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High Efficiency Method for Gene Transfer in Normal Pituitary Gonadotropes: Adenoviral-Mediated Expression of G Protein-Coupled Receptor Kinase 2 Suppresses Luteinizing Hormone Secretion¹

Department of Physiology and Biophysics, University of Alabama, Birmingham, Alabama 35294-0005

Address all correspondence and requests for reprints to: Dr. Jimmy D. Neill, Department of Physiology and Biophysics, 812 McCallum Building, 1918 University Boulevard, University of Alabama, Birmingham, Alabama 35294-0005. E-mail: neill{at}uab.edu

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 ß-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 ß-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-ß-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-ß-galactosidase infected 96–99% of the cells based on X-Gal staining. Uninfected and adeno-ß-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 Cß 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.

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