The C-Terminal Tail of the Rat Lutropin/Choriogonadotropin (CG) Receptor Independently Modulates Human (h)CG-Induced Internalization of the Cell Surface Receptor and the Lysosomal Targeting of the Internalized hCG-Receptor Complex

Mikiko Kishi 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 analysis of 21 progressive truncations of the C-terminal tail of the rat LH/CG receptor (rLHR) revealed the presence of a region delineated by residues 628–649 that, when removed, enhanced the degradation of the internalized human (h)CG. The analysis of these truncations also revealed the presence of a region delineated by residues 624–631 that, when removed, enhanced the rate of internalization of hCG. Since there is little overlap between these two regions, we conclude that the structural features of the rLHR that mediate internalization and degradation of the internalized hormone are different. Detailed analyses of cells expressing a truncation at Y637 (designated rLHR-t637) showed that the enhanced degradation of hCG observed in the these cells is due to an increase in the rate of transfer of the internalized hCG-rLHR complex from the endosomes to the lysosomes rather than to the enhanced dissociation of the hCG-rLHR complex in the lysosomes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The LH/CG receptor (LHR) is a member of the rhodopsin-like subfamily of G protein-coupled receptors (GPCRs) (1, 2). Like other members of this family, the LHR is internalized after agonist binding by a pathway that requires receptor activation (3, 4, 5, 6) and phosphorylation (7, 8). This pathway has been extensively characterized using biochemical approaches (8, 9, 10, 11, 12) as well as by electron (13) and confocal (14) microscopy. The results from all of these approaches are consistent and show that the internalization of the agonist-LHR complex proceeds via clathrin-coated pits (13) and is dependent on the participation of the nonvisual arrestins and dynamin (8, 10, 11, 12). Whereas most internalized GPCRs recycle back to the plasma membrane from the endosomal compartment, the agonist-LHR complex is instead routed to the lysosomes (9, 13, 14). Once in the lysosomes the hCG-LHR complex dissociates, and both subunits of the hormone are degraded (15). Although the lysosomal degradation of the LHR has not been directly measured, its degradation has been inferred from the lack of recycling of the internalized receptor, and the net loss of cell surface and total LHR (i.e. down-regulation) caused by agonist-induced activation (9, 13, 16, 17).

Since there is virtually no information available about the structural features of the LHR that target the internalized agonist-LHR complex to the lysosomes, the studies presented here were designed to address this question. We focused our attention on the C-terminal cytoplasmic tail of the rat (r) LHR because previous experiments from this and other laboratories have shown that mutation or removal of certain residues present in the C-terminal tail of the LHR can have pronounced effects on the targeting of the LHR precursor to the plasma membrane, and on the agonist-induced cAMP accumulation, uncoupling, internalization, and down-regulation of the cell surface LHR (11, 17, 18, 19, 20, 21).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Effect of Truncations of the Cytoplasmic Tail of the rLHR on Agonist-Induced Internalization and on the Degradation of the Internalized hCG
To study the effects of the C-terminal region of the rLHR on the trafficking of the rLHR, we prepared 21 progressive C-terminal truncations. Each deletion mutant was transiently transfected in 293 cells, and internalization was measured during a 30-min incubation at 37 C with a concentration of 125I-hCG equivalent to the Kd. The results are expressed as the internalization index, which is defined as the ratio of internalized/surface-bound ligand (22). This index can be readily used to approximate the rate of internalization because a plot of the internalization index vs. time is linear for approximately 60 min, and the slope of this plot gives the rate of internalization (10, 11, 22). The results presented (Fig. 1Go) show that two truncations (at residues 663 and 654) induced a slight (<2-fold) enhancement in internalization, but truncations of their flanking residues (i.e. residues 664, 655, or 653) did not. Truncations starting at residue 631 up to residue 624 enhance the internalization of hCG approximately 2-fold, however. Additional truncations, starting at residue 622, were poorly expressed (if at all) at the cell surface and could not be analyzed. The cell surface expression of all the C-terminal constructs shown in Fig. 1Go was similar to that of rLHR-wt, however.



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Figure 1. Effect of Truncations of the C-Terminal Tail of the rLHR on the Receptor-Mediated Internalization of 125I-hCG

293 Cells were transiently transfected with the indicated constructs, and the internalization index was measured at the end of a 30-min incubation with 125I-hCG as described in Materials and Methods. The total (i.e. surface + internalized) amount of radioactivity associated with the cells at this time point was 10,000–20,000 cpm/well. The mature rLHR-wt is 674 residues long (34 ), and the different C-terminal truncations are designated as txxx(X) where xxx denotes the position and (X) denotes the identity of the C terminus. Each value represents the average ± SEM of 3–50 independent transfections. The absence of an error bar indicates that the SEM is too small to be shown. The dashed line across the figure highlights the internalization index displayed by cells expressing rLHR-wt. The two arrows highlight the two mutants that were later chosen for further analysis. The asterisks denote statistically significant differences (P < 0.05 from rLHR-wt).

 
The internalization of hCG mediated by three of these truncated mutants (rLHR-t653, rLHR-t631 and rLHR-t628) has been previously measured (17, 18). As in previous reports, the data presented in Fig. 1Go show that rLHR-t631 and rLHR-t628 internalize hCG at a faster rate than rLHR-wt. In contrast to previous publications that reported a slower rate of internalization for rLHR-t653 (17, 18), however, Fig. 1Go now shows that this mutant internalizes hCG at about the same rate as rLHR-wt. The reason for this discrepancy is simple: we have found that the rLHR-t653 construct used in previous experiments had an additional mutation (V497A in the second extracellular loop) that went unnoticed until now, and this additional mutation is actually responsible for the decreased rate of internalization previously reported for rLHR-t653. The rLHR-t653 plasmid used for the present experiments does not have the additional mutation that went previously unnoticed. Additional experiments with rLHR-V497A are now underway to determine how a mutation of an extracellular amino acid affects the rate of internalization.

Because the internalized rLHR-receptor complex is delivered to the lysosomes in the intact form (9, 13), measurements of the degradation of the internalized hCG can be used to indirectly (but conveniently) measure the targeting of the internalized hCG-receptor complex to the lysosomes using a protocol that allows for the measurement of hormone degradation in a manner that is independent of the rate of internalization (3, 23, 24). Therefore, we transfected 293 cells with each of the truncated receptors and subjected the transiently transfected cells to a two-incubation procedure. During the first incubation the cells were allowed to internalize 125I-hCG for 2 h at 37 C. The cells were then washed with a neutral buffer to remove the free 125I-hCG and with an acidic buffer to remove the surface-bound 125I-hCG (t = 0). Due to the acid release, more than 90% of the cell-associated radioactivity is located intracellularly at t = 0 regardless of the plasmid used to transfect the cells at this point (data not shown). A second incubation at 37 C was performed to allow the cells to process the internalized agonist; at the end of this incubation (t = 2 h) the medium was assayed for degraded and undegraded hormone released, and the cells were used to determine the amount of 125I-hCG that remained cell-associated.

Since internalization was stopped from occurring at t = 0, all the degraded and undegraded 125I-hCG found at t = 2 h is derived from the 125I-hCG that was internalized during the first incubation. Under these conditions, cells expressing rLHR-wt release approximately 40% of the radioactivity that was initially internalized back to the medium as degraded hormone (Fig. 2Go). The data presented in Fig. 2Go also show that most truncations of the C-terminal tail have no effect on degradation, whereas others enhance degradation. We did not, however, find any truncations that inhibited degradation. A truncation at residue 652 enhances degradation but truncations at residue 653 or 651 do not. Truncations of residues 649–628 also serve to delineate a broader region of the C-terminal tail that enhances degradation. The maximal stimulatory effect on degradation was noted upon removal of the 628–649 region in particular when the C-terminal tail of the rLHR was truncated at position 637. In cells expressing rLHR-t637, the amount of degraded hormone reached a maximum of approximately 60%.



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Figure 2. Effect of Truncations of the C-Terminal Tail of the rLHR on the Degradation of the Internalized 125I-hCG

Cells were transiently transfected with the indicated constructs and incubated with 125I-hCG for 2 h at 37 C. At this time the cells were washed with a neutral buffer (to remove the free hormone) and briefly treated with an isotonic pH 3 buffer (to remove the surface-bound hormone). They were then incubated for another 2 h at 37 C. At the end of this incubation the medium was collected and used to measure the undegraded and degraded hormone that were released back into the medium, and the cells were used to determine the amount of radioactivity that remained cell-associated. The degraded hormone released is expressed as a percent of the sum of the radioactivity present in each of these three compartments. At the end of the second 2-h incubation the amount of undegraded hormone released back into the medium accounted for at most 5% of the radioactivity present in all compartments, and this low percentage was not affected by any of the truncations tested (data not shown). The internalized radioactivity present at the beginning of the 2-h incubation was 10–20,000 cpm/well. The mature rLHR-wt is 674 residues long (34 ), and the different C-terminal truncations are designated as txxx(X) where xxx denotes the position and (X) denotes the identity of the C terminus. Each value represents the mean ± SEM of 3–10 independent transfections. The absence of an error bar indicates that the SEM is too small to be shown. The dashed line across the figure highlights the degradation of 125I-hCG measured in cells expressing rLHR-wt. The two arrows highlight the two mutants that were later chosen for further analysis. The asterisks denote statistically significant differences (P < 0.05 from rLHR-wt).

 
Lastly, it is worth emphasizing that a comparison of the data presented in Figs. 1Go and 2Go readily demonstrates that the effects of truncations of the C-terminal tail on internalization and degradation are not co- linear, even with severe truncations of the C-terminal tail that affect both of these processes. For example, a truncation at residue 637 enhanced the degradation of the internalized hCG but clearly had no effect on internalization of the surface-bound hormone, whereas truncations at residues 624 and 626 enhanced the internalization of hCG but had no effect on the degradation of the internalized hormone.

Since the aim of this study was to delineate structural features of the rLHR that affect degradation of the internalized hormone, we performed a more complete time course of degradation in cells expressing rLHR-wt, rLHR-t655, a truncation mutant that does not affect degradation, and rLHR-t637, the mutant with the more pronounced effect on degradation. These data, presented in Fig. 3Go, clearly document the enhanced degradation of the internalized 125I-hCG mediated by rLHR-t637. In the rest of the experiments presented here, we further characterized the trafficking of the internalized hCG mediated by rLHR-t637.



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Figure 3. Time Course of Degradation of the Internalized 125I-hCG by 293 Cells Transiently Transfected with rLRH-wt, rLHR-t655, and rLHR-t637

Cells were transiently transfected with the indicated constructs and incubated with 125I-hCG for 2 h at 37 C. At this time (time = 0) the cells were washed with a neutral buffer (to remove the free hormone) and briefly treated with an isotonic pH 3 buffer (to remove the surface-bound hormone). They were then placed back in warm medium and incubated at 37 C. At the indicated times the medium was collected and used to measure the undegraded and degraded hormone that were released back into the medium, and the cells were used to determine the amount of radioactivity that remained cell-associated. The radioactivity released as degraded hormone and the radioactivity that remained cell associated are expressed as a percent of the sum of the radioactivity present in each of these three compartments. The radioactivity released as undegraded hormone accounted for at most 5% of the sum of these three compartments and is not shown. The internalized radioactivity present at t = 0 was 10,000-20,000 cpm/well. Each value represents the mean ± SEM of four independent transfections. The absence of an error bar indicates that the SEM is too small to be shown. The asterisks denote statistically significant differences (P < 0.05 from rLHR-wt).

 
Trafficking of the Internalized 125I-hCG Receptor Complex between the Endosomes and Lysosomes
The enhanced degradation of the internalized 125I-hCG mediated by rLHR-t637 could be due to the increased delivery of 125I-hCG to the lysosomes and/or to the enhanced degradation of the 125I-hCG that accumulates in the lysosomes. These possibilities were tested by measuring the trafficking of the internalized 125I-hCG-rLHR complex between the endosomes and lysosomes. To facilitate analysis of the 125I-hCG-rLHR complex (see below), the experiments described below were done using modified forms of the rLHR-wt and rLHR-t637 (designated myc-rLHR-wt and myc-rLHR-t637) containing the myc-epitope at the N terminus (25). The addition of the myc-epitope to the rLHR was previously shown to have no effect on the rate of internalization of the hCG-receptor complex (25), and additional experiments (not presented) showed that the intracellular trafficking of the complex formed by hCG and the myc-tagged rLHR-wt is similar to that of the complex formed with the nontagged rLHR-wt.

Three independent but complementary experimental approaches, subcellular fractionation (9), electron microscopy (13), and confocal microscopy (14), have been used to track the fate of the hormone and the receptor during the receptor-mediated endocytosis of hCG. The results obtained with these three approaches are internally consistent, and they all show that the internalized hCG-rLHR complex is delivered to the lysosomes in the intact form. Whereas the microscopic approaches provide formal and unambiguous data about the subcellular location of the complex, the subcellular fraction approach is much more amenable to quantitation, and this analysis was chosen for the studies described below. Cells were homogenized and the postnuclear supernatant was fractionated on a Percoll gradient (9, 26). Figure 4Go shows that biochemical markers for plasma membranes (i.e. 125I-hCG bound to the surface of 293 cells transfected with the rLHR) and endosomes (i.e. internalized 125I-transferrin) migrate toward the top of the gradient, whereas a biochemical marker for lysosomes (ß-hexosaminidase activity) migrates to the bottom of the gradient. Thus the gradient chosen can separate endosomes from lysosomes but cannot separate plasma membranes from endosomes. The lack of separation of plasma membranes and endosomes is not a problem for our experiments because the surface-bound 125I-hCG can be released from the cells by a brief exposure to pH 3 (see Materials and Methods) at the beginning of the experiment (see below). Thus, any 125I-hCG migrating in the position of plasma membranes/endosomes would have to be located in the endosomal compartment rather than the plasma membrane.



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Figure 4. Distribution of Biochemical Markers on Percoll Gradients

Postnuclear supernatants of 293 cells were prepared and fractionated on Percoll gradients as described in Materials and Methods. The top panel shows the distribution of the 125I-hCG bound to the cell surface of 293 cells transiently transfected with rLHR-wt. The middle panel shows the distribution of 125I-transferrin internalized by the endogenous transferrin receptor of 293 cells. The bottom panel shows the distribution of endogenous ß-hexosaminadase activity present in 293 cells. The results of a representative experiment are shown.

 
To measure the transit of the internalized 125I-hCG from endosomes to lysosomes, we first incubated 293 cells expressing myc-rLHR-wt or myc-rLHR-t637 with leupeptin and 125I-hCG for 15 min at 37 C. The presence of leupeptin, a lysosomal enzyme inhibitor, does not affect the rate of internalization of the hCG-receptor complex or the trafficking of the internalized complex to the lysosomes, but it prevents lysosomal degradation and allows the undegraded hormone to accumulate in the lysosomes (9, 15). At this point (t = 0) the cells were washed to remove the surface-bound hormone, treated with a pH 3 buffer to release the surface-bound hormone, and then reincubated at 37 C (in the presence of leupeptin) to allow processing of the internalized hormone while preventing degradation. At t = 0 and at two subsequent time points thereafter, the cells were homogenized and the postnuclear supernatants were analyzed on Percoll gradients to track the movement of the internalized 125I-hCG between the endosomes and lysosomes. The representative experiment shown in Fig. 5Go and the summary shown in Fig. 6Go show that at t = 0 most of the radioactivity derived from internalized 125I-hCG is present in endosomes, and that the radioactivity present in this compartment decreases with time, at the expense of an increase in the radioactivity present in the lysosomes. These results also show that the transit of the internalized 125I-hCG from endosomes to lysosomes is faster in cells expressing myc-rLHR-t637 than in cells expressing myc-rLHR-wt. Thus, whereas at t = 0 cells expressing myc-rLHR-wt or myc-rLHR-t637 displayed similar amounts of 125I-hCG in the endosomes and lysosomes, the transfer of internalized 125I-hCG from endosomes to lysosomes at the 90-min time point in cells expressing myc-rLHR-t637 was equivalent to that detected in cells expressing rLHR-wt at the 270-min time point (Fig. 6Go).



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Figure 5. Analysis of the Distribution of the Internalized 125I-hCG Present in 293 Cells Expressing myc-rLHR-wt or myc-rLHR-t637

Cells were transiently transfected with the indicated constructs and incubated with 125I-hCG and leupeptin for 20 min at 37 C. At this time (time = 0) the cells were washed with a neutral buffer (to remove the free hormone) and briefly treated with an isotonic pH 3 buffer (to remove the surface-bound hormone). They were then placed back in medium containing leupeptin and incubated at 37 C. At the indicated times the cells were collected, homogenized, and analyzed on Percoll gradients as described in Materials and Methods. The results of a representative experiment are shown in which the radioactivity present in each fraction is expressed as % of the total radioactivity present in the gradient. The total amount of radioactivity present in each gradient was 10,000–20,000 cpm.

 


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Figure 6. Quantitative Analysis of the Transfer of the Internalized 125I-hCG between Endosomes and Lysosomes in 293 Cells Expressing myc-rLHR-wt or myc-rLHR-t637

Cells were transiently transfected with the indicated constructs and incubated with 125I-hCG and leupeptin for 20 min at 37 C. At this time (time = 0) the cells were washed with a neutral buffer (to remove the free hormone) and briefly treated with an isotonic pH 3 buffer (to remove the surface-bound hormone). They were then placed back in medium containing leupeptin and incubated at 37 C. At the indicated times the cells were collected, homogenized, and analyzed on Percoll gradients as described in Materials and Methods and shown in Fig. 5Go. The radioactivity associated with endosomes (fractions 3–15) and lysosomes (fractions 28–36) was calculated as a percent of the total radioactivity present in the gradient and plotted as a function of the length of time of the second incubation. Each value shows the mean ± SEM of six independent transfections. The absence of an error bar indicates that the SEM is too small to be shown. The asterisks denote statistically significant differences (P < 0.05 from rLHR-wt).

 
The nature of the 125I-hCG radioactivity associated with the endosomes and lysosomes was next determined by immunoprecipitation with a monoclonal antibody (9E10) to the myc epitope. Since the rLHR used for these experiments is tagged with the myc epitope, any 125I-hCG radioactivity immunoprecipitated by the 9E10 antibody could be safely considered to be receptor-bound rather than free. As shown in Fig. 7Go, approximately 75% and 50% of the 125I-hCG present in the endosomes and lysosomes, respectively, of 293 cells transfected with the myc-rLHR-wt or myc-rLHR-t637 can be immunoprecipitated with an antibody to the myc epitope. We conclude that most of the 125I-hCG present in the endosomes and lysosomes of cells expressing myc-rLHR-wt or myc-rLHR-t637 is receptor-bound. This conclusion is in agreement with previous biochemical and morphological data obtained with the endogenous or transfected LHR (9, 13, 14).



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Figure 7. Distribution of Internalized, Receptor-Bound 125I-hCG in Endosomes and Lysosomes of 293 Cells Expressing myc-rLHR-wt or myc-rLHR-t637

Cells were transiently transfected with the indicated constructs and incubated with 125I-hCG and leupeptin for 20 min at 37 C. At this time (time = 0) the cells were washed with a neutral buffer (to remove the free hormone) and briefly treated with an isotonic pH 3 buffer (to remove the surface-bound hormone). They were then placed back in medium with leupeptin and incubated at 37 C. At the indicated times the cells were collected, homogenized, and analyzed on Percoll gradients as described in Materials and Methods and shown in Fig. 5Go. The fractions containing the endosomes (fractions 3–15) and lysosomes (fractions 28–36) were pooled, solubilized with detergent, and immunoprecipitated with an antibody (9E10) to the myc-epitope. The immunoprecipitated radioactivity was considered to be receptor bound and was expressed as % of the total radioactivity present in each fraction. Each of the samples used for immunoprecipitation contained about 1,000 cpm. Each value shows the mean ± SEM of three independent transfections. The absence of an error bar indicates that the SEM is too small to be shown. The receptor-bound radioactivity associated with lysosomes at t = 0 was not measured because of the low amount of radioactivity associated with this fraction at this time point (cf. Figs. 5Go and 6Go). Control samples in which equivalent amounts of free 125I-hCG were immunoprecipitated as described above revealed that only about 10% of the free 125I-hCG was immunoprecipitated. In contrast, when cells transiently transfected with myc-rLHR-wt were incubated with 125I-hCG at 4 C followed by detergent solubilization, about 70% of the solubilized radioactivity could be immunoprecipitated using the method described above.

 
Since there is no difference in the proportion of 125I-hCG that is associated with the endosomes or lysosomes of cells expressing myc-rLHR-wt or myc-rLHR-t637, yet the lysosomal accumulation of the 125I-hCG-receptor complex and the degradation of 125I-hCG is faster in the latter, we conclude that the increased degradation of the internalized 125I-hCG mediated by rLHR-t637 is due to an increase in the rate of transfer of the internalized 125I-hCG-rLHR complex from endosomes to lysosomes.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Whereas much has been recently learned about the molecular and cellular basis of the agonist-induced internalization of GPCRs in general (reviewed in Refs. 27, 28, 29) and the LHR in particular (5, 8, 10, 11, 12), little is known about the structural features of these receptors that modulate other aspects of their cellular trafficking. Since the internalized LHR is one of the few GPCRs that is targeted to the lysosomal degradation pathway rather than to the recycling pathway (9, 13), it provides a good opportunity to define the structural features of GPCRs that are involved in lysosomal targeting. With this in mind we analyzed a series of truncations of the C-terminal tail of the rLHR for agonist-induced internalization, for the transit of the internalized hCG- receptor complex to lysosomes, and for the lysosomal degradation of hCG.

C-Terminal truncations at C663 and A654 enhanced agonist-induced internalization, but truncations at Q664 or S652 or additional truncations between these two residues did not affect internalization (Fig. 1Go). Thus, the enhanced internalization mediated by truncations at C663 and A654 appear to be due to the creation of new C-terminal sequences rather than to the removal of a given region. The nature of the C terminus itself does not appear to be important at least for the alanine residue since two other truncations (at residues 636 and 626) also create C-terminal alanines, but only one of them, the truncation at residue 626, enhances internalization (Fig. 1Go). Progressive truncations starting at Arg631 and continuing to Arg624 enhanced agonist-induced internalization about 2-fold, and they serve to define a region of approximately 8 residues that enhances internalization when removed (shown by the top horizontal bar in Fig. 8Go).



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Figure 8. Amino Acid Sequence of the C-Terminal Tail of the rLHR

The amino acid sequence of the relevant portion of the C-terminal tail (starting at residue 621) of the rLHR (accession number P16235, SWISS-PROT databank) is shown. The asterisks at the top and the bottom of the amino acid sequence show the truncations that enhanced the internalization of hCG and the degradation of the internalized hCG, respectively. Likewise, the top and the bottom dark horizontal bars denote the regions of the rLHR that, when removed, enhance the internalization of hCG and the degradation of the internalized hCG, respectively (see Figs. 1Go and 2Go). The arrow indicates the C terminus of rLHR-t637, the truncated rLHR that was extensively characterized in the experiments presented herein. The four serine residues that become phosphorylated upon agonist stimulation (7 8 30 41 ) are enclosed in rectangles.

 
Two of the truncations examined here (rLHR-t631 and rLHR-t628) have been previously shown to enhance the internalization of hCG (17, 18). The internalization indexes reported here (Fig. 1Go) can be readily translated into t1/2’s of about 100 min for rLHR-wt and about 50 min for rLHR-t631 and rLHR-t628. These values are comparable to those previously measured in stably transfected cells lines (17) and are consistent with other reports from this laboratory showing that the rate of internalization of hCG mediated by the rLHR-wt or mutants thereof is the same in stably and transiently transfected 293 cells (5, 10, 11, 12, 17).

Previous results from this laboratory have shown that the agonist-induced phosphorylation of the rLHR maps to S635, S639, S649, and S652 (enclosed in rectangles in Fig. 8Go) in the C-terminal of the rLHR (7, 8, 30). We have also shown that the agonist-induced internalization of the rLHR is impaired by the simultaneous mutation of these four residues (7) or by the individual mutation of S635, S639, or S649, but not by the individual mutation of S652 (8). Thus, while truncations of the C-terminal tail up to S649 were not expected to remove phosphorylation sites that influence internalization, all truncations upstream of S649 would remove at least one of the phosphate acceptors that influences internalization and would thus be expected to impair internalization. As shown here, however, truncations that remove only S649 (i.e. rLHR-t643) S649 and S639 (i.e. rLHR-t637 and rLHR-t636), or S649, S639, and S635 (i.e. rLHR-t632) have no effect on internalization (Fig. 1Go). Moreover, as already noted above, more severe truncation enhances internalization (Fig. 1Go). These findings suggest that, in addition to the phosphorylation sites, the C-terminal tail of the rLHR may contain other structural features that (directly or indirectly) influence endocytosis independently of receptor phosphorylation. Other experiments have already shown that the internalization of phosphorylation-deficient mutants of the rLHR can be rescued by cotransfection of cells with one of the nonvisual arrestins (8). Thus, while phosphorylation may be necessary for the agonist-induced internalization of the rLHR, it is certainly not sufficient. In the absence of rLHR phosphorylation agonist- induced internalization can occur normally (or even at enhanced rates) by overexpression of one of the nonvisual arrestins (8) or by truncation of the C-terminal tail as shown herein.

The effect of C-terminal truncations on the degradation of the internalized hCG revealed that a truncation at Ser652 enhanced degradation, but truncations at flanking residues, A653 and P651, did not (Figs. 2Go and 8Go). Thus, the enhanced degradation mediated by rLHR-t652 appears to be due to the creation of a new C-terminal sequence rather than to the removal of a given region. Although another truncation that enhances degradation (i.e. rLHR-t649) also terminates in a serine residue, there are additional truncations that enhance degradation but do not terminate in a serine residue (Figs. 2Go and 8Go). Thus, the nature of the C terminus itself does not appear to be an important determinant of degradation. Progressive truncations starting at Ser649 and continuing to Leu628 enhanced the degradation of the internalized hormone, and they serve to define a region of approximately 22 residues that enhances degradation when removed (Figs. 2Go and 8Go). The most dramatic effects on degradation were observed using truncations that did not affect internalization (such as rLHR-t643 and rLHR-t637), whereas some truncations that enhanced internalization had no effect on degradation (such as rLHR-t624 and rLHR-t626). Thus, the regions of the C-terminal tail that modulate these two aspects of the trafficking of the rLHR overlap to some extent, but are clearly not co-linear (Fig. 8Go).

To our knowledge this is the first report showing that the C-terminal tail of GPCRs influences the rate of transit of internalized receptors among different compartments of the endocytic pathway. We have shown here that the removal of residues 628–649 (Figs. 2Go and 8Go) enhances the degradation of the internalized hCG by promoting the transfer of the hormone receptor complex from the endosomes to the lysosomes. Other investigators have already shown that the C-terminal tail of some GPCRs such as the thrombin, substance P, ß2-adrenergic, arginine-vasopressin type 2, and TSH and LH receptors (14, 31, 32, 33) have a great influence on the sorting of these internalized GPCRs to the degradation or recycling pathways. Thus, whereas the internalized substance P receptor recycles from endosomes to the plasma membrane, the internalized thrombin receptor is targeted to the lysosomes. However, the fate of chimeras of these two receptors is dictated by the origin of the C-terminal tail (31). Likewise, whereas the internalized TSH receptor recycles from endosomes to the plasma membrane, the internalized LH receptor is targeted to the lysosomes. In this case too, the fate of chimeras of these two receptors is dictated by the origin of the C-terminal tail (14). The 628–649 region of the rLHR identified here as influencing degradation of the internalized hormone is enriched in basic and polar amino acids, but it does not contain known protein-targeting motifs. It is also not known if the 628–649 region directly affects trafficking of the internalized receptor or if it does so indirectly by interacting with another intracellular region(s) of the rLHR.

Previous results from this (9) and other laboratories (13, 14) have shown that once internalized, the hCG-LHR complex traverses the endosomal compartment in an intact form and is delivered to the lysosomes without dissociation. Once in the lysosomes, the complex dissociates and the free hormone (and presumably the receptor) are degraded (9). While such a pathway is best documented using microscopy (13, 14), these methods are not readily amenable to quantitation. We carefully quantitated this process in MA-10 cells a number of years ago by assessing the state of the internalized hormone (i.e. free vs. receptor-bound and intact vs. degraded) in endosomes and lysosomes separated by Percoll gradient centrifugation (9). We have now used similar protocols to determine the fate of the internalized hCG in transfected 293 cells and to begin to understand the reasons why some C-terminal truncations of the rLHR result in enhanced degradation of the internalized hCG (Figs. 2Go and 3Go). The results obtained here with 293 cells expressing the rLHR-wt document the trafficking of the hCG-LHR complex from endosomes to lysosomes ( Figs. 4–6GoGoGo) and recapitulate the results previously reported by us and others in target or transfected cells (9, 13, 14). We have previously shown that approximately 99% and 60% of the 125I-hCG present in the endosomes and lysosomes, respectively, of MA-10 cells treated with leupeptin is receptor bound (9). Here we show that about 75% and 50% of the 125I-hCG present in the endosomes and lysosomes, respectively, of transfected 293 cells treated with leupeptin is receptor bound (Fig. 6Go). Whereas the quantitative results are somewhat different in these two cell lines, the data clearly show that a substantial portion of the hCG present in these two fractions is receptor bound. These small differences may also be due to the methodology used. In previous experiments we used polyethylene glycol to precipitate the receptor-bound 125I-hCG whereas in the current experiments we used immunoprecipitation with an antibody to the epitope-tagged rLHR to quantitate the receptor-bound 125I-hCG. Our quantitative biochemical studies, together with the microscopic approaches used by Milgrom and co-workers (13, 14), clearly show that most of the internalized 125I-hCG-LHR complex is routed to the lysosomes rather than recycled back to the plasma membrane in MA-10 cells, porcine Leydig cells, transfected 293 cells, or transfected mouse L cells. More importantly, the biochemical approaches described here allowed us to conclude that the enhanced degradation of 125I-hCG detected in 293 cells expressing rLHR-t637 (Fig. 3Go) is due to an increase in the rate of transfer of the internalized 125I-hCG-LHR complex from the endosomes to the lysosomes ( Figs. 4–6GoGoGo) rather than to the increased dissociation of the internalized 125I-hCG-LHR complex.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids and Cells
A full-length cDNA encoding the rLHR in pcDNAI/Neo has been described (34). Truncations of the C-terminal tail of the rLHR were constructed using PCR strategies to introduce a stop codon in the position immediately following the new desired C terminus. The identity of all mutants was verified by automated DNA sequencing (performed by the DNA core of The Diabetes and Endocrinology Research Center of the University of Iowa). All transient transfections in 293 cells were done using the rLHR-wt and mutants thereof subcloned in the pcDNAI/Neo. Some experiments ( Figs. 5–7GoGoGo) were done using rLHR-wt and rLHR-t637 that were tagged with the myc-epitope. These constructs (designated myc-rLHR-wt and myc-rLHR-t637) have the myc-epitope at the N terminus, and the preparation and characterization of the parent construct have been described (25).

Human embryonic kidney (293) cells (CRL 1573) were obtained from the American Type Culture Collection (Manassas, VA) and maintained in DMEM containing 10 mM HEPES, 10% newborn calf serum, and 50 µg/ml gentamicin, pH 7.4. Transient transfections were done using the calcium phosphate method of Chen and Okayama (35). Cells were plated in 35-mm wells and transfected with not more than 2 µg of plasmid DNA when 70–80% confluent. After an overnight incubation, the cells were washed and used 24 h later. Plasmids were prepared using QIAGEN kits.

Internalization Assays
The endocytosis of 125I-hCG was measured using intact cells incubated with 40 ng/ml 125I-hCG at 37 C for 30 min as described previously (10, 11, 12, 22, 23, 24). All internalization data are expressed as an internalization index, which is defined as the ratio of the internalized to surface-bound hormone (22). This index is used because under the assay conditions used here, plots of the internalization index against time are linear for up to about 60 min and can be used to calculate a rate constant and a half-life for internalization (10, 11, 22).

Degradation of the Internalized Hormone
This was measured using previously established protocols (3, 11, 23, 24). Briefly, transfected cells were washed and then allowed to bind and internalize 40 ng/ml 125I-hCG for 2 h at 37 C. At this point the cells were placed on ice, washed to remove the free hormone, and treated with an acidic buffer to remove the hormone bound to the cell surface. The cells were then placed back in warm assay medium, and a second incubation (at 37 C) was performed to allow the cells to process and degrade the hormone that had been internalized during the first incubation. At the end of the second incubation (which lasted from 1 to 4 h as described in the figure legends) the dishes were placed on ice, the medium was saved, and the undegraded and degraded hormone were measured by precipitation with trichloracetic acid (15). The cells were solubilized with NaOH to determine the amount of hormone that remained cell-associated.

Fate of the Internalized Hormone
Transfected cells were washed and then allowed to bind and internalize 125I-hCG for 20 min at 37 C in Waymouth’s MB752/1 containing 1 mg/ml BSA and 20 mM HEPES, pH 7.4 (assay medium) and 200 µM leupeptin. At this point (t = 0 in the figures) the cells were placed on ice and washed two to three times with 2-ml portions of cold HBSS containing 1 mg/ml BSA (wash medium). 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 (15). This buffer was removed and the cells were washed once more with the same acid buffer and then once with cold assay medium. The cells were then placed back in 1 ml of warm assay medium containing 200 µM leupeptin, and a second incubation (lasting 90 or 270 min) at 37 C was conducted to allow the cells to process the hormone that had been internalized during the first incubation. In the presence of leupeptin, however, the internalized hormone is not degraded and it accumulates in the lysosomes (9, 15). At the indicated times the dishes were placed on ice, the cells were washed twice with cold homogenization buffer (0.25 M sucrose, 10 mM HEPES, 1 mM EDTA, pH 7.4), scraped into a small volume of the same buffer, and collected by centrifugation at 4 C. The cells were then resuspended in cold homogenization buffer, and they were lysed by forcing them through a 21-gauge needle 10 times. Postnuclear supernatants were prepared by centrifuging the homogenates at 800 x g for 10 min at 4 C. The supernatants were saved, and the pellets were rehomogenized and centrifuged again. The two supernatants were combined and a 2-ml aliquot was thoroughly mixed with 8 ml of a Percoll solution (with a density of 1.046 g/ml) prepared in homogenization buffer (9, 26). These mixtures were then centrifuged at 33,000 x g for 20 min (70.1 Ti rotor in a L7–80 ultracentrifuge, Beckman Coulter, Inc., Palo Alto, CA) at 4 C. The contents of the gradients were then collected from the top (250 µl/fraction), and aliquots of each fraction were assayed for radioactivity with the aid of a {gamma}-counter.

These Percoll gradients were calibrated by analyzing the migration of biochemical markers for plasma membranes, endosomes, and lysosomes. Since a 4 C incubation of cells with 125I-hCG prevents internalization (9, 13, 15), we used the 125I-hCG radioactivity associated with 293 cells transiently transfected with rLHR-wt and allowed to bind 125I-hCG at 4 C as a marker for plasma membranes. Since internalized transferrin localizes only to endosomes (36, 37, 38), we used the 125I-transferrin radioactivity internalized by 293 cells during a 20-min incubation at 37 C as a marker for endosomes. Lastly, ß-hexosaminidase activity (measured as described in Ref. 39) was used as a marker for lysosomes.

Analysis of Receptor-Bound 125I-hCG
After separation by Percoll gradient centrifugation (cf. Figs. 4Go and 5Go) the fractions containing the resolved endosomes and lysosomes (fractions 3–15 and 28–36, respectively) from cells expressing rLHR-wt or rLHR-t637 were pooled. Each pool received 200 µl of protease inhibitors (Complete protease inhibitor cocktail from Roche Molecular Biochemicals, Indianapolis, IN) and 500 µl of 10% NP-40. The volume was adjusted to 5 ml with homogenization buffer, and the samples were incubated for 45 min at 4 C. The solubilized samples were then centrifuged at 100,000 x g for 60 min at 4 C to remove the Percoll and insoluble material.

Duplicate aliquots of the supernatants were used to determine the total amount of 125I-hCG present, and duplicate aliquots (500 µl) were mixed with an antibody to the myc-epitope (9E10) that was previously bound to protein G-Sepharose (see below). This mixture was rotated overnight at 4 C, and the beads were recovered by centrifugation (4 C) and washed two times with 0.1% NP-40, 20 mM HEPES, 0.15 M NaCl, pH 7.4). The beads were then counted in a {gamma} counter to determine the amount of 125I-hCG that was bound to the receptor. Control samples in which equivalent amounts of free 125I-hCG were immunoprecipitated as described above revealed that only about 10% of the free 125I-hCG was immunoprecipitated. In contrast, when cells transiently transfected with myc-rLHR-wt were incubated with 125I-hCG at 4 C followed by detergent solubilization, approximately 70% of the solubilized radioactivity could be immunoprecipitated using the method described above.

Prebinding of the 9E10 antibody to the protein G-Sepharose was accomplished by mixing 50 µl of a 20-fold dilution of 9E10 antibody (i.e. a concentrated cell culture supernatant of the 9E10 hybridoma cell line) with 25 µl of protein G-agarose (50% slurry in 0.1% NP-40, 20 mM HEPES, 0.15 M NaCl, and 1 mM EDTA, pH 7.4) for at least 3 h at 4 C. The bound antibody was recovered by centrifugation at 4 C, and the agarose beads were washed twice with the same solution before mixing them with the lysate (see above).

Hormones and Supplies
Purified hCG (CR-127, ~13,000 U/mg) was kindly provided by the National Hormone and Pituitary Agency of the National Institute of Diabetes and Digestive and Kidney Diseases. Partially purified hCG (~3,000 U/mg) was purchased from Sigma (St. Louis, MO), and it was used only to correct for nonspecific binding. 125I-hCG was prepared as previously described (40). Percoll and 125I-transferrin were purchased from Amersham Pharmacia Biotech. Cell culture supplies and reagents were obtained from Corning, Inc. (Corning, NY) and Life Technologies, Inc., respectively. All other chemicals were obtained from commonly used suppliers.


    ACKNOWLEDGMENTS
 
We thank Dr. Deborah L. Segaloff for critically reading this manuscript and Professor Masatomo Mori (First Department of Internal Medicine, Gunma University) for his support.


    FOOTNOTES
 
Address requests for reprints to: Dr. Mario Ascoli, Department of Pharmacology, 2–319A BSB 51 Newton Road, The University of Iowa, Iowa City, Iowa 52242-1109.

This work was supported by NIH Grant CA-40629 to M.A. The services and facilities provided by the Diabetes and Endocrinology Research Center of the University of Iowa (supported by NIH Grant DK-25295) are also gratefully acknowledged.

Received for publication November 30, 1999. Revision received February 1, 2000. Accepted for publication March 2, 2000.


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