title: Regulation of G(q/11)alpha by the gonadotropin-releasing hormone receptor.
abstract: Evidence from use of pertussis and cholera toxins and from NaF suggested the involvement of G proteins in GnRH regulation of gonadotrope function. We have used three different methods to assess GnRH receptor regulation of G(q/11)alpha subunits (G(q/11)alpha). First, we used GnRH-stimulated palmitoylation of G(q/11)alpha to identify their involvement in GnRH receptor-mediated signal transduction. Dispersed rat pituitary cell cultures were labeled with [9,10-(3)H(N)]-palmitic acid and immunoprecipitated with rabbit polyclonal antiserum made against the C-terminal sequence of G(q/11)alpha. The immunoprecipitates were resolved by 10% SDS-PAGE and quantified. Treatment with GnRH resulted in time-dependent (0-120 min) labeling of G(q/11)alpha. GnRH (10(-12), 10(-10), 10(-8), or 10(-6) g/ml) for 40 min resulted in dose-dependent labeling of G(q/11)alpha compared with controls. Cholera toxin (5 microg/ml; activator of G(i)alpha), pertussis toxin (100 ng/ml; inhibitor of G(i)alpha actions) and Antide (50 nM; GnRH antagonist) did not stimulate palmitoylation of G(q/11)alpha above basal levels. However, phorbol myristic acid (100 ng/ml; protein kinase C activator) stimulated the palmitoylation of G(q/11)alpha above basal levels, but not to the same extent as 10(-6) g/ml GnRH. Second, we used the ability of the third intracellular loop (3i) of other seven-transmembrane segment receptors that couple to specific G proteins to antagonize GnRH receptor-stimulated signal transduction and therefore act as an intracellular inhibitor. Because the third intracellular loop of alpha1B-adrenergic receptor (alpha1B 3i) couples to G(q/11)alpha, it can inhibit G(q/11)alpha-mediated stimulation of inositol phosphate (IP) turnover by interfering with receptor coupling to G(q/11)alpha. Transfection (efficiency 5-7%) with alpha1B 3i cDNA, but not the third intracellular loop of M1-acetylcholine receptor (which also couples to G(q/11)alpha), resulted in 10-12% inhibition of maximal GnRH-evoked IP turnover, as compared with vector-transfected GnRH-stimulated IP turnover. The third intracellular loop of alpha2A adrenergic receptor, M2-acetylcholine receptor (both couple to G(i)alpha), and D1A-receptor (couples to G(s)alpha) did not inhibit IP turnover significantly compared with control values. GnRH-stimulated LH release was not affected by the expression of these peptides. Third, we assessed GnRH receptor regulation of G(q/11)alpha in a PRL-secreting adenoma cell line (GGH(3)1') expressing the GnRH receptor. Stimulation of GGH(3)1' cells with 0.1 microg/ml Buserelin (a metabolically stable GnRH agonist) resulted in a 15-20% decrease in total G(q/11)alpha at 24 h following agonist treatment compared with control levels; this action of the agonist was blocked by GnRH antagonist, Antide (10(-6) g/ml). Neither Antide (10(-6) g/ml, 24 h) alone nor phorbol myristic acid (0.33-100 ng/ml, 24 h) mimicked the action of GnRH agonist on the loss of G(q/11)alpha immunoreactivity. The loss of G(q/11)alpha immunoreactivity was not due to an effect of Buserelin on cell-doubling times. These studies provide the first direct evidence for regulation of G(q/11)alpha by the GnRH receptor in primary pituitary cultures and in GGH3 cells.
authors.affiliation: Department of Physiology and Pharmacology, Oregon Health Sciences University, Portland 97201, USA.
authors.forename: D
authors.initials: D
authors.lastname: Stanislaus
authors.forename: J A
authors.initials: JA
authors.lastname: Janovick
authors.forename: S
authors.initials: S
authors.lastname: Brothers
authors.forename: P M
authors.initials: PM
authors.lastname: Conn
mesh_terms: D000818:Animals; D002064:Buserelin / Q000494:pharmacology; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000494:pharmacology; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D010168:Palmitates / Q000493:pharmacokinetics; D037342:Pertussis Toxin; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018340:Receptors, Adrenergic, alpha-1 / Q000378:metabolism; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism; D017447:Receptors, Dopamine D1 / Q000378:metabolism; D011966:Receptors, LHRH / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: C547718:Adra1b protein, rat; C547770:Adra2a protein, rat; D009842:Oligopeptides; D010168:Palmitates; D018340:Receptors, Adrenergic, alpha-1; D018341:Receptors, Adrenergic, alpha-2; D017447:Receptors, Dopamine D1; D011966:Receptors, LHRH; D010566:Virulence Factors, Bordetella; C098593:dopamine D1A receptor; D002772:Cholera Toxin; C057022:iturelix; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate; D002064:Buserelin