Desensitization of Gonadotropin-releasing Hormone Action in αT3-1 Cells Due to Uncoupling of Inositol 1,4,5-Trisphosphate Generation and Ca2+ Mobilization

Craig A Mcardle,Gary B Willars,Robert C Fowkes,Stefan R Nahorski,James S Davidson,Wyn Forrest-Owen

doi:10.1074/jbc.271.39.23711

Abstract

Gonadotropin-releasing hormone (GnRH) acts via a G-protein coupled receptor on gonadotropes to increase cytosolic Ca2+ and stimulate gonadotropin secretion. Sustained exposure causes desensitization of these effects, but the GnRH receptor has no C-terminal tail and does not undergo rapid (<5 min) desensitization. Nevertheless, pretreatment of alphaT3-1 cells with GnRH reduced the spike Ca2+ response to GnRH and decreased the GnRH effect on inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) by 30-50%. Ca2+-free medium with or without thapsigargin also decreased GnRH-stimulated Ins(1,4,5)P3 generation, implying that attenuation of the Ca2+ response underlies the Ins(1,4,5)P3 reduction rather than vice versa. Intracellular Ca2+ pool depletion cannot explain desensitization of the Ca2+ response because pool depletion and repletion were faster (half-times, <1 min) than the onset of and recovery from desensitization (half-times 10-20 min and 4-6 h). Moreover, 1-h GnRH pre-treatment attenuated the spike Ca2+ response to GnRH but not that to ionomycin, and brief GnRH exposure in Ca2+-free medium reduced the response to ionomycin more effectively in controls than in desensitized cells. GnRH pretreatment also attenuated the Ca2+ response to PACAP38. This novel form of desensitization does not reflect uncoupling of GnRH receptors from their immediate effector system but rather a reduced efficiency of mobilization by Ins(1,4,5)P3 of Ca2+ from an intact intracellular pool.

Highlights

Studies over recent years have revealed a general scheme for homologous desensitization of GPCRs1 involving rapid uncoupling of receptors from their G-protein, subsequent sequestration of receptors from the plasma membrane, and internalization followed by proteolytic degradation [1]
Sustained exposure to Gonadotropin-releasing hormone (GnRH) reduces GnRH-stimulated gonadotropin secretion, and this underlies the suppression of the reproductive system, which is exploited in the major clinical applications of GnRH analogues [14, 15]
Gonadotropin-releasing hormone is released from the hypothalamus in a pulsatile manner and elicits pulsatile gonadotropin secretion

Summary

Introduction

Studies over recent years have revealed a general scheme for homologous desensitization of GPCRs1 involving rapid uncoupling of receptors from their G-protein, subsequent sequestration of receptors from the plasma membrane, and internalization followed by proteolytic degradation [1]. As shown (Fig. 6), GnRH caused a rapid increase in Ins[1,4,5]P3 levels in control cells (pool intact) stimulated in Ca2ϩ-free medium.

Results

Conclusion