Rat gonadotropin-releasing hormone receptor expressed in insect cells induces activation of adenylyl cyclase

Abstract

Increasing evidence exist that multiple G proteins mediate the effects of gonadotropin-releasing hormone (GnRH) on the synthesis and release of pituitary gonadotropins. In the present study, we have expressed the rat GnRH receptor (GnRH-R) in insect cells, by infection with a recombinant baculovirus. Under the conditions used, insect cells expressed, 48 h post-infection, a maximum of 7800 +/- 650 receptors/cell which bound GnRH agonist [D-Trp6]GnRH with a Kd = 0.52 +/- 0.06 nM indicating characteristics similar to those of the natural receptor. No binding was observed in non-infected cells or cells infected with wild-type baculovirus. In presence of GnRH, GnRH-R expressing cells elicited a time- and dose-dependent production of inositol trisphosphate, with a maximum level reached within 30 min and an EC50 = 5 nM. These recombinant insect cells also produced cAMP in response to GnRH. However, in contrast to other heterologous systems, or rat pituitary gonadotropes wherein GnRH induced a weak and delayed elevation of cAMP, in insect cells the rise of cAMP was comparatively rapid, attaining a maximum level after 2 h, and the EC50 was 5 nM. Finally, a clear activation of adenylyl cyclase (AC) in response to GnRH was shown for the first time by measuring the conversion of [alpha-32P]ATP into labeled cAMP, using membrane preparations from GnRH-R expressing insect cells. These data demonstrate that rat GnRH-R has the potential for dual coupling to both phosphoinositidase C and AC and suggest a major influence of the host cell for this coupling and/or its expression, probably in relation with the G protein repertoire and preference. This notion could be extended to several target cells other than pituitary gonadotropes that normally express the GnRH-R in mammals, including hippocampal, Leydig, granulosa, placental and GnRH-secreting hypothalamic cells.