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)

Mammalian ras genes substitute for the yeast RAS gene, and their products activate adenylate cyclase in yeast cells, although the direct target protein of mammalian ras p21s remains to be identified. ras p21s undergo posttranslational processing, including prenylation, proteolysis, methylation, and palmitoylation, at their C-terminal regions. We have previously reported that the posttranslational processing of Ki-ras p21 is essential for its interaction with one of its GDP/GTP exchange proteins named smg GDS. In this investigation, we have studied whether the posttranslational processing of Ki- and Ha-ras p21s is critical for their stimulation of yeast adenylate cyclase in a cell-free system. We show that the posttranslationally fully processed Ki- and Ha-ras p21s activate yeast adenylate cyclase far more effectively than do the unprocessed proteins. The previous and present results suggest that the posttranslational processing of ras p21s is important for their interaction not only with smg GDS but also with the target protein.
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PMID:The posttranslational processing of ras p21 is critical for its stimulation of yeast adenylate cyclase. 140 40

GDP-dissociation stimulators (GDSs) are the key element for the regeneration of the active state of ras proteins, but despite intensive investigations, little is so far known about their functional and structural properties, particularly in mammals. A growing number of genes from various organisms have been postulated to encode GDSs on the basis of sequence similarity with the Saccharomyces cerevisiae CDC25 gene, whose product acts as a GDS of RAS proteins. However, except for CDC25 and the related SDC25 C-domain, no biochemical evidence of ras GDS activity for these CDC25-like proteins has yet been available. We show that the product of a recently isolated mouse CDC25-like gene (CDC25Mm) can strongly enhance (more than 1000 times) the GDP release from both human c-Ha-ras p21 and yeast RAS2 in vitro. As a consequence, the CDC25Mm induces a rapid formation of the biologically active Ras.GTP complex. This GDS is much more active on the GDP than on the GTP complex and has a narrow substrate specificity, since it was found to be inactive on several ras-like proteins. The mouse GDS can efficiently substitute for yeast CDC25 in an in vitro adenylylcyclase assay on RAS2 cdc25 yeast membranes. Our results show that a cloned GDP to GTP exchange factor of mammalian ras belongs to the novel family of CDC25-like proteins.
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PMID:A mouse CDC25-like product enhances the formation of the active GTP complex of human ras p21 and Saccharomyces cerevisiae RAS2 proteins. 144 67

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

The Saccharomyces cerevisiae ras-like gene RSR1 is particularly closely related to the mammalian gene Krev-1 (also known as smg21A and rap1A). RSR1 was originally isolated as a multicopy suppressor of a cdc24 mutation, which causes an inability to bud or establish cell polarity. Deletion of RSR1 itself does not affect growth but causes a randomization of bud position. We have now constructed mutant alleles of RSR1 encoding proteins with substitutions of Val for Gly at position 12 (analogous to constitutively activated Ras proteins) or Asn for Lys at position 16 (analogous to a dominant-negative Ras protein). rsr1Val-12 could not restore a normal budding pattern to an rsr1 deletion strain but could suppress a cdc24 mutation when overexpressed. rsr1Asn-16 could randomize the budding pattern of a wild-type strain even in low copy number but was not lethal even in high copy number. These and other results suggest that Rsr1p functions only in bud site selection and not in subsequent events of polarity establishment and bud formation, that this function involves a cycling between GTP-bound and GDP-bound forms of the protein, and that the suppression of cdc24 involves direct interaction between Rsr1p[GTP] and Cdc24p. Functional homology between Rsr1p and Krev-1 p21 was suggested by the observations that expression of the latter protein in yeast cells could both suppress a cdc24 mutation and randomize the budding pattern of wild-type cells. As Krev-1 overexpression can suppress ras-induced transformation of mammalian cells, we looked for effects of RSR1 on the S. cerevisiae Ras pathway. Although no suppression of the activated RAS2Val-19 allele was observed, overexpression of rsr1Val-12 suppressed the lethality of strains lacking RAS gene function, apparently through a direct activation of adenyl cyclase. This interaction of Rsr1p with the effector of Ras in S. cerevisiae suggests that Krev-1 may revert ras-induced transformation of mammalian cells by affecting the interaction of ras p21 with its effector.
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PMID:RSR1, a ras-like gene homologous to Krev-1 (smg21A/rap1A): role in the development of cell polarity and interactions with the Ras pathway in Saccharomyces cerevisiae. 173 42

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

Monoclonal antibody Y13-259 to ras p21 was shown to bind to the highly conserved residues in the region 63-73 and to neutralize ras action in the Saccharomyces cerevisiae adenylate cyclase system. Inhibition of adenylate cyclase activity in isolated membranes by antibody Y13-259 occurred after a lag period of 6 min. This lag corresponded to the time necessary for binding of antibody Y13-259 to the membranes in a ras-dependent manner. The mechanism of inhibition appeared to be steric in nature because antibody Y13-259 neutralized ras p21 bound to a stable GTP analogue. Monoclonal antibodies Y13-4 and Y13-128 also inhibited yeast adenylate cyclase activity, and the epitopes for both the these antibodies were localized to ras region 65-75. However, the ras residues essential for binding of antibodies Y13-4 and Y13-128 to ras p21 (positions 65, 66, 68 and 75) were different from those essential for binding of antibody Y13-259 (positions 63, 65, 66, 67, 70 and 73). These results indicate that residues 63-75 constitute a major neutralizing epitope on ras p21.
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PMID:Inhibition of yeast adenylate cyclase by antibodies to ras p21. 245 12

It is well known that many of thyroid carcinoma are capable of responding to TSH, but our studies shown that there are some alteration in this responsiveness. The adenylate cyclase responsiveness to TSH was usually greater in thyroid carcinoma than in adjacent histologically normal thyroid tissue. The level of increased response of adenylate cyclase were correlated with the level of enhanced expression of ras oncogene product p21 assessed by Western blotting analysis. The TSH induced desensitization of adenylate cyclase was not observed in some differentiated carcinoma. This loss of desensitization may be reflect the change in ADP-ribosylable Gi protein. In the differentiated carcinoma, the capacity of EGF receptor was higher than that in normal thyroid. The EGF binding to cultured carcinoma cells did not increase in response to TSH. These altered properties of transmembrane control in human thyroid carcinoma may be related to the neoplastic growth.
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PMID:[Transmembrane controls in cultured human thyroid carcinoma]. 256 2

Leukemic cell growth in the marrow microenvironment may be modulated by stromal cell products, including stimulatory growth factors and the inhibitory regulator prostaglandin E. The production of both of these stromal cell products induced by cytokine mediators appears to be closely linked. Cyclic AMP (cAMP) is an intracellular second messenger that inhibits myeloid cell proliferation and is produced in myeloid leukemia cells on stimulation of adenylate cyclase enzyme by prostaglandin E1 (PGE1). Cells expressing the product of an RAS oncogene have been observed to display diminished hormone-stimulated adenylate cyclase of membranes. If this observation were applicable to myeloid cells, a potentially important mode for leukemia cells expressing p21 RAS to escape inhibitory regulation within the hematopoietic microenvironment would be identified. We studied an interleukin-3 (IL-3)-dependent myeloid cell line, NFS/N1.H7, and a derivative line transfected with H-RAS codon 12 (T24) oncogene, H7 Neo Ras.F3, for inhibition of proliferation by PGE1, 1 microM, alone or in combination with pertussis toxin, which inactivates Gi, an inhibitory regulatory guanosine triphosphate (GTP)-binding protein of adenylate cyclase. NFS/N1.H7 cells were inhibited in interleukin-3-dependent proliferation (dose range, IL-3 10 to 100 U/mL) by PGE1 79 +/- 11%, by pertussis toxin 51 +/- 9%, and by the combination 92 +/- 2%, whereas H7 Neo RAS.F3 was inhibited 51 +/- 7%, 6 +/- 2%, or 58 +/- 9% by PGE1, pertussis toxin, and the combination, respectively. These differences in capacity for inhibition by adenylate cyclase agonists between RAS-transfectant cells (lower inhibition) versus parent cells (greater inhibition) were all highly significant (P less than .0005). Intracellular cAMP formed on PGE1 stimulation of pertussis-intoxicated cells was 150% lower in RAS-transfectant cells than in parent cells. The adenylate cyclase activity of membranes from pertussis-intoxicated RAS-transfected cells was 1.5 to two times lower than that of pertussis-intoxicated parent-cell membranes on Mg2+-dependent activation by hormone and/or guanine nucleotide. However, very similar adenylate cyclase activity was observed in oncogenic p21 RAS-containing membranes compared with parental membranes under conditions of direct activation by 4 mM Mn2+ and forskolin, where inhibitory or stimulatory G-protein influences are minimal. These studies showed diminished adenylate cyclase activity in mutant RAS-bearing myeloid-cell membranes compared with parent-cell membranes independent of the pertussis toxin-sensitive G protein, Gi.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Effector function for RAS oncogene in interleukin-3-dependent myeloid cells involves diminished efficacy of prostaglandin E1-mediated inhibition of proliferation. 267 12

The plasma membrane-bound mammalian ras proteins of relative molecular mass 21,000 (ras p21) share biochemical and structural properties with other guanine nucleotide-binding regulatory proteins (G-proteins). Oncogenic ras p21 variants result from amino acid substitutions at specific positions that cause p21 to occur predominantly complexed to GTP in vivo. Recently, a GTPase activating protein (GAP) with cytosolic activity has been discovered that stimulates the GTPase activity of normal but not of oncogenic ras p21. GAP might be either a negative regulatory agent which acts further upstream in the regulatory pathway or the downstream target of ras p21. We have identified a protein from bovine brain with apparent relative molecular mass 125,000 that has GAP activity. Here, using pure GAP in a kinetic competition assay, we show that GAP interacts preferentially with the active GTP complexes of both normal and oncogenic Harvey (Ha) ras p21 compared with the inactive GDP complexes. We also report the cloning and sequencing of the complementary DNA for bovine GAP. Regions of GAP share amino acid similarity with the noncatalytic domain of adenylate cyclase from the yeast Saccharomyces cerevisiae and with regions conserved between phospholipase C-148, the crk oncogene product and the nonreceptor tyrosine kinases.
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PMID:Cloning of bovine GAP and its interaction with oncogenic ras p21. 284 90

Reconstitution of purified mu opioid receptors with purified guanine nucleotide-binding regulatory proteins (G proteins) was investigated. mu opioid receptors were purified by 6-succinylmorphine AF-AminoTOYOPEARL 650M affinity chromatography and by PBE isoelectric chromatography. The purified mu opioid receptor (pI 5.6) migrated as a single Mr 58,000 polypeptide by NaDodSO4/PAGE, a value identical to that obtained by affinity cross-linking purified mu receptors. When purified mu receptors were reconstituted with purified Gi, the G protein that mediates the inhibition of adenylate cyclase, the displacement of [3H]naloxone (a mu opioid antagonist) binding by [D-Ala2,MePhe4,Gly-ol5]enkephalin (a mu opioid agonist) was increased 215-fold; this increase was abolished by adding 100 microM (guanosine 5'-[gamma-thio]triphosphate. Similar increases in agonist displacement of [3H]naloxone binding (33-fold) and its abolition by guanosine 5'-[gamma-thio]triphosphate were observed with Go, the G protein of unknown function, but not with the v-Ki-ras protein p21. In reconstituted preparations with Gi or Go, neither [D-Pen2,D-Pen5]enkephalin (a delta opioid agonist; where Pen is penicillamine) nor U-69,593 (a kappa opioid agonist) showed displacement of the [3H]naloxone binding. In addition, the mu agonist stimulated both [3H]guanosine 5'-[beta,gamma-imido]triphosphate binding (in exchange for GDP) and the low-Km GTPase in such reconstituted preparations, with Gi and Go but not with the v-Ki-ras protein p21, in a naloxone-reversible manner. The stoichiometry was such that the stimulation of 1 mol of mu receptor led to the binding of [3H]guanosine 5'-[beta,gamma-imido]triphosphate to 2.5 mol of Gi or to 1.37 mol of Go. These results suggest that the purified mu opioid receptor is functionally coupled to Gi and Go in the reconstituted phospholipid vesicles.
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PMID:Reconstitution of rat brain mu opioid receptors with purified guanine nucleotide-binding regulatory proteins, Gi and Go. 284 1


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