The lack of gonadotrophin-releasing hormone (GnRH) receptor desensitisation in alpha T3-1 cells is not due to GnRH receptor reserve or phosphatidylinositol 4,5-bis-phosphate pool size

The phospholipase C (PLC)-activating gonadotrophin-releasing hormone (GnRH) receptor is thought not to rapidly desensitise in alpha T3-1 cells. This e xtremely unusual characteristic raises the concern that it might be a featu re of the cell type, rather than the receptor per se. Here we have used vid eo imaging to establish whether the effects of endogenous PLC-activating G- protein coupled receptors (GPCRs) on Ca2+ ion concentration [Ca2+](i) desen sitise in these cells. Oxytocin, endothelin-l, methacholine, and UTP all ca used [Ca2+](i) increases which underwent rapid homologous desensitisation i n that they were transient and responses to repeat stimuli were attenuated whereas subsequent responses to GnRH were not. To test whether receptor res erve obscures functional desensitisation of GnRH receptors, a photoaffinity antagonist (Pant-l), was used to effect a partial and irreversible recepto r blockade. UV crosslinking in medium with 1000 nM Pant-1 reduced GnRH rece ptor number to 20 +/- 5% and reduced maximal buserelin-stimulated [H-3]IPx accumulation to 57 +/- 5%, demonstrating removal of receptor reserve. In co ntrol alpha T3-1 cells the initial rate of GnRH-stimulated [H-3]IPx accumul ation was maintained for at least 5 min and GnRH caused a sustained increas e in Ins(1,4,5)P-3 mass (confirming the resistance of GnRH receptors to des ensitisation) and Pant-1 pre-treatment reduced the magnitude of these respo nses without altering. their temporal profiles. In alpha T3-1 cells stably transfected with recombinant human muscarinic receptors (alpha T3-1/M3), re sponses to methacholine were characteristic of desensitising GPCRs (transie nt Ins(1,4,5)P-3 and curvilinear [H-3]IPx responses) and were unaltered by Pant-1. To test the relevance of phospholipid pool size, alpha T3-1/M3 cell s were pre-treated with GnRH or methacholine in medium with LiCl (to deplet e PtdIns(4,5)P-2 pools). These pre-treatments reduced subsequent responses to methacholine and GnRH comparably, indicating access to a shared PtdIns(4 ,5)P-2 pool. Partial depletion of this pool (GnRH pre-treatment in medium w ith LiCl) reduced the magnitude of the [H-3]IPx and Ins(1,4,5)P-3 responses to methacholine and GnRH, without altering their temporal profiles. Thus t he GnRH receptor does not undergo rapid homologous desensitisation in alpha T3-1 cells in spite of the fact that they can desensitise other endogenous (and recombinant) PLC-activating GPCRs, and the lack of desensitisation ca nnot be attributed to the existence of GnRH receptor reserve or access to a n atypically large or rapidly re-cycled PtdIns(4,5)P-2 pool. This unique fu nctional characteristic (mammalian GnRH receptors are the only PLC-activati ng GPCRs known not to rapidly desensitise) almost certainly therefore refle cts the atypical structure of these receptors (mammalian GnRH receptors are the only PLC-activating GPCRs known to lack C-terminal tails). (C) 1999 El sevier Science Ireland Ltd. All rights reserved.