0013-7227/91/1286-2837$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 6 Printed in U.S.A.
The Slow Rate of Internalization of Deglycosylated Human Chorionic Gonadotropin Is Not Due to Its Inability to Stimulate Cyclic Adenosine Monophosphate Accumulation* SALLY R. HOELSCHERt, M. R. SAIRAM, AND MARIO ASCOLI Department of Pharmacology (S.R.H., M.A.), University of Iowa College of Medicine, Iowa City, Iowa 52242; and the Reproduction Research Laboratory (M.R.S.), Clinical Research Institute of Montreal, Montreal, Quebec, Canada
than that of hCG. Two sets of experiments were performed which led to the conclusion that the slower internalization of deglycosylated hCG is not due to its inability to stimulate cAMP production. First, the rate of internalization of deglycosylated hCG in MA-10 cells is not increased in the presence of a cAMP analog. Second, there is little or no difference in the rate of internalization of hCG in a subclone of MA-10 cells [designated MA-10(K3)] that express a cAMP-resistant phenotype. {Endocrinology 128: 2837-2843, 1991)
ABSTRACT. Using a clonal strain of cultured Leydig tumor cells (designated MA-10), we have compared the internalization and degradation of human CG (hCG) and deglycosylated hCG. Deglycosylated hCG is a derivative of hCG which retains the ability to bind to the LH/CG receptor, but is unable to activate adenylyl cyclase and thus stimulate cAMP production. A comparison of the fates of the receptor-bound hormones during a single round of endocytosis showed that deglycosylated hCG is internalized slower than hCG. It was also noted that the rate of degradation of the internalized deglycosylated hCG is slower
I
N A SERIES of experiments recently published (1), we found that there are two distinct pathways for the LH/CG-induced down-regulation of the LH/CG receptor. These two pathways are receptor-mediated endocytosis and a cAMP-dependent reduction in receptor mRNA. Receptor-mediated endocytosis appears to be responsible for the "fast phase" of LH/CG-induced down-regulation of its receptor, while the reduction in receptor mRNA occurs more slowly. In this previous study, deglycosylated human CG (hCG) was used to test the involvement of cAMP in receptor mRNA reduction. Deglycosylated hCG is a derivative of hCG that binds to the LH/CG receptor with normal or increased affinity, but has a reduced ability to activate adenylyl cyclase and stimulate cAMP production (reviewed in Ref. 2). Based on the hypothesis that the decrease in receptor mRNA is cAMP dependent, it would be expected that deglycoReceived January 10,1991. Address correspondence and reprint requests to: Dr. Mario Ascoli, Department of Pharmacology, University of Iowa, College of Medicine, Iowa City, Iowa 52242-1109. * This work was supported by grants from the National Institutes of Health: CA-40629 (to M.A.) and DK-25295 (Diabetes and Endocrinology Research Center of the University of Iowa), funds from the Roy J. Carver Charitable Trust (to M.A.), and a grant from the Medical Research Council of Canada (to M.R.S.). t Supported by a University of Iowa Fellowship.
sylated hCG would not be able to induce a reduction in receptor mRNA since this hormone is unable to stimulate cAMP. When experiments were performed to test this hypothesis, deglycosylated hCG was unable to induce a reduction in receptor mRNA. This result was as expected and together with results of other experiments supports the conclusion that this pathway of LH/CGinduced down-regulation of the LH/CG receptor is cAMP dependent. Surprisingly, however, it was also observed in these experiments that although deglycosylated hCG was still capable of reducing [125I]hCG binding, the binding decreased at a much slower rate than that observed with comparable concentrations of hCG. Since the fast phase of LH/CG-induced down regulation of the LH/CG receptor appears to be due to increased internalization of the hormone-receptor complex, it was hypothesized that perhaps the rate of internalization of deglycosylated hCG is slower than that of hCG. The studies presented herein were designed to determine whether the internalization and degradation of deglycosylated hCG is slower than that of hCG. We also investigated the possible effects of cAMP on the internalization and degradation of these two hormones since deglycosylated hCG is unable to activate adenylyl cyclase and increase cAMP levels.
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INTERNALIZATION AND DEGRADATION OF hCG AND DEGLYCOSYLATED hCG
Materials and Methods Cells The origin and handling of MA-10 and MA-10(K3) cells have been described (3, 4). Stock cultures of MA-10 cells were maintained in growth medium (Waymouth MB752/1 modified to contain 1.1 g/liter NaHCO3, 20 mM HEPES, 50 Mg/ml gentamycin, and 15% horse serum, pH 7.4). Stock cultures of MA-10(K3) cells were maintained in growth medium supplemented with 200 Mg/ml G418 (a geneticin analog) as described elsewhere (4). Standard experimental protocol Cells were plated on day 0 in 6 X 35-mm wells at a density of 2.5-3.5 x 105 cells per well and maintained at 37 C in a humidified atmosphere containing 2.5% CO2. The medium was changed on day 2 or 3, and all experiments were performed on day 3 or day 4, respectively. Experiments were initiated by washing the wells twice with 2-ml portions of warm assay medium (Waymouth MB752/1 modified to contain 1.1 g/liter NaHCO3, 20 mM HEPES, 50 Mg/ml gentamycin, and 1 mg/ml BSA, pH 7.4). Incubations were performed at 37 C in a humidified atmosphere containing 2.5% CO2 in a volume of 2 ml assay medium. When MA-10(K3) cells were used they were cultured without G418 for 3-4 days before the experiment. One day before the experiment, the medium was changed, the concentration of serum was decreased to 5% horse serum and ZnSO4 (final concentration = 100 nM) was added (4). This concentration of ZnSO4 was maintained throughout the experiment. In experiments with 8-bromo-cAMP (8-Br-cAMP), cells were preincubated with 8-Br-cAMP (final concentration = 500 fiM) for 15 min at 37 C. This concentration of 8-Br-cAMP was maintained throughout the experiment. Fate of the receptor-bound hormones during one round of endocytosis Dishes were washed as described above. The 125I-labeled hormone was then added (final concentration = 40 ng/ml), and the dishes were incubated for 15 min at 37 C. The dishes were placed on ice and quickly washed five times with 2-ml portions of ice cold Hank's balanced salt solution containing 1 mg/ml BSA (wash buffer). Dishes were either processed immediately, or allowed to continue to incubate in 2-ml assay medium at 37 C. At the times indicated, the dishes were placed on ice, the medium was removed, and the dishes were washed once with 2 ml cold wash buffer. The cells were then incubated at 4 C with 1 ml cold 50 mM glycine/100 mM NaCl, pH 3, for 4 min and washed once with 1 ml of the same solution. The cells were solubilized with 100 ^1 1 N NaOH and collected with a cotton swab. The radioactivity that was removed by the acid wash was taken as a measure of the surface-bound hormone, the radioactivity that remained associated with the cells was considered to be internalized hormone (5). The medium and buffer washes were combined and precipitated with 10% trichloroacetic acid, and the soluble and insoluble radioactivities were considered to be degraded and undegraded hormone, respectively (6).
Endo• 1991 Voll28«No6
Rate of degradation of the internalized hormone Cells were washed as described above. Cells were then incubated with 125I-labeled hormone for 2 h at 37 C. The dishes were placed on ice, washed five times with 2-ml portions of cold wash buffer, incubated for 4 min with cold 50 mM glycine/100 mM NaCl, pH 3 (to remove the surface-bound hormone), washed once with the same solution, once with cold wash buffer, and once with cold assay medium. Dishes were either processed immediately, or the incubation was continued for up to 2 h at 37 C in 2 ml assay medium. At the times indicated, the dishes were placed on ice, the medium was saved, and the cells were washed once with 2 ml cold wash buffer. The medium and buffer washes were combined and precipitated with 10% trichloroacetic acid to determine the amounts of degraded and undegraded hormone released. The cells were solubilized with 100 ^1 1 N NaOH as described above to determine the amount of radioactivity that remained internalized.
After preincubation with 125I-labeled hormone and removal of surface-bound hormone (see above), the remaining cellassociated radioactivity was considered to be internalized hormone. Hormone degradation and release of degradation products in the medium were reflected by a corresponding decrease in cell-associated radioactivity. The rate constant of degradation (kh) was calculated using a semilog plot of the internalized hormone vs. time. (7-9). Slopes were calculated by linear regression and had correlation coefficients of at least 0.96. Rate of hormone internalization The rate of internalization (ke) was calculated from the rate of disappearance of receptor-bound hormone from the cell surface measured in the experiments dealing with the fate of the receptor-bound hormones during one round of endocytosis (see above). The rate of internalization was calculated from a plot of the log of surface-bound radioactivity us. time. The slopes were calculated by linear regression and had correlation coefficients of at least 0.93. Analysis of data Three dishes were used for each point shown. Two dishes contained the 125I-labeled hormone only (total binding), and the third dish contained the 125I-labeled hormone together with 25 IU/ml crude hCG (nonspecific binding). Specific binding was calculated by subtracting the nonspecific binding from the total binding. The nonspecific binding of [125I]hCG was at most 15% of the total binding, and the nonspecific binding of [I25I] deglycosylated hCG, although higher, was at most 24% of the total binding. Hormones and supplies Purified hCG (batch CR-125, 11,900 IU/mg) was obtained from the National Institutes of Health. Deglycosylated hCG was prepared as described previously (10). These hormones were iodinated as previously described (11). The specific activity of the [125I]hCG was approximately 19.4 iiCi/^g and the specific activity of the [125I]deglycosylated hCG was approximately 8.9 nQ\/ng. Crude hCG (3,000 IU/mg), 8-Br-cAMP, and BSA were obtained from Sigma Chemical Co. (St. Louis, MO). Na[125I] was
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INTERNALIZATION AND DEGRADATION OF hCG AND DEGLYCOSYLATED hCG obtained from New England Nuclear Co. (Boston, MA). Cell culture supplies and plasticware were from GIBCO (Grand Island, NY) and Corning (Corning, NY), respectively. All other supplies and materials were obtained from commonly used suppliers.
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Results Comparison of the internalization and degradation patterns of hCG and deglycosylated hCG In initial experiments we assessed a single round of endocytosis of the 125I-labeled hormone by preincubating MA-10 cells with [125I]hCG or [125I]deglycosylated hCG for 15 min at 37 C, washing to remove the free hormone, and reincubating the cells in hormone-free medium for up to 4 h at 37 C. Using this experimental design, one can follow the amount of surface-bound and internalized hormone associated with the cells and the amounts of degraded and undegraded hormone which appear in the medium over time. This represents a single round of endocytosis of the prebound hormone, because the free hormone is removed after the initial binding period. We had previously assessed a single round of endocytosis of hCG by preincubating MA-10 cells with 125I-labeled hormone for 2 h at 4 C (7). The preincubation was done at 4 C because virtually no internalization of hormone occurs at this temperature (5). In the experiments presented here, the preincubations were performed at 37 C in an effort to decrease the nonspecific binding of [125I] deglycosylated hCG. The results are essentially the same as those obtained with a 4 C preincubation, since during a 15-min preincubation at 37 C there is little internalization of the hormone. Thus, as can be seen in Fig. 1, approximately 95% of the cell-associated hormone is localized at the cell surface at time zero. The results presented in Fig. 1 illustrate that with [125I]hCG the surface-bound hormone disappears quickly and is accumulated within the cell. The levels of internalized [125I]hCG reach a maximum at 1-2 h and then decline as degradation products begin to appear in the medium. The appearance of degraded [125I]hCG in the medium displays a lag of about 1 h and then increases linearly. A small amount of undegraded [125I]hCG (less than 10% of the hormone initially bound) was also found in the medium, although this is not shown in this figure. Within 4 h, 95% of the [125I]hCG which was initially bound has been internalized and/or degraded. These results are consistent with previous data which show that hCG is internalized and ultimately degraded in the lysosomes (5,12). The rate of internalization of [125I] deglycosylated hCG during endocytosis is markedly different from [125I]hCG (Fig. 1). Although the surface-bound [125I]deglycosylated hCG also disappears from the surface, this occurs at a
hours FlG. 1. Comparison of the internalization and degradation patterns of hCG and deglycosylated hCG (dghCG) in MA-10 cells. Cells were preincubated with 40 ng/ml [125I]hCG (upper panel) or 40 ng/ml [125I] dghCG (lower panel). At the end of the incubation time (time = 0 in the figure), the cells were washed to remove free hormone. Cells were either processed immediately, or incubation was continued for up to 4 h in hormone-free medium. Amounts of radioactivity associated with the cell surface (circles), internalized (squares), and degraded (triangles) hormone were determined at the times indicated. The amounts of hormone bound at time = 0 were 0.41 ± 0.03 and 0.69 ± 0.2 ng/well for [125I]hCG and [125I]deglycosylated hCG, respectively. Each point represents the average ± range of two independent experiments.
much slower rate than that seen with [125I]hCG. After 4 h only 50% of the [125I]deglycosylated hCG which was initially bound has been internalized. Levels of internalized [125I]deglycosylated hCG appear to plateau after about 1 h. The accumulation of degradation products of [125I]deglycosylated hCG is also lower than that seen with [125I]hCG. This could be attributable either to the decreased internalization, or to a difference in the actual rates of degradation of these two hormones. From these data it can be concluded that the internalization of [125I]deglycosylated hCG is much slower than the internalization of [125I]hCG. It is not clear from this
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INTERNALIZATION AND DEGRADATION OF hCG AND DEGLYCOSYLATED hCG
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data whether the rate of degradation of [125I]deglycosylated hCG is also slower than that of [125I]hCG. Effect of cAMP Because deglycosylated hCG is unable to stimulate cAMP production (2), experiments were done to determine whether the lack of cAMP stimulation was responsible for the decreased rate of internalization of [125I] deglycosylated hCG. Two approaches were used to test this possibility. The first approach involved studying the internalization and degradation of deglycosylated hCG in the presence of a cAMP analog. Experiments were performed as described in Materials and Methods except that cells were preincubated with 500 /xM 8-Br-cAMP for 15 min before addition of 125I-labeled hormone. This concentration of 8-Br-cAMP was then maintained throughout the experiment. This high concentration of 8-Br-cAMP was chosen because 1) it elicits maximal stimulation of steroidogenesis and 2) the increase in progesterone production seen with this concentration of cAMP is similar to that seen with the concentrations of hCG used in these experiments (4). Results shown in Fig. 2 show that there was little or no difference between the disappearance of the surface-bound [125I]deglycosylated hCG in MA-10 cells which received the hormone alone or in cells which received [125I]deglycosylated hCG plus 8-Br-cAMP. Lev-
Endo«1991 Voll28«No6
els of internalized hormone and degraded hormone in the medium were also similar in the cells that received 8-Br-cAMP. The second approach used a subclone of MA-10 cells (MA-10(K3)) that express a cAMP-resistant phenotype (4). MA-10(K3) cells were obtained by transfection of MA-10 cells with an expression vector that encodes for a mutant form of the type I regulatory subunit of the cAMP-dependent protein kinase under the transcriptional control of the metallothionein promoter (13). Thus, incubation of MA-10(K3) cells with Zn2+ leads to the expression of a cAMP-resistant phenotype. In the experiments presented in Fig. 3, MA-10 cells and MA10(K3) cells were preincubated with Zn2+, and then were incubated with [125I]hCG. Amounts of surface-bound, internalized and degraded hormone in the medium were then measured over the course of 4 h. There was little or no difference between MA-10 cells and MA-10(K3) cells in any of the parameters measured. If anything, internalization is slightly faster in MA-10(K3) cells (Fig. 1), which is opposite of what would be expected if cAMP was involved in the internalization of hCG. Taken together, these results indicate that the slower rate of internalization of deglycosylated hCG as compared to hCG is not related to the inability of deglycosylated hCG to stimulate cAMP accumulation. This is shown by the lack of effect of a cAMP analog on the internalization and degradation of deglycosylated hCG, and the fact that there was no change in the internali-
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hours FIG. 2. Effect of 8-Br-cAMP on internalization and degradation of deglycosylated hCG. Experiments were performed as described in the legend to Fig. 1, except that experiments were performed in the absence (closed symbols) or presence (open symbols) of 500 nM 8-Br-cAMP. Preincubation with 8-Br-cAMP was performed for 15 min before beginning the experiments, and 500 nM 8-Br-cAMP was present in the medium throughout the duration of the experiments. Each point represents the average ± SEM of three independent experiments.
hours FIG. 3. Internalization and degradation of hCG in MA-10 and MA10(K3) cells. Experimental design was as described in the legend to Fig. 1. Experiments were performed using MA-10 cells (closed symbols) or MA-10(K3) cells (open symbols). Each point represents the average ± SEM of three independent experiments.
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INTERNALIZATION AND DEGRADATION OF hCG AND DEGLYCOSYLATED hCG zation and degradation pattern of hCG when tested in a cell line with a cAMP resistant phenotype. Rate of internalization From visually comparing the graphs in Fig. 1 it can be seen that there is a difference in the rate of internalization of [125I]hCG and [125I] deglycosylated hCG. In order to further quantify this data, the actual rates of internalization (ke) were calculated from the rate of disappearance of receptor-bound hormone from the surface as described in Materials and Methods. The rate of hormone internalization can be approximated from the rate of disappearance of the surface-bound hormone only if the binding affinity of the hormone is high enough so that there is little or no dissociation of the hormone from the receptor during the course of the experiment. It has been shown previously in this (7, 14) and other (15-17) laboratories that once hCG is receptor bound, little or no dissociation of hCG occurs. Therefore, the disappearance of surface-bound hormone can be directly correlated with hormone internalization. These data can then be used to calculate the rate of internalization of the hormone. The results of these calculations are displayed in Table 1. These same data can also be expressed as the half-life of internalization, with [125I]hCG having a half-life of 61 ± 3 min and [125I]deglycosylated hCG having a half-life of 252 ± 10 min. Therefore, the rate of internalization of [125I]deglycosylated hCG is 4 times slower than the rate of internalization of [125I]hCG. Rates of internalization were also calculated for deglycosylated hCG in MA-10 cells incubated with 8-BrcAMP and for hCG in MA-10(K3) cells. These results (Table 1) further illustrate that there is little or no difference between the internalization of deglycosylated hCG in MA-10 cells incubated with or without 8-BrcAMP, or between hCG in MA-10 and MA-10(K3) cells. This supports the conclusion that cAMP accumulation is not responsible for the slower rate of internalization of deglycosylated hCG. The rate of internalization of hCG in MA-10 cells has been previously determined in two separate studies in this laboratory to be about 0.04 min"1 (7, 18). These results differ from the rate of internalization of hCG in MA-10 cells which we report here. It should be pointed TABLE 1. Rate of internalization (ke) Hormone 125
[ I]hCG [125I]dghCG
Cell
Additions
MA-10 MA-10(K3) MA-10 MA-10
none none none 8-Br-cAMP
ke (min *) 0.012 ± 0.014 ± 0.0028 ± 0.0032 ±
0.0007 0.0006 0.0001 0.0001
5 3 5 3
The rate constants for internalization were calculated as described in Materials and Methods. Each number represents the average ± SEM of the indicated number of experiments.
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out that a different method was previously used to determine the rate of internalization than that which was used here. From recent experiments, however, the rate of internalization of hCG calculated using the other method was found to be similar to that shown in Table 1. We are not presently able to account for this difference. Rate of degradation It can be seen from Fig. 1 that the rate of accumulation of degradation products is slower with [125I]deglycosylated hCG than with [125I]hCG. Although the slower rate of internalization of [125I]deglycosylated hCG may be sufficient to explain the accumulation of fewer degradation products, this may not necessarily be the case. It is possible that the accumulation of fewer degradation products may also be related to a difference in the rates of degradation of these two hormones. For this reason, the rate of degradation was measured directly in order to determine whether the slower rate of accumulation of degradation products of [125I]deglycosylated hCG in the medium, as compared to [125I]hCG (shown in Fig. 1), was related solely to the difference in the rates of internalization, or whether there is also a difference in the rates of degradation of hCG and deglycosylated hCG. The rate constant of degradation (kh) was calculated from the rate of disappearance of internalized hormone after removal of surface-bound hormone as described in Materials and Methods. Figure 4 illustrates the decrease of internalized radioactivity over time and the corresponding increase of degraded hormone in the medium. The amount of undegraded hormone in the medium is also shown. As mentioned previously, the undegraded hormone in the medium accounts for a small percentage of hormone initially bound. The rates of degradation are displayed in Table 2. These same data can also be expressed as the half-life of degradation, with [l25I]hCG having a half-life of 55 min and [125I]deglycosylated hCG having a half-life of 88 min. These data show that [125I]deglycosylated hCG is degraded at a rate which is approximately 1.5 times slower than the rate of degradation of [125I]hCG. The difference in the rates of degradation is not as large as the difference in the rates of internalization of the two hormones. Therefore, it appears that the slower accumulation of degradation products of [125I]deglycosylated hCG (see Fig. 1) may be a combination of both a decreased rate of degradation and a decreased rate of internalization as compared to hCG. Discussion The original objective of this study was to explain the observation from a previous study (1) that deglycosylated
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INTERNALIZATION AND DEGRADATION OF hCG AND DEGLYCOSYLATED hCG
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T3
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30
60
90
120
100 -
120 FIG. 4. Rates of degradation of internalized hCG and deglycosylated hCG in MA-10 cells. Cells were preincubated with 40 ng/ml [125I]hCG (closed symbols) or 40 ng/ml [125I]deglycosylated hCG (open symbols) for 2 h at 37 C. At the end of this incubation time (time = 0 in the figure), the cells were washed to remove the free hormone, treated with acid to remove the surface-bound hormone, and then incubated at 37 C in hormone-free medium. At the times indicated, the amount of radioactivity that remained associated with the cells (upper panel), and the amounts of acid soluble and insoluble radioactivity released into the medium (lower panel) were determined. Each point represents the average ± SEM of three independent experiments. TABLE 2. Rate of degradation (kh)
Hormone
kh (mhT 1 )
[125I]hCG [125I]dghCG
0.012 ± 0.0004 0.008 ± 0.0008
The rates of degradation were calculated from semilog plots of the data presented in Fig. 4. Each number represents the average ± SEM of the indicated number of experiments.
hCG induces down-regulation of the LH/CG receptor much more slowly than hCG. The studies presented herein illustrate that the rate of internalization of deglycosylated hCG is 4 times slower than that of hCG. This conclusion is supported by the results presented in Fig. 1 and the calculated rates of internalization. The slower rate of internalization of deglycosylated hCG as compared to hCG would explain the slower induction of down-regulation of the LH/CG receptor elicited by de-
Endo• 1991 Voll28«No6
glycosylated hCG, since the initial phase of down-regulation appears to be due to receptor-mediated endocytosis (1). It was observed in initial experiments that the accumulation of degradation products of deglycosylated hCG was slower than seen with hCG. In a previous publication Rebois and Fishman (19) showed that deglycosylated hCG was degraded slower than hCG in another clonal strain of cultured Leydig tumor cells (MLTC-1) similar to MA-10 cells. Similar observations have also been made in cultured rat granulosa cells (20). Thus, although our results are in agreement with these previous observations, our experiments are more detailed and allow us to conclude that the slower rate of accumulation of degradation products of deglycosylated hCG as compared to hCG is due to a combination of a decreased rate of internalization and a decreased rate of degradation of deglycosylated hCG. Since the rate of internalization of deglycosylated hCG is 4 times slower than hCG, while the rate of degradation of deglycosylated hCG is only 1.5 times slower than hCG, it is probable that the slower accumulation of degradation products is mainly due to the difference in internalization rates. These data also support previous observations in pseudopregnant rats where the labeled deglycosylated hCG and deglycosylated LH were found to be retained in the ovary for a longer time than the native hormones (21). In addition, the studies presented support the conclusion that the slower internalization of deglycosylated hCG as compared to hCG is not due to the inability of deglycosylated hCG to stimulate cAMP production. Two points illustrate this conclusion. The rate of internalization of deglycosylated hCG is not increased by incubating MA-10 cells with 8-Br-cAMP. Second, there is little or no difference in the rate of internalization of hCG in a subclone of MA-10 cells (designated MA10(K3)) that express a cAMP-resistant phenotype. In this respect it is interesting to note that other hormonereceptor complexes that are unable to elicit intracellular signals are also internalized at a slower rate than their normal counterparts. For example, the rate of internalization of EGF in cell lines expressing mutant EGF receptors that lack tyrosine kinase activity is slower than in cells expressing the wild type receptor (22). Although it is clear that the rate of internalization of deglycosylated hCG cannot be accounted for by the lack of cAMP accumulation, the molecular basis of this phenomenon is not currently understood. A potential explanation is that there is a difference in the conformation of the hormone-receptor complex and/or the ability of this complex to associate with other membrane proteins (such as Gs for example) (2). This question may now be experimentally approached by site-directed mutagenesis of the LH/CG receptor (23, 24).
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INTERNALIZATION AND DEGRADATION OF hCG AND DEGLYCOSYLATED hCG In summary, this study fulfilled its original objective of explaining the slower down-regulation of the LH/CG receptor induced by deglycosylated hCG as compared with hCG (1). In addition, we have shown that the internalization and degradation of deglycosylated hCG is slower than hCG and that this effect is not mediated by cAMP. Finally, the slower accumulation of degradation products of deglycosylated hCG as compared to hCG is probably due to a combination of a decreased rate of internalization and a decreased rate of degradation.
Acknowledgments We wish to thank Bruce D'Souza for expert assistance with cell culture. We also thank Deborah L. Segaloff for critical reading of the manuscript.
References 1. Wang H, Segaloff D, Ascoli M 1991 Lutropin/choriogonadotropin down-regulates its receptor by both receptor-mediated endocytosis and a cAMP dependent reduction in receptor mRNA. J Biol Chem 266:780-785 2. Sairam MR 1989 Role of carbohydrates in glycoprotein hormone signal transduction. FASEB J 3:1915-1926 3. Ascoli M 1981 Characterization of several clonal lines of cultured Leydig tumor cells: gonadotropin receptors and steroidogenic responses. Endocrinology 108:88-95 4. Wang H, Ascoli M 1990 Reduced gonadotropin responses in a novel clonal strain of Leydig tumor cells established by transfection of MA-10 cells with a mutant gene of the type I regulatory subunit of the cAMP-dependent protein kinase. Mol Endocrinol 4:80-90 5. Ascoli M 1982 Internalization and degradation of receptor-bound human choriogonadotropin in Leydig tumor cells: fate of the hormone subunits. J Biol Chem 257:13306-13311 6. Ascoli M, Puett D 1978 Degradation of receptor-bound human choriogonadotropin by murine Leydig tumor cells. J Biol Chem 253:4892-4899 7. Ascoli M, Segaloff D 1987 On the fates of receptor-bound ovine luteinizing hormone and human chorionic gonadotropin in cultured Leydig tumor cells. Demonstration of similar rates of internalization. Endocrinology 120:1161-1172 8. Wiley HS, Cunningham DD 1981 A steady state model for analyzing the cellular binding, internalization and degradation of polypeptide ligands. Cell 25:433-440 9. Wiley HS, Cunningham DD 1982 The endocytotic rate constant. A cellular parameter for quantitating receptor-mediated endocytosis. J Biol Chem 257:4222-4229
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10. Manjunath P, Sairam MR 1982 Biochemical, biological, and immunological properties of chemically deglycosylated human choriogonadotropin. J Biol Chem 257:7109-7115 11. Ascoli M, Puett D 1978 Gonadotropin binding and stimulation of steroidogenesis in Leydig tumor cells. Proc Natl Acad Sci USA 75:99-102 12. Ascoli M 1984 Lysosomal accumulation of the hormone receptor complex during receptor-mediated endocytosis of human choriogonadotropin. J Biol Chem 99:1242-1250 13. Clegg CH, Correll LA, Cadd GG, McKnight GS 1987 Inhibition of intracellular cAMP-dependent protein kinase using mutant genes of the regulatory type I subunit. J Biol Chem 262:13111-13119 14. Ascoli M 1983 An improved method for the solubilization of stable gonadotropin receptors. Endocrinology 113:2129-2134 15. Ketelslegers J-M, Knott GD, Catt KJ 1975 Kinetics of gonadotropin binding by receptors of the rat testis. Analysis by a nonlinear curve-fitting method. Biochemistry 14:3075-3083 16. Lee CY, Ryan RJ 1973 Interaction of ovarian receptors with human luteinizing hormone and human chorionic gonadotropin. Biochemistry 12:4609-4615 17. Huhtaniemi IT, Catt KJ 1981 Differential binding affinities of rat testis luteinizing hormone (LH) receptors for human chorionic gonadotropin, human LH, and ovine LH. Endocrinology 108:19311938 18. Lloyd CE, Ascoli M 1983 On the mechanisms involved in the regulation of the cell-surface receptors for human choriogonadotropin and mouse epidermal growth factor in cultured Leydig tumor cells. J Biol Chem 96:521-526 19. Rebois RV, Fishman PH 1983 Deglycosylated human chorionic gonadotropin: an antagonist to desensitization and down-regulation of the gonadotropin receptor-adenylate cyclase system. J Biol Chem 258:12775-12778 20. Zor U, Sairam MR, Shentzer P, Azrad A, Amsterdam A 1984 The role of carbohydrate moiety of the gonadotropin molecules in transduction of biological signal. In: McKerns KW, Noar Z (eds) Hormonal Control of the Hypothalamo-Pituitary Gonadal Axis. Plenum Press, New York, pp 235-248 21. Sairam MR, Kato K, Manjunath P, Sairam J, Dobias-Goff M 1984 Blockade of gonadotropin action at the receptor. In: Saxena BB, Catt KJ, Birnbaumer L (eds) Hormone Receptors in Growth and Reproduction. Raven Press, New York, pp 185-200 22. Glenney Jr JR, Chen WS, Lazar CS, Walton GM, Zokas LM, Rosenfeld MG, Gill GN 1988 Ligand-induced endocytosis of the EGF receptor is blocked by mutational inactivation and by microinjection of anti-phosphotyrosine antibodies. Cell 52:675-684 23. McFarland KC, Sprengel R, Phillips HS, Kohler M, Rosemblit N, Nikolics K, Segaloff DL, Seeburg PH 1989 Lutropin-choriogonadotropin receptor: An unusual member of the G protein-coupled receptor family. Science 245:494-499 24. Loosfelt H, Misrahi M, Atger M, Salesse R, Thi MTVH-L, Jolivet A, Guiochon-Mantel A, Sar S, Jallal B, Gamier J, Milgrom E 1989 Cloning and sequencing of porcine LH-hCG receptor cDNA: variants lacking transmembrane domain. Science 245:525-528
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Vol. 128, No. 6 Printed in U.S.A.
The Slow Rate of Internalization of Deglycosylated Human Chorionic Gonadotropin Is Not Due to Its Inability to Stimulate Cyclic Adenosine Monophosphate Accumulation* SALLY R. HOELSCHERt, M. R. SAIRAM, AND MARIO ASCOLI Department of Pharmacology (S.R.H., M.A.), University of Iowa College of Medicine, Iowa City, Iowa 52242; and the Reproduction Research Laboratory (M.R.S.), Clinical Research Institute of Montreal, Montreal, Quebec, Canada
than that of hCG. Two sets of experiments were performed which led to the conclusion that the slower internalization of deglycosylated hCG is not due to its inability to stimulate cAMP production. First, the rate of internalization of deglycosylated hCG in MA-10 cells is not increased in the presence of a cAMP analog. Second, there is little or no difference in the rate of internalization of hCG in a subclone of MA-10 cells [designated MA-10(K3)] that express a cAMP-resistant phenotype. {Endocrinology 128: 2837-2843, 1991)
ABSTRACT. Using a clonal strain of cultured Leydig tumor cells (designated MA-10), we have compared the internalization and degradation of human CG (hCG) and deglycosylated hCG. Deglycosylated hCG is a derivative of hCG which retains the ability to bind to the LH/CG receptor, but is unable to activate adenylyl cyclase and thus stimulate cAMP production. A comparison of the fates of the receptor-bound hormones during a single round of endocytosis showed that deglycosylated hCG is internalized slower than hCG. It was also noted that the rate of degradation of the internalized deglycosylated hCG is slower
I
N A SERIES of experiments recently published (1), we found that there are two distinct pathways for the LH/CG-induced down-regulation of the LH/CG receptor. These two pathways are receptor-mediated endocytosis and a cAMP-dependent reduction in receptor mRNA. Receptor-mediated endocytosis appears to be responsible for the "fast phase" of LH/CG-induced down-regulation of its receptor, while the reduction in receptor mRNA occurs more slowly. In this previous study, deglycosylated human CG (hCG) was used to test the involvement of cAMP in receptor mRNA reduction. Deglycosylated hCG is a derivative of hCG that binds to the LH/CG receptor with normal or increased affinity, but has a reduced ability to activate adenylyl cyclase and stimulate cAMP production (reviewed in Ref. 2). Based on the hypothesis that the decrease in receptor mRNA is cAMP dependent, it would be expected that deglycoReceived January 10,1991. Address correspondence and reprint requests to: Dr. Mario Ascoli, Department of Pharmacology, University of Iowa, College of Medicine, Iowa City, Iowa 52242-1109. * This work was supported by grants from the National Institutes of Health: CA-40629 (to M.A.) and DK-25295 (Diabetes and Endocrinology Research Center of the University of Iowa), funds from the Roy J. Carver Charitable Trust (to M.A.), and a grant from the Medical Research Council of Canada (to M.R.S.). t Supported by a University of Iowa Fellowship.
sylated hCG would not be able to induce a reduction in receptor mRNA since this hormone is unable to stimulate cAMP. When experiments were performed to test this hypothesis, deglycosylated hCG was unable to induce a reduction in receptor mRNA. This result was as expected and together with results of other experiments supports the conclusion that this pathway of LH/CGinduced down-regulation of the LH/CG receptor is cAMP dependent. Surprisingly, however, it was also observed in these experiments that although deglycosylated hCG was still capable of reducing [125I]hCG binding, the binding decreased at a much slower rate than that observed with comparable concentrations of hCG. Since the fast phase of LH/CG-induced down regulation of the LH/CG receptor appears to be due to increased internalization of the hormone-receptor complex, it was hypothesized that perhaps the rate of internalization of deglycosylated hCG is slower than that of hCG. The studies presented herein were designed to determine whether the internalization and degradation of deglycosylated hCG is slower than that of hCG. We also investigated the possible effects of cAMP on the internalization and degradation of these two hormones since deglycosylated hCG is unable to activate adenylyl cyclase and increase cAMP levels.
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INTERNALIZATION AND DEGRADATION OF hCG AND DEGLYCOSYLATED hCG
Materials and Methods Cells The origin and handling of MA-10 and MA-10(K3) cells have been described (3, 4). Stock cultures of MA-10 cells were maintained in growth medium (Waymouth MB752/1 modified to contain 1.1 g/liter NaHCO3, 20 mM HEPES, 50 Mg/ml gentamycin, and 15% horse serum, pH 7.4). Stock cultures of MA-10(K3) cells were maintained in growth medium supplemented with 200 Mg/ml G418 (a geneticin analog) as described elsewhere (4). Standard experimental protocol Cells were plated on day 0 in 6 X 35-mm wells at a density of 2.5-3.5 x 105 cells per well and maintained at 37 C in a humidified atmosphere containing 2.5% CO2. The medium was changed on day 2 or 3, and all experiments were performed on day 3 or day 4, respectively. Experiments were initiated by washing the wells twice with 2-ml portions of warm assay medium (Waymouth MB752/1 modified to contain 1.1 g/liter NaHCO3, 20 mM HEPES, 50 Mg/ml gentamycin, and 1 mg/ml BSA, pH 7.4). Incubations were performed at 37 C in a humidified atmosphere containing 2.5% CO2 in a volume of 2 ml assay medium. When MA-10(K3) cells were used they were cultured without G418 for 3-4 days before the experiment. One day before the experiment, the medium was changed, the concentration of serum was decreased to 5% horse serum and ZnSO4 (final concentration = 100 nM) was added (4). This concentration of ZnSO4 was maintained throughout the experiment. In experiments with 8-bromo-cAMP (8-Br-cAMP), cells were preincubated with 8-Br-cAMP (final concentration = 500 fiM) for 15 min at 37 C. This concentration of 8-Br-cAMP was maintained throughout the experiment. Fate of the receptor-bound hormones during one round of endocytosis Dishes were washed as described above. The 125I-labeled hormone was then added (final concentration = 40 ng/ml), and the dishes were incubated for 15 min at 37 C. The dishes were placed on ice and quickly washed five times with 2-ml portions of ice cold Hank's balanced salt solution containing 1 mg/ml BSA (wash buffer). Dishes were either processed immediately, or allowed to continue to incubate in 2-ml assay medium at 37 C. At the times indicated, the dishes were placed on ice, the medium was removed, and the dishes were washed once with 2 ml cold wash buffer. The cells were then incubated at 4 C with 1 ml cold 50 mM glycine/100 mM NaCl, pH 3, for 4 min and washed once with 1 ml of the same solution. The cells were solubilized with 100 ^1 1 N NaOH and collected with a cotton swab. The radioactivity that was removed by the acid wash was taken as a measure of the surface-bound hormone, the radioactivity that remained associated with the cells was considered to be internalized hormone (5). The medium and buffer washes were combined and precipitated with 10% trichloroacetic acid, and the soluble and insoluble radioactivities were considered to be degraded and undegraded hormone, respectively (6).
Endo• 1991 Voll28«No6
Rate of degradation of the internalized hormone Cells were washed as described above. Cells were then incubated with 125I-labeled hormone for 2 h at 37 C. The dishes were placed on ice, washed five times with 2-ml portions of cold wash buffer, incubated for 4 min with cold 50 mM glycine/100 mM NaCl, pH 3 (to remove the surface-bound hormone), washed once with the same solution, once with cold wash buffer, and once with cold assay medium. Dishes were either processed immediately, or the incubation was continued for up to 2 h at 37 C in 2 ml assay medium. At the times indicated, the dishes were placed on ice, the medium was saved, and the cells were washed once with 2 ml cold wash buffer. The medium and buffer washes were combined and precipitated with 10% trichloroacetic acid to determine the amounts of degraded and undegraded hormone released. The cells were solubilized with 100 ^1 1 N NaOH as described above to determine the amount of radioactivity that remained internalized.
After preincubation with 125I-labeled hormone and removal of surface-bound hormone (see above), the remaining cellassociated radioactivity was considered to be internalized hormone. Hormone degradation and release of degradation products in the medium were reflected by a corresponding decrease in cell-associated radioactivity. The rate constant of degradation (kh) was calculated using a semilog plot of the internalized hormone vs. time. (7-9). Slopes were calculated by linear regression and had correlation coefficients of at least 0.96. Rate of hormone internalization The rate of internalization (ke) was calculated from the rate of disappearance of receptor-bound hormone from the cell surface measured in the experiments dealing with the fate of the receptor-bound hormones during one round of endocytosis (see above). The rate of internalization was calculated from a plot of the log of surface-bound radioactivity us. time. The slopes were calculated by linear regression and had correlation coefficients of at least 0.93. Analysis of data Three dishes were used for each point shown. Two dishes contained the 125I-labeled hormone only (total binding), and the third dish contained the 125I-labeled hormone together with 25 IU/ml crude hCG (nonspecific binding). Specific binding was calculated by subtracting the nonspecific binding from the total binding. The nonspecific binding of [125I]hCG was at most 15% of the total binding, and the nonspecific binding of [I25I] deglycosylated hCG, although higher, was at most 24% of the total binding. Hormones and supplies Purified hCG (batch CR-125, 11,900 IU/mg) was obtained from the National Institutes of Health. Deglycosylated hCG was prepared as described previously (10). These hormones were iodinated as previously described (11). The specific activity of the [125I]hCG was approximately 19.4 iiCi/^g and the specific activity of the [125I]deglycosylated hCG was approximately 8.9 nQ\/ng. Crude hCG (3,000 IU/mg), 8-Br-cAMP, and BSA were obtained from Sigma Chemical Co. (St. Louis, MO). Na[125I] was
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INTERNALIZATION AND DEGRADATION OF hCG AND DEGLYCOSYLATED hCG obtained from New England Nuclear Co. (Boston, MA). Cell culture supplies and plasticware were from GIBCO (Grand Island, NY) and Corning (Corning, NY), respectively. All other supplies and materials were obtained from commonly used suppliers.
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Results Comparison of the internalization and degradation patterns of hCG and deglycosylated hCG In initial experiments we assessed a single round of endocytosis of the 125I-labeled hormone by preincubating MA-10 cells with [125I]hCG or [125I]deglycosylated hCG for 15 min at 37 C, washing to remove the free hormone, and reincubating the cells in hormone-free medium for up to 4 h at 37 C. Using this experimental design, one can follow the amount of surface-bound and internalized hormone associated with the cells and the amounts of degraded and undegraded hormone which appear in the medium over time. This represents a single round of endocytosis of the prebound hormone, because the free hormone is removed after the initial binding period. We had previously assessed a single round of endocytosis of hCG by preincubating MA-10 cells with 125I-labeled hormone for 2 h at 4 C (7). The preincubation was done at 4 C because virtually no internalization of hormone occurs at this temperature (5). In the experiments presented here, the preincubations were performed at 37 C in an effort to decrease the nonspecific binding of [125I] deglycosylated hCG. The results are essentially the same as those obtained with a 4 C preincubation, since during a 15-min preincubation at 37 C there is little internalization of the hormone. Thus, as can be seen in Fig. 1, approximately 95% of the cell-associated hormone is localized at the cell surface at time zero. The results presented in Fig. 1 illustrate that with [125I]hCG the surface-bound hormone disappears quickly and is accumulated within the cell. The levels of internalized [125I]hCG reach a maximum at 1-2 h and then decline as degradation products begin to appear in the medium. The appearance of degraded [125I]hCG in the medium displays a lag of about 1 h and then increases linearly. A small amount of undegraded [125I]hCG (less than 10% of the hormone initially bound) was also found in the medium, although this is not shown in this figure. Within 4 h, 95% of the [125I]hCG which was initially bound has been internalized and/or degraded. These results are consistent with previous data which show that hCG is internalized and ultimately degraded in the lysosomes (5,12). The rate of internalization of [125I] deglycosylated hCG during endocytosis is markedly different from [125I]hCG (Fig. 1). Although the surface-bound [125I]deglycosylated hCG also disappears from the surface, this occurs at a
hours FlG. 1. Comparison of the internalization and degradation patterns of hCG and deglycosylated hCG (dghCG) in MA-10 cells. Cells were preincubated with 40 ng/ml [125I]hCG (upper panel) or 40 ng/ml [125I] dghCG (lower panel). At the end of the incubation time (time = 0 in the figure), the cells were washed to remove free hormone. Cells were either processed immediately, or incubation was continued for up to 4 h in hormone-free medium. Amounts of radioactivity associated with the cell surface (circles), internalized (squares), and degraded (triangles) hormone were determined at the times indicated. The amounts of hormone bound at time = 0 were 0.41 ± 0.03 and 0.69 ± 0.2 ng/well for [125I]hCG and [125I]deglycosylated hCG, respectively. Each point represents the average ± range of two independent experiments.
much slower rate than that seen with [125I]hCG. After 4 h only 50% of the [125I]deglycosylated hCG which was initially bound has been internalized. Levels of internalized [125I]deglycosylated hCG appear to plateau after about 1 h. The accumulation of degradation products of [125I]deglycosylated hCG is also lower than that seen with [125I]hCG. This could be attributable either to the decreased internalization, or to a difference in the actual rates of degradation of these two hormones. From these data it can be concluded that the internalization of [125I]deglycosylated hCG is much slower than the internalization of [125I]hCG. It is not clear from this
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INTERNALIZATION AND DEGRADATION OF hCG AND DEGLYCOSYLATED hCG
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data whether the rate of degradation of [125I]deglycosylated hCG is also slower than that of [125I]hCG. Effect of cAMP Because deglycosylated hCG is unable to stimulate cAMP production (2), experiments were done to determine whether the lack of cAMP stimulation was responsible for the decreased rate of internalization of [125I] deglycosylated hCG. Two approaches were used to test this possibility. The first approach involved studying the internalization and degradation of deglycosylated hCG in the presence of a cAMP analog. Experiments were performed as described in Materials and Methods except that cells were preincubated with 500 /xM 8-Br-cAMP for 15 min before addition of 125I-labeled hormone. This concentration of 8-Br-cAMP was then maintained throughout the experiment. This high concentration of 8-Br-cAMP was chosen because 1) it elicits maximal stimulation of steroidogenesis and 2) the increase in progesterone production seen with this concentration of cAMP is similar to that seen with the concentrations of hCG used in these experiments (4). Results shown in Fig. 2 show that there was little or no difference between the disappearance of the surface-bound [125I]deglycosylated hCG in MA-10 cells which received the hormone alone or in cells which received [125I]deglycosylated hCG plus 8-Br-cAMP. Lev-
Endo«1991 Voll28«No6
els of internalized hormone and degraded hormone in the medium were also similar in the cells that received 8-Br-cAMP. The second approach used a subclone of MA-10 cells (MA-10(K3)) that express a cAMP-resistant phenotype (4). MA-10(K3) cells were obtained by transfection of MA-10 cells with an expression vector that encodes for a mutant form of the type I regulatory subunit of the cAMP-dependent protein kinase under the transcriptional control of the metallothionein promoter (13). Thus, incubation of MA-10(K3) cells with Zn2+ leads to the expression of a cAMP-resistant phenotype. In the experiments presented in Fig. 3, MA-10 cells and MA10(K3) cells were preincubated with Zn2+, and then were incubated with [125I]hCG. Amounts of surface-bound, internalized and degraded hormone in the medium were then measured over the course of 4 h. There was little or no difference between MA-10 cells and MA-10(K3) cells in any of the parameters measured. If anything, internalization is slightly faster in MA-10(K3) cells (Fig. 1), which is opposite of what would be expected if cAMP was involved in the internalization of hCG. Taken together, these results indicate that the slower rate of internalization of deglycosylated hCG as compared to hCG is not related to the inability of deglycosylated hCG to stimulate cAMP accumulation. This is shown by the lack of effect of a cAMP analog on the internalization and degradation of deglycosylated hCG, and the fact that there was no change in the internali-
•o
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hours FIG. 2. Effect of 8-Br-cAMP on internalization and degradation of deglycosylated hCG. Experiments were performed as described in the legend to Fig. 1, except that experiments were performed in the absence (closed symbols) or presence (open symbols) of 500 nM 8-Br-cAMP. Preincubation with 8-Br-cAMP was performed for 15 min before beginning the experiments, and 500 nM 8-Br-cAMP was present in the medium throughout the duration of the experiments. Each point represents the average ± SEM of three independent experiments.
hours FIG. 3. Internalization and degradation of hCG in MA-10 and MA10(K3) cells. Experimental design was as described in the legend to Fig. 1. Experiments were performed using MA-10 cells (closed symbols) or MA-10(K3) cells (open symbols). Each point represents the average ± SEM of three independent experiments.
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INTERNALIZATION AND DEGRADATION OF hCG AND DEGLYCOSYLATED hCG zation and degradation pattern of hCG when tested in a cell line with a cAMP resistant phenotype. Rate of internalization From visually comparing the graphs in Fig. 1 it can be seen that there is a difference in the rate of internalization of [125I]hCG and [125I] deglycosylated hCG. In order to further quantify this data, the actual rates of internalization (ke) were calculated from the rate of disappearance of receptor-bound hormone from the surface as described in Materials and Methods. The rate of hormone internalization can be approximated from the rate of disappearance of the surface-bound hormone only if the binding affinity of the hormone is high enough so that there is little or no dissociation of the hormone from the receptor during the course of the experiment. It has been shown previously in this (7, 14) and other (15-17) laboratories that once hCG is receptor bound, little or no dissociation of hCG occurs. Therefore, the disappearance of surface-bound hormone can be directly correlated with hormone internalization. These data can then be used to calculate the rate of internalization of the hormone. The results of these calculations are displayed in Table 1. These same data can also be expressed as the half-life of internalization, with [125I]hCG having a half-life of 61 ± 3 min and [125I]deglycosylated hCG having a half-life of 252 ± 10 min. Therefore, the rate of internalization of [125I]deglycosylated hCG is 4 times slower than the rate of internalization of [125I]hCG. Rates of internalization were also calculated for deglycosylated hCG in MA-10 cells incubated with 8-BrcAMP and for hCG in MA-10(K3) cells. These results (Table 1) further illustrate that there is little or no difference between the internalization of deglycosylated hCG in MA-10 cells incubated with or without 8-BrcAMP, or between hCG in MA-10 and MA-10(K3) cells. This supports the conclusion that cAMP accumulation is not responsible for the slower rate of internalization of deglycosylated hCG. The rate of internalization of hCG in MA-10 cells has been previously determined in two separate studies in this laboratory to be about 0.04 min"1 (7, 18). These results differ from the rate of internalization of hCG in MA-10 cells which we report here. It should be pointed TABLE 1. Rate of internalization (ke) Hormone 125
[ I]hCG [125I]dghCG
Cell
Additions
MA-10 MA-10(K3) MA-10 MA-10
none none none 8-Br-cAMP
ke (min *) 0.012 ± 0.014 ± 0.0028 ± 0.0032 ±
0.0007 0.0006 0.0001 0.0001
5 3 5 3
The rate constants for internalization were calculated as described in Materials and Methods. Each number represents the average ± SEM of the indicated number of experiments.
2841
out that a different method was previously used to determine the rate of internalization than that which was used here. From recent experiments, however, the rate of internalization of hCG calculated using the other method was found to be similar to that shown in Table 1. We are not presently able to account for this difference. Rate of degradation It can be seen from Fig. 1 that the rate of accumulation of degradation products is slower with [125I]deglycosylated hCG than with [125I]hCG. Although the slower rate of internalization of [125I]deglycosylated hCG may be sufficient to explain the accumulation of fewer degradation products, this may not necessarily be the case. It is possible that the accumulation of fewer degradation products may also be related to a difference in the rates of degradation of these two hormones. For this reason, the rate of degradation was measured directly in order to determine whether the slower rate of accumulation of degradation products of [125I]deglycosylated hCG in the medium, as compared to [125I]hCG (shown in Fig. 1), was related solely to the difference in the rates of internalization, or whether there is also a difference in the rates of degradation of hCG and deglycosylated hCG. The rate constant of degradation (kh) was calculated from the rate of disappearance of internalized hormone after removal of surface-bound hormone as described in Materials and Methods. Figure 4 illustrates the decrease of internalized radioactivity over time and the corresponding increase of degraded hormone in the medium. The amount of undegraded hormone in the medium is also shown. As mentioned previously, the undegraded hormone in the medium accounts for a small percentage of hormone initially bound. The rates of degradation are displayed in Table 2. These same data can also be expressed as the half-life of degradation, with [l25I]hCG having a half-life of 55 min and [125I]deglycosylated hCG having a half-life of 88 min. These data show that [125I]deglycosylated hCG is degraded at a rate which is approximately 1.5 times slower than the rate of degradation of [125I]hCG. The difference in the rates of degradation is not as large as the difference in the rates of internalization of the two hormones. Therefore, it appears that the slower accumulation of degradation products of [125I]deglycosylated hCG (see Fig. 1) may be a combination of both a decreased rate of degradation and a decreased rate of internalization as compared to hCG. Discussion The original objective of this study was to explain the observation from a previous study (1) that deglycosylated
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INTERNALIZATION AND DEGRADATION OF hCG AND DEGLYCOSYLATED hCG
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T3
Iro c
E a
u 75
30
60
90
120
100 -
120 FIG. 4. Rates of degradation of internalized hCG and deglycosylated hCG in MA-10 cells. Cells were preincubated with 40 ng/ml [125I]hCG (closed symbols) or 40 ng/ml [125I]deglycosylated hCG (open symbols) for 2 h at 37 C. At the end of this incubation time (time = 0 in the figure), the cells were washed to remove the free hormone, treated with acid to remove the surface-bound hormone, and then incubated at 37 C in hormone-free medium. At the times indicated, the amount of radioactivity that remained associated with the cells (upper panel), and the amounts of acid soluble and insoluble radioactivity released into the medium (lower panel) were determined. Each point represents the average ± SEM of three independent experiments. TABLE 2. Rate of degradation (kh)
Hormone
kh (mhT 1 )
[125I]hCG [125I]dghCG
0.012 ± 0.0004 0.008 ± 0.0008
The rates of degradation were calculated from semilog plots of the data presented in Fig. 4. Each number represents the average ± SEM of the indicated number of experiments.
hCG induces down-regulation of the LH/CG receptor much more slowly than hCG. The studies presented herein illustrate that the rate of internalization of deglycosylated hCG is 4 times slower than that of hCG. This conclusion is supported by the results presented in Fig. 1 and the calculated rates of internalization. The slower rate of internalization of deglycosylated hCG as compared to hCG would explain the slower induction of down-regulation of the LH/CG receptor elicited by de-
Endo• 1991 Voll28«No6
glycosylated hCG, since the initial phase of down-regulation appears to be due to receptor-mediated endocytosis (1). It was observed in initial experiments that the accumulation of degradation products of deglycosylated hCG was slower than seen with hCG. In a previous publication Rebois and Fishman (19) showed that deglycosylated hCG was degraded slower than hCG in another clonal strain of cultured Leydig tumor cells (MLTC-1) similar to MA-10 cells. Similar observations have also been made in cultured rat granulosa cells (20). Thus, although our results are in agreement with these previous observations, our experiments are more detailed and allow us to conclude that the slower rate of accumulation of degradation products of deglycosylated hCG as compared to hCG is due to a combination of a decreased rate of internalization and a decreased rate of degradation of deglycosylated hCG. Since the rate of internalization of deglycosylated hCG is 4 times slower than hCG, while the rate of degradation of deglycosylated hCG is only 1.5 times slower than hCG, it is probable that the slower accumulation of degradation products is mainly due to the difference in internalization rates. These data also support previous observations in pseudopregnant rats where the labeled deglycosylated hCG and deglycosylated LH were found to be retained in the ovary for a longer time than the native hormones (21). In addition, the studies presented support the conclusion that the slower internalization of deglycosylated hCG as compared to hCG is not due to the inability of deglycosylated hCG to stimulate cAMP production. Two points illustrate this conclusion. The rate of internalization of deglycosylated hCG is not increased by incubating MA-10 cells with 8-Br-cAMP. Second, there is little or no difference in the rate of internalization of hCG in a subclone of MA-10 cells (designated MA10(K3)) that express a cAMP-resistant phenotype. In this respect it is interesting to note that other hormonereceptor complexes that are unable to elicit intracellular signals are also internalized at a slower rate than their normal counterparts. For example, the rate of internalization of EGF in cell lines expressing mutant EGF receptors that lack tyrosine kinase activity is slower than in cells expressing the wild type receptor (22). Although it is clear that the rate of internalization of deglycosylated hCG cannot be accounted for by the lack of cAMP accumulation, the molecular basis of this phenomenon is not currently understood. A potential explanation is that there is a difference in the conformation of the hormone-receptor complex and/or the ability of this complex to associate with other membrane proteins (such as Gs for example) (2). This question may now be experimentally approached by site-directed mutagenesis of the LH/CG receptor (23, 24).
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INTERNALIZATION AND DEGRADATION OF hCG AND DEGLYCOSYLATED hCG In summary, this study fulfilled its original objective of explaining the slower down-regulation of the LH/CG receptor induced by deglycosylated hCG as compared with hCG (1). In addition, we have shown that the internalization and degradation of deglycosylated hCG is slower than hCG and that this effect is not mediated by cAMP. Finally, the slower accumulation of degradation products of deglycosylated hCG as compared to hCG is probably due to a combination of a decreased rate of internalization and a decreased rate of degradation.
Acknowledgments We wish to thank Bruce D'Souza for expert assistance with cell culture. We also thank Deborah L. Segaloff for critical reading of the manuscript.
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