Homologous up-regulation of the gonadotropin-releasing hormone receptor in alpha T3-1 cells is associated with unchanged receptor messenger RNA (mRNA) levels and altered mRNA activity.
In primary cultures of rat pituitary, GnRH receptor has been reported to up-regulate when exposed to GnRH. The molecular basis of this regulation could, in theory, involve modulation of gene transcription, RNA processing or stability, translation, and/or receptor degradation. The role of altered biosynthesis was investigated in the mouse gonadotrope cell line, alpha T3-1 cells, by studying the homologous up-regulation of GnRH receptor binding, mRNA levels, and mRNA activity. After GnRH exposure, ligand binding was performed on alpha T3-1 cell membranes. To correlate changes in receptor number and measurements of biosynthetic activity, GnRH receptor mRNA levels were quantitated by solution hybridization/RNase protection assay and Northern blot analysis, and the capacity of RNA from treated cells to direct the synthesis of new receptors (mRNA activity) was evaluated using a Xenopus oocyte-based bioassay. GnRH agonist radioligand binding assay results showed that exposure of alpha T3-1 cells to 10(-10) or 10(-8) M GnRH for 20 min induced an approximately 50% increase in the number of GnRH receptors, similar to previously reported results in rat pituitary primary culture. Despite the increase in receptor number, however, cytosolic and total GnRH receptor mRNA levels assayed by solution hybridization/RNase protection assay with GnRH receptor cRNA probes and by Northern blot analysis were not altered. In contrast to the unchanging mRNA levels, the measurements of mRNA activity paralleled the changes observed at the binding level. alpha T3-1 RNA from treated and control cells were injected into Xenopus oocytes, and the GnRH-induced Cl- current was quantified 48 h later by voltage clamp recording of the response to GnRH.(ABSTRACT TRUNCATED AT 250 WORDS)
Tsutsumi
M
M
Fishberg Center in Neurobiology, Department of Neurology, New York, New York 10029.
Laws
S C
SC
Sealfon
S C
SC
D000818:Animals; D019204:GTP-Binding Proteins / Q000378:metabolism; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology*; D051379:Mice; D008822:Mice, Transgenic; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D009865:Oocytes / Q000378:metabolism; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000378:metabolism*; D010911:Pituitary Neoplasms / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D011966:Receptors, LHRH / Q000096:biosynthesis* / Q000235:genetics; D014407:Tumor Cells, Cultured; D015854:Up-Regulation; D014982:Xenopus laevis
D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D011966:Receptors, LHRH; D007987:Gonadotropin-Releasing Hormone; D019204:GTP-Binding Proteins