High efficiency method for gene transfer in normal pituitary gonadotropes: adenoviral-mediated expression of G protein-coupled receptor kinase 2 suppresses luteinizing hormone secretion.
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.
Neill
J D
JD
Department of Physiology and Biophysics, University of Alabama, Birmingham 35294-0005, USA. neill@uab.edu
Musgrove
L C
LC
Duck
L W
LW
Sellers
J C
JC
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
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