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Regulation of G_q/11 by the Gonadotropin-Releasing Hormone Receptor

Department of Physiology and Pharmacology (D.S., P.M.C.),Oregon Health Sciences University, Portland, Oregon 97201,
Oregon Regional Primate Research Center,(D.S., J.A.J., S.B., P.M.C.), Beaverton, Oregon 97006

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 subunits(G_q/11). First, we used GnRH-stimulated palmitoylation of G_q/11 to identify their involvement in GnRH receptor-mediated signal transduction. Dispersed rat pituitary cell cultures were labeled with [9,10-³H(N)]-palmitic acid and immunoprecipitated with rabbit polyclonal antiserum made against the C-terminal sequence of G_q/11. 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. GnRH (10^-12, 10^-10, 10^-8, or 10^-6 g/ml) for 40 min resulted in dose-dependent labeling of G_q/11 compared with controls. Cholera toxin (5 µg/ml; activator of G_s), pertussis toxin (100 ng/ml; inhibitor of G_i actions) and Antide (50 nM; GnRH antagonist) did not stimulate palmitoylation of G_q/11 above basal levels. However, phorbol myristic acid (100 ng/ml; protein kinase C activator) stimulated the palmitoylation of G_q/11 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 (3_i) 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 _1B-adrenergic receptor (_1B3_i) couples to G_q/11, it can inhibit G_q/11-mediated stimulation of inositol phosphate (IP) turnover by interfering with receptor coupling to G_q/11. Transfection (efficiency 5–7%) with _1B3_i cDNA, but not the third intracellular loop of M₁-acetylcholine receptor (which also couples toG_q/11), 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 _2A-adrenergic receptor, M₂-acetylcholine receptor (both couple to G_i), and D_1A-receptor (couples to G_s) 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 in a PRL-secreting adenoma cell line (GGH₃1') expressing the GnRH receptor. Stimulation of GGH₃1' cells with 0.1 µg/ml Buserelin (a metabolically stable GnRH agonist) resulted in a 15–20% decrease in total G_q/11 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 immunoreactivity. The loss of G_q/11 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 by the GnRH receptor in primary pituitary cultures and in GGH₃ cells.

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