Abstract
Gonadotropins (follicle-stimulating hormone (FSH), luteinizing hormone, and human chorionic gonadotropin) and beta-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 microM 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 cotransfected 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 gonadotropin 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.
Highlights
Gonadotropins (follicle-stimulating hormone (FSH), The gonadotropins FSH’ and luteinizing hormoneregulate luteinizing hormone, and human chorionic gonadotro- a number of ovarian functions, includingfolliculargrowth, pin) and &adrenergic agonists have been shown to ovulation, and corpus luteum function
Primary cultures of rat granulosa cells were transfected with the plasmid POMC-CAT-150, which expresses the chloramphenicol acetyltransferase (CAT)reporter gene under the regulatioonf the rat POMC 5’-flanking region
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 wasunaffected by either goandotropin treatmenotcrotransfectionwith
Summary
The abbreviations used are: FSH, follicle-stimulating hormone; hFSH, human follicle-stimulating hormone; CAT, chloramphenicol acetyltransferase;POMC, proopiomelanocortin; CRE,cAMP regulatory element; ACTH, adrenocorticotropic hormone; AP, activating. A in the regulation of gene expression have utilized peptide Cetus Instruments) andligation into a plasmid containinga promotinhibitors containing the kinase pseudosubstrate site found erless CAT gene (XhoIIHindIII digest of POMC-CAT-704).The in theregulatory subunit [10,11,12,13, 17]. This issue has pMtR(A,B) plasmidwas a gift from Dr.S. McKnight at theUniversity beenexamined using an expression plasmidthat overexpresses a mutated regulatory subunit witha much lower affinity for CAMP; this mutant acts as an inhibitor of the kinase [14, 27].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
