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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.
GnRH, GnRH receptor
INTRODUCTION
Gonadotropin-releasing hormone and its receptor (GnRH-R) are key mediators of the reproductive axis by virtue of their regulation of the synthesis and release of LH and FSH [1]. The GnRH-R is coupled to phospholipase C via G proteins, and its stimulation by GnRH induces the production of inositol phosphates and diacylglycerol that results in the elevation of cytosolic calcium and the activation of protein kinase C to initiate a variety of cellular responses including hormone secretion [1]. Receptor activation is followed by extensive internalization of GnRH-R complex via endocytosis [1, 2]. This event that leads to decreased receptor numbers on the cell surface (down-regulation) may be responsible for the loss of gonadotroph sensitivity to GnRH [1–4].
Internalization of murine [5–7], rat [8–12], chicken [13], human [5, 13, 14], and mutant GnRH-R [5–7, 10, 13, 14] has been measured by using radioligand [5–7, 9–14] and microscopy [8, 11, 12]. Human GnRH-R undergoes GnRH-induced internalization much more rapidly than murine receptor [5, 6], and chicken GnRH-R is internalized 12-fold more rapidly than the human receptor [13]. Furthermore, it is reported that mutations in the structure of GnRH-R change the rates of internalization [5–7, 10, 13, 14]; most notably, addition of a longer intracellular C-terminus accelerates rate of internalization [13]. Recently, construction of a fusion protein comprised of murine (m)GnRH-R linked via its C-terminus to green fluorescent protein (GFP; GnRH-R-GFP) has been reported [15, 16]. This molecule represents an important tool for evaluating receptor synthesis, transport to the plasma membrane and for visually evaluating kinetics of internalization of the mGnRH-R. The mGnRH-R-GFP recapitulates ligand binding and activation of second messengers of the native receptor. However, whether addition of GFP to the C-terminus of the GnRH-R alters its rate of internalization is not known. Recently, a clonal murine gonadotroph cell line (LßT2 cells) was developed that expresses GnRH-R and both 
and ß subunits of LH, and the cell secretes LH in response to GnRH [17–19]. Therefore, to determine if fusion of GFP to the mGnRH-R influences internalization, rates of internalization of mGnRH-R-GFP in CHO cells were compared to mGnRH-R in LßT2 cells. The homology of amino acids between the sheep and mGnRH-R is 86%. However, the ovine (o)GnRH-R contains one additional amino acid in the second extracellular loop, and this structure resembles that of the human (h)GnRH-R more closely than the murine receptor [20, 21]. The hGnRH receptor is internalized more rapidly than the mGnRH-R [5, 6]. The kinetics of oGnRH-R internalization have yet to be examined. Therefore, the internalization kinetics of oGnRH-R in normal pituitary cells were also compared with those of these mGnRH-Rs to determine if the kinetics of oGnRH-R internalization more closely resembles that of the hGnRH-R or mGnRH-R.
MATERIALS AND METHODS
Cells and Cell Culture
Cell lines Construction of the Chinese hamster ovary (CHO) cell line expressing the GnRH-R fusion protein was described previously [15]. The LßT2 cells were provided by Dr. Pamela Mellon of the University of California, San Diego, CA. Cells were maintained in Dulbecco's minimum essential medium (DMEM; Sigma Chemical Co., St. Louis, MO) containing 2 mM glutamine (Sigma), 100 IU penicillin/ml, 100 µg streptomycin/ml, 10% fetal bovine serum (FBS; Gemini Bio-Products, Inc., Calabasas, CA), and 1% nonessential amino acids (Life Technologies, Inc., Grand Island, NY) in a 5% CO2, 37°C humidified environment. Cells in 1 ml of cell suspension media were plated in 24-well dishes (Falcon; Becton Dickinson Labware, Franklin Lake, NJ) at a density of 1 x 105 and 7.5 x 105 cells/well, respectively, for CHO cells and LßT2 cells. The CHO cells were cultured for 1 day after plating, and LßT2 cells for 1 or 2 days.
Sheep pituitary cells Anterior pituitary glands were obtained from five ovariectomized, crossbred ewes (Rambouillets x Columbia, 4–5 yr old) and dispersed into single cells as described by Gregg et al. [22]. Briefly, the ewes were euthanized with an overdose of sodium pentobarbital, and the pituitary glands were removed aseptically. Pituitary cells were dispersed enzymatically using collagenase, hyaluronidase, and deoxyribonuclease and suspended in culture medium (DMEM supplemented with 10% horse serum [Gemini Bio-Products, Inc.], 2.5% FBS, 1% nonessential amino acids, 100 IU/ml penicillin, and 100 µg/ml streptomycin). Cells (2 x 106) in 3 ml media were plated in 6-well dishes (Falcon; Becton Dickinson Labware) and cultured for 3 days at 37°C in a humidified atmosphere of 5% CO2. To maximize the numbers of GnRH-R, 40 nM estradiol-17ß (Sigma) was added 12 h before the start of the experiment [22].
Measurement of GnRH-R Internalization
Chinese hamster ovary cells and LßT2 cells D-Ala6–desGly10-GnRH-Pro9-ethylamide ([D-Ala6]GnRH) was radioiodinated using a glucose-oxidase procedure and reaction products were separated on QAE Sephadex as described by Wagner et al. [23]. Internalization of 125I-labeled [D-Ala6]GnRH was measured by a modification of the acid-wash procedure of Pawson et al. [13]. Cultured CHO cells and LßT2 cells in 24-well dishes were washed once with 1 ml ice-cold DMEM containing 0.1% BSA (assay media); 2 x 105 cpm of 125I-labeled [D-Ala6]GnRH (72 fmol) in 100 µl assay media were added to each well in the presence or absence of 72 pmol (1000-fold excess) unlabeled [D-Ala6]GnRH in 50 µl of assay media (final concentration of 125I-labeled [D-Ala6]GnRH in the assay media was 0.48 nM). Cells were incubated on ice for 4 h and then warmed to 37°C for 0, 10, 20, 30, 60, 90, or 120 min. Internalization of radioligand was stopped by cooling the cells to 0°C and rapidly washing three times with 1 ml ice-cold PBS. Acid-sensitive radioligand binding, representing cell-surface binding, was removed by the addition of 1 ml ice-cold acid solution (150 mM NaCl, 50 mM acetic acid, pH 2.8) for 12 min. After removal of the acid wash, cells were washed twice with 1 ml ice-cold PBS and then were solubilized with 0.5 ml of NaOH/SDS (0.2 M NaOH, 1% SDS) to measure acid-resistant (internalized) radioligand. Nonspecific binding (binding in the presence of 72 pmol unlabeled [D-Ala6]GnRH) was determined and subtracted at each time point. Surface-bound radioligand was expressed as percentage of initial values. Internalized radioligand was expressed as percentage of total cell-associated label (internalized radioligand/internalized radioligand plus surface radioligand) at each time point.
To determine if any of the radioactivity in the media resulted from degradation and excretion of internalized radioligand, immunoprecipitable radioligand was evaluated in CHO cells using an antiserum specific for [D-Ala6]GnRH. In this experiment, 140 µl media in the well was removed at each time point, and radioactivity and immunoprecipitable radioligand in the extracellular media were evaluated. Appearance of nonimmunoprecipitable counts was taken to mean that 125I-labeled [D-Ala6]GnRH had been degraded by the cells and excreted into the medium.
Sheep pituitary cells Assay procedures were the same as those in CHO cells and LßT2 cells except that 1 x 106 cpm of 125I-labeled [D-Ala6]GnRH in 500 µl assay media were added to each well in the presence or absence of 360 pmol of unlabeled [D-Ala6]GnRH in 250 µl of assay media. Cells were washed with 3 ml assay media or ice-cold PBS. Acid-sensitive radioligand was removed by 1 ml ice-cold acid solution, and cells were solubilized with 1 ml of NaOH/SDS.
Effect of estradiol on internalization of GnRH-R in sheep pituitary cells Pituitary cells from three ovariectomized ewes were dissociated and cultured as described above with the following differences: cells in half of the wells in the culture plates were treated with 40 nM estradiol-17ß 12 h prior to the internalization studies and half remained untreated. Assay procedures to assess the rates of internalization were as described above.
Statistical Analysis
Experiments were performed in triplicate and were replicated a minimum of four times. All values presented are mean ± SD for four or five experiments. Data were subjected to the general linear models procedure of SAS, and differences between groups were determined by Tukey's test [24]. Differences were considered significant at P < 0.05.
RESULTS
Changes in Surface-Bound Radioligand and Internalized Radioligand in CHO Cells and LßT2 Cells
Changes in amounts of surface-bound 125I-labeled [D-Ala6]GnRH in CHO cells, LßT2 cells and ovine pituitary cells are shown in Figure 1. Surface-bound radioligand began to decrease as soon as the cells were warmed to 37°C, and had decreased significantly by 20 min to 72% of the initial value for CHO cells and 74% for LßT2 cells (P < 0.05). The findings were similar in sheep pituitary cells, but it was 30 min before the amount of surface-bound radioligand had decreased significantly (P < 0.05). A steady-state level of surface-bound radioligand was achieved after 60 min in both CHO cells and LßT2 cells as well as ovine pituitary cells (38%, 41%, and 41% of the initial value, respectively; P < 0.05). There were no significant differences in the percentage of surface-bound radioligand between CHO cells, LßT2 cells, or ovine anterior pituitary cells at any of the times evaluated. Absolute amounts of surface-bound radioligand at time 0 averaged 3.36, 0.79, and 3.00 fmol/105 cells, and those at 60 min after warming of the cells were 1.09, 0.31, and 1.15 fmol/105 cells for CHO cells, LßT2 cells, and ovine anterior pituitary cells, respectively (Table 1).
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Amounts of 125I-labeled [D-Ala6]GnRH internalized by CHO cells, LßT2 cells, and ovine pituitary cells are shown in Figure 2. Internalization of radioligand began immediately after warming the cells to 37°C, and internalized ligand was greater at 20 min after than just prior to warming for CHO cells and LßT2 cells (18% of surface counts at time 0 and 15%, respectively; P < 0.05). As for disappearance of radioligand from the cell surface, the amount of 125I-labeled [D-Ala6]GnRH internalized by ovine anterior pituitary cells did not reach significance until 30 min after warming the cells. A steady-state maximum of internalization was reached after 60 min in each of the cell types (CHO cells [29%], LßT2 cells [28%], and ovine anterior pituitary cells [40%], respectively). There were no significant differences between CHO cells and LßT2 cells at any of the times evaluated, but the steady-state amount of radioligand internalized by ovine pituitary cells was higher (P < 0.05) than that in either CHO cells or LßT2 cells. Absolute amounts of internalized radioligand 60 min after warming of the cells averaged 0.43, 0.12, and 0.84 fmol/105 cells for CHO cells, LßT2 cells, and ovine anterior pituitary cells, and the amount of radioligand internalized by LßT2 cells was less than that in the other cell types (P < 0.05; Table 1).
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Radioactivity in the extracellular media of CHO cells increased immediately after warming the cells to 37°C (P < 0.05), and a steady-state maximum of radioactivity was reached after 60 min (Fig. 3). The percentage of immunoprecipitable radioactivity in the extracellular media at each time point ranged from 98% to 101% when compared to the value obtained immediately prior to warming the samples. Therefore, the increase in radioactivity in the extracellular medium appears to occur as a result of dissociation from the membrane rather than from degradation and excretion of internalized ligand.
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Effect of Estradiol-17ß on Internalization of 125I-Labeled [D-Ala6]GnRH in Ovine Pituitary Cells
To determine if treatment with estradiol altered the rate at which pituitary cells internalized 125I-labeled [D-Ala6]GnRH, dissociated ovine anterior pituitary cells were cultured in control media or in media containing 40 nM estradiol for 12 h prior to beginning the experiment. Treatment of cells with estradiol increased the number of GnRH-Rs on the pituitary cells by 
100% (data not shown), but did not alter the rate at which receptors were internalized (Fig. 4).
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DISCUSSION
In this study we compared the rates of internalization of mGnRH-R fused via its C-terminus to GFP and expressed in CHO cells to those of native mGnRH-R in LßT2 cells. Differences in rates of internalization of GnRH-R between mouse and sheep were also evaluated.
Surface-bound radioligand decreased quickly after warming CHO cells, LßT2 cells, and sheep pituitary cells to 37°C. However, this decline was not entirely associated with receptor internalization. Amounts of cell-associated radioligand (surface-bound radioligand plus internalized radioligand) 60 min after warming of CHO cells, LßT2 cells, and sheep pituitary cells were 45%, 54%, and 67%, respectively, of the amount of each initial surface-bound radioligand (Table 1). Therefore, 55%, 46%, and 33% of radioligand initially bound to the cells could not be accounted for by intracellular radioligand or radioligand remaining bound to the surface of the cells 60 min after warming. There are two possible explanations for the loss of radioactivity: 1) the 125I-labeled [D-Ala6]GnRH was internalized, degraded, and excreted into the extracellular medium; or 2) radioligand simply dissociated from receptor during the experiment. To distinguish between these possibilities we immunoprecipitated radioactivity in the extracellular medium of CHO cells. Radioactivity in the extracellular media increased immediately after warming the cells to 37°C. Compared to the value obtained immediately prior to warming the samples, virtually all of the radioligand (98–101%) could be immunoprecipitated at all times after warming the samples to 37°C. Therefore, at least in the CHO cells (and presumably in LßT2 cells and sheep pituitary cells) it appears that dissociation from the receptor accounts for the majority of radioactivity not present on the cell membrane or within the cell. Thus, intracellular degradation and excretion of degraded products does not appear to occur with the GnRH agonist or within the time frame employed for these experiments. In the present experiment, cells were incubated on ice for 4 h to maximize binding of radioligand to GnRH-R and then quickly warmed to 37°C to permit internalization. This abrupt change in the temperature might induce acute dissociation of radioligand from the receptors. Whether there is rapid dissociation of GnRH superagonist from receptor under physiological conditions in vivo is not known.
As noted above, the percentage of receptors internalized was greater for the oGnRH-R than for the mGnRH-R in these studies. Because ovine pituitary cells had been treated with estradiol to maximize cell surface receptors prior to internalization studies, one could argue that the reason for the higher degree of internalization in the sheep pituitary cells was due to treatment with estradiol. To examine this possibility, we compared both the rate and extent of internalization in ovine anterior pituitary cells not treated with estradiol to those that had been exposed to estradiol for 12 h prior to initiating internalization. Treatment with estradiol did not alter either the rate or percentage of GnRH-R that were internalized. Two conclusions can be made from this study: 1) enhanced sensitivity of the pituitary gland to GnRH after administration of estradiol is not due to a decreased rate of disappearance of GnRH-R from the cell membrane, and 2) the percentage of GnRH-R that are internalized does not appear to depend on the total number of receptors on the cell surface.
Rates of internalization of radioligand in murine GnRH-R-GFP expressed in CHO cells were similar to those of native receptor in Cos-1 [5] and Cos-7 cells [6, 7] and were identical to those measured in situ in LßT2 cells. Thus, rates of internalization of mGnRH-R appear to be independent of the cell line into which it is transfected. Our results also show that fusion to GFP does not alter the rate of GnRH-R internalization. Therefore, constructs of the mGnRH-R fused via its C-terminus to GFP recapitulate not only binding and second messenger transduction of the native receptor [15] but are also internalized with similar kinetics. Recently [25], it was shown that introduction of the intracellular C-terminus of either thyrotropin-releasing hormone receptor or catfish GnRH-R accelerated internalization of the rat GnRH-R. That C-terminal fusion to GFP did not influence internalization in the present study suggests that the presence of an intracellular C-terminus in itself has little impact on kinetics of internalization of the GnRH-R. At the concentration of radioligand used, the initial surface-bound radioligand is indicative of the number of receptors present on the cells; therefore, the number of receptors was 4.25 times higher in CHO cells than in LßT2 cells. However, the percentage of receptors internalized in each cell line was similar. Thus, it appears that the number of receptors expressed by the cell does not affect the rate of internalization but does affect the amount of ligand internalized. Our supposition that the number of receptors expressed by the cell does not alter rate of internalization is in accordance with a recent report [12].
The time course for internalization of oGnRH-R was similar to that of mGnRH-R. However, a greater percentage of oGnRH-R was internalized than murine receptor in either CHO cells or LßT2 cells. The reason for the greater degree of internalization of oGnRH-R is unknown but presumably reflects differences between cells or arises due to structural differences among the receptors. With regard to the latter, there is 86% homology of amino acids between oGnRH-R and mGnRH-R [20, 21]. However, oGnRH-R contains one additional amino acid at position 191 (Glu 191) in the second extracellular loop. Human GnRH-R also has an extra residue (Lys) at position 191, and deletion of this residue slows the rate of internalization and decreases the percentage of receptors internalized [5]. Mutation of Lys 191 in hGnRH-R to Glu, Gln, Arg, or Ala did not influence either the rate of internalization or percentage of receptor internalized [5]. Therefore, it appears that regardless of identity, the extra amino acid in the second extracellular loop may be responsible for the differing degrees of internalization noted between oGnRH-R and mGnRH-R.
Average numbers of receptors, calculated from amounts of radioligand on surface of the cells just before warming (initial value at Time 0), were 20 000, 4702, and 17 857 receptors/cell, respectively, in CHO cells, LßT2 cells, and sheep pituitary cells (for this calculation it was assumed that 10% of ovine pituitary cells were gonadotropes [26]). Average numbers of receptors internalized at 60 min after warming the cells were 2559, 714, and 5000 receptors/cell, respectively, in CHO cells, LßT2 cells, and sheep pituitary cells. The number of ligand-bound receptors on the surface of the cells after 60 min was 6458, 1845, and 6845 receptors/cell. Therefore, after 60 min of incubation at 37°C, there were 10 953, 2143, and 6012 receptors/cell, respectively, in CHO cells, LßT2 cells, and sheep pituitary cells that could not be accounted for by radioligand. This probably represents surface receptors from which the radioligand dissociated, and internalized receptors from which the radioligand was removed, degraded, and exocytosed. As discussed above, dissociation of ligand appeared to account for the majority of receptors that were not associated with ligand binding.
One caveat of the data obtained in this study relates to the dissociation of radioligand that was bound to the receptor prior to warming cells to 37°C. As indicated above, 33–55% of radioligand initially bound appears to dissociate from the receptor within 60 min. These studies employed a superagonist of GnRH (D-Ala6-GnRH) that has a much higher affinity for the receptor than native GnRH. Therefore, it might be expected that the rate of dissociation of native GnRH from receptor would be much higher than was observed for the superagonist [23]. If this is true, then under normal physiological conditions when the receptor is only exposed to GnRH for a short time (i.e., a GnRH pulse) dissociation may represent the primary mechanism of receptor deactivation. On the other hand, when prolonged exposure of the receptor to high concentrations of GnRH occurs (i.e., the preovulatory surge), then internalization may also be an important means for deactivating the gonadotrope.
It has been demonstrated that after internalization, the GnRH-R is freed of agonist and transported back to the membrane, whereas the agonist is retained inside the cell [12]. The appearance of free receptors on the membrane results from not only the recycling of previously internalized receptors but also the transport to the membrane of newly synthesized protein. Furthermore, it is known that some internalized receptors undergo degradation in the cell. These parameters were not evaluated in this study; therefore, further experiments are needed to clarify the relationship between internalization kinetics of GnRH-R and recycled, newly synthesized, and degraded receptors. Because we have demonstrated that internalization rate of GnRH-R-GFP expressed in CHO cells is similar to wild-type GnRH-R, it may be possible to examine these parameters in the CHO cells by using confocal microscopy.
In conclusion, we have demonstrated that internalization kinetics of mGnRH-R-GFP in CHO cells, GnRH-R in LßT2 cells, and oGnRH-R in sheep pituitary cells are similar, but the percentage of oGnRH-R internalized is greater than the mGnRH-R.
FOOTNOTES
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
Accepted: October 31, 2000.
Received: April 17, 2000.
REFERENCES
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