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)α subunits (G(q/11)α). First, we used GnRH-stimulated palmitoylation of G(q/11)α to identity 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)α. 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)α. GnRH (10-12, 10-10, 10-8, or 10-6 g/ml) for 40 min resulted in dose-dependent labeling of G(q/11)α compared with controls. Cholera toxin (5 μg/ml; activator of G(s)α), pertussis toxin (100 ng/ml; inhibitor of G(i)α actions) and Antide (50 nm; GnRH antagonist) did not stimulate palmitoylation of G(q/11)α above basal levels. However, phorbol myristic acid (100 ng/ml; protein kiness C activator) stimulated the palmitoylation of G(q/11)α 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 α(1B)-adrenergic receptor (α(1B)3(i)) couples to G(q/11)α. It can inhibit G(q/11)α-mediated stimulation of inositol phosphate (IP) turnover by interfering with receptor coupling to G(q/11)α. Transfection (efficiency 5-7%) with α(1b)3(i) cDNA, but not the third intracellular loop of M1-acetylcholine receptor (which also couples to G(q/11)α), 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 α(2A)-adrenergic receptor, M2- acetylcholine receptor (both couple to G(i)α), and D(1A)-receptor (couples to G(s)α) 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)α 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)α 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)α immunoreactivity. The loss of G(q/11)α 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)α by the GnRH receptor in primary pituitary cultures and in GGH3 cells.
ASJC Scopus subject areas
- Molecular Biology