REGULATION OF G(Q 11)ALPHA BY THE GONADOTROPIN-RELEASING-HORMONE RECEPTOR/

Evidence from use of pertussis and cholera toxins and from NaF suggest ed the involvement of G proteins in GnRH regulation of gonadotrope fun ction. We have used three different methods to assess GnRH receptor re gulation of G(q/11)alpha subunits (G(q/11)alpha). First, we used GnRH- stimulated palmitoylation of G(q/11)alpha to identify their involvemen t in GnRH receptor-mediated signal transduction. Dispersed rat pituita ry cell cultures were labeled with [9,10(-3)H(N)]-palmitic acid and im munoprecipitated with rabbit polyclonal antiserum made against the C-t erminal 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 labelin g of G(q/11)alpha compared with controls. Cholera toxin (5 mu g/ml; ac tivator of G(s) alpha), pertussis toxin (100 ng/ml; inhibitor of G(i) alpha actions) and Antide (50 nM; GnRH antagonist) did not stimulate p almitoylation of G(q/11)alpha above basal levels. However, phorbol myr istic acid (100 ng/ml; protein kinase C activator) stimulated the palm itoylation of G(q/11)alpha above basal levels, but not to the same ext ent as 10(-6) g/ml GnRH. Second, we used the ability of the third intr acellular loop (3) 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. B ecause the third intracellular loop of alpha(1B)-adrenergic receptor ( alpha(1B)3(i)) couples to G(q/11)alpha, it can inhibit G(q/11)alpha-me diated stimulation of inositol phosphate (IP) turnover by interfering with receptor coupling to G(q/11)alpha. Transfection (efficiency 5-7%) with alpha(1B)3(i) cDNA, but not the third intracellular loop of M-1- acetylcholine receptor (which also couples to G(alpha/11)alpha), resul ted in 10-12% inhibition of maximal GnRH-evoked IP turnover, as compar ed with vector-transfected GnRH-stimulated IP turnover. The third intr acellular loop of alpha(2A)-adrenergic receptor, M-2-acetylcholine rec eptor (both couple to G(i) alpha), and D-1A-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 t he GnRH receptor. Stimulation of GGH(3)1' cells with 0.1 mu g/ml Buser elin (a metabolically stable GnRH agonist) resulted in a 15-20% decrea se 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 an tagonist, Antide (10(-6) mu/ml). Neither Antide (10(-6) g/ml, 24 h) al one nor phorbol myristic acid (0.33-100 ng/ml, 24 h) mimicked the acti on of GnRH agonist on the loss of G(q/11)alpha immunoreactivity. The l oss of G(q/11)alpha immunoreactivity was not due to an effect of Buser elin on cell-doubling times. These studies provide the first direct ev idence for regulation of G(q/11)alpha by the GnRH receptor in primary pituitary cultures and in GGH(3) cells.