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Regular Article |
a Animal Reproduction and Biotechnology Laboratory, Department of Physiology, Colorado State University, Fort Collins, Colorado 80523
b Laboratory of Animal Breeding and Reproduction, Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan
ABSTRACT
Rates of internalization of the murine GnRH receptor fused via its C-terminus to green fluorescent protein (GnRH-R-GFP) were examined in Chinese hamster ovary cells (CHO cells) and compared to those of native murine GnRH-R in a clonal murine gonadotroph cell line (LßT2 cells). The resulting rates of internalization of murine receptors were then compared with those of sheep GnRH-R in ovine gonadotrophs. Cells were incubated with radioiodinated [D-Ala6]GnRH on ice for 4 h to allow binding of the ligand to GnRH-R, then cells were warmed to 37°C to permit internalization. Surface-bound radioligand began to decrease as soon as the cells were warmed and had decreased significantly within 20 min. A steady-state level of surface-bound radioligand was achieved after 60 min in both CHO cells and LßT2 cells (38% and 41%, respectively, of initial value; P < 0.05). Internalization of radioligand began immediately after warming the cells to 37°C, and a significant proportion of surface ligand had been internalized by 20 min. A steady-state maximum of internalization was reached after 60 min in both CHO cells and LßT2 cells (29% and 28%, respectively, of total cell-associated ligand; P < 0.05). Changes in surface-bound radioligand and internalized radioligand in sheep pituitary cells were similar to those in CHO cells and LßT2 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 LßT2 cells (P < 0.05). In a separate experiment, the effect of estradiol on the rate of internalization of GnRH-R in ovine pituitary cells was examined. Although treatment of ovine pituitary cells with estradiol approximately doubled the number of GnRH receptors, it did not alter either the rate or extent of receptor internalization. These results show that rates of internalization of recombinant murine GnRH-R-GFP in CHO cells and native murine and ovine GnRH-R in LßT2 cells and in sheep pituitary cells, respectively, are similar, but amounts of ovine GnRH-R internalized are greater than those for murine GnRH-R. Further, the rate of internalization of occupied receptor is similar in gonadotroph and nongonadotroph cells, and the addition of GFP to the C-terminus of the murine GnRH-R does not alter the rate of internalization.
1 Supported by National Institutes of Health grant CA75662 (T.M.N.). The first author, T.H., thanks the Ministry of Education, Science, Sports and Culture of Japanese Government for awarding him a grant from the Overseas Research Culture of Fellowship.
2 Correspondence. FAX: 970 491 3557; tnett{at}cvmbs.colostate.edu
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