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 Gq/11α subunits(Gq/11α). First, we used GnRH-stimulated palmitoylation of Gq/11α 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 Gq/11α. The immunoprecipitates were resolved by 10% SDS-PAGE and quantified. Treatment with GnRH resulted in time-dependent (0–120 min) labeling of Gq/11α. GnRH (10−12, 10−10, 10−8, or 10−6 g/ml) for 40 min resulted in dose-dependent labeling of Gq/11α compared with controls. Cholera toxin (5 μg/ml; activator of Gsα), pertussis toxin (100 ng/ml; inhibitor of Giα actions) and Antide (50 nm; GnRH antagonist) did not stimulate palmitoylation of Gq/11α above basal levels. However, phorbol myristic acid (100 ng/ml; protein kinase C activator) stimulated the palmitoylation of Gq/11α 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 α1B-adrenergic receptor (α1B3i) couples to Gq/11α, it can inhibit Gq/11α-mediated stimulation of inositol phosphate (IP) turnover by interfering with receptor coupling to Gq/11α. Transfection (efficiency 5–7%) withα 1B3i cDNA, but not the third intracellular loop of M1-acetylcholine receptor (which also couples toGq/11α), 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 α2A-adrenergic receptor, M2-acetylcholine receptor (both couple to Giα), and D1A-receptor (couples to Gsα) 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 Gq/11α 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 Gq/11α 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 Gq/11α immunoreactivity. The loss of Gq/11α immunoreactivity was not due to an effect of Buserelin on cell-doubling times. These studies provide the first direct evidence for regulation of Gq/11α by the GnRH receptor in primary pituitary cultures and in GGH3 cells.
β 2 -Adrenergic Receptor Conformational Response to Fusion Protein in the Third Intracellular Loop
