lipase B was found to have 27% sequence identity and 50% sequence homology with the N-terminal domain (8) of the VPAC(1) receptor together with a good alignment of the hydrophobic clusters. A model of the N-terminal domain of VPAC(1) receptor was thus constructed by homology. It indicated the presence of a putative signal sequence in the N-terminal extremity. Moreover, residues (Glu(36), Trp(67), Asp(68), Trp(73), and Gly(109)) which were shown to be crucial for VIP binding are gathered around a groove that is essentially negatively charged. New putatively important residues for VIP binding were suggested from the model analysis. Site-directed mutagenesis and stable transfection of mutants in CHO cells indicated that Pro(74), Pro(87), Phe(90), and Trp(110) are indeed important for VIP binding and activation of adenylyl cyclase activation. Combination of molecular modeling and directed mutagenesis provided the first partial three-dimensional structure of a VIP-binding domain, constituted of an electronegative groove with an outspanning tryptophan shell at one end, in the N-terminal extracellular region of the human VPAC(1) receptor.
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PMID:The human VPAC1 receptor: three-dimensional model and mutagenesis of the N-terminal domain. 1112 60

The peptide apelin, recently isolated from bovine stomach tissue extracts, has been identified as an endogenous ligand of the human putative receptor protein related to the angiotensin receptor AT(1) (APJ). In this article, we report cloning of the rat apelin receptor cDNA. The sequence shares 90% identity with the human APJ receptor and 31% with the rat AT(1A) angiotensin receptor. Subsequently a stable CHO cell line expressing the receptor fused at its C-terminal part with the enhanced green fluorescent protein (EGFP) was established, allowing to verify its cell surface distribution and to determine the affinity of various apelin and angiotensin fragments on the cloned receptor. As shown for the human APJ receptor, the rat apelin receptor expressed in the cell line was negatively coupled to adenylate cyclase. The apelin fragment K17F (Lys(1)-Phe-Arg-Arg-Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe(17)) inhibited forskolin-stimulated cAMP production at sub-nanomolar concentrations whereas angiotensin II and angiotensin III were inactive. N-terminal elongation of K17F with a tyrosine or the N-terminal deletion of the first four amino acids did not modify the inhibitory action of K17F on cAMP production. In contrast, deletion of the first seven amino acids of K17F or substitution of phenylalanine by an alanine residue at the C-terminus completely abolished the activity of the peptide. In situ hybridization analysis of apelin receptor mRNA expression in the adult rat brain showed intense labeling in the hypothalamus, especially in the supraoptic and the paraventricular nuclei. The anterior and intermediate lobes of the pituitary were also highly labeled, as well as the pineal gland. Labeling was also found in extrahypothalamic structures such as the piriform cortex, the nucleus of the lateral olfactory tract, the central grey matter, the pars compacta of the substantia nigra, the dorsal raphe nucleus, the entorhinal cortex, the dentate gyrus and the Ammon's horn. The hypothalamic and hypophyseal distribution of the receptor suggests an involvement of apelin in the control of neuro- and adenohypophyseal hormone release, whereas its presence in the pineal gland and in discrete higher brain structures points out to possible roles in the regulation of circadian rhythms and of water and food intake behavior.
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PMID:Cloning, pharmacological characterization and brain distribution of the rat apelin receptor. 1114 23

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) binding to their common receptor stimulates second messenger accumulation, receptor phosphorylation, and internalization. LLC-PK(1) cells expressing a green fluorescent protein-tagged PTH/PTHrP receptor show time- and dose-dependent receptor internalization. The internalized receptors colocalize with clathrin-coated pits. Internalization is stimulated by PTH analogs that bind to and activate the PTH/PTHrP receptor. Cell lines expressing a mutant protein kinase A regulatory subunit that is resistant to cAMP and/or a mutant receptor (DSEL mutant) that does not activate phospholipase C internalize their receptors normally. In addition, internalization of the wild-type receptor and the DSEL mutant is stimulated by the PTH analog [Gly(1),Arg(19)]hPTH-(1-28), which does not stimulate phospholipase C. Forskolin, IBMX, and the active phorbol ester, phorbol-12-myristate-13-acetate, did not promote receptor internalization or increase PTH-induced internalization. These data indicate that ligand-induced internalization of the PTH/PTHrP receptor requires both ligand binding and receptor activation but does not involve stimulation of adenylate cyclase/protein kinase A or phospholipase C/protein kinase C.
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PMID:Parathyroid hormone receptor internalization is independent of protein kinase A and phospholipase C activation. 1150 Mar 10

The structural gene for anthrax edema factor (EF) was expressed in Escherichia coli under the control of a powerful T5 promoter to yield the 89-kDa recombinant protein that reacted with anti-EF antibodies. Recombinant EF was purified to homogeneity by a two-step procedure involving metal chelate affinity chromatography and cation-exchange chromatography. From 1 liter of culture, 2.5 mg of biologically active EF was easily purified. This is the first report of purification of anthrax EF from E. coli. EF purified from E. coli was biologically and functionally as active as its Bacillus anthracis counterpart. The recombinant protein could compete with lethal factor for binding to protective antigen. Sequence analysis revealed a stretch of seven amino acids, Val Tyr Tyr Glu Ile Gly Lys, present both in EF (residues 136 to 142) and lethal factor (residues 147 to 153). To investigate the role of these seven residues in binding to protective antigen, the residues were individually mutated to alanine in EF. Mutations in residues Tyr137, Tyr138, Ile140, and Lys142 of EF specifically blocked its interaction with anthrax protective antigen. The adenylate cyclase activity of the mutants remained unaffected. The results suggested that residues Tyr137, Tyr138, Ile140, and Lys142 are required for binding of EF to anthrax protective antigen, which facilitates its entry into susceptible cells.
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PMID:Purification of anthrax edema factor from Escherichia coli and identification of residues required for binding to anthrax protective antigen. 1155 1


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