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:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The rate of transcription of the beta 2-adrenergic receptor gene is increased in response to beta-adrenergic agonist stimulation of the receptor at the cell surface. This effect is mediated by stimulation of adenylyl cyclase and elevation of intracellular cAMP levels. We have previously shown that this responsiveness to cAMP resides in the 5'-flanking region of the human beta 2-adrenergic receptor gene (Collins, S., Bouvier, M., Bolanowski, M. A., Caron, M. G., and Lefkowitz, R. J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4853-4857). A 34-base pair sequence derived from the beta 2-adrenergic receptor promoter region (-70 to -37 base pairs), containing the sequence GTACGTCA, confers responsiveness to cAMP when present in either orientation 5' to the thymidine kinase promoter on the chloramphenicol acetyltransferase reporter gene. Overexpression of the catalytic subunit of protein kinase A fully substituted for forskolin in inducing expression through this sequence, indicating that the cAMP induction is mediated through this kinase. Mutations within the GTACGTCA sequence completely abolished the stimulation. A 43-kDa transcription factor (cAMP response element-binding protein) confers cAMP responsiveness through binding to specific sequences. In gel mobility shift assays, purified cAMP response element-binding protein bound to the 34-base pair oligonucleotide from the beta 2-adrenergic receptor gene with an affinity similar to that for the well-characterized cAMP response element from the human glycoprotein hormone alpha-subunit gene, and failed to bind to mutated elements. Thus, positive autoregulation of the beta 2-adrenergic receptor gene appears to occur through receptor-mediated stimulation of adenylyl cyclase, with consequent activation of cAMP response element-binding protein and stimulation of beta 2-adrenergic receptor gene transcription. These results demonstrate a novel mechanism by which a receptor (beta 2-adrenergic receptor) stimulatory for adenylyl cyclase can exert positive feedback regulation on its own expression.
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PMID:A cAMP response element in the beta 2-adrenergic receptor gene confers transcriptional autoregulation by cAMP. 217 52

In addition to conveying cellular responses to an effector molecule, receptors are often themselves regulated by their effectors. We have demonstrated that epinephrine modulates both the rate of transcription of the beta 2-adrenergic receptor (beta 2AR) gene and the steady-state level of beta 2AR mRNA in DDT1MF-2 cells. Short-term (30 min) exposure to epinephrine (100 nM) stimulates the rate of beta 2AR gene transcription, resulting in a 3- to 4-fold increase in steady-state beta 2AR mRNA levels. These effects are mimicked by 1 mM N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate (Bt2cAMP) or foskolin but not by phorbol esters. The half-life of the beta 2AR mRNA after addition of actinomycin D (46.7 +/- 10.2 min; mean +/- SEM; n = 5) remained unchanged after 30 min of epinephrine treatment (46.8 +/- 10.6 min; mean +/- SEM; n = 4), indicating that a change in transcription rate is the predominant factor responsible for the increase of beta 2AR mRNA. Whereas brief exposure to epinephrine or Bt2cAMP does not significantly affect the total number of cellular beta 2ARs (assessed by ligand binding), continued exposure results in a gradual decline in beta 2AR number to approximately 20% (epinephrine) or approximately 45% (Bt2cAMP) of the levels in control cells by 24 hr. Similar decreases in agonist-stimulated adenylyl cyclase activity are observed. This loss of receptors with prolonged agonist exposure is accompanied by a 50% reduction in beta 2AR mRNA. Transfection of the beta 2AR promoter region cloned onto a reporter gene (bacterial chloramphenicol acetyltransferase) allowed demonstration of a 2- to 4-fold induction of transcription by agents that elevate cAMP levels, such as forskolin or phosphodiesterase inhibitors. These results establish the presence of elements within the proximal promoter region of the beta 2AR gene responsible for the transcriptional enhancing activity of cAMP and demonstrate that beta 2AR gene expression is regulated by a type of feedback mechanism involving the second messenger cAMP.
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PMID:cAMP stimulates transcription of the beta 2-adrenergic receptor gene in response to short-term agonist exposure. 247 35

A cAMP-dependent reporter gene has been used in transiently transfected human choriocarcinoma (JEG-3) cells to examine the second messenger coupling of the human alpha 2-adrenergic receptor subtypes. The reporter gene consists of a cAMP response element linked to the gene for chloramphenicol acetyltransferase (CAT). Plasmids encoding the alpha 2-C10 (alpha 2A), alpha 2-C2 (alpha 2B), or alpha 2-C4 (alpha 2C) receptor subtypes were co-transfected with a plasmid containing the reporter gene, and the ability of alpha 2 receptor agonists to influence forskolin-stimulated CAT expression was examined. For alpha 2-C10, agonists had a biphasic effect on forskolin-stimulated CAT expression. Thus, low (nanomolar) concentrations of agonist inhibited CAT expression by approximately 60%, whereas high (micromolar) concentrations reversed this inhibition and could even potentiate CAT expression by as much as 140%. A significantly different pattern of coupling was observed for the other alpha 2 receptor subtypes. For alpha 2-C4, agonists only inhibited forskolin-stimulated CAT expression, whereas for alpha 2-C2 only potentiation of expression was seen. Each of these responses was specifically blocked by alpha 2- but not alpha 1- or beta-adrenergic receptor antagonists. For alpha 2-C4, the inhibition of forskolin-stimulated CAT expression was prevented by pretreatment of the cells with pertussis toxin. This was also true for the inhibition obtained with alpha 2-C10. The potentiation of CAT expression, however, was not prevented by pertussis toxin pretreatment in cells transfected with either alpha 2-C2 or alpha 2-C10. In this transient expression system, each alpha 2-adrenergic receptor subtype had access to the same complement of G proteins, adenylyl cyclase, and other second messengers. It would appear, therefore, that the potential for the activation of unique intracellular responses exists even among closely related receptor subtypes.
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PMID:Selective coupling of alpha 2-adrenergic receptor subtypes to cyclic AMP-dependent reporter gene expression in transiently transfected JEG-3 cells. 823 31

The hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), can efficiently increase cAMP levels in pituitary cells and release a number of pituitary hormones, suggesting an important physiological role for this peptide in pituitary function. Exposure of GH3 rat pituitary cells to PACAP results in increases in cellular cAMP levels, PRL promoter activity, and PRL messenger RNA levels. We have employed this system to further characterize PACAP regulation of PRL gene expression. RT-PCR analysis showed that GH3 cells express transcripts for two PACAP receptors, PACAP-R-hop1 and VIP2. As the former can couple PACAP to increases in both cAMP and inositol phosphates, we investigated whether either pathway mediates PACAP action on the PRL promoter. Our observations that TRH, but not PACAP, increases the intracellular Ca2+ concentration in GH3 cell cultures and that the optimal concentrations of TRH and PACAP have additive effects on transient expression of a PRL-CAT construct imply that the inositol trisphosphate-Ca2+ pathway is not significantly involved in PACAP action on the PRL promoter. Four kinase inhibitors exhibited similar profiles of inhibition of the activity on PRL-chloramphenicol acetyltransferase (PRL-CAT) of either the adenylyl cyclase activator forskolin (FSK) or PACAP, suggesting a transcriptional role for protein kinase A (PKA). The observations that coexpression of the dominant PKA inhibitor RAB completely blocked either FSK or PACAP action on PRL-CAT and that these actions of FSK and PACAP were completely nonadditive imply that the cAMP-PKA pathway plays a dominant role in PACAP regulation of PRL gene expression. Coexpression of low levels of KCREB, a cAMP response element (CRE)-binding protein (CREB) dominant inhibitor, partially blocked regulation of PRL-CAT activity by PACAP, but not TRH, implying that PACAP action is mediated at least in part by a CREB family member that can dimerize with CREB. The PRL promoter contains an asymmetric sequence at positions -99/-92 resembling a canonical CRE and termed here the CRE-like element (CLE). Mutation of either the left or right 4 bp of the CLE yielded a strong decrease in the response to either FSK or PACAP, but not to TRH. These data imply that PACAP and TRH employ independent pathways to regulate the PRL promoter, and that PACAP action is exerted virtually entirely via a cAMP/PKA-mediated pathway that is strongly dependent upon an intact CLE sequence and at least partially dependent upon the activity of a CREB-related protein.
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PMID:Pituitary adenylate cyclase-activating polypeptide regulates prolactin promoter activity via a protein kinase A-mediated pathway that is independent of the transcriptional pathway employed by thyrotropin-releasing hormone. 862

Transcriptional regulation of the rat tyrosine hydroxylase (TH) gene by prostaglandin E2 (PGE2) was investigated in human neuroblastoma SK-N-BE(2)C cells. Prostaglandins increased intracellular cAMP in the presence of 3-isobutyl-1-methylxanthine (IBMX), a cAMP phosphodiesterase inhibitor. Among the prostaglandins tested for their cAMP raising property PGE2 was the most effective. The results suggest that the cells express adenylyl cyclase-linked prostanoid receptors that have a higher affinity for PGE2 than for any other naturally occurring prostaglandin. The treatment of cells with PGE2 increased the TH gene expression approximately 2-fold, even though the cAMP accumulation induced by PGE2 alone was almost negligible. Simultaneous treatment with PGE2 and IBMX enhanced the gene expression concomitantly with a marked accumulation of cAMP. Transient transfection assays with 5' upstream serially deleted constructs of the rat TH gene promoter region fused to the chloramphenicol acetyltransferase (CAT) gene revealed that a cAMP response element (CRE) located at -45 to -38 from the start of the TH gene was essential for the enhancement of TH gene expression by PGE2. Site-directed mutagenesis and specific deletion within the sequence of the CRE motif abolished the transcriptional enhancement by PGE2. In addition, a protein kinase A (PKA) inhibitor, H89, specifically blocked the PGE2 effect on TH gene expression. Northern blot analysis revealed that the increase in TH gene transcription with PGE2 is associated with an elevated TH mRNA level. Gel retardation and competition assays confirmed that the binding of nuclear factors to the CRE site was sequence specific and was augmented by PGE2. Our data indicate that PGE2 enhances transcription of the TH gene mediated by the CRE motif through the activation of PKA. They also suggest that the signal flow from the adenylyl cyclase-linked prostanoid receptor to the nucleus is efficient although cAMP accumulation is not prominent.
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PMID:Transcriptional enhancement of tyrosine hydroxylase by prostaglandin E2 in SK-N-BE(2) C cells. 880 26

Increased expression of the inhibitory G protein Gi alpha-2 is assumed to contribute to desensitization of adenylyl cyclase in human heart failure. The mechanisms of upregulation involve increases in myocardial Gi alpha-2 protein, mRNA and gene transcriptional activity. To elucidate these mechanisms in more detail, the 5' flanking region of the human Gi alpha-2 gene (-1214/+115 bp) was cloned upstream of the bacterial chloramphenicol acetyltransferase (CAT) gene and transfected in embryonic chick cardiomyocytes. CAT activity was measured 48 h after transfection. Unstimulated activity of the -1214/+115 bp construct was about 10-fold higher than activity of the basal CAT-construct (pGEMCAT). 5' deletion from -1214/+115 to -85/+115 bp upstream of the transcriptional start site increased, further stepwise deletions to 46/+115 gradually decreased promotor activity. Deletion from -46/+115 to -33/+115 bp completely abolished promotor activity. Stimulation of cardiomyocytes that had been transfected with the -1214/+115 CAT-construct with isoprenaline (10 microM), forskolin (10 microM), forskolin (10 microM) plus IBMX (10 microM) or dibutyryl-cAMP (1 mM) for 24 h induced an increase in CAT activity to 139 +/- 12% (n = 9), 211 +/- 18% (n = 12), 256 +/- 20% (n = 5) and 198 +/- 28% (n = 7) of unstimulated values, respectively. We conclude: 1) In chicken cardiomyocytes a sequence element of 52 bp between -85 and -33 bp is necessary to provide basal Gi alpha-2 promotor activity. 2) Elevation of cAMP has a stimulatory effect on the human Gi alpha-2 promotor, thereby offering a mechanism for beta-adrenoceptor-mediated increases in Gi alpha-2 in the heart.
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PMID:Regulation of the human Gi alpha-2 gene promotor activity in embryonic chicken cardiomyocytes. 895 43

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