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Research ArticleArticle

Prostaglandin E-Prostanoid-3 Receptor Activation of Cyclic AMP Response Element-Mediated Gene Transcription

Laurent P. Audoly, Lijun Ma, Igor Feoktistov, Stephanie K. de Foe, Matthew D. Breyer and Richard M. Breyer
Journal of Pharmacology and Experimental Therapeutics April 1999, 289 (1) 140-148;
Laurent P. Audoly
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Lijun Ma
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Igor Feoktistov
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Stephanie K. de Foe
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Matthew D. Breyer
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Richard M. Breyer
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Abstract

The prostaglandin E-prostanoid (EP)3 receptor signals primarily through the inhibitory G protein Gi, thereby decreasing intracellular cAMP levels. To study the signal transduction properties of the rabbit EP3 receptor, five splice variants were expressed in HEK293tsA201 cells: 72A, 74A, 77A, 80A and the novel splice variant NT, which lacks the C-terminal sequence. The ability of the EP3 receptor splice variants to modulate expression of a β-galactosidase reporter gene under the control of a promoter containing cAMP response elements (CRE) was assessed. Each splice variant induced sulprostone-mediated increase in β-galactosidase enzymatic activity with EC50 ranging from 0.8 nM for the NT splice variant to 3.1 nM for the 77A splice variant. Substitution of either Asp338 with Ala, or Arg329 with Ala or Glu in the 77A splice variant resulted in a loss of receptor-evoked increases in β-galactosidase activity, whereas substitution of Lys300 with alanine had no effect on signal transduction. These phenotypes correlate with the inhibition of cAMP generation by direct cAMP measurement. Signal transduction was insensitive to pretreatment of cells with pertussis toxin, suggesting that a nonGi/Go pathway is activated by the EP3 receptor. Direct measurement of second messenger levels confirmed that there was no increase in cAMP levels mediated by the 77A splice variant, however, there was a modest increase in intracellular Ca2+. Partial blockade of the reporter activity with kinase inhibitors demonstrates that CRE activation is mediated in part by a Ca2+-dependent kinase pathway. These data suggest that the EP3 receptor signals through a novel cAMP response element binding protein/CRE pathway.

Footnotes

  • Send reprint requests to: Richard M. Breyer, Ph.D., Vanderbilt University, Division of Nephrology, S3223 MCN, 1161 21st Ave. S., Nashville TN 37323-2372. E-mail:rich.breyer{at}mcmail.vanderbilt.edu

  • ↵1 Support for this project was provided in part by National Institutes of Health Grants DK-46205 and DK-37097 (R.M.B. and M.D.B.)

  • ↵2 Current affiliation: Division of Nephrology, Duke University Medical School.

  • Abbreviations:
    fura-2 AM
    acetoxymethyl ester of fura-2
    CRE
    cAMP response element
    CREB
    cAMP response element binding protein
    HA
    hemagglutinin
    PGE2
    prostaglandin E2
    PK
    protein kinase
    RIA
    radioimmunoassay
    Ptx
    pertussis toxin
    ELISA
    enzyme-linked immunosorbent assay
    UAS
    upstream activating sequence
    EP
    E-prostanoid
    PCR
    polymerase chain reaction
    DMEM
    Dulbecco’s modified Eagle’s medium
    FBS
    fetal bovine serum
    IBMX
    3-isobutyl-1-methylxanthine
    • Received July 29, 1998.
    • Accepted October 30, 1998.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 289 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 289, Issue 1
1 Apr 1999
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Research ArticleArticle

Prostaglandin E-Prostanoid-3 Receptor Activation of Cyclic AMP Response Element-Mediated Gene Transcription

Laurent P. Audoly, Lijun Ma, Igor Feoktistov, Stephanie K. de Foe, Matthew D. Breyer and Richard M. Breyer
Journal of Pharmacology and Experimental Therapeutics April 1, 1999, 289 (1) 140-148;

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Research ArticleArticle

Prostaglandin E-Prostanoid-3 Receptor Activation of Cyclic AMP Response Element-Mediated Gene Transcription

Laurent P. Audoly, Lijun Ma, Igor Feoktistov, Stephanie K. de Foe, Matthew D. Breyer and Richard M. Breyer
Journal of Pharmacology and Experimental Therapeutics April 1, 1999, 289 (1) 140-148;
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