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)

To assess whether receptor binding is sufficient to initiate vasopressin receptor endocytosis in cells expressing the vasopressin V1 or V2 receptors, we synthesized a novel fluorescent-labeled vasopressin analog, [1-(beta-mercapto-beta, beta-cyclopentamethylene propionic acid), 2-(O-ethyl)-D-tyrosine, 4-valine, 8-lysine-N6-carboxytetramethylrhodamine] vasopressin (R-CLVP), that binds to vasopressin receptors but does not activate intracellular events such as the mobilization of intracellular calcium or the activation of adenylate cyclase. We compared the manner in which this analog was endocytosed in cells expressing V1 (A-10, rat smooth muscle cells) or V2 (LLC-PK1, porcine kidney cells) receptors with that of a full agonist, [1-(beta-mercaptopropionic acid), 8-lysine-N6-carboxytetramethylrhodamine] vasopressin (R-MLVP) [Lutz et al. (1990) J. Biol. Chem. 265, 4657-4663; Lutz et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87,6507-6511]. We showed that R-CLVP bound to both types of receptors with good affinity. It failed to increase cyclic AMP concentrations in LLC-PK1 cells and did not increase the mobilization of intracellular calcium in A-10 cells. It bound to the surface of both these cell types in a diffuse manner and it did not undergo receptor endocytosis in either cell type. In contrast, R-MLVP, an agonist that bound to both receptor subtypes and elicited changes in intracellular cyclic AMP and calcium, bound to the surface of these cells in a diffuse manner at early times after exposure, and rapidly underwent endocytosis. We conclude that binding of vasopressin to its receptors alone is insufficient to cause receptor endocytosis, and other events distal to the receptor are required to initiate endocytosis. R-CLVP should be a useful analog in determining the factors responsible for initiating receptor endocytosis.
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PMID:A vasopressin analog that binds but does not activate V1 or V2 vasopressin receptors is not internalized into cells that express V1 or V2 receptors. 130 61

Albright's hereditary osteodystrophy is an autosomal dominant disorder characterized by a short stature, brachydactyly, subcutaneous ossifications, and reduced expression or function of the alpha subunit of the stimulatory G protein (Gs alpha) of adenylate cyclase, which is necessary for the action of parathyroid and other hormones that use cyclic AMP as an intracellular second messenger. We identified a unique Gs alpha protein in erythrocytes from two related patients with Albright's hereditary osteodystrophy and reduced Gs alpha bioactivity. The Gs alpha variant was recognized by a carboxyl terminal-specific Gs alpha antiserum but not by polyclonal antiserums specific for the amino terminus of Gs alpha. To investigate the molecular basis for this structurally abnormal Gs alpha protein, we studied the Gs alpha gene by restriction-endonuclease analysis. DNA from the two patients had an abnormal restriction-fragment pattern when digested with Ncol, which was consistent with loss of an Ncol restriction site in exon 1 of one Gs alpha allele. Amplification of a 260-base-pair region that includes exon 1 of the Gs alpha gene and direct sequencing of the amplified DNA revealed an A-to-G transition at position +1 in one Gs alpha allele from each of the two patients. This mutation converts the initiator ATG (methionine) codon to GTG (valine), blocking initiation of translation at the normal site. Translation of the abnormal Gs alpha messenger RNA would result in the synthesis of a truncated Gs alpha molecule lacking the amino terminus. We conclude that in at least some patients with Albright's hereditary osteodystrophy, the disease is caused by a single-base substitution in the Gs alpha gene and is thus due to an inherited mutation in a human G protein.
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PMID:Mutation in the gene encoding the stimulatory G protein of adenylate cyclase in Albright's hereditary osteodystrophy. 210 29

A truncated, 432 residue long, Bordetella pertussis adenylate cyclase expressed in Escherichia coli was analyzed for intrinsic fluorescence properties. The two tryptophans (Trp69 and Trp242) of adenylate cyclase, each situated in close proximity to residues important for catalysis or binding of calmodulin (CaM), produced overlapping fluorescence emission bands upon excitation at 295 nm. CaM, alone or in association with low concentrations of urea, induced important modifications in the spectra of adenylate cyclase such as shifts of the maxima and change in the shape of the bands. From these changes and from the fluorescence spectrum of a modified form of adenylate cyclase, in which a valine residue was substituted for Trp242, it was deduced that, upon binding of CaM to the wild-type adenylate cyclase, only the environment of Trp242 was affected. The fluorescence maximum of this residue, which is more exposed to the solvent than Trp69 in the absence of CaM, is shifted by 13 nm to shorter wavelength upon interaction of protein with its activator. Trypsin cleaved adenylate cyclase into two fragments, one carrying the catalytic domain, and the second carrying the CaM-binding domain (Ladant et al., 1989). The isolated peptides conserved most of the environment around their single tryptophan residues, as in the intact adenylate cyclase, which suggests that the two domains of truncated B. pertussis adenylate cyclase also conserved most of their three-dimensional structure in the isolated forms.
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PMID:Intrinsic fluorescence of a truncated Bordetella pertussis adenylate cyclase expressed in Escherichia coli. 226 68

Radioligand binding and functional assays were employed to demonstrate the existence of somatostatin receptors in the murine neuroblastoma clone N1E-115. Saturation experiments with [125I][Tyr11]somatostatin-14 indicated the presence of a single class of binding sites in membranes prepared from N1E-115 cells (Kd = 83 pM; Bmax = 21,000 receptors/cell). Somatostatin-14, somatostatin-28 and L363586 (cyclo(N-Me-ALA-TYR-D-TRP-LYS-VAL-PHE] all displaced the 125I-ligand monophasically in N1E-115 cells (Ki values were 28, 82 and 34 pM, respectively), which contrasted with the binding heterogeneity apparent with L363586 in rat brain membranes. The binding of [125I][Tyr11]somatostatin-14 was reduced by GppNHp, indicating that N1E-115 somatostatin receptors interacted with guanine nucleotide binding protein(s). Somatostatin agonists decreased by 30-50% the levels of [3H]cyclic AMP induced in intact cells by forskolin, prostaglandin E1, or vasoactive intestinal polypeptide. The EC50 values for inhibition of the [3H]cyclic AMP response to PGE1 by L363586, somatostatin-14, and somatostatin-28 were 0.24, 0.63 and 1.0 nM, respectively. Pertussis toxin treatment of N1E-115 cells reduced both binding to the receptor and the functional response to somatostatin-14. These data suggest that a single class of somatostatin receptors in N1E-115 cells are linked to the inhibition of adenylate cyclase through a Gi protein.
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PMID:Biochemical evidence for somatostatin receptors in murine neuroblastoma clone N1E-115. 256 62

Neurohypophysial hormones stimulate the motility of tunica albuginea, epididymis, and vas deferens acting through oxytocin (OT) and V1 vasopressin receptors. To test the hypothesis that these hormones are involved also in the regulation of seminal vesicle physiology, we studied binding of [3H]OT and [3H] arginine vasopressin ([3H]AVP) to porcine seminal vesicle membranes. Neurohypophysial hormones bind to two different classes of sites. The first class shows low capacity (35 fmol per mg of protein) and a very high affinity (Kd less than 1 nM) for both the labeled ligands. The second class is characterized by a high capacity (2000 fmol per mg of protein) and a high affinity for AVP (Kd approximately equal to 2.5 nM), whereas OT has 160 times lower affinity. Lysine vasopressin and the V1 antagonist [1-deaminopenicillamine, 2-(O-methyl)tyrosine]Arg8-vasopressin compete with high affinity with [3H]AVP binding, whereas the V2 agonist [1-deamino,4-valine]D-Arg8-vasopressin (dVDAVP) is 110 times less potent than AVP. The OT agonist [Thr4,Gly7]OT and the OT antagonist [1(beta-mercapto-beta, beta-cyclopentamethylene propionic acid), 2-(O-ethyl)tyrosine, 8-ornithine]vasotocin failed to affect [3H]AVP binding. These findings seem to suggest that AVP interacts with the V1 vasopressin isoreceptor in porcine seminal vesicle membranes. However, AVP stimulates adenylate cyclase activity in a dose-dependent fashion with an EC50 of 14 nM, whereas OT or dVDAVP has no effect at 100 nM. Moreover, a well-characterized V1 vasopressin antagonist, [1-(beta-mercapto-beta, beta-cyclopentamethylene propionic acid),2-(O-methyl)tyrosine]Arg8-vasopressin [d(CH2)5Tyr(Me)AVP], competes with [3H]AVP binding with an IC50 of 0.17 microM. These pharmacological properties are distinct from the previously described V1 and V2 vasopressin receptors and indicate the presence of a new class of AVP receptors. Although this vasopressin isoreceptor shares some pharmacological characteristics with the V1 (pressor) isoreceptor, it has low affinity for the V1 antagonist d(CH2)5-Tyr(Me)AVP and is linked to the adenylate cyclase system. The extremely high density of AVP receptors in porcine seminal vesicles (2 pmol per mg of protein) is comparable to the density of V2 vasopressin receptors in porcine renal medulla, suggesting a physiological role for vasopressin in the seminal vesicle.
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PMID:Identification and characterization of a vasopressin isoreceptor in porcine seminal vesicles. 294 37

We studied the effect of an arginine vasopressin (AVP) analogue, (1-[beta-mercapto-beta, beta-cyclopentamethylenepropionic acid],2-O-ethyltyrosine, 4-valine)AVP(d[CH2]5Tyr[Et]VAVP), on the stimulation of adenylate cyclase by various hormones in the isolated nephron segments and 3H-AVP binding to renal papillary membranes from the rat. The net water flux across the renal cortical collecting tubules of the rabbit was also examined. We found that d(CH2)5Tyr(Et)VAVP significantly inhibited adenylate cyclase activation by AVP in cortical, medullary, and papillary collecting tubules and in the medullary thick ascending limb. In contrast, the AVP analogue did not alter the stimulation of adenylate cyclase by parathyroid hormone in the cortical thick ascending limb, by glucagon in the medullary thick ascending limb, and by calcitonin in cortical collecting tubules. In addition, d(CH2)5Tyr(Et)VAVP blocked [3H]AVP binding to renal papillary membranes. The enhanced net water transport induced by AVP in isolated, perfused rabbit cortical collecting tubules also was completely blocked by this AVP analogue. These results indicate that d(CH2)5Tyr(Et)VAVP specifically antagonizes the cellular action of AVP on the medullary thick ascending limb and on the cortical, medullary, and papillary collecting tubules. Evidence is also presented for competitive antagonism as the cellular mechanism of action.
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PMID:Effects of vasopressin antagonist on vasopressin binding, adenylate cyclase activation, and water flux. 299 81

Beta adrenergic receptor agonists and forskolin stimulated cyclic AMP (cAMP) accumulation in cultured rat aortic smooth muscle cells (A-10). Furthermore, these cells display a high density of vasopressin receptors of the vascular (V1) subtype. Addition of vasopressin to these cells inhibited beta adrenergic agonist- and forskolin-stimulated cAMP accumulation by 30 to 40% and by 25 to 35%, respectively. The extent of inhibition was dependent on the concentration of vasopressin used. Half-maximal inhibition of cAMP accumulation by isoproterenol occurred at 8 X 10(-10) M vasopressin. Basal cAMP levels were not affected. The inhibition by arginine vasopressin was mediated by V1 receptors because the V2 renal receptor subtype selective agonists (1-deamino, 8-D-arginine)vasopressin and (1-deamino,4-valine,8-D-arginine)vasopressin were ineffective. Of the antagonists tested, the V1-selective antagonist [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid),2-(O-methyl)tyrosine,8-arginine]vasopressin was more potent than the mixed V1/V2 antagonist [1-beta-mercapto--beta, beta-cyclopentamethylenepropionic acid), 2-D-(O-ethyl)tyrosine,4-valine 8-arginine]vasopressin. The V2-selective antagonist [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid),2-D-isoleucine,4-valine,8-arginine]vasopressin displayed minimal ability to block the vasopressin-mediated inhibitory effect. These data demonstrate that in rat aortic smooth muscle cells V1 receptors are negatively coupled to adenylate cyclase. The studies presented suggest that the vasoconstrictor activity of vasopressin might involve inhibition of beta adrenergic receptor-mediated vascular relaxation through inhibition of cAMP accumulation.
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PMID:Vascular vasopressin receptors mediate inhibition of beta adrenergic receptor-induced cyclic AMP accumulation. 300 35

The ligand specificity of rat adenohypophyseal vasopressin receptors was directly compared to that of peripheral receptors of the V1 and V2 types. For this purpose a series of 15 recently designed vasopressin antagonists was used. The affinities of these antagonists for rat adenohypophyseal membranes were deduced from the determination of the concentration-dependent inhibition of [3H]vasopressin binding. In parallel experiments the corticotropin (or anti-corticotropin)-releasing activities of the tested peptides were determined on freshly dispersed rat adenohypophyseal cells. All peptides tested which were found to be antagonists of the vasopressor and antidiuretic responses to vasopressin in vivo behaved as antagonists of vasopressin-induced corticotropin release. There was a close correlation between the relative affinities of the analogues tested for binding to adenohypophyseal membranes and their relative potencies in inhibiting vasopressin-induced corticotropin release, indicating that the detected vasopressin-binding sites are the receptors involved in the vasopressin effect on corticotropin secretion. No correlation could be demonstrated between anti-corticotropin-releasing activities and either anti-antidiuretic or antivasopressor potencies of the antagonists tested. A direct comparison of the ligand specificities of adenohypophyseal receptors on the one hand, and V1 (hepatic) and V2 (renal) receptors on the other hand, showed that most of the antagonists discriminated very efficiently between adenohypophyseal and either hepatic or renal receptors. The selectivity index reaches values as high as 260,000 for desGly(NH2)9 [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid), 2-D-O-ethyl-tyrosine, 4-valine] arginine vasopressin. It is concluded that adenohypophyseal receptors represent a novel type of vasopressin receptors. Based on the observation that adenohypophyseal receptors, like hepatic or vascular V1 receptors, do not appear to be coupled to adenylate cyclase, we propose that adenohypophyseal receptors could be designated as V1b receptors as opposed to the V1a receptors previously characterized on liver and blood vessels.
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PMID:Vasopressin antagonists allow demonstration of a novel type of vasopressin receptor in the rat adenohypophysis. 301

Activating mutations (valine 19 or leucine 68) were introduced into the Saccharomyces cerevisiae RAS1 and RAS2 genes. In addition, a deletion was introduced into the wild-type gene and into an activated RAS2 gene, removing the segment of the coding region for the unique C-terminal domain that lies between the N-terminal 174 residues and the penultimate 8-residue membrane attachment site. At low levels of expression, a dominant activated phenotype, characterized by low glycogen levels and poor sporulation efficiency, was observed for both full-length RAS1 and RAS2 variants having impaired GTP hydrolytic activity. Lethal CDC25 mutations were bypassed by the expression of mutant RAS1 or RAS2 proteins with activating amino acid substitutions, by expression of RAS2 proteins lacking the C-terminal domain, or by normal and oncogenic mammalian Harvey ras proteins. Biochemical measurements of adenylate cyclase in membrane preparations showed that the expression of RAS2 proteins lacking the C-terminal domain can restore adenylate cyclase activity to cdc25 membranes.
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PMID:Regulatory function of the Saccharomyces cerevisiae RAS C-terminus. 330 71

To understand the molecular mechanism of action of the novel class of diuretic agents, the antidiuretic hormone antagonists ["aquaretics" (specific water-losing activity as caused by vasopressin antagonists, as distinguished from the saluresis of conventional diuretics)], in the dog studies were made of the properties of the vasopressin-responsive adenylate cyclase system and the antagonist potencies of the vasopressin analogs [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid), 2-(O-ethyl)tyrosine,4-valine,8-arginine]vasopressin; [1-(beta-mercapto-beta-mercapto-beta, beta-cyclopentamethylenepropionic acid), 2-D-phenylalanine,4-valine,8-arginine]vasopressin; and [1-(beta-mercapto-beta, beta-cyclopenta-methylenepropionic acid), 2-D-(O-ethyl)tyrosine,4-valine,8-arginine]vasopressin (SK&F 100398, 101071 and 101498, respectively) using plasma membranes prepared from cortex, medulla and papilla of dog kidney. It was observed that the greatest sensitivity for vasopressin was in the papilla (concentration of 8-arginine vasopressin required for 50% activation of adenylate cyclase [Kact] was 2.0 X 10(-9)M, 1.1 X 10(-9)M and 5.1 X 10(-10) M in the cortex, medulla and papilla, respectively). The addition of 10(-5)M GTP did not alter the Kact of the cortex but enhanced 10-fold the vasopressin sensitivity of the papilla to 5.2 X 10(-11) M. The vasopressin analogs were competitive antagonists of vasopressin-stimulated adenylate cyclase of cortex and papilla with the greatest potency for the papillary enzyme (Ki in papilla was 3.6 X 10(-9)M, 4.6 X 10(-9)M and 1.0 X 10(-9)M for SK&F 100398, 101071 and 101498, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Antidiuretic hormone antagonists and aquaresis in dogs: different vasopressin sensitivity and antagonist potency in renal cortex and papilla. 396 88


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