Rat gonadotropin-releasing hormone receptor expressed in insect cells induces activation of adenylyl cyclase.
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.
Delahaye
R
R
Endocrinologie Cellulaire et Moléculaire de la Reproduction, Université P. & M. Curie, URA CNRS 1449, Paris, France.
Manna
P R
PR
Bérault
A
A
Berreur-Bonnenfant
J
J
Berreur
P
P
Counis
R
R
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002999:Clone Cells / Q000166:cytology / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D015870:Gene Expression / Q000235:genetics / Q000502:physiology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007313:Insecta / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D011966:Receptors, LHRH / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology
D011966:Receptors, LHRH; D011994:Recombinant Proteins; D015544:Inositol 1,4,5-Trisphosphate; D000242:Cyclic AMP; D000262:Adenylyl Cyclases