TRANSLATIONAL REGULATION OF THE GONADOTROPIN-RELEASING-HORMONE RECEPTOR IN ALPHA-T-3-1 CELLS

Prolonged exposure of the GnRH receptor to high concentrations of GnRH leads to receptor down-regulation. The role of altered receptor biosy nthesis in this agonist-induced receptor down-regulation was investiga ted in the mouse gonadotrope cell line, alpha T-3-1 cells. After expos ure to 1 mu M GnRH for 24 h, the number of GnRH receptor-binding sites in alpha T-3-1 cells decreased to 25 +/- 6% of the control levels. No corresponding changes were observed in GnRH receptor messenger RNA (m RNA) using either quantitative ribonuclease protection/solution hybrid ization assay or Northern blot analysis. However, when the ability of this RNA to direct the synthesis of functional GnRH receptors was exam ined by quantitative assessment of the voltage clamp response in Xenop us oocytes, GnRH-induced currents in oocytes injected with RNA isolate d from down-regulated cells was reduced to 40 +/- 13% of the response obtained after the injection of RNA from control alpha T-3-1 cells. Th us, although GnRH receptor mRNA levels were not altered, the ability o f cellular RNA isolated from alpha T-3-1 cells to direct the synthesis of functional GnRH receptors was regulated in concert with receptor b inding. To investigate the possibility that GnRH receptor mRNA transla tional efficiency was reduced, the distribution of polyribosome-associ ated GnRH receptor mRNA was studied. polyribosome-associated mRNA was separated by linear sucrose gradient, and GnRH receptor mRNA distribut ion was determined by ribonuclease protection assay. GnRH receptor mRN A distribution shifted from the largest to smaller polyribosome and mo nosome fractions in cells exposed to GnRH compared to controls. The we ighted mean of GnRH receptor mRNA distribution among eight fractions s hifted from fraction 5.924 +/- 0.06 in control polysomes to fraction 5 .45 +/- 0.219 for polysomes from down-regulated cells (P < 0.05). Thes e results demonstrate that GnRH receptor down-regulation is accompanie d by decreased GnRH receptor mRNA translation in the absence of any ch ange in GnRH receptor mRNA levels. These data suggest that decreased e fficiency of GnRH receptor mRNA translation contributes to the down-re gulation of this receptor in alpha T-3-1 cells.