title: High efficiency method for gene transfer in normal pituitary gonadotropes: adenoviral-mediated expression of G protein-coupled receptor kinase 2 suppresses luteinizing hormone secretion.
abstract: The level of LH secretion is determined by both alterations in gonadotrope responsiveness and alterations in GnRH secretion. The molecular mechanisms underlying gonadotrope responsiveness are unknown, but may include G protein-coupled receptor kinases (GRKs). Typically, GRKs phosphorylate the intracellular regions of seven-transmembrane receptors permitting beta-arrestin to bind, which prevents receptor activation of its G protein. Previously, we reported that heterologous expression of GRK2, -3, and -6 in GnRH receptor-expressing COS cells by complementary DNA transfection suppressed GnRH-stimulated inositol trisphosphate production, and that coexpression of GRK2 and beta-arrestin-2 was more inhibitory than either expressed alone. Here, we have investigated the effect of GRK2 on GnRH-stimulated LH secretion using adenovirus-mediated gene transfer in normal pituitary gonadotropes. Pituitary cells were infected with adeno-GRK2 or adeno-beta-galactosidase constructs at a multiplicity of infection of 60 (number of viral particles per cell). Seventy-two hours later, GRK2 expression was measured by enzyme-linked immunosorbent assay, and GnRH-stimulated LH secretion (10(-7) M GnRH-A for 90 min) was assayed by RIA. Adeno-beta-galactosidase infected 96-99% of the cells based on X-Gal staining. Uninfected and adeno-beta-galactosidase-infected cells exhibited endogenous GRK immunoreactivity of about 0.5 (OD405), and LH secretion of 14.8-17.7 ng/ml. Adeno-GRK2-infected cells showed a GRK2 immunoreactivity of about 2.5 (OD405) and LH secretion of 2.5 ng/ml. Therefore, adeno-GRK2 infection resulted in a 5-fold increase in the GRK2 OD405 value, which was accompanied by an 80-85% decrease in GnRH-stimulated LH secretion. GnRH-stimulated inositol trisphosphate production by gonadotropes also was inhibited, suggesting a site of action for GRK2 at phospholipase Cbeta or earlier in the signal transduction pathway. The significance of these findings is 2-fold: 1) adenoviral-mediated gene transfer permits investigation of the regulatory role of gene products in the cell of interest, the gonadotrope, rather than in heterologous cell systems; and 2) additional, stronger evidence is provided that supports a role for GRKs in setting the responsiveness of GnRH receptor signaling.
authors.affiliation: Department of Physiology and Biophysics, University of Alabama, Birmingham 35294-0005, USA. neill@uab.edu
authors.forename: J D
authors.initials: JD
authors.lastname: Neill
authors.forename: L C
authors.initials: LC
authors.lastname: Musgrove
authors.forename: L W
authors.initials: LW
authors.lastname: Duck
authors.forename: J C
authors.initials: JC
authors.lastname: Sellers
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D054769:G-Protein-Coupled Receptor Kinase 2; D018014:Gene Transfer Techniques; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology; D007986:Luteinizing Hormone / Q000378:metabolism*; D010902:Pituitary Gland / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011966:Receptors, LHRH / Q000378:metabolism; D051552:beta-Adrenergic Receptor Kinases
chemical_list: D011966:Receptors, LHRH; D007987:Gonadotropin-Releasing Hormone; D007986:Luteinizing Hormone; D017868:Cyclic AMP-Dependent Protein Kinases; C496569:Grk2 protein, rat; D051552:beta-Adrenergic Receptor Kinases; D054769:G-Protein-Coupled Receptor Kinase 2