Role of the Rate of Internalization of the Agonist-Receptor Complex on the Agonist-Induced Down-Regulation of the Lutropin/ Choriogonadotropin Receptor

Kazuto Nakamura, Maria de Fatima M. Lazari1, Shenghua Li, Chandrashekhar Korgaonkar and Mario Ascoli

Department of Pharmacology The University of Iowa College of Medicine Iowa City, Iowa 52242-1109


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The extent of agonist-induced down-regulation of the LH/CG receptor (LHR) in human kidney 293 cells transfected with the rat LHR (rLHR) is much lower than in two Leydig tumor cell lines (MA-10 and R2C) that express the rodent LHR endogenously. This difference can not be attributed to differences in the recycling of internalized receptors, or in the replenishment of new receptors at the cell surface. It can be correlated, however, with the half-life of internalization of the bound agonist, which is approximately 60 min in Leydig tumor cells and about 100 min in transfected 293 cells. To determine whether the rate of internalization of the bound agonist affects down-regulation, we compared these two parameters in 293 cells expressing four rLHR mutants that enhance internalization and three mutants that impair internalization. We show that all four mutations of the rLHR that enhanced internalization enhanced down-regulation, while only one of the three mutations that impaired internalization impaired down-regulation. In addition, cotransfections of 293 cells with the rLHR-wt and three constructs that enhanced internalization (G protein-coupled receptor kinase 2, ß-arrestin, and arrestin-3) increased down-regulation, while a related construct (visual arrestin) that had no effect on internalization also had no effect on down-regulation. We conclude that the rate of internalization of the agonist-LHR complex is the main determinant of the extent of down-regulation of the LHR.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Previous biochemical and morphological studies have delineated the internalization pathway followed by the agonist-LH/CG receptor (LHR) complex. In the absence of agonist the LHR is randomly distributed on the cell surface, but upon agonist-induced activation, the agonist-LHR complex clusters in coated pits and is internalized into endosomes (1, 2). These initial steps of internalization are facilitated by the agonist-induced phosphorylation of the LHR and require the participation of dynamin as well as that of a nonvisual arrestin (3, 4), which serves the function of an adaptor molecule between the receptor and clathrin (5). The same active conformation of the LHR that is involved in G protein activation seems to be involved in endocytosis because 1) the internalization of the agonist-bound rat (r) or mouse (m) LHR is faster than that of the free mLHR or rLHR (6, 7); 2) the internalization of the complex formed between the mLHR and a weak partial agonist is much slower than that of the agonist-mLHR complex (8); 3) inactivating mutations of the rLHR impair the endocytosis of the agonist-rLHR complex (7, 9); and 4) activating mutations of the rLHR enhance basal and/or agonist-stimulated endocytosis (7).

In contrast to many other agonist-receptor complexes that dissociate in the acidic environment (pH 5–6) that prevails in endosomes (10), the agonist-LHR complex is rather insensitive to dissociation in this environment (1), and the agonist-LHR complex internalized into endosomes is delivered to the lysosomes in the intact form (i.e without dissociation of the agonist and receptor; see Refs. 1, 2). The more acidic environment that prevails in lysosomes as well as proteolysis promote the dissociation of the agonist-receptor complex, and both subunits of the agonist are eventually degraded to single amino acids (11). The lysosomal degradation of the LHR has not been formally documented, however. The net result of this pathway is to target the cell surface LHR for lysosomal degradation and as such, the endocytosis of the agonist-LHR complex is quantitatively responsible for the down-regulation of the cell surface LHR that occurs when mouse Leydig tumor cells are exposed to agonist (12).

Many of the newer experiments that have examined the agonist-induced internalization and subsequent down-regulation of the LHR have been performed with transfected 293 cells. While side-to-side comparisons using the same methodology have not been made, a perusal of the studies conducted using 293 cells expressing the recombinant LHR suggest that the extent of agonist-induced down-regulation is less in 293 cells expressing the recombinant LHR than in mouse Leydig tumor cells expressing the endogenous LHR (3, 6, 12, 13, 14, 15, 16). The experiments presented here were thus designed to determine the basis of this difference.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Agonist-Induced Internalization and Down-Regulation of the LHR in Leydig Tumor Cells and Transfected 293 Cells
The experiments summarized in Fig. 1Go were performed to compare agonist-induced internalization and down-regulation of the LHR in Leydig tumor cells and transfected 293 cells under the same experimental conditions.



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Figure 1. Agonist-Induced Internalization and Down-Regulation of the rLHR in Leydig Tumor Cells and in 293 Cells Stably Transfected with Different Truncations of the rLHR

MA-10 and R2C cells, as well as clonal lines of 293 cells expressing the indicated truncations of the rLHR, were used. Down-regulation was measured after incubation of the cells with or without 100 ng/ml hCG for 24 h at 37 C. The cells were then washed to remove the free hormone, briefly treated with a pH 3 buffer to remove any receptor-bound hormone, and then used to measure [125I]hCG during a 4-h incubation at 4 C with 100 ng/ml [125I]hCG as described in Materials and Methods. The results of these experiments are shown in the top panel and are expressed as percent of the binding activity detected in cells incubated without hCG but otherwise treated under identical conditions. The t1/2 values of internalization of [125I]hCG were measured as described in Materials and Methods and are shown in the bottom panel. Each number represents the average ± SEM of three to five independent experiments. The asterisks indicate differences that are statistically significant (P < 0.05) from 293L(wt-12) cells.

 
The results shown in the top panel of Fig. 1Go show that incubation of a mouse Leydig tumor cell line (MA-10) or a rat Leydig tumor cell line (R2C) with a saturating concentration of hCG for 24 h (conditions that lead to maximal loss of cell surface LHR) results in the down-regulation of at least 95% of the cell surface LHR. Under the same experimental conditions, however, the down-regulation of the LHR in 293 cells stably transfected with the rLHR-wt [i.e. 293L(wt-12) cells] is only approximately 25%. In Fig. 1Go we also analyzed 293 cells stably transfected with three different truncations of the cytoplasmic tail of the rLHR that have been previously reported to affect internalization and/or down-regulation (13, 14). The extent of agonist-induced down-regulation of the LHR (~75%) in 293L(t631–1) and 293L(t628–1) cells is more pronounced than that detected in 293L(wt-12) cells but not as high as that detected in MA-10 or R2C cells. Lastly, the extent of agonist-induced down-regulation of the LHR in 293L(t653–6) cells (~25%) was comparable to that of 293 cells expressing rLHR-wt.

Since the down-regulation of cell surface receptors that occurs upon agonist-induced activation is due to the balance of internalization/degradation of the agonist-occupied receptor, the recycling of the internalized receptor, and the synthesis/externalization of new receptors, we attempted to measure these parameters in MA-10 cells and in 293 cells stably transfected with the rLHR-wt or mutants thereof.

The results presented in the bottom panel of Fig. 1Go show that the t1/2 of internalization of the agonist-receptor complex is approximately 50 min in Leydig tumor cells and about 100 min in 293L(wt-12) cells. As expected (13), truncation of the C-terminal tail of the rLHR at residue 653 increased the t1/2 of internalization, and truncations at residues 631 or 628 reduced the t1/2 of internalization in transfected 293 cells. Under our standardized assay conditions the t1/2 of internalization of the agonist-LHR complex in 293L(t631–1) or 293L(t628–1) cells was comparable to that measured in MA-10 or R2C cells (i.e. ~50 min).

Recycling of internalized receptors was next measured in MA-10, 293L(wt-12), and 293L(t631–1) cells. In these experiments cells were first allowed to bind and internalize a saturating concentration of hCG during a 2-h incubation at 37 C. At this point the surface-bound hCG was removed with a mild acid buffer, and the cells were reincubated at 37 C in hormone-free medium for up to 2 h (to allow for the recycling of internalized receptors at the cell surface) before measuring [125I]hCG binding at 4 C. The results summarized in Fig. 2Go show that MA-10, 293L(wt-12), and 293L(t631–1) behave similarly in that there is little or no replenishment of the cell surface receptor within 2 h of hormone removal. Longer times were not examined because in other systems recycling of internalized receptors occurs within minutes (see Refs. 17, 18 for recent examples). Moreover, at longer times it is difficult to distinguish recycling of internalized receptors from externalization of newly synthesized receptors without the aid of protein synthesis inhibitors. While protein synthesis inhibitors can be readily used in 293 cells without adverse effects on cell viability, they could not be used in MA-10 cells without compromising cell viability.



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Figure 2. Recovery of [125I]hCG Binding after Removal of the Surface-Bound Hormone

The indicated cell lines were incubated with 100 ng/ml hCG for 2 h at 37 C. At the end of this incubation (labeled t = 0 in the figure and marked with an arrow), the cells were washed to remove the free hormone and briefly incubated in a pH 3 buffer to remove the receptor-bound hormone (see Materials and Methods). The cells were then reincubated in hormone-free medium at 37 C and the binding of [125I]hCG was measured (see Materials and Methods) as a function of time after hormone removal. Results are expressed as percent of the binding activity detected in cells incubated without hCG but otherwise treated under identical conditions. Each point represents the mean ± SEM of three independent experiments.

 
We have previously shown that after proteolytic destruction of the cell surface rLHR expressed in transfected cells, the complement of cell surface rLHR can be readily replenished by the externalization of an intracellular rLHR precursor and by the synthesis of new receptors (19). Thus, the rate of replenishment of cell surface receptors after proteolysis can be used to determine whether there is a difference in the rates of externalization/synthesis of the rLHR-wt and rLHR-t631. The results of these experiments are presented in Fig. 3Go and show that the rate of replenishment of the LHR at the cell surface is the same in 293L(wt-12) and 293L(t631–1) cells.



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Figure 3. Recovery of [125I]hCG Binding after Proteolysis of 293L(wt-12) and 293L(t631–1) Cells

The indicated cell lines were exposed to protease XIV (250 µg/ml) for 30 min at 4 C and washed as described in Materials and Methods. At this point (t = 0 in the figure), the cells were further incubated at 37 C for the indicated times. The binding of [125I]hCG to intact cells was then measured as described in Materials and Methods and expressed relative to the 24-h time point. Each point represents the average ± SEM of three independent experiments.

 
From the data presented in Fig. 3Go it can be calculated (7) that the cell surface LHR is replenished with a half-life of approximately 400 min in 293L(wt-12) and 293L(t631–1) cells. This half-life is comparable to those previously reported by us in 293L(wt-12) cells (i.e. ~460 min; see Refs. 7, 19) and in MA-10 cells (i.e. ~400 min; see Ref. 6).

Taken together, these data suggest that the difference in the extent of down-regulation of the LHR detected in 293L(wt-12) and MA-10 cells is not due to differences in the recycling of the internalized receptor or the replenishment of new receptors at the cell surface. Since a decrease in the transcription of the endogenous LHR gene does not contribute to the hCG-induced down-regulation of the LHR in MA-10 cells (see Discussion and Ref. 12), the differences in the magnitude of down-regulation between MA-10 and 293L(wt-12) cells cannot be accounted for by the absence of a transcriptional effect on the rLHR cDNA that is stably incorporated into the 293 cell genome. Lastly, the enhanced agonist-induced down-regulation detected in 293L(t631–1) cells, when compared with 293L(wt-12) cells, also cannot be explained by differences in the recycling of the internalized receptor or the replenishment of new receptors at the cell surface.

Mutations of the rLHR That Affect the Rate of Internalization of the Agonist-Receptor Complex Affect the Extent of Agonist-Induced Down-Regulation in Transfected 293 Cells
In the next series of experiments we tested the hypothesis that the rate of internalization of the hCG-LHR complex is an important determinant of the extent of down-regulation of the LHR. We considered this hypothesis because 1) the experiments described above excluded changes in recycling of the internalized receptors or in the replenishment of new receptor at the cell surface as being important determinants of down-regulation; and 2) the two truncations of the rLHR that enhanced down-regulation in 293 cells also enhanced internalization (cf. Fig. 1Go). It is also possible, however, that truncations of the C-terminal tail affect down-regulation by other mechanisms and that there is no cause-effect relationship between the rate of internalization and the extent of down-regulation in 293 cells.

To more directly establish a causal effect between internalization and down-regulation, we took advantage of several previously established clonal lines of 293 cells that are stably transfected with point mutations of the rLHR that affect the rate of internalization of the agonist-rLHR complex. An aspartic-to-asparagine mutation in codon 383 in TM2 (D383N) and a tyrosine-to-phenylalanine mutation in codon 524 of TM5 (Y524F) of the rLHR have been previously shown to impair signal transduction as well as the internalization of hCG (7, 9), while an aspartic-to-tyrosine mutation in codon 556 of TM6 (D556Y) and a leucine-to-arginine mutation in codon 435 of TM3 (L435R) of the rLHR have been previously shown to induce constitutive activation and to enhance the internalization of hCG (7, 20, 21).

When compared with 293 cells expressing rLHR-wt, the extent of agonist-induced down-regulation is higher in 293 cells expressing rLHR-D556Y or rLHR-L435R, the two constitutively active mutants that internalize hCG at a faster rate than rLHR-wt (Fig. 4Go). Note that although the t1/2 of internalization of hCG is shorter in 293Lmyc-(L435R-2) cells than in 293L(D556Y-6) (~7 and ~ 37 min, respectively) the magnitude of down-regulation is greater in 293L(D556Y-6) cells than in 293L(mycL435R-2) cells. hCG induces approximately 80% down-regulation in 293L(D556Y-6) cells compared with about 30% in 293L(wt-12) cells, and about 55% down-regulation in 293Lmyc(L:435R-2) cells compared with approximately 10% in 293Lmyc(wt-11) cells. This difference may very well be explained by the finding that new rLHR-L435R is replenished at the cell surface at a faster rate than rLHR-D556Y or rLHR-wt (7). This faster rate of replenishment will, of course, balance the increased rate of internalization of the hCG-receptor complex and ultimately reduce the extent of down-regulation observed at steady state.



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Figure 4. Agonist-Induced Internalization and Down-Regulation of the rLHR in 293 Cells Stably Transfected with Signaling-Impaired and Constitutively Active Mutants of the rLHR

Stably transfected, clonal lines of 293 cells expressing rLHR-wt [293L(wt-12)] or the myc-tagged rLHR-wt [293Lmyc(wt-11)], as well as the indicated clonal cell lines expressing several point mutations of the rLHR constructed in the context of the otherwise unmodified rLHR or the myc-tagged rLHR, were used. The t1/2 values of internalization of [125I]hCG in these cell lines were measured as described in Materials and Methods and are shown in the top panel. To measure down-regulation after multiple rounds of endocytosis, the different cell lines were incubated with or without 100 ng/ml hCG for 18–24 h at 37 C. The cells were then washed to remove the free hormone, briefly treated with a pH 3 buffer to remove any receptor-bound hormone, and then used to measure [125I]hCG during a 4-h incubation at 4 C with 100 ng/ml [125I]hCG as described in Materials and Methods. The results of these experiments are expressed as percent of the binding activity detected in cells incubated without hCG but otherwise treated under identical conditions. Each bar shows the average ± SEM of at least three experiments. *, Significantly different (P < 0.05) from 293L(wt-12) cells. **, Significantly different (P < 0.05) from 293Lmyc(wt-11) cells.

 
The data presented in Fig. 4Go also show results obtained with two signaling-impairing mutations that also impair internalization. 293 cells expressing rLHR-Y524F, a mutant that internalizes the bound agonist with a half-life that is about 2-fold slower than rLHR-wt, display a normal degree of down-regulation while cells expressing rLHR-D383N, a mutant that internalizes the bound agonist with a half-life that is about 3-fold slower than rLHR-wt, display no down-regulation.

Thus, four mutations that enhance the rate of agonist-induced internalization of the recombinant rLHR (i.e. rLHR-t631, rLHR-t628, rLHR-D556Y, and rLHR-L435R) also enhance the extent of agonist-induced down-regulation of the rLHR expressed in 293 cells, while only one (i.e. rLHR-D383N) of the three mutations that reduce the rate of agonist-induced internalization (i.e. rLHR-D383N, rLHR-Y524F, and rLHR-t653) reduce the extent of agonist-induced down-regulation. It should also be noted that only two (rLHR-D556Y and rLHR-L435R) mutations that enhance internalization induce constitutive activation, and that only two (rLHR-D383N and rLHR-Y524F) mutations that impair internalization impair signal transduction (7, 9, 14).

Cotransfections of 293 Cells with the rLHR-wt and Nonvisual Arrestins or G Protein-Coupled Receptor Kinase 2 (GRK2) Enhance the Internalization of the hCG-LHR Complex and the hCG-Induced Down-Regulation of the Cell Surface rLHR
While the experiments summarized above suggest a relationship between the rate of internalization of hCG and the extent of down-regulation of the cell surface rLHR, this suggestion is based entirely on the study of receptor mutants. Thus we sought to also examine this issue using manipulations that affect the endocytosis of the rLHR-wt. We chose to use cotransfections of 293 cells with the rLHR-wt and GRK2 or arrestins because the GRK-catalyzed phosphorylation of the rLHR and the interaction of the rLHR with the nonvisual arrestins are expected to enhance the internalization of the hCG-LHR complex (4, 5, 22), while the interaction of the rLHR with visual arrestin is not expected to enhance internalization (5).

We (23) and others (24) have previously shown the presence of GRK2 and nonvisual arrestins in mock-transfected 293 cells. The results presented in Fig. 5AGo document again the presence of endogenous GRK2 in 293 cells and show the increased expression of this kinase after transfection with a GRK2 expression vector.2 The results presented in Fig. 5BGo document the enhanced expression of visual arrestin, arrestin 3, and ß-arrestin in 293 cells transfected with the appropriate expression vectors.



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Figure 5. Expression of GRK2 and Arrestins in Transiently Transfected 293 Cells

293 cells were transiently transfected with 10 µg/dish of an empty expression vector, or expression vectors for GRK2, visual arrestin, ß-arrestin, or arrestin-3 as indicated. These amounts of expression vectors were shown to be maximally effective on expression and internalization (c.f. Table 1Go). Cell lysates were prepared, and the indicated amounts of lysate protein were resolved on SDS gels and electrophoretically transferred to polyvinylidenefluoride membranes. GRK2 and arrestins were detected using the 3A10 and F4C1 monoclonal antibodies, respectively, as described in Materials and Methods. Proteins were ultimately visualized using the ECL system of detection as described in Materials and Methods. Only the relevant portions of the blots of a representative experiment are shown.

 
The results presented in Table 1Go show the effects of GRK2 and arrestin cotransfections on the internalization of the hCG-rLHR complex and subsequent hCG-induced down-regulation of the cell surface rLHR. Cotransfection with GRK2 or ß-arrestin resulted in a reduction (~2-fold) in the t1/2 of internalization of the hCG-rLHR complex and an increase (~2-fold) in the magnitude of down-regulation of the rLHR. Cotransfection with arrestin-3 resulted in a reduction (~5-fold) in the t1/2 of internalization of the hCG-rLHR complex and an increase (~4-fold) in the magnitude of down-regulation of the rLHR. Since visual arrestin lacks a clathrin-binding domain (5), we did not expect this construct to increase the internalization of the hCG-rLHR. As shown in Table 1Go, this was found to be the case, and the lack of effect of visual arrestin on internalization was accompanied by a lack of effect on down-regulation.


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Table 1. Effects of Arrestins or GRK-2 on the Internalization and Down-Regulation of the rLHR

 
Taken together, the results presented with target and transfected cells, mutations of the rLHR that affect the rate of internalization of the transfected receptor, or with manipulations that affect the rate of internalization of the transfected rLHR-wt suggest that the rate of internalization of the agonist-LHR complex is an important determinant of the extent of agonist-induced down-regulation of the cell surface LHR.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Previous results from this (1, 3, 4, 6, 7, 8, 9, 12, 14, 26) and other (13, 15, 16) laboratories have suggested that there are differences in the agonist-induced internalization and down-regulation of the LHR expressed in transfected 293 cells and in mouse Leydig tumor cells. The experiments presented here were designed to determine the basis of this difference.

Using standardized assay conditions we clearly showed that the extent of agonist-induced down-regulation of the LHR is indeed much lower in 293 cells expressing rLHR-wt than in rat or Leydig tumor cells (Fig. 1Go). This decreased down-regulation does not appear to be due to differences in the fate of the internalized agonist-receptor complex because 1) this complex has been shown to accumulate in the lysosomes in target and transfected cells (1, 2); and 2) target or transfected cells degrade more than 90% of the internalized agonist during each round of endocytosis (3, 9, 11). Here we show that these differences cannot be accounted for by differences in the extent of recycling of the internalized receptor because this is minimal in Leydig tumor cells and in 293 cells expressing rLHR-wt (Fig. 2Go).

The possibility that differences in the extent of hCG-induced down-regulation of the LHR in target and 293 cells are partially due to an hCG-induced reduction in the transcription of the endogenous LHR gene expressed in MA-10 or R2C cells has already been excluded. While it is clear that hCG, acting through cAMP as a second messenger, decreases the transcription of the endogenous LHR gene and the levels of LHR mRNA in MA-10 cells (12, 27, 28), this effect is quantitatively unimportant to down-regulation. Thus, the magnitude of hCG-induced down-regulation of the LHR is unaffected in a subclone of MA-10 cells that express a cAMP-resistant phenotype and do not respond to hCG with a decrease in LHR mRNA (12). The effects of hCG on the levels of mRNA transcribed from the LHR cDNA incorporated into the 293 cell genome have not been examined, but we have shown that cAMP tends to increase LHR mRNA without affecting the levels of cell surface LHR in transfected 293 cells (28). While a cAMP-induced increase in LHR mRNA in transfected cells could theoretically attenuate the extent of the hCG-induced down-regulation of the LHR in these cells, this does not appear to be the case. If a cAMP-induced increase in LHR mRNA was an important attenuator of down-regulation in transfected 293 cells, one would expect an enhancement of down-regulation in 293 cells expressing signaling-impairing mutations of the LHR and perhaps even an impairment of down-regulation in 293 cells expressing constitutively active mutants of the rLHR. In fact, the data presented here document the opposite effect in that signaling-impairing mutations attenuate down-regulation while mutations that induced constitutive activation enhance it (Fig. 4Go and Refs. 7, 9). The three truncations of the cytoplasmic tail of the rLHR that impair or enhance internalization and down-regulation (Fig. 1Go) have little or no effect on hCG-induced cAMP accumulation (14, 29). Overexpression of arrestin-3, a manipulation that enhances internalization and down-regulation of the rLHR-wt (Table 1Go), also has no effect on hCG-induced cAMP accumulation (4). Lastly, the rate of replenishment of new receptors at the cell surface, a rate that is affected by the transport of the LHR precursor to the cell surface and by the synthesis of new LHR (19), is also not responsible for differences in the extent of down-regulation between target and transfected cells because this rate is very similar in 293 cells expressing rLHR-wt and in MA-10 cells (Fig. 3Go and Refs. 6, 7).

The data presented here argue that the difference in the extent of agonist-induced down-regulation of the LHR observed in rodent Leydig tumor cells and 293 cells expressing the rLHR-wt is due mostly to differences in the rate at which these cells internalize the agonist-receptor complex. Thus, there is a positive correlation between the rate of internalization of the agonist-LHR complex and the extent of down-regulation in three different cell lines (MA-10, R2C and transfected 293 cells) that express the mouse or rat LHR-wt (Fig. 1Go). Second, all mutations of the rLHR that enhance the rate of internalization of the agonist-rLHR complex in transfected 293 cells also enhance the extent of agonist-induced down-regulation of the rLHR in these cells (Figs. 1Go and 4Go). Third, three manipulations that enhance the rate of internalization of the agonist-rLHR complex in transfected 293 cells (i.e. cotransfections with GRK2, arrestin-3, or ß-arrestin) also enhance the extent of down-regulation of the rLHR-wt in these cells. Lastly, cotransfection with a related construct that has little or no effect on internalization (visual arrestin) does not enhance down-regulation (Table 1Go).

If the rate of internalization is important to the extent of down-regulation, one would predict that manipulations that slow down internalization should also impair down-regulation. This has been found to be the case only in some instances, however. For example, the slow rate of internalization of a weak partial agonist in MA-10 cells (8) is accompanied by a reduction in the extent of down-regulation (12). In contrast, when using transfected 293 cells, where the rate of internalization of the agonist-receptor complex is already slow compared with that of target cells (cf. Fig. 1Go), mutations of the rLHR that increase the t1/2 of internalization by less than about 2-fold (i.e rLHR-t653 and rLHR-Y524F) are not accompanied by a reduction in the extent of down-regulation (Figs. 1Go and 4Go). One mutation that increased the t1/2 of internalization by 3- to 4-fold (rLHR-D383N, see Fig. 4Go) did abolish down-regulation, however. Taken together with the results discussed above, these data suggest that the extent of down-regulation in 293 cells stays fairly constant when the t1/2 of internalization is between 100 and 200 min (cells expressing rLHR-wt, rLHR-t653, or rLHR-Y524F), but it is enhanced when the t1/2 of internalization is shorter than about 100 min (cells expressing rLHR-t631, rLHR-t628, rLHR-D556Y, and rLHR-L435R or cells expressing rLHR-wt but overexpressing GRK2, ß-arrestin, or arrestin-3), and is impaired when the t1/2 of internalization is longer than about 200 min (cells expressing rLHR-D383N).

Results of experiments presented above and elsewhere (3, 4, 7, 13) have shown that the activation and phosphorylation of the rLHR facilitate the internalization of the agonist-LHR complex and have identified some cellular proteins involved in this process. The importance of receptor activation is readily demonstrated by the slower rate of internalization of a complex formed between the mLHR-wt and weak partial agonist (8) and by the findings that rLHR mutations that impair signal transduction internalize agonist at a slow rate while mutations of the rLHR that induce constitutive activation internalize agonist at a fast rate (7, 9). The importance of receptor phosphorylation is also readily demonstrated by the findings that a phosphorylation-deficient mutant of the rLHR internalizes agonist at a slow rate (3, 4) while overexpression of GRKs, a manipulation that enhances LHR phosphorylation, also stimulate the internalization of the agonist-LHR complex (Table 1Go). Thus, GRKs are among the cellular proteins that may affect internalization. Dynamin and nonvisual arrestins also participate in the internalization of the agonist-LHR complex as judged by the finding that their specific dominant-negative mutants (4) inhibit the internalization of this complex and by the finding that overexpression of nonvisual arrestins stimulate the endocytosis of the agonist-LHR complex (Table 1Go and Ref. 4). It is therefore possible that the differences in the rate of internalization of the agonist-LHR complex reported here between MA-10/R2C cells and 293 cells expressing the recombinant rLHR could be due to differences in the expression of GRKs, nonvisual arrestins, dynamin (and perhaps other as yet unidentified proteins that participate in internalization) between these cells. While antibodies to all these proteins are available, we could not use them to accurately measure their relative levels in MA-10, R2C, and 293 cells because these cell lines are from different species (mouse, rat, and human, respectively), and the interspecies cross-reactivity of all these antibodies is not known.

When using a single cell line (i.e. 293 cells), the rate of internalization of the agonist-rLHR complex can be readily manipulated by mutations that affect receptor activation (Fig. 4Go and Refs. 7, 9), by mutations that affect receptor phosphorylation (3, 4), and by overexpression of GRKs or nonvisual arrestins or their dominant-negative mutants (Table 1Go and Ref. 4). Other structural features of the LHR must be involved in internalization, however, as illustrated by the findings that some truncations of the C-terminal tail of the rLHR (such as rLHR-t653) impair endocytosis, while others (such as rLHR-t631 and rLHR-t628) enhance endocytosis (Fig. 4Go and Refs. 13, 14). The structural features of the rLHR removed by these truncations appear to be more important than receptor activation, because receptor activation (as measured by second messenger generation) is not impaired by any of these three truncations (14). They are also more important than phosphorylation because the sites phosphorylated in response to agonist stimulation are four serine residues (serine635, serine639, serine649, and serine652) located between residues 631 and 653 (3, 4, 14). Thus, agonist-induced phosphorylation of rLHR-t653 is normal or only slightly reduced when compared with rLHR-wt while the agonist-induced phosphorylation of rLHR-t631 and rLHR-t628 are undetectable (14).

Further experiments on the effects of truncations or mutations of the C-terminal tail of the LHR on internalization and on its interaction with nonvisual arrestins may provide important information about how this region of the LHR affects internalization.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids and Cells
The cloning of the rat luteal LHR cDNA and the template plasmid containing the full-length coding region plus portions of the 5'- and 3'-untranslated regions of the wild-type rLHR cDNA have been previously described (30). Mutations were introduced using PCR strategies, and the sequence of the entire region of each mutant cDNA generated by PCR was verified by automated DNA sequencing. The mutant and wild-type rLHR cDNAs were subcloned into pcDNA1-Neo or pcDNA 3.1 (Invitrogen, San Diego, CA) for transfection.

Expression vectors encoding for visual arrestin (in pBC12B1), ß-arrestin, and arrestin-3 (both in pcDNA3.1) have been described previously (31) and were generously provided by J. L. Benovic (Thomas Jefferson University, Philadelphia, PA). A full-length GRK2 (32) was subcloned into pcDNA1.1/Amp for expression studies.

Transient transfections of human embryonic kidney (293) cells were done using calcium phosphate as described by Chen and Okayama (33). After an overnight incubation the cells were washed, placed back in culture medium, and used 24 h later (4, 34).

The establishment and properties of clonal cell lines of 293 cells stably transfected with rLHR-wt, designated 293L(wt-12); rLHR-t631, designated 293L(t631–1); rLHR-t653, designated 293L(t653–6); rLHR-t628, designated 293L(t628–1); rLHR-D383N, designated 293L(D383N-9); rLHR-Y524F, designated 293L(Y524F-22); and rLHRD556Y, designated 293L(D556Y-6), have been described previously (7, 14, 29). Clonal lines of 293 cells stably transfected with a myc-tagged rLHR-wt, designated 293Lmyc(wt-11); and with a myc-tagged rLHR-L435R mutant, designated 293Lmyc(L435R-2), have also been described (7, 19). All of these cell lines express between 100,000 and 200,000 cell surface receptors per cell (7, 14, 19, 29).

The origin and handling of MA-10 cells, a clonal strain of mouse Leydig tumor cells that were adapted to culture in this laboratory and express the LHR endogenously, have been described (35). R2C cells are a clonal strain of rat Leydig tumor cells (36) available from the American Type Culture Collection (Manassas, VA). These cells were maintained using the same culture conditions previously described for MA-10 cells (35).

Internalization Assays
The endocytosis of [125I]hCG was measured as follows (6, 26). Cell monolayers (in 35-mm wells), were washed twice with assay medium (Waymouths MB752/1 containing 1 mg/ml BSA and 20 mM HEPES, pH 7.4) and then preincubated in 1 ml of assay medium for 60 min at 37 C. Each well then received 40 ng/ml [125I]hCG with or without 25 IU/ml of crude hCG (to correct for nonspecific binding), and the incubation was continued at 37 C. At 5- to 10-min intervals, groups of cells were placed on ice and washed twice with 2-ml aliquots of cold HBSS containing 1 mg/ml BSA. The surface-bound hormone was then released by incubating the cells in 1 ml of cold 50 mM glycine, 150 mM NaCl, pH 3, for 2–4 min (11). The acidic buffer was removed, and the cells were washed once more with another aliquot of the same buffer. The acid buffer washes were combined and counted, and the cells were solubilized with 100 µl of 0.5 N NaOH, collected with a cotton swab, and counted to determine the amount of internalized hormone. The endocytotic rate constant (ke) was calculated from the slope of the line obtained by plotting the internalized radioactivity against the integral of the surface-bound radioactivity (7, 26, 37, 38). The half-life of internalization (t1/2) is defined as 0.693/ke.

Assay of Receptor Down-Regulation
These assays were done with minor modifications of previously described methods (1). Cell monolayers (in 35-mm wells) were incubated at 37 C with a saturating concentration of hCG (100 ng/ml) for 24 h. The cells were then cooled to 4 C for 30 min, washed twice with 2-ml aliquots of cold HBSS containing 1 mg/ml BSA (to remove the free hormone), treated with 1 ml of cold 50 mM glycine, 150 mM NaCl, pH 3, for 2–4 min to remove the residual surface-bound hormone, and then washed twice with 2-ml aliquots of assay medium. Each well then received 100 ng/ml 125I-hCG with or without 25 IU/ml of crude hCG (to correct for nonspecific binding), and the cells were incubated for 4 h at 4 C. The free hormone was then removed by washing three times with 2-ml aliquots of cold HBSS containing 1 mg/ml BSA, and the cells were solubilized with 100 µl of 0.5 N NaOH, collected with a cotton swab, and counted to determine the amount of bound hormone. All binding data were then expressed as percent of the radioactivity detected in cells incubated without hCG, but otherwise treated under identical conditions. For the stably transfected 293 cell lines, 100% binding varied between 100,000 and 200,000 cpm/well. For MA-10 and R2C cells and for the transiently transfected 293 cells, 100% binding varied between 20,000 and 40,000 cpm/well. Since a saturating concentration of [125I]hCG was used, only a small fraction (<10%) of the added [125I]hCG was specifically bound to the cells.

Measurement of the Replenishment of the Cell Surface rLHR
Since proteolysis of intact cells under mild conditions has been shown to destroy the cell surface rLHR, the rate of replenishment of the rLHR at the cell surface can be measured by following the time course of recovery of [125I]hCG binding to intact cells at 4 C as a function of time after removal of the protease (7, 19). Cells (plated in 100-mm dishes) were placed on ice and washed twice with 4-ml portions of cold HBSS. The cell surface rLHR was then proteolyzed by incubating the cells on ice for 30–45 min in cold HBSS supplemented with 250 µg/ml of Protease type XIV (19). Protease activity was quenched by the addition of 4 ml of Waymouths MB752/1 medium supplemented with 20 mM HEPES, 15% horse serum, 1 mM phenylmethyl sulfonylfluoride, 2 mM EDTA, and 5 mM N-ethylmaleimide. The cells were then scraped from the plate and collected by centrifugation. The pellet was resuspended in the same medium, and the cells were collected by centrifugation again and resuspended in DMEM supplemented with 10% newborn calf serum, 20 mM HEPES, 50 µg/ml gentamicin, pH 7.4. The cells were then distributed into 35-mm wells and placed in a CO2 incubator at 37 C to allow the cell surface rLHR to recover. At predetermined times the cells were used for [125I]hCG binding assays as described above, and the amount of hormone bound was expressed relative to the last time point used (i.e. 24 h). The amount of [125I]hCG bound at this time point varied between 100,000 and 200,000 cpm/well for the two 293 cell lines used.

Immunoblots
Cells were lysed in a solution containing 1% Triton X-100, 200 mM NaCl, 20 mM HEPES, 1 mM EDTA, pH 7.4, during a 30-min incubation at 4 C. The lysates were clarified by centrifugation (100,000 x g for 30 min), and the amount of protein present in the supernatants was measured using the DC protein assay from Bio-Rad Laboratories, Inc. (Hercules, CA). The lysates were resolved on SDS gels and electrophoretically transferred to polyvinylidenefluoride membranes as described elsewhere (39). After blocking (39), expression of the different proteins was determined by incubating the blots overnight with the indicated concentrations of the primary antibodies listed below. Horseradish peroxidase-labeled secondary antibodies were then used during a 1-h incubation at a final dilution of 1:5,000, and the proteins were finally visualized using the Enhanced Chemiluminescence (ECL) system of detection from Amersham Pharmacia Biotech (Arlington Heights, IL).

GRK2 was detected using a mouse monoclonal antibody (3A10, at a final dilution of 1:100) (40). Visual arrestin, ß-arrestin, and arrestin-3 were detected with a mouse monoclonal antibody (F4C1; final dilution, 1:2,000) directed against an epitope common to all known arrestins (41).

Other Methods
Statistical analysis (t test with two-sided P values) was performed using Instat (GraphPad Software, Inc., San Diego, CA).

Hormones and Supplies
Purified hCG (CR-127, ~12,000 IU/mg) was obtained from the National Hormone and Pituitary Agency of the NIDDK (Baltimore, MD). This material was used for iodinations and in the experiments designed to measure down-regulation. [125I]hCG was prepared using the purified hCG as described previously (42), to give a specific radioactivity of 25,000–30,000 cpm/ng. Crude hCG (~3,000 IU/mg) was obtained from Sigma Chemical Co. (St. Louis, MO) and was used exclusively for the correction of nonspecific binding (see above). Cell culture supplies and reagents were obtained from Corning, Inc. (Corning, NY) and Life Technologies, Inc. (Gaithersburg, MD), respectively. Human kidney 293 cells and the rat Leydig tumor cell line (R2C) were purchased from the American Type Culture Collection. All other materials were obtained from commonly used suppliers.


    ACKNOWLEDGMENTS
 
We thank Ann Martin and JoEllen Fabritz for expert technical assistance. We also wish to thank Drs. Deborah L. Segaloff and John Koland for reading the manuscript and Dr. Jeff Benovic (Thomas Jefferson University, Philadelphia, PA) for generously providing us with the expression vectors for GRKs and arrestins and the antibodies to detect them. Lastly, we thank Dr. Steve Wiley (The University of Utah, Salt Lake City, UT) for providing us with the spreadsheet to calculate ke.


    FOOTNOTES
 
Address requests for reprints to: Dr. Mario Ascoli, Department of Pharmacology, 2–319A BSB, The University of Iowa College of Medicine, Iowa City, Iowa 52241-1109.

This work was supported by NIH Grants CA-40629 (to M.A.) and DK-25295, which supports The Diabetes and Endocrinology Research Center of The University of Iowa. K.N. was partially supported by a fellowship from the Lalor Foundation. M. de F.M.L. was supported by a fellowship from the Fudaçao de Amparo A Pesquisa Do Estado de Sao Paulo, Brazil (FAPESP, 96/1454–8).

1 Present address: Laboratorio de Farmacologia, Instituto Butantan, Avenue Dr. Vital, Brazil 1500, 05503–900, Sao Paulo, Brazil. Back

2 The presence of a high molecular weight form of GRK2 in transfected cells has been previously noted by us (23 ) and others (25 ). Although the identity of this band has not been established, it has been suggested that it represents an incompletely processed form of GRK2 (25 ). Back

Received for publication March 2, 1999. Revision received May 11, 1999. Accepted for publication May 14, 1999.


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