Specific inhibition of protein kinase a in granulosa cells abolishes gonadotropin regulation of the proopiomelanocortin promoter

Steven L. Young, Robert Searles, Alan H. Kaynard, Michael H. Melner

    Research output: Contribution to journalArticle

    8 Citations (Scopus)

    Abstract

    Gonadotropins (follicle-stimulating hormone (FSH), luteinizing hormone, and human chorionic gonadotropin) and β-adrenergic agonists have been shown to stimulate expression of the proopiomelanocortin (POMC) gene in ovarian granulosa cells. The current studies investigate the intracellular mechanisms by which gonadotropins regulate gene expression. Primary cultures of rat granulosa cells were transfected with the plasmid POMC-CAT-150, which expresses the chloramphenicol acetyltransferase (CAT) reporter gene under the regulation of the rat POMC 5′-flanking region. CAT activity was stimulated by treatment of the cells with either 20 ng/ml FSH or 1 μM isoproterenol. To assess the role of protein kinase A (ATP:protein phosphotransferase; EC 2.7.1.37) in the gonadotropin and adrenergic response, an expression vector, MtR-AB, encoding a mutant RI regulatory subunit was co-transfected with POMC-CAT-150. The mutant protein kinase A regulatory subunit encoded by MtR-AB lacks functional cAMP-binding sites but effectively binds and specifically inhibits the catalytic activity of protein kinase A. The results of this analysis demonstrated that gonadotropin and adrenergic agonist stimulation of the POMC-CAT reporter construct in primary cultures of rat granulosa cells were abolished by cotransfection with MtR-AB; whereas a control SV40-promoter construct was unaffected by either goandotropin treatment or cotransfection with MtR-AB. Basal expression directed by the POMC promoter was also decreased by cotransfection with the MtR-AB, implying that basal expression from the POMC promoter may also depend on protein kinase A. Deletion analysis of the POMC sequence indicated regions (-40 to -33 and +4 to +63) important for basal and FSH-stimulated expression. These studies suggest that both gonadotropin and adrenergic stimulation of the POMC promoter are mediated by protein kinase A and that regions proximal to the promoter are essential for gonadotropin-regulated expression from the promoter.

    Original languageEnglish (US)
    Pages (from-to)15839-15844
    Number of pages6
    JournalJournal of Biological Chemistry
    Volume266
    Issue number24
    StatePublished - 1991

    Fingerprint

    Pro-Opiomelanocortin
    Granulosa Cells
    Gonadotropins
    Protein Kinases
    Chloramphenicol O-Acetyltransferase
    Cyclic AMP-Dependent Protein Kinases
    Follicle Stimulating Hormone
    Rats
    Adrenergic Agonists
    Adrenergic Agents
    Genes
    Gene Expression
    5' Flanking Region
    Mutant Proteins
    Chorionic Gonadotropin
    Luteinizing Hormone
    Reporter Genes
    Isoproterenol
    Gene expression
    Sequence Analysis

    ASJC Scopus subject areas

    • Biochemistry

    Cite this

    Specific inhibition of protein kinase a in granulosa cells abolishes gonadotropin regulation of the proopiomelanocortin promoter. / Young, Steven L.; Searles, Robert; Kaynard, Alan H.; Melner, Michael H.

    In: Journal of Biological Chemistry, Vol. 266, No. 24, 1991, p. 15839-15844.

    Research output: Contribution to journalArticle

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    abstract = "Gonadotropins (follicle-stimulating hormone (FSH), luteinizing hormone, and human chorionic gonadotropin) and β-adrenergic agonists have been shown to stimulate expression of the proopiomelanocortin (POMC) gene in ovarian granulosa cells. The current studies investigate the intracellular mechanisms by which gonadotropins regulate gene expression. Primary cultures of rat granulosa cells were transfected with the plasmid POMC-CAT-150, which expresses the chloramphenicol acetyltransferase (CAT) reporter gene under the regulation of the rat POMC 5′-flanking region. CAT activity was stimulated by treatment of the cells with either 20 ng/ml FSH or 1 μM isoproterenol. To assess the role of protein kinase A (ATP:protein phosphotransferase; EC 2.7.1.37) in the gonadotropin and adrenergic response, an expression vector, MtR-AB, encoding a mutant RI regulatory subunit was co-transfected with POMC-CAT-150. The mutant protein kinase A regulatory subunit encoded by MtR-AB lacks functional cAMP-binding sites but effectively binds and specifically inhibits the catalytic activity of protein kinase A. The results of this analysis demonstrated that gonadotropin and adrenergic agonist stimulation of the POMC-CAT reporter construct in primary cultures of rat granulosa cells were abolished by cotransfection with MtR-AB; whereas a control SV40-promoter construct was unaffected by either goandotropin treatment or cotransfection with MtR-AB. Basal expression directed by the POMC promoter was also decreased by cotransfection with the MtR-AB, implying that basal expression from the POMC promoter may also depend on protein kinase A. Deletion analysis of the POMC sequence indicated regions (-40 to -33 and +4 to +63) important for basal and FSH-stimulated expression. These studies suggest that both gonadotropin and adrenergic stimulation of the POMC promoter are mediated by protein kinase A and that regions proximal to the promoter are essential for gonadotropin-regulated expression from the promoter.",
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