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 protei n-coupled receptor kinases (GRKs). Typically, GRKs phosphorylate the intrac ellular regions of seven-transmembrane receptors permitting beta-arrestin t o 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-sti mulated inositol trisphosphate production, and that coexpression of GRK2 an d 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. Pituit ary cells were infected with adeno-GRK2 or adeno-beta-galactosidase constru cts at a multiplicity of infection of 60 (number of viral particles per cel l). 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-inf ected 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 GR K2 immunoreactivity of about 2.5 (OD405) and LH secretion of 2.5 ng/ml. The refore, adeno-GRK2 infection resulted in a 5-fold increase in the GRK2 OD40 5 value, which was accompanied by an 80-85% decrease in GnRH-stimulated LH secretion. GnRH-stimulated inositol trisphosphate production by gonadotrope s also was inhibited, suggesting a site of action for GRK2 at phospholipase C beta or earlier in the signal transduction pathway. The significance of these findings is 2-fold: 1) adenoviral-mediated gene transfer permits inve stigation of the regulatory role of gene products in the cell of interest, the gonadotrope, rather than in heterologous cell systems; and 2) additiona l, stronger evidence is provided that supports a role for GRKs in setting t he responsiveness of GnRH receptor signaling.