Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:4.6.1.1 (adenylate cyclase)
 19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have identified and characterized receptors for the amino-terminal domains of PTH and PTH-like peptide (PLP) on an immortalized human keratinocyte cell line, RHEK-1. Binding of both PLP-(1-34) and PTH-(1-34) to the RHEK-1 cells was consistent with a two-site model; affinities and capacities for each site were similar for the two peptides. Both peptides also stimulated adenylate cyclase activity with an equal ED50 in this cell line. Pertussis toxin pretreatment enhanced this peptide-mediated enzyme activity, suggesting linkage of the receptor to an inhibitory guanyl nucleotide-binding protein (Gi). Adenylate cyclase activity was diminished by both homologous [PLP-(1-34)] and heterologous [epidermal growth factor (EGF)] effectors. Malignant conversion of the immortalized cells with an activated H-ras oncogene to produce the RHEK-ras cell line was associated with a reduction in binding at both PLP/PTH and EGF receptors as well as a postreceptor defect in PLP/PTH-stimulated adenylate cyclase activity. The defect in enzyme activity appeared to be due in part to a decrease in the activity of the stimulatory guanyl nucleotide-binding protein (Gs), but not to an increase in Gi activity. Activation of the keratinocyte amino-terminal PLP/PTH receptor resulted in a small increase in [3H]thymidine incorporation, which was associated with an increase in cell numbers. This mitogenic effect was enhanced in the presence of EGF and was markedly reduced when cells were cultured in a high extracellular calcium environment. These studies demonstrate that the amino-terminal region of PLP and PTH activates adenylate cyclase-linked receptors, which are associated with mitogenesis, in RHEK-1 cells and suggest that this cell line represents a suitable model in which to examine the actions of PLP in keratinocytes.
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PMID:Identification and functional characterization of adenylate cyclase-linked receptors for parathyroid hormone-like peptides on immortalized human keratinocytes. 130 43

Many animal and in vitro experiments have shown that the supplementation of diet with vitamin E within a certain dose range reduced the risk of chemical- and radiation-induced cancers. In vitro studies revealed that alpha-tocopheryl succinate (TS) induced differentiation and growth-inhibition in certain animal and human tumor cells in culture, whereas alpha-tocopherol (alpha-T), alpha-tocopheryl acetate (alpha-TA) and alpha-tocopheryl nicotinate (alpha-TN) were ineffective, alpha-TS also reduced basal and ligand-stimulated adenylate cyclase activity, and expression of c-myc and H-ras oncogenes in certain tumor cells in culture. The relative efficacy of various forms of vitamin E in cancer prevention in animal or human models has not been evaluated. Human epidemiologic studies utilizing retrospective and prospective case-control experimental designs are not suitable for evaluating the role of vitamin E in cancer prevention due to several inherent problems associated with these methodologies. Intervention trials utilizing vitamin E with appropriate biological and statistical rationales are most suitable for testing the role of vitamin E in cancer prevention in humans. Some human trials utilizing vitamin E alone or in combination with other nutrients are in progress.
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PMID:Vitamin E and cancer prevention: recent advances and future potentials. 840 88

We have constructed mutants of the alpha subunit of Gs in an attempt to identify sites in the protein that are important for its interaction with adenylylcyclase. Some residues specific for those G proteins that activate adenylylcyclase were replaced with residues characteristic of Gi alpha. Mutant proteins were expressed in Escherichia coli, and two of these were purified to homogeneity and characterized in detail. Mutation of trp263, leu268, or arg269 caused a significant loss of the capacity of Gs alpha to stimulate adenylylcyclase, and the triple mutant had less than 1% of the ability of wild type Gs alpha to activate the enzyme. Guanine nucleotide binding and GTP hydrolysis by the mutant proteins were unaltered, as was guanosine 5'-3-O-(thio)triphosphate-induced enhancement of intrinsic tryptophan fluorescence. Mutant proteins also appeared to have a reduced affinity for the G protein beta gamma subunit complex. Secondary structure analysis and comparison with the structure of p21ras suggests that the region of Gs alpha that we have identified is part of a loop that may be involved in interaction of the protein with adenylylcyclase. Although these residues are essential for activation of adenylylcyclase, they are not sufficient to do this when placed in the context of another G protein alpha subunit.
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PMID:Expression and analysis of Gs alpha mutants with decreased ability to activate adenylylcyclase. 165 37

We previously showed that the proliferative response of a serum- and interleukin-3 (IL-3)-dependent murine myeloid cell line, NFS/N1-H7, was partially inhibited by pertussis toxin as a result of toxin-induced increased adenylate cyclase activity. In the present studies, we examined the role of the phosphoinositide cycle in the proliferative response of these cells and demonstrated that there was no change in PIP (phosphatidylinositol bisphosphate)-specific phospholipase C activity in response to IL-3 alone. However, serum caused a pertussis toxin-insensitive increase in PIP2-specific phospholipase C activity as reflected by decreased cellular levels of 32P-labelled PIP2. Proliferation of a subline selected from val-12-mutant H-ras-transfected NFS-H7 cells, clone E5, was insensitive to pertussis toxin, occurred in the absence of serum but remained serum-stimulatable and absolutely dependent on IL-3. This val-12 mutant ras-expressing cell line showed an increase in 32P-labelled PIP (phosphatidylinositol phosphate) in response to serum whereas the parent cell line did not. Membrane fractions from 32P-labelled ras-transfected cells displayed higher GTP gamma S-, GTP-, or F(-)-stimulated PIP2-specific phospholipase C activity compared to membranes from the parent cell line. Thus serum-dependence and adenylate cyclase-mediated pertussis toxin-sensitivity of the parent cell line was bypassed by val-12 mutant ras p21, possibly as a result of increased PIP2-specific phospholipase C activity.
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PMID:Expression of val-12 mutant ras p21 in an IL-3-dependent murine myeloid cell line is associated with loss of serum-dependence and increases in membrane PIP2-specific phospholipase C activity. 165 97

In Saccharomyces cerevisiae, the product of the CDC25 gene controls the RAS-mediated production of adenosine 3',5'-monophosphate (cAMP). In vivo the carboxyl-terminal third of the CDC25 gene product is sufficient for the activation of adenylate cyclase. The 3'-terminal part of SCD25, a gene of S. cerevisiae structurally related to CDC25, can suppress the requirement for CDC25. Partially purified preparations of the carboxy-terminal domain of the SCD25 gene product enhanced the exchange rate of guanosine diphosphate (GDP) to guanosine triphosphate (GTP) of pure RAS2 protein by stimulating the release of GDP. This protein fragment had a similar effect on the human c-H-ras-encoded p21 protein. Thus, the SCD25 carboxyl-terminal domain can enhance the regeneration of the active form of RAS proteins.
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PMID:Enhancement of the GDP-GTP exchange of RAS proteins by the carboxyl-terminal domain of SCD25. 218 57

Injection of human H-ras protein induces maturation of Xenopus oocytes; that is, progression from prophase to metaphase of meiosis. The oncogenic protein encoded by H-rasval12 is nearly a 100-fold more potent than the protein encoded by the wild-type gene. We do not observe any measurable increase or decrease in cyclic AMP concentration in injected oocytes, and the effects of H-ras protein are only partially blocked by cholera toxin. Our results suggest that not all, if any, of the effects of H-rasval12 protein in this system are mediated by adenylate cyclase.
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PMID:ras proteins can induce meiosis in Xenopus oocytes. 241 66

Transducin, the GTP-binding protein of the retinal light-sensitive phosphodiesterase system, and Gs and Gi, regulatory proteins of the hormone-sensitive adenylate cyclase, are members of a family of guanyl nucleotide-binding proteins termed G proteins that are important in signal transduction. To probe relationships within this family of G proteins, monoclonal antibodies were prepared against the alpha-subunit of bovine transducin (T alpha). Three of four monoclonal antibodies were specific for T alpha and did not cross-react with other G proteins. One, MAB1, cross-reacted strongly with the alpha-subunit of Gi (Gi alpha) purified from rabbit liver and, to a lesser extent, with the alpha-subunit of Go (Go alpha) purified from bovine brain and the proto-oncogene product H-ras p21. All four monoclonal antibodies recognized epitopes on a 23-kDa tryptic peptide fragment of T alpha which is derived from the N-proximal region. The three monoclonal antibodies that recognized only T alpha inhibited rhodopsin-stimulated GTP binding and hydrolysis by transducin, whereas MAB1 had no significant effect in these assays. These studies demonstrate that, within the 23-kDa tryptic peptide of T alpha, there is a domain(s) unique to T alpha that is involved in GTP binding and hydrolysis and another domain which is highly conserved in T alpha and to a lesser extent in other G proteins. Prior studies have identified regions involved in nucleotide binding and hydrolysis that are homologous in all G proteins. The observations reported here are consistent with the conclusion that the G proteins may have in addition unique regions involved in these functions.
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PMID:Structural and functional characterization of guanyl nucleotide-binding proteins using monoclonal antibodies to the alpha-subunit of transducin. 242 38

We report a class of interfering mutants of the human H-ras gene capable of inhibiting phenotypes arising from the expression of the activated RAS2 gene, RAS2val19, in the yeast Saccharomyces cerevisiae. All these mutants encode unprocessed H-ras proteins that remain in the cytoplasm. One of the mutants, H-rasarg186, was examined in detail. H-rasarg186 protein is a competitive inhibitor of RAS2val19 protein. It does not interfere with processing and membrane localization of RAS2val19, nor does it appear to compete with RAS protein for its proposed regulator, the CDC25 protein. By several criteria the RAS2val19 adenylate cyclase interaction is unaffected by H-rasarg186. We infer from our results that H-rasarg186 protein interferes with an alternative function of RAS2val19.
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PMID:Mutants of H-ras that interfere with RAS effector function in Saccharomyces cerevisiae. 268 34

The c-Ha-ras oncogene has been implicated as a causative agent in the development of tumors in humans as well as mice. The molecular nature of the ras-induced tumorigenic process remains unclear, however. To address this question directly we have constructed a cell line which carries a zinc-inducible metallothionein-ras hybrid oncogene, transformant 212. Upon exposure to zinc for 24-48 hr, 212 cells assume a highly transformed morphology, concomitant with the induction of ras-expression. Natural killer cells constitute a subpopulation of lymphoid effector cells which have for a long time been hypothesized to be involved in the earliest stages of antitumor surveillance. Central to this hypothesis is the prediction that NK sensitivity arises during cellular transformation. By carrying out cytotoxicity assays against the 212 transformant, we showed that, indeed, increased sensitivity to NK-mediated lysis correlated with expression of the ras oncogene, which is consistent with the above hypothesis. We then addressed the question of the biochemical mechanism of ras-induced transformation. Owing to their similarity to G proteins, regulatory elements interposed between cell-surface receptors and their effector enzymes, it has been postulated that p21, the ras oncogene protein, mediates its transforming effects by constitutive activation of proliferative signal transduction pathways. We studied the effect of ras expression on the regulation of adenylate cyclase (A.C.), key enzyme of one such major pathway. We found that ras expression correlated with a dampening of responsiveness of A.C. to several stimuli, including hormones such as isoproterenol and other agents such as GMP-PNP, forskolin and fluoride-ion. Accumulation of cAMP as measured by RIA in intact cells, as basal or in response to stimulation of A.C. activity with forskolin, was also decreased (approximately 10-fold) with ras expression. Because the regulation of calcium, another important second messenger is dependent, in part, upon cAMP and GTP-binding proteins, we investigated the possible influence of ras expression on the intracellular concentration of calcium. Steady-state intracellular free Ca2+ concentration, as measured by fluorimetry, was indeed increased by approximately 50-125% in association with ras expression. Finally, we studied the possible influence of p21ras on protein kinase C (PKC), which is a key enzyme in the important signal transduction pathway of phosphatidylinositol lipid turnover. We assessed PKC activity directly, in a cell-free system, by measuring the ability of the enzyme to transfer radiolabelled phosphate from gamma-32P-ATP to histone, and exogenous substrate.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The cell biology of ras-induced transformation: insights from studies utilizing an inducible hybrid oncogene system. 284 54

Balb/3T3 murine fibroblasts transformed by transfection with the EJ/T24 human bladder carcinoma oncogene were assayed in terms of adenylate cyclase response and hydrolysis of polyphosphoinositides dependent on specific agents. Transformed cells were much less responsive to beta-adrenergic agonists in rising cAMP than normal cells. They are instead much more sensitive to muscarinic receptor agonists, inducing a rapid intracellular accumulation of inositol phosphates. These results suggest that the functional alteration of the cell membrane caused by the product of the point mutated H-ras oncogene concerns in 3T3 fibroblasts both inhibitory and stimulatory effects, respectively on adenylate cyclase and phosphoinositide-phosphodiesterase.
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PMID:Transformation of BALB/3T3 cells with EJ/T24/H-ras oncogene inhibits adenylate cyclase response to beta-adrenergic agonist while increases muscarinic receptor dependent hydrolysis of inositol lipids. 286 65


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