Transient transfection of GGH3-1' 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 receptor-mediated signal transduction, GGH3-1' cells were transfected with plasmids PRK5-beta ARK1 (495-689) containing complementary DNA (cDNA) of the carboxyl-terminal (Gly495-Leu689) of beta-adrenergic receptor kinase 1 (beta ARK1). GGH3-1' cells are GH3 cells that have been stably transfected with rat GnRH receptor cDNA. The carboxyl region of beta ARK1 (Gly495-Leu689) binds to the beta gamma complex and thereby inhibits its action. Twenty-four hours after stimulation, PRL release, cAMP release, and inositol phosphate (IP) production were measured in these cells and in control cells transfected with vector PRK5 cDNA 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 analog (Buserelin; 10(-7) M), maximal responses were inhibited (66.1% for PRL release, 52.3% for cAMP release, and 79.1% for IP production). Scatchard analysis of GnRH analog binding was also performed in the two groups of transfected cells. No significant differences in Kd or receptor numbers were found between beta ARK1-(495-689)-transfected cells and control cells containing the vector alone. These data suggest a role for the beta gamma complex in mediation of cAMP release, IP production, and hormone release in response to agonist occupancy of the GnRH receptor.
Guo
C H
CH
Oregon Regional Primate Research Center, Beaverton 97006, USA.
Janovick
J A
JA
Kuphal
D
D
Conn
P M
PM
D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000096:biosynthesis; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics* / Q000502:physiology; D018076:DNA, Complementary / Q000235:genetics*; D019204:GTP-Binding Proteins / Q000502:physiology; D015870:Gene Expression; D007987:Gonadotropin-Releasing Hormone / Q000235:genetics; D007295:Inositol Phosphates / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics; D011388:Prolactin / Q000378:metabolism*; D051381:Rats; D011966:Receptors, LHRH / Q000235:genetics* / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases
D018076:DNA, Complementary; D007295:Inositol Phosphates; D010446:Peptide Fragments; D011966:Receptors, LHRH; D007987:Gonadotropin-Releasing Hormone; D011388:Prolactin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D019204:GTP-Binding Proteins