TRANSIENT TRANSFECTION OF GGH(3)-1' CELLS [GH3 CELLS STABLY TRANSFECTED WITH THE GONADOTROPIN-RELEASING-HORMONE (GNRH) RECEPTOR COMPLEMENTARY DEOXYRIBONUCLEIC-ACID] WITH THE CARBOXYL-TERMINAL OF BETA-ADRENERGIC-RECEPTOR KINASE-1 BLOCKS PROLACTIN-RELEASE - EVIDENCE FOR A ROLE OF THE G-PROTEIN BETA-GAMMA-SUBUNIT COMPLEX IN GNRH SIGNAL-TRANSDUCTION

G proteins consist of heterotrimeric alpha-, beta-, and gamma-subunits . To assess the role of the beta gamma-subunit complex in GnRH recepto r-mediated signal transduction, GGH(3)-1' cells were transfected with plasmids PRK5-beta ARK1(495-689) containing complementary DNA (cDNA) o f the carboxyl-terminal (Gly(495)-Leu(689)) of beta-adrenergic recepto r kinase 1 (beta ARK1). GGH(3)-1' cells are GH(3) cells that have been stably transfected with rat GnRH receptor cDNA. The carboxyl region o f beta ARK1 (Gly(495)-Leu(689)) binds to the beta gamma complex and th ereby inhibits its action. Twenty-four hours after stimulation, PRL re lease, cAMP release, and inositol phosphate (IP) production were measu red in these cells and in control cells transfected with vector PRK5 c DNA alone. In cells expressing the beta ARK1-(495-689) sequence there was inhibition of basal PRL release (69.3%), cAMP release (61.2%), and IP production (75.5%) compared to cells containing vector only. When cells expressing the beta ARK1 fragment were stimulated with a GnRH an alog (Buserelin; 10(-7) M), maximal responses were inhibited (66.1% fo r PRL release, 52.3% for cAMP release, and 79.1% for IP production). S catchard analysis of GnRH analog binding was also performed in the two groups of transfected cells. No significant differences in K-d or rec eptor numbers were found between beta ARK1-(495- 689)-transfected cell s and control cells containing the vector alone. These data suggest a role for the beta gamma complex in mediation of cAMP release, IP produ ction, and hormone release in response to agonist occupancy of the GnR H receptor.