Internalization rates of murine and ovine gonadotropin-releasing hormone receptors

Rates of internalization of the murine GnRH receptor fused via ifs C-termin us to green fluorescent protein (GnRH-R-GFP) were examined in Chinese hamst er ovary cells (CHO cells) and compared to those of native murine GnRH-R in a clonal murine gonadotroph cell line (L beta T2 cells). The resulting rat es of internalization of murine receptors were then compared with those of sheep GnRH-R in ovine gonadotrophs. Cells were incubated with radioiodinate d [D-Ala(6)]GnRH on ice for 4 h to allow binding of the ligand to GnRH-R, t hen cells were warmed to 37 degreesC to permit internalization. Surface-bou nd radioligand began to decrease as soon as the cells were warmed and had d ecreased significantly within 20 min. A steady-state level of surface-bound radioligand was achieved after 60 min in both CHO cells and L beta T2 cell s (38% and 41%, respectively of initial value; P < 0.05). Internalization o f radioligand began immediately after warming the cells to 37<degrees>C, an d a significant proportion of surface ligand had been internalized by 20 mi n. A steady-state maximum of internalization was reached after 60 min in bo th CHO cells and L beta T2 cells (29% and 28%, respectively, of total cell- associated ligand; P < 0.05). Changes in surface-bound radioligand and inte rnalized radioligand in sheep pituitary cells were similar to those in CHO cells and L<beta>T2 cells, but the amount of radioligand internalized after 60 min (40% of total cell-associated ligand) was 1.4 times higher than in CHO cells and L beta T2 cells (P < 0.05). In a separate experiment, the eff ect of estradiol on the rate of internalization of GnRH-R in ovine pituitar y cells was examined. Although treatment of ovine pituitary cells with estr adiol approximately doubled the number of GnRH receptors, it did not alter either the rate or extent of receptor internalization. These results show t hat rates of internalization of recombinant murine GnRH-R-GFP in CHO cells and native murine and ovine GnRH-R in L<beta>T2 cells and in sheep pituitar y cells, respectively, are similar, but amounts of ovine GnRH-R internalize d are greater than those for murine GnRH-R. Further,the rate of internaliza tion of occupied receptor is similar in gonadotroph and nongonadotroph cell s, and the addition of GFP to the C-terminus of the murine GnRH-R does not alter the rate of internalization.