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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We placed the cDNAs encoding one of the short types of alpha s (alpha s-1) with Asp-Ser in positions 70 and 71 and one of the long types of alpha s (alpha s-2) in which Asp-Ser are substituted with a string of 16 amino acids, into the pGEM-3 transcription vector downstream from its T7 RNA polymerase promoter, obtained transcripts and translated the mRNAs using a rabbit reticulocyte lysate system, to determine if the molecules would be synthesized and, if so, whether they would be active as assessed in cyc- reconstitution assays. The translation products obtained from both alpha s RNAs were a mixture of primarily three polypeptides of which one (approximately 40-50% of total) represented the complete translation product and the other two appeared to be due to internal translation starts at Met60, before the splice difference between the RNAs, and the other at the first Met after the splice difference. Lysates incubated with short or long alpha s RNA when added to cyc- membranes reconstituted fluoride and GTP[gamma S]-stimulated activities. Thus, in vitro synthesized alpha s subunits are active in interacting both with guanine nucleotides and the adenylyl cyclase enzyme. On incubation without and with the receptor agonist isoproterenol, using GTP as sole added guanine nucleotide, both types of alpha s subunits reconstituted the isoproterenol-stimulated adenylyl cyclase activity. Thus, the synthetic alpha s also interact with receptors, and by inference with beta-gamma dimers, shown previously to be needed for activation by receptor. Quantitative assays in which the activity of the synthetic alpha s-1 was compared to that of native purified human erythrocyte type-1 Gs, indicated that the two products are equipotent within a 2-fold margin of error. Thus, the lysate made fully active alpha s subunits, and alpha s subunits require no post-translational modifications dependent on microsomal processes. This approach may be useful in studying biological functions of other cloned alpha subunits of G proteins.
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PMID:Reticulocyte lysates synthesize an active alpha subunit of the stimulatory G protein Gs. 283 88

Previous studies with U937 cells, a human monocyte cell line, have shown that the activity of cyclic nucleotide phosphodiesterase 4 (PDE4) is increased by agents that elevate cyclic AMP content. The present experiments were conducted to determine 1) whether an increase in PDE4 steady-state message and/or protein accompanies the up-regulation of PDE4 activity and 2) whether the up-regulation changes the functional responses of U937 cells to activators of adenylyl cyclase. To up-regulate PDE4 activity, U937 cells were treated for 4 h with a combination of 1 microM salbutamol, a beta-adrenoceptor agonist, and 30 microM rolipram, a PDE4 inhibitor. Cells were washed extensively to remove drugs and used immediately in various experimental protocols. Reverse transcriptase-polymerase chain reactions conducted with primers specific for the four PDE4 subtypes suggested that pretreatment with salbutamol and rolipram increased steady-state mRNA levels of PDE4A and PDE4B, but not PDE4C or PDE4D. Immunoblot analyses using two rabbit polyclonal antibodies, one directed against human recombinant PDE4A and PDE4D and a second directed against human recombinant PDE4B, revealed bands of immunoreactivity corresponding to approximately 125 kDa (PDE4A) and approximately 70 kDa (PDE4B), respectively, that increased in intensity after cells were treated with salbutamol and rolipram. As demonstrated in both time course and concentration-response studies with prostaglandin E2 (PGE2), an agent that activates adenylyl cyclase by a non-beta-adrenoceptor-mediated mechanism, cAMP accumulation was substantially decreased in cells in which PDE4 activity had been up-regulated. The difference in PGE2-stimulated cAMP accumulation between control and PDE4 up-regulated cells was greatly reduced in the presence of rolipram, consistent with the notion that an increase in PDE4 activity was responsible for the heterologous desensitization. Functionally, up-regulation of PDE4 markedly decreased the ability of PGE2 to inhibit LTD4-induced Ca2+ mobilization in intact cells. A hypothetical implication of these results is that increasing PDE4 activity in vivo by administering beta-adrenoceptor agonists could exacerbate inflammatory processes by decreasing the activity of endogenous anti-inflammatory agents such as PGE2.
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PMID:Salbutamol up-regulates PDE4 activity and induces a heterologous desensitization of U937 cells to prostaglandin E2. Implications for the therapeutic use of beta-adrenoceptor agonists. 755 25

Studies were performed to determine the mechanism underlying deficient arginine vasopressin (AVP)-stimulated adenylyl cyclase activity in chronic renal failure (CRF). As compared to control, principal cells cultured from the inner medullary collecting tubule of rats previously made uremic by 5/6 nephrectomy fail to accumulate cAMP when stimulated with AVP. CRF cells do respond normally to forskolin or cholera toxin and the defect in AVP responsiveness is not prevented by treatment with pertussis toxin, by cyclooxygenase inhibition, or by inhibition or down-regulation of protein kinase C. In contrast to their lack of responsiveness to AVP, CRF cells respond normally to other agonists of adenylyl cyclase such as isoproterenol or prostaglandin E2. Plasma membranes prepared from the inner medullae of CRF rats exhibit a marked decrease in apparent AVP receptor number but no change in the apparent number of beta adrenergic receptors. Reverse transcriptase PCR of total RNA from the inner medullae of CRF animals reveals virtual absence of V2 receptor mRNA; mRNA for alpha s is present in normal abundance. These studies indicate that AVP resistance in CRF is due, at least in part, to selective down-regulation of the V2 receptor as a consequence of decreased V2 receptor mRNA.
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PMID:Vasopressin resistance in chronic renal failure. Evidence for the role of decreased V2 receptor mRNA. 761 8

Using a combination of polymerase chain reaction and genomic library screening we have cloned a human gene for a subtype of the somatostatin (SST) receptor (SSTR) termed human SSTR5 (hSSTR5), which is located on chromosome 16. The predicted amino acid sequence of hSSTR5 displays 75% sequence identity with a recently identified rat SSTR [Mol. Pharmacol. 42:939-946 (1992)], suggesting that it is the human homologue of this receptor. hSSTR5 consists of a 363-residue polypeptide exhibiting a putative seven-transmembrane domain topology typical of G protein-coupled receptors. The receptor displays considerable sequence identity to hSSTR1 (42%), hSSTR2 (48%), hSSTR3 (47%), and hSSTR4 (46%). Membranes prepared from COS-7 cells transiently expressing the hSSTR5 gene bound 125I-Leu8,D-Trp22,Tyr25-SST-28 (125I-LTT-SST-28) with high affinity and in a saturable manner. SST-14, SST-28, and various synthetic SST peptide agonists produced dose-dependent inhibition of radioligand binding with the following rank order of potency: LTT-SST-28 > SST-28 > D-Trp8-SST-14 > SST-14 approximately RC-160 approximately BIM 23014 > MK-678 > SMS 201-995. hSSTR5 bound SST-28 with a 12.6-fold greater affinity (Ki = 0.19 nM), compared with SST-14 (Ki = 2.24 nM), indicating that the receptor is SST-28 selective. Addition of GTP, guanosine-5'-O-(3-thio)triphosphate, Na+ ions, or pertusis toxin greatly reduced 125I-LTT-SST-28 binding, thereby indicating that hSSTR5 is coupled to pertussis toxin-sensitive G proteins. Both SST-14 and SST-28 displayed dose-dependent inhibition of forskolin-stimulated cAMP accumulation, consistent with functional coupling of the receptor to adenylyl cyclase inhibition. Northern blot analysis of SSTR5 mRNA revealed a 2.4-kilobase transcript in normal rat pituitary and GH3 rat pituitary tumor cells and a 4.0-kilobase transcript in normal human pituitary. Reverse transcriptase polymerase chain reaction revealed expression of the hSSTR gene in fetal human pituitary and hypothalamus but not in human cerebral cortex. In situ hybridization of the rat pituitary showed that SSTR5 mRNA is selectively localized in the anterior lobe. SSTR5 mRNA was not expressed in four human pituitary tumors (somatotroph adenoma, prolactinoma, and chromophobe adenomas) or in a human insulinoma. Although hSSTR5 displays approximately 75% sequence identity with rat SSTR5, the two receptors display significantly different pharmacological profiles, especially with respect to their binding affinities for the SST analogue SMS 201-995.
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PMID:Molecular cloning, functional characterization, and chromosomal localization of a human somatostatin receptor (somatostatin receptor type 5) with preferential affinity for somatostatin-28. 790 5

Biologic responses to peptide calciotropic hormones, such as parathyroid hormone (PTH) and calcitonin, exhibit desensitization. As with most hormones, however, the mechanisms of desensitization are not completely understood. For the beta 2-adrenergic receptor (beta 2AR) system, which is coupled to adenylyl cyclase via the stimulatory guanine nucleotide-binding regulatory (G5) protein, homologous desensitization is mediated in part by a receptor-specific kinase (beta ARK) and a soluble cofactor (beta-arrestin). Recently, this system has been reported to be involved in rapid homologous desensitization of the PTH/parathyroid hormone receptor protein (PTHrP) receptor. We have identified the presence of this system in bone using reverse-transcriptase PCR. Nucleotide sequence of PCR fragments from ROS 17/2.8 cells revealed 100% identity with rat brain beta ARK1 and beta-arrestin 1 sequences. Northern analyses with RNA from ROS 17/2.8, UMR 106-H5 cells, and primary cultures of nontransformed neonatal rat calvariae demonstrated two mRNA species of 4 and 2.6 kilobases (kb) for beta ARK and 7.5 kb for beta-arrestin, comparable to those found in bovine brain. beta ARK-like activity was demonstrated in cytosolic extracts of the UMR 106-H5 cells by assessing phosphorylation of the retinal photoreceptor, rhodopsin, by the extracts. Phosphorylation was enhanced with light-activated rhodopsin and by bovine brain G beta gamma subunits; heparin inhibited phosphorylation. These findings are characteristic of beta ARK. Expression of beta-arrestin in the UMR 106-H5 cells was confirmed by immunoblot. Thus, osteoblastic cells express proteins, beta ARK, and beta-arrestin, which may regulate desensitization of calciotropic hormone receptors.
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PMID:Beta-adrenergic receptor kinase-like activity and beta-arrestin are expressed in osteoblastic cells. 872 79

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel neuropeptide that produces its biological effects by interacting with G protein-coupled receptors. Molecular cloning of the PACAP receptor revealed the existence of five splice variant receptor forms differing in the third intracellular loop region, with four variants activating both adenylyl cyclase and phosphoinositide phospholipase C and one variant activating only adenylyl cyclase (Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., Bockaert, J., Seeburg, P. H., and Journot, L. (1993) Nature 365, 170-175). Here, we report cloning of a novel PACAP receptor variant, designated PACAPR TM4 (transmembrane domain IV), that differs from the previously cloned short form of the PACAP receptor (PACAPR) primarily by discrete sequences located in transmembrane domains II and IV. Reverse transcriptase-polymerase chain reaction and primer extension analyses demonstrated tissue-specific differential expression of mRNAs encoding PACAPR TM4 and splice variant forms of the PACAP receptor. PACAPR TM4 and PACAPR possess identical intracellular domains, implicated as primary determinants of G protein recognition by rhodopsin-like receptors. However, unlike the PACAPR, PACAPR TM4 does not activate either adenylyl cyclase or phosphoinositide phospholipase C in response to PACAP in either transient or stable expression systems. However, PACAP stimulates increases in [Ca2+]i in cells expressing PACAPR TM4 by activating L-type Ca2+ channels, a response not elicited by stimulation with vasoactive intestinal polypeptide. The signaling phenotype of PACAPR TM4 is characteristic of the PACAP receptor involved in regulation of insulin secretion from pancreatic beta islets, a tissue expressing transcripts for PACAPR TM4 but not for PACAPR or its longer splice variant forms. These findings are consistent with a role of PACAPR TM4 in the physiological control of insulin release by PACAP in beta-islet cells. The finding that PACAPR TM4 has a unique signaling phenotype, although it possesses intracellular domains identical to those of the PACAPR, suggests that receptor-G protein recognition by rhodopsin-like receptors can be determined by sequences other than those located in intracellular receptor domains.
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PMID:Molecular cloning of a novel variant of the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor that stimulates calcium influx by activation of L-type calcium channels. 894 80

1. 5-Hydroxytryptamine (5-HT) elicited a dose-dependent stimulation of intracellular adenosine 3': 5'-cyclic monophosphate (cyclic AMP) accumulation in cultured astrocytes derived from neonatal rat (Sprague Dawley) thalamic/hypothalamic area with a potency (pEC50) of 6.68 +/- 0.08 (mean +/- s.e. mean). 2. In order to characterize the 5-HT receptor responsible for the cyclic AMP accumulation the effects of a variety of compounds were investigated on basal cyclic AMP levels (agonists) and 5-carboxamidotryptamine (5-CT) stimulated cyclic AMP levels (antagonists). The rank order of potency for the agonists investigated was 5-CT (pEC50 = 7.81 +/- 0.09) > 5-methoxytryptamine (5-MeOT) (pEC50 = 6.86 +/- 0.36) > 5-HT (pEC50 = 6.68 +/- 0.08). The following compounds, at concentrations up to 10 microM, did not affect basal cyclic AMP levels 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), cisapride, sumatriptan, DOI and RU 24969. The rank order of potency of antagonists was methiothepin (pKi = 7.98 +/- 0.25) > mesulergine (pKi = 7.58 +/- 0.18) > ritanserin (pKi = 7.20 +/- 0.24) > clozapine (pKi = 7.03 +/- 0.19) > mianserin (pKi = 6.41 +/- 0.19). The following compounds, at concentrations up to 10 microM, were inactive: ketanserin, WAY100635, GR127935. This pharmacological profile is consistent with that of 5-HT7 receptor subtype-mediated effects. 3. The cultured astrocytes exhibited regional heterogeneity in the magnitude of cyclic AMP accumulation (Emax). Cells cultured from the thalamic/hypothalamic area had significantly higher Emax values (588 +/- 75% and 572 +/- 63% of basal levels for 5-CT and 5-HT, respectively) compared to brainstem (274 +/- 51% and 318 +/- 46%, respectively) and colliculus astrocytes (244 +/- 15% and 301 +/- 24%, respectively). No significant differences in pEC50 (for either 5-HT or 5-CT) values were observed. 4. Reverse transcriptase-polymerase chain reaction (RT-PCR) with primers specific for the 5-HT7 receptor confirmed expression of messenger RNA for this receptor subtype by the cultured astrocytes derived from all regions investigated. Primers specific for the 5-HT6 receptor also amplified a cDNA fragment from the same samples. 5. From these findings, we conclude that astrocytes cultured from a number of brain regions express functional 5-HT receptors positively coupled to adenylyl cyclase and that the level of receptor expression or the efficiency of receptor coupling is regionally-dependent. The pharmacological profile of the receptor on thalamic/hypothalamic astrocytes suggests that the 5-HT7 receptor is the dominant receptor that is functionally expressed even though astrocyte cultures have the capacity to express both 5-HT6 and 5-HT7 receptor messenger RNA.
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PMID:Identification of 5-hydroxytryptamine receptors positively coupled to adenylyl cyclase in rat cultured astrocytes. 903 57

The identification of adenylyl cyclase isoenzymes in mammalian host cells is important for the interpretation of data obtained from cell lines heterologously expressing G-protein coupled receptors. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to amplify adenylyl cyclase cDNAs from Chinese hamster ovary (CHO) and mouse fibroblast (B82) cells. The isolated fragments were identified by restriction analyses and by sequencing. We found mRNAs for adenylyl cyclases VI and VII in CHO and adenylyl cyclases IX and VII in B82 cells.
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PMID:Identification of adenylyl cyclase isoenzymes in CHO and B82 cells. 965 50

The literature describing the expression of 5-HT receptor subtypes by astrocytes is controversial and incomplete. It is clear that primary cultures of astrocytes express receptors of the 5-HT2 family coupled to phospholipase C and of the 5-HT7 receptor family positively coupled to adenylyl cyclase. Cultured astrocytes have also been reported to express receptors of the 5-HT1 family, although the exact subtypes present are unknown. In the present study we have investigated which of the known rat G-protein coupled 5-HT receptor mRNAs are expressed by cultured astrocytes. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed expression of 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT5B, 5-HT6 and 5-HT7 receptor mRNAs in astrocytes derived from 2-day old rats and cultured for 10-12 days. Messenger RNAs for 5-HT4 and 5-HT5A receptors were not detected. The functional expression of 5-HT1 receptor subtypes was investigated by measuring the ability of 5-HT1 receptor agonists: 8-OH-DPAT (5-HT1A receptors), RU24969 (5-HT1A, 5-HT1B, 5-HT1D, and 5-HT1F receptors) or sumatriptan (5-HT1B, 5-HT1D, and 5-HT1F receptors) to modulate forskolin or isoproterenol stimulated cAMP production. These compounds, at concentrations up to 10 microM, did not significantly attenuate cAMP production. These results indicate that although astrocytes express mRNA for each of the five 5-HT1 receptor subtypes which have been isolated from the rat, these receptors are not coupled to the inhibition of adenylyl cyclase.
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PMID:Cultured astrocytes express messenger RNA for multiple serotonin receptor subtypes, without functional coupling of 5-HT1 receptor subtypes to adenylyl cyclase. 979 56

Extracellular ATP can function as a glial trophic factor as well as a neuronal transmitter. In astrocytes, mitogenic signalling by ATP is mediated by metabotropic P(2Y) receptors that are linked to the extracellular signal regulated protein kinase (Erk) cascade, but the types of P(2Y) receptors expressed in astrocytes have not been defined and it is not known whether all P(2Y) receptor subtypes are coupled to Erk by identical or distinct signalling pathways. We found that the P(2Y) receptor agonists ATP, ADP, UTP and 2-methylthioATP (2MeSATP) activated Erk and its upstream activator MAP/Erk kinase (Mek). cRaf-1, the first kinase in the Erk cascade, was activated by 2MeSATP, ADP and UTP but, surprisingly, cRaf-1 was not stimulated by ATP. Furthermore, ATP did not activate B-Raf, the major isoform of Raf in the brain, nor other Mek activators such as Mek kinase 1 (MekK1) and MekK2/3. Reverse transcriptase-polymerase chain reaction (RT - PCR) studies using primer pairs for cloned rat P(2Y) receptors revealed that rat cortical astrocytes express P(2Y(1)), a receptor subtype stimulated by ATP and ADP and their 2MeS analogues, as well as P(2Y(2)) and P(2Y(4)), subtypes in rats for which ATP and UTP are equipotent. Transcripts for P(2Y(6)), a pyrimidine-preferring receptor, were not detected. ATP did not increase cyclic AMP levels, suggesting that P(2Y(11)), an ATP-preferring receptor, is not expressed or is not linked to adenylyl cyclase in rat cortical astrocytes. These signal transduction and RT - PCR experiments reveal differences in the activation of cRaf-1 by P(2Y) receptor agonists that are inconsistent with properties of the P(2Y(1)), P(2Y(2)) and P(2Y(4)) receptors shown to be expressed in astrocytes, i.e. ATP=UTP; ATP=2MeSATP, ADP. This suggests that the properties of the native P(2Y) receptors coupled to the Erk cascade differ from the recombinant P(2Y) receptors or that astrocytes express novel purine-preferring and pyrimidine-preferring receptors coupled to the ERK cascade.
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PMID:P(2Y) purinoceptor subtypes recruit different mek activators in astrocytes. 1069 92


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