Identification of Two Distinct Structural Motifs That, When Added to the C-Terminal Tail of the Rat LH Receptor, Redirect the Internalized Hormone-Receptor Complex from a Degradation to a Recycling Pathway

Mikiko Kishi, Xuebo Liu, Takashi Hirakawa, David Reczek, Anthony Bretscher and Mario Ascoli

Department of Pharmacology (M.K., X.L., T.H., M.A.), The University of Iowa College of Medicine, Iowa City, Iowa 52242-1109; and Department of Molecular Biology and Genetics (D.R., A.B.), Cornell University, Ithaca, New York 14853-2703

Address all correspondence and requests for reprints to: Dr. Mario Ascoli, Department of Pharmacology, 2–319B BSB, 51 Newton Road, The University of Iowa, Iowa City, IA 52242-1109. E-mail: mario-ascoli{at}uiowa.edu


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We show that most of the internalized rat LH receptor is routed to a lysosomal degradation pathway whereas a substantial portion of the human LH receptor is routed to a recycling pathway. Chimeras of these two receptors identified a linear amino acid sequence (GTALL) present near the C terminus of the human LH receptor that, when grafted onto the rat LH receptor, redirects most of the rat LH receptor to a recycling pathway. Removal of the GTALL sequence from the human LH receptor failed to affect its routing, however.

The GTALL sequence shows homology with the C-terminal tetrapeptide (DSLL) of the ß2-adrenergic receptor, a motif that has been reported to mediate the recycling of the internalized ß2-adrenergic receptor by binding to ezrin-radixin-moesin-binding phosphoprotein-50. Addition of the DSLL tetrapeptide to the C terminus of the rat LH receptor also redirects most of the internalized rat LH receptor to a recycling pathway but, like the recycling of the human LH receptor, this rerouting is not mediated by ezrin-radixin-moesin-binding phosphoprotein-50.

We conclude that most of the internalized rat LH receptor is degraded because its C-terminal tail lacks motifs that promote recycling and that two distinct, but homologous, motifs (DSLL at the C terminus or GTALL near the C terminus) can reroute the internalized rat LH receptor to a recycling pathway that is independent of ezrin-radixin-moesin-binding phosphoprotein-50.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
INTERNALIZATION OF G protein-coupled receptors (GPCRS) is a ubiquitous response that follows agonist-induced activation (1, 2, 3). Most GPCRs are internalized via clathrin-coated pits by a pathway that requires the formation of a complex between the agonist-activated and phosphorylated GPCR and a family of proteins known as the nonvisual or ßarrestins (1, 2, 3). Once internalized, most GPCRs are recycled back to the plasma membrane (1, 2, 3), but a few are routed to the lysosomes and targeted for degradation (4, 5, 6, 7, 8).

The ß2-adrenergic receptor (ß2AR) is a prototypical GPCR that is sorted to the recycling pathway (9, 10, 11). The C-terminal tetrapeptide of the wild-type ß2AR (DSLL) has been shown (12, 13) to mediate the binding of this receptor to the PDZ domains of ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) (see Refs. 14 and 15), also known as Na+/H+-exchange regulatory factor (NHERF) (see Ref. 16). EBP50 is an abundant phosphoprotein composed of two N-terminal PDZ domains and a C-terminal domain that binds ezrin, a component of the cortical cytoskeleton (17). Cao and co-workers (11) recently showed that mutation of the DSLL motif of the ß2-AR or overexpression of a C-terminally truncated form of EBP50 that cannot bind ezrin results in the rerouting of the internalized ß2-AR from the recycling pathway to a lysosomal degradation pathway. Although these experiments identified the molecular basis of the sorting of the internalized ß2-AR, it is not yet known whether the recycling or lysosomal targeting of other GPCRs is also mediated by their interaction with, or lack of interaction with, EBP50, respectively. This is an important issue because most internalized GPCRs are routed to a recycling pathway (1, 2, 3), but there is only one other GPCR (the P2Y1 purinergic receptor) that has a C-terminal sequence (D-S/T-x-L) that promotes EBP50 binding (13).

The rat (r), mouse (m), and porcine (p) LH receptors (LHRs) are among the few GPCRs that recycle poorly after internalization. These receptors are routed mostly to a lysosomal degradation pathway that has been particularly well characterized using biochemical (4, 18, 19, 20) and microscopic approaches (5, 7). Thus, it is now known that the complex formed by the r, m, or pLHR and one of its agonists [human CG (hCG)] is internalized via clathrin-coated pits (5) by a pathway that requires the involvement of a nonvisual arrestin and dynamin (18, 19). The r, m, or pLHR-hCG complex is resistant to dissociation by the mild acidic pH that prevails in the endosomes (4), and a substantial proportion of the internalized complex is routed to the lysosomes where it dissociates before degradation (4, 5, 7, 20). By promoting the accumulation of the hCG-LHR complex in a compartment where it can be degraded, this pathway is ultimately responsible not only for the degradation of hCG (21) but also for the net loss of cell surface LHR that ensues after exposure of rodent or porcine target cells or cells expressing the recombinant rodent or porcine LHR to agonists (22, 23, 24). Surprisingly, however, the fate of the highly related human (h) LHR is different from that of the rLHR. As shown herein, a substantial portion of the hCG-hLHR complex is routed to a recycling pathway rather than to a degradation pathway.

Since most of the internalized rLHR is routed to a lysosomal degradation pathway, this GPCR provides an ideal model system to test the hypothesis that the DSLL-mediated interaction of GPCRs with EBP50 is sufficient to target internalized GPCRs to the recycling pathway. The experiments presented herein were initially designed as a classical gain-of-function approach to test this hypothesis. Since the data obtained supported a role for the DSLL motif, but excluded the involvement of EPB50, we performed additional experiments that ultimately resulted in the identification of a structural motif (GTALL) present near the C terminus of the hLHR that, when grafted onto the C-terminal tail of the rLHR, also redirects the internalized rLHR to a recycling pathway. Like the DSLL-induced recycling, the GTALL-induced recycling of the rLHR was also found to be independent of EBP50.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Addition of a DSLL Motif to the C-Terminal Tail of the rLHR Promotes Binding to EBP50 and Reroutes the Internalized Agonist-rLHR Complex from a Lysosomal Degradation to a Recycling Pathway
The C-terminal tetrapeptide of the rLHR (ALTH, cf. Fig. 1Go) does not conform to the consensus sequence (D-S/T-x-L) necessary for binding EBP50. In view of the recent results of Cao and co-workers (11), we initially postulated that the well documented routing of the internalized agonist-rLHR complex to the lysosomes (4, 5, 7) could be due to the lack of an interaction of the rLHR with EBP50. This hypothesis was tested by using a gain-of-function approach involving the analysis of two mutants of the rLHR in which their C-terminal tails were extended by similar sequences (DSLL or DSLA) that were predicted to support or not support, respectively, the binding of the rLHR to EBP50. Initial experiments (not presented) showed that extension of the C-terminal tail of the rLHR by addition of the DSLL or DSLA sequences had no effect on the expression of the receptor. Human kidney 293 cells transiently transfected with epitope (N-terminal myc)-tagged versions of wild-type rLHR (rLHR-wt), rLHR-DSLL, or rLHR-DSLA bound equivalent amounts of 125I-hCG (~100 fmol/10 6 cells) and internalized the bound 125I-hCG at similar rates (t 1/2 of 100–120 min).



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Figure 1. Amino Acid Sequence Alignment of the C-Terminal Tails of the Rat, Mouse, Porcine, and hLHR

The amino acid sequences of the different species of LHR were taken from Refs. 33 39 40 , and 46 . The partial sequences shown start at the an NPXXY motif present in transmembrane helix 7 (TM-7) that is highly conserved among GPCRs of the rhodopsin/ß 2-AR subfamily of GPCRs (47 ). Dots indicate gaps introduced for optimal alignment. The partial box at the left end of the sequences shows the cytoplasmic end of TM-7. The two palmitoylated cysteines that are believed to form an additional point of membrane attachment are also enclosed in a box. The residues shown in bold are divergent between the groups of receptors that display little recycling (i.e. the p, m, and rLHR) and the single receptor (hLHR) that recycles more. The serine residues marked with the asterisk below the rLHR sequence or hLHR sequences become phosphorylated upon hCG stimulation (41 48 49 ). The boxes outlined with double lines highlight the rLHR residues that were substituted with the corresponding hLHR residues in the rLHR-mt1, -mt2, and -mt3 mutants shown in Table 1Go. For example, in rLHR-mt2 the QPIPP sequence of the rLHR was substituted for the GTALL sequence of the hLHR. The amino acids shaded in gray were removed by truncation of the rLHR at residue 664 (i.e. rLHR-t664 in Table 1Go) and the hLHR at residue 686 (i.e. hLHR-t686 in Table 1Go).

 
Figure 2AGo shows that the rLHR-wt or rLHR-DSLA mutants do not bind to EBP50, but the rLHR-DSLL mutant does. The three bands of rLHR detected in the pull-down assays (~68 kDa, ~85 kDa, and ~165 kDa) correspond to an immature intracellular precursor, the mature cell surface receptor, and an aggregate/oligomer of the immature intracellular precursor, respectively (25, 26, 27). The data presented in Fig. 2BGo document that the interaction of rLHR-DSLL with EBP50 occurs through the PDZ domain(s) rather than the ezrin-radixin-moesin (ERM) binding domain of EBP50. Thus, a glutathione-S-transferase (GST) fusion protein of the ERM binding domain of EBP50 (i.e. residues 242–358, see Ref. 28) is not capable of forming a complex with the DSLL mutant of the rLHR.



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Figure 2. Association of rLHR-DSLL with EBP50

A, Detergent lysates of 293 cells transiently transfected with rLHR-wt, rLHR-DSLL, or rLHR-DSLA were prepared and partially purified on a lectin column as described in Materials and Methods. Aliquots of the partially purified lysates containing the same amount of solubilized protein were allowed to bind to GST or GST-EBP50 that had been previously bound to glutathione agarose. The bound proteins were eluted, and visualized on Western blots developed using a monoclonal antibody to the myc epitope (9E10) and the ECL visualization system. B, Detergent lysates of 293 cells transiently transfected with rLHR-DSLL were prepared and partially purified on a lectin column as described in Materials and Methods. Aliquots of the partially purified lysates containing the same amount of solubilized protein were allowed to bind to GST, GST-EBP50, or GST-EBP50(242–358) that had been previously bound to glutathione agarose. After the bound proteins were eluted, they were visualized on Western blots developed using a monoclonal antibody to the myc epitope (9E10) and the ECL visualization system. The results of a representative experiment are shown in each panel.

 
Since most of the internalized 125I-hCG remains bound to the rLHR in endosomes and lysosomes (4, 5, 7, 20), measurements of the fate of the internalized 125I-hCG can be used to indirectly (but conveniently) assess the targeting of the internalized hCG-receptor complex. Thus, to study the fate of the internalized receptor cells expressing rLHR-wt, rLHR-DSLL or rLHR-DSLA were allowed to bind and internalize 125I-hCG for 2 h at 37 C. After removal of the free and bound 125I-hCG, the fate of the intracellular 125I-hCG was followed by reincubating the cells at 37 C in medium without any added hormone. The data summarized in Fig. 3Go show that cells expressing rLHR-wt quickly recycle approximately 15% of the internalized 125I-hCG to the cell surface and eventually degrade about 70% of the internalized hormone by the end of a 4-h incubation. In contrast, cells expressing rLHR-DSLL quickly recycle about 45% of the internalized 125I-hCG to the cell surface and degrade approximately 40% of the internalized 125I-hCG during a 4-h incubation. The behavior of rLHR-DSLA was intermediate between that of rLHR-wt and rLHR-DSLL. These cells recycled about 25% and degraded approximately 55% of the internalized hormone.



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Figure 3. Fate of the Internalized 125I-hCG in Cells Transiently Transfected with rLHR-wt, rLHR-DSLL, or rLHR-DSLA

Transiently transfected 293 cells were incubated with a saturating concentration of 125I-hCG for 2 h at 37 C. After washing to remove the free hormone, the surface-bound hormone was released by a brief exposure of the cells to an isotonic pH 3 buffer, and the cells were placed in hormone free medium at 37 C (t = 0 in the figure) as described in Materials and Methods. At the times indicated the medium was removed and saved. The cells were washed with cold medium and they were briefly exposed again to the isotonic pH 3 buffer, thus releasing any internalized hormone that had recycled back to the surface. The acid-stripped cells were solubilized with NaOH. The radioactivity that remained associated with the cells after the acid elution (top panel) and the radioactivity released by the acid treatment (middle panel) were subsequently quantitated in a {gamma}-counter. The saved medium was used to determine the amount of degraded and undegraded 125I-hCG released as described in Materials and Methods. Only the degraded 125I-hCG is shown (lower panel) because the amount of undegraded 125I-hCG released into the medium was low (<5% of the initial counts per min) in cells expressing any of these receptors. Three 35-mm wells were used for each time point. Two of them contained 125I-hCG only, and the third also contained an excess of nonlabeled hCG. The radioactivity associated with the third well was used to correct for nonspecific binding. Each point represents the mean ± SE of three independent transfections. The absence of an error bar indicates that the SEM is too small to be shown. Results are expressed as % of the total radioactivity present at t = 0 (10,000–20,000 cpm/well in individual experiments).

 
Dominant-Negative Mutants of EBP50 Do Not Interfere with the Trafficking of the rLHR-DSLL Mutant
A dominant-negative approach was next used to determine whether the functional effects described above are due to an interaction between the rLHR-DSLL and endogenous EBP50. The presence of EBP50 in 293 cells could be readily documented by Western blots (Fig. 4AGo). Overexpression of the hemagglutinin (HA)-tagged forms of the wild-type EBP50 or a modified form of EPB50 (designated EBP50{Delta}) lacking the ERM-binding domain (11) could also be readily demonstrated using antibodies to the HA epitope (Fig. 4BGo) or to an N-terminal epitope of EBP50 (Fig. 4CGo). Expression of a FLAG-tagged form of the ERM binding domain of EBP50 (designated ERM-BD) was also readily demonstrated (Fig. 4CGo).1



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Figure 4. Detection of Endogenous EBP50 and of Several Transfected Constructs

A, Endogenous EBP50 was detected in lysates of untransfected 293 cells using a monoclonal antibody to residues 128–249 of human EBP50. B and C, 293 cells were transiently transfected with an empty vector, HA-EBP50, or HA-EBP50{Delta} as indicated. The expression of the transfected constructs was ascertained using the 12CA5 monoclonal antibody to the HA-epitope (panel B) or a monoclonal antibody to residues 128–249 of human EBP50 (panel C). In panel C the endogenous EBP50 is not visible in the cells transfected with the empty vector simply because of the short exposure used. D, 293 cells were transiently transfected with an empty vector or a construct encoding for the FLAG-tagged version of the ERM binding domain of EBP50 (ERM-BD). The expression of the transfected constructs was ascertained using the M2 monoclonal antibody to the FLAG-epitope. The results of a representative experiment are shown.

 
The functional effects of these constructs on the trafficking of the internalized 125I-hCG were measured in cells cotransfected with rLHR-wt or rLHR-DSLL. Whereas we did not expect EBP50 to have an effect on the trafficking of the hCG internalized by cells expressing rLHR-wt, we were surprised to find that overexpression of EBP50 did not further increase the recycling or further prevent the degradation of the 125I-hCG internalized by cells expressing rLHR-DSLL (Fig. 5Go). We were also surprised by the finding that a construct of EBP50 that lacks the ERM binding domain (i.e. EBP50{Delta}) did not prevent the recycling or promote the degradation of the 125I-hCG internalized by cells expressing rLHR-DSLL (Fig. 6Go) because this same construct has been previously shown to prevent the recycling and to promote the degradation of the internalized ß 2-AR in 293 cells (11).2 An additional EBP50 construct (designated ERM-BD) that lacks the two PDZ domains and is also expected to interfere with the functions of the endogenous EBP50 failed to prevent the recycling or promote the degradation of the 125I-hCG internalized by cells expressing rLHR-DSLL (Fig. 5Go).



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Figure 5. Effect of Several Constructs on the Fate of the 125I-hCG Internalized by Cells Cotransfected with rLHR-wt or rLHR-DSLL

Cells were cotransfected with combinations of rLHR-wt or rLHR-DSLL and HA-EBP50, HA-EBP50{Delta}, or the FLAG-tagged version of the ERM binding domain of EBP50 (ERM-EBP50) as indicated. The fate of the internalized 125I-hCG was measured as described in the legend to Fig. 3Go, except that only the 2 h time point was analyzed. The top panel shows the proportion of internalized hormone that remained cell associated, the middle panel shows the proportion of the internalized hormone that recycled to the membrane, and the lower panel shows the proportion of internalized hormone that was degraded and released into the medium. Results represent the mean ± SE of three independent transfections and are expressed as % of the total radioactivity present at t = 0 (10,000–20,000 cpm/well in individual experiments). The absence of an error bar indicates that the error is too small to be shown.

 


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Figure 6. Fate of the Internalized 125I-hCG in Cells Transiently Transfected with rLHR-wt or hLHR-wt

The fate of the internalized 125I-hCG was determined in transiently transfected cells exactly as described in the legend to Fig. 3Go. Results are expressed as percent of the total radioactivity present at t = 0 (10,000–20,000 cpm/well in individual experiments) and they represent the mean ± SE of three independent transfections. The absence of an error bar indicates that the SEM is too small to be shown.

 
When Grafted onto the C-Terminal Tail of the rLHR, a Motif Present in the C-Terminal Tail of the hLHR Reroutes the Internalized Agonist-rLHR Complex from a Lysosomal Degradation to a Recycling Pathway
There are two hypotheses that are consistent with the results presented above. First it is possible that the DSLL motif added to the C terminus of the rLHR may promote the interaction of this receptor with a protein distinct from EBP50 that also has some affinity for the DSLA motif and can, therefore, route the internalized hCG-receptor complex to the recycling pathway. Alternatively, it is also possible that the addition of the DSLL or DSLA motifs to the C-terminus of the rLHR disrupts the interaction of the rLHR with a protein(s) that normally route the internalized hormone-rLHR complex to a lysosomal degradation pathway.

Since the amino acid sequence of the human (h) LHR and the rLHR are highly homologous (~87% identity, see Ref. 29), but they internalized hCG at vastly different rates (19), we speculated that the intracellular routing of the hLHR and rLHR may also be different. If this were the case we could then take advantage of the availability of several hLHR/rLHR chimeras (19) to differentiate between the two hypotheses proposed above. As shown in Fig. 6Go, we found that the fate of internalized agonist-hLHR complex is indeed different from that of the agonist-rLHR complex. When compared with cells expressing rLHR-wt, cells expressing hLHR-wt recycle more and degrade less of the internalized hormone. The extent of recycling of the internalized agonist-hLHR complex detected in cells expressing hLHR-wt is in fact very similar to that detected with rLHR-DSLL (cf. Fig. 3Go).

The results presented in Table 1Go show that replacing the C-terminal tail of the rLHR with that of the hLHR (i.e. the rrh chimera) or replacing the serpentine domain and the C-terminal tail of the rLHR with those of the hLHR (i.e. the rhh chimera) increased recycling and decreased degradation in such a way that the routing of these two chimeras is closer to that of hLHR-wt than to that of rLHR-wt. Although the routing of the rrh chimera is not identical to that of the rhh chimera, it is still reasonable to conclude that the C-terminal tail of the hLHR contains sufficient structural information to reroute the rLHR from a degradation to a recycling pathway. Surprisingly, however, the complementary manipulation had little or no effect on the routing of the hLHR. As shown in Table 1Go, the recycling and degradation of hCG mediated by the hhr and the hrr chimeras more closely resembles the hLHR-wt than the rLHR-wt.


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Table 1. Fate of the Internalized 125I-hCG in Cells Transiently Transfected with the rLHR, hLHR, and Mutants Thereof

 
In the next series of experiments we concentrated on identifying the structural features present in the C-terminal tail of the hLHR that promote the recycling of the rLHR. The alignment of the amino acid sequences of the C-terminal tail of three different species of the LHR that are routed mostly to the lysosomes (rat, mouse, and porcine, see Refs. 4, 5, 7 , and 18, 19, 20) and one that recycles more (human LHR) show that there are only 10 amino acid residues that are different between the hLHR and the r, m, and pLHR group (these are shown in bold in Fig. 1Go). Since most of these divergent residues are present near the C terminus, additional mutants focused on this region. We prepared and analyzed three mutants of the rLHR (designated rLHR-mt1, -mt2, and -mt3) in which short amino acid sequences near the C terminus of the rLHR were substituted with the corresponding sequences of the hLHR as shown in Fig. 1Go. These results are summarized in Table 1Go and show that rLHR-mt1 and rLHR-mt2 enhance the recycling and decrease the degradation of the internalized hCG-rLHR complex whereas rLHR-mt3 has no effect on recycling or degradation. We conclude from these experiments that, when grafted onto the rLHR, the GTALL sequence present near the C terminus of the hLHR (see Fig. 1Go) is responsible for rerouting the rLHR from a degradation to a recycling pathway.

To confirm that the rerouting of the rLHR was caused by the addition of the GTALL sequence of the hLHR rather than by the removal of the QPIPP sequence of the rLHR (the sequences exchanged in rLHR-mt2, see Fig. 1Go) we analyzed two additional mutants in which the relevant portions of the C-terminal tail of the rLHR and the hLHR were removed by truncation of their C-terminal tails at residues 664 and 686, respectively (see Fig. 1Go). As shown in Table 1Go, the removal of the QPIPP sequence of the rLHR caused by truncation at residue 664 had no effect on the fate of the internalized hCG-rLHR complex. Lastly, the removal of the GTALL motif by truncation of the hLHR at residue 686 also had little or no effect on the fate of the internalized hLHR (Table 1Go).

The hLHR-wt and rLHR-mt2 Recycle by an EBP50-Independent Pathway
Because of the involvement of EBP50 in the recycling of the internalized ß 2-AR (11), it was important to test whether this protein is also involved in the recycling of the hLHR-wt and rLHR-mt2.

The pull-down assays shown in Fig. 7Go show that, in contrast to rLHR-DSLL, the binding of hLHR-wt and rLHR-mt2 to EBP50 is minimal or undetectable. The functional assays summarized in Fig. 8Go also show that overexpression of EBP50 or two distinct dominant negative mutants of EBP50 has no effect on the trafficking of the hCG internalized by the hLHR-wt or by rLHR-mt2.



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Figure 7. Lack of Association of hLHR-wt and rLHR-mt2 with EBP50

Detergent lysates of 293 cells transiently transfected with hLHR-wt, rLHR-mt2, or rLHR-DSLL were prepared and partially purified on a lectin column as described in Materials and Methods. Aliquots of the partially purified lysates containing the same amount of solubilized protein were allowed to bind to GST or GST-EBP50 that had been previously bound to glutathione agarose. After the bound proteins were eluted, they were visualized on Western blots developed using a monoclonal antibody to the myc epitope (9E10) and the ECL visualization system. The results of a representative experiment are shown.

 


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Figure 8. Effect of Several Constructs on the Fate of the 125I-hCG Internalized by Cells Cotransfected with hLHR-wt or rLHR-mt2

Cells were cotransfected with combinations of hLHR-wt or rLHR-mt2 and HA-EBP50, HA-EBP50{Delta}, or the FLAG-tagged version of the ERM binding domain of EBP50 (ERM-EBP50) as indicated. The fate of the internalized 125I-hCG was measured as described in the legend to Fig. 3Go, except that only the 2 h time point was analyzed. The top panel shows the proportion of internalized hormone that remained cell associated; the middle panel shows the proportion of the internalized hormone that recycled to the membrane, and the lower panel shows the proportion of internalized hormone that was degraded and released into the medium. Results represent the mean ± SE of three independent transfections and are expressed as % of the total radioactivity present at t = 0 (10,000–20,000 cpm/well in individual experiments). The absence of an error bar indicates that the error is too small to be shown.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The biochemical data presented here on the fate of the hCG internalized by the rLHR are in agreement with previous results obtained using electron microscopy (5), confocal microscopy (7), and biochemical approaches (4, 20, 21) in target or transfected cells that express the rodent or porcine LHR. When considered together, they show that most of the 125I-hCG-rLHR complex internalized by 293 cells transfected with rLHR-wt is routed to the lysosomal degradation pathway and that there is little recycling of the internalized hormone or receptor. Surprisingly, the new data presented here also show that the complex formed by hCG and the highly homologous hLHR is routed differently. In this case a substantial portion of the internalized complex is recycled to the membrane rather than routed to the lysosomes for degradation.

More importantly, the results presented here define two discrete but distinct experimental manipulations that reroute a substantial portion of the internalized hCG-rLHR complex from a lysosomal degradation pathway to a recycling pathway. These are 1) the addition of a DSLL sequence to the C terminus of the rLHR; and 2) substitution of a QPIPP sequence present near the C terminus of the rLHR with the corresponding sequence (GTALL) of the hLHR.

The data presented here with rLHR-DSLL are interesting in view of the recent results of Cao and co-workers (11) with the ß2-AR. They used a loss-of-function approach to show that the interaction of the C-terminal DSLL sequence of the ß 2-AR with the PDZ domains of EBP50 is necessary for the recycling of the internalized ß2-AR. The data presented here utilize a classical gain-of-function approach to test whether the DSLL sequence is sufficient to promote the recycling of a GPCR (the rLHR) that is normally routed mostly to a lysosomal degradation pathway. We show that the DSLL sequence promotes the association of the rLHR with EBP50 and is indeed sufficient to reroute a substantial portion of the internalized rLHR to a recycling pathway. Importantly, however, our data also show that the forced interaction of the rLHR-DSLL with EBP50 is not responsible for the rerouting of the internalized hCG-rLHR complex. This conclusion is supported by three different findings. First, modification of the rLHR with a C-terminal sequence (DLSA) that does not support EBP50 binding (12, 13) does not promote the association of the rLHR with EBP50 but it has a weak effect on rerouting. Second, over expression of EBP50 does not further enhance the recycling of the hCG internalized by the rLHR-DSLL. Third, and perhaps more importantly, overexpression of two dominant-negative mutants of EBP50 does not override the rerouting of the internalized hCG-rLHR complex induced by addition of the DSLL motif to the rLHR. The discrepant effects on the involvement of EBP50 in the recycling of the rLHR-DSLL mutant (this paper) and the ß2-AR (11) do not appear to be due to differences in the cell types used, as both sets of experiments were done using 293 cells. Also, the constructs used by us to overexpress the wt EBP50 or one of its dominant negative mutants (EBP50D) are identical to those used in the ß2-AR studies (11). One reason for this discrepancy includes the possibility that the binding affinity of the DSLL motif to EBP50 (or other PDZ domain-containing proteins) could be dependent on other structural features of the protein containing the DSLL motif. Since it is now known that there is at least one other PDZ domain-containing protein (designated CAP70) that can recognize the consensus sequence (D-S/T-x-L) necessary for binding EBP50 (30), it is possible that the DSLL-induced recycling of the rLHR is mediated by CAP70 or by other as-yet-unidentified proteins that recognize the DSLL motif.

When considered together the results presented here with rLHR-DSLL, rLHR-DSLL, and the rLHR/hLHR mutants exclude the hypothesis that the hCG-rLHR complex is actively routed to the lysosomes because the rLHR has structural motifs that target it to the lysosomes. Our results instead are consistent with the conclusion that the internalized hCG-rLHR complex is routed mostly to the lysosomes because the rLHR lacks a sorting motif that is necessary for recycling. Thus, if the C-terminal tail of the rLHR contained sorting motifs that routed the internalized hCG-rLHR complex to the lysosomes, one would expect that truncations of the C-terminal tail of the rLHR would reroute it to a recycling pathway. As shown here and elsewhere (20), however, progressive truncations of the C-terminal tail of the rLHR do not reroute the internalized hCG-rLHR complex to a recycling pathway. If anything, severe truncations tend to enhance transfer of the complex to the lysosomes (20). Second, and perhaps more importantly, a substantial portion of the internalized hCG-rLHR complex can be rerouted from a degradation to a recycling pathway by the addition of a DSLL motif at the C terminus (as discussed above) or by substituting a QPIPP sequence present near the C terminus of the rLHR with the corresponding sequence (GTALL) of the hLHR. This latter effect is due to the addition of the GTALL sequence rather than to the removal of the QPIPP sequence, because as noted above, progressive truncations of the C-terminal tail of the rLHR do not reroute the internalized hCG-rLHR complex from a lysosomal degradation to a recycling pathway.

The behavior of the hhr chimera and hLHR-t686 clearly show that exchanging the GTALL motif of the hLHR for the QPIPP motif of the rLHR (as it occurred in the hhr chimera) or removing the GTALL motif from the C-terminal tail of the hLHR (as it occurred in hLHR-t686) do not redirect the internalized hCG-hLHR complex from a recycling to a degradation pathway. We can thus conclude that although the GTALL motif is sufficient to redirect a substantial portion of the rLHR from a degradation to a recycling pathway (see above), this motif is not necessary to promote the default sorting of the hLHR to a recycling pathway. This finding is consistent with two distinct hypotheses. First, it is possible that the sorting of the internalized hLHR to a recycling pathway is completely independent of the GTALL motif. Alternatively, the sorting of the internalized hLHR may occur by a pathway that involves redundant motifs, one of which is the GTALL motif. If redundant sorting motifs do exist in the hLHR, the GTALL motif would be neither necessary nor sufficient to promote the recycling of this receptor. Conversely, if the rLHR does not have any motifs that promote recycling, then grafting the GTALL motif on its C-terminal tail would be sufficient to reroute most of the internalized rLHR to a recycling pathway as shown herein.

One way to differentiate between these two hypotheses would be to search for additional structural motifs of the hLHR that participate in recycling. A comparison of the trafficking of the hLHR-wt with the hrr and hhr chimeras suggest that, if the hLHR has redundant structural features that promote recycling, these features must be located in the extracellular domain. Since the extracellular domain of the LHR is the main determinant of ligand binding affinity (31), we propose that the additional feature of the LHR that may participate in recycling is the binding affinity of this receptor for hCG. We specifically propose that the routing of the rLHR to a degradation pathway may require two features, a high binding affinity for hCG and the absence of a GTALL motif in the C-terminal tail. We further propose that the routing of the hLHR to a recycling pathway may require only one of two features, a low binding affinity for hCG or the presence of a GTALL motif in its C-terminal tail. This proposal is best illustrated by the data shown in Table 2Go where we summarize the hCG binding affinity, the presence/absence of the GTALL motif, and the fate of the internalized hCG-receptor complexes in cells expressing the most informative LHR mutants characterized here. These data show that the only form of the LHR that is routed mostly to a degradation pathway (i.e. the rLHR-wt) binds hCG with a high affinity and does not have a GTALL motif in its C-terminal tail. The presence of a GTALL motif in mutants that have the extracellular domain of the rLHR and bind hCG with a high affinity (i.e. rrh, rLHR-mt1, and rLHR-mt2) results in rerouting of a substantial amount of the internalized receptors to a recycling pathway. The deletion of the GTALL motif (as it occurred in hLHR-t686) or the substitution of the GTALL motif in the hLHR (as it occurred in the hhr and hrr chimeras) does not affect routing, and the only common feature among these forms of the hLHR is the low hCG binding affinity caused by the presence of the extracellular domain of the hLHR. This is illustrated in Table 2Go with the hLHR-wt, as well as the hhr and hrr chimeras. The second hypothesis proposed above is, therefore, consistent with all the available data. The proposal that hCG binding affinity may contribute to the fate of the internalized hCG-LHR complex is also consistent with previous reports showing that all three members of the family of the LHR that display minimal recycling (i.e. rat, porcine, and mouse) bind hCG with high affinity (~0.2 nM; this paper and Refs. 19, 32 , and 33). Moreover, ligand binding affinity has already been shown to be an important determinant of the fate of internalized growth factor-receptor complexes (34, 35).


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Table 2. Properties of Several hLHR and rLHR Mutants

 
Although the precise locations of the two sorting motifs (DSLL and GTALL) that reroute the internalized hCG-rLHR complex to the recycling pathway are not identical, they both are located in the C-terminal tail, a finding that agrees with the perceived importance of this region in the intracellular routing of other GPCRs (8, 10, 11, 36). We do not yet know whether the DSLL or GTALL motifs redirect the internalized hCG-rLHR complex to a recycling pathway by the same or by different mechanisms, but it is clear that this rerouting is not mediated by EBP50. Although addition of the DSLL tetrapeptide to the C-terminal tail of the rLHR promotes the binding of this receptor to EBP50, the presence of the GTALL motif in the C-terminal tail of the rLHR does not promote EBP50 binding. More importantly, dominant negative constructs of EBP50 do not interfere with the DSLL- or GTALL-induced rerouting of the internalized hCG-rLHR complex to a recycling pathway. These are important observations because of the paucity of information about the structural features of GPCRs that contribute to intracellular sorting and the identity of the cellular proteins that mediate the sorting of internalized receptors. In this respect it is interesting to note that the DSLL and GTALL motifs share some structural features. They both have a phosphate acceptor (DSLL and GTALL) and a downstream dileucine motif (DSLL and GTALL). Given the involvement of phosphorylation (10, 11, 37) and dileucine motifs (38) in protein trafficking, it is possible that both of these features are needed to sort the internalized rLHR to a recycling pathway. Future studies will examine this possibility. Furthermore, the availability of different constructs of the LHR that are routed to the lysosomes or to a recycling pathway should also now allow us to identify proteins that may be involved in the sorting of the internalized LHR and to determine whether the fate of the internalized LHR has an effect on its biological functions.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids and Cells
Full-length cDNAs encoding for the hLHR and rLHR (39, 40) were subcloned into pcDNAI/Neo (rLHR) or pcDNA 3.1(hLHR) for expression. The preparation and characterization of myc-rLHR-wt and myc-hLHR-wt, modified forms of the LHR containing the myc epitope at the N terminus, have also been described (26, 41). The different mutants of the rLHR and hLHR used here were constructed by standard PCR strategies using the myc-rLHR-wt or myc-hLHR-wt as templates. The wild-type and mutant rLHR cDNAs were subcloned into the eukaryotic expression vector pcDNAI/Neo (Invitrogen, San Diego, CA) for transfection. The preparation of the rLHR/hLHR chimeras and the preparation of C-terminally truncated mutants of the rLHR (rLHR-t664) and the hLHR (hLHR-t686) have also been described (19, 20).

Bacterial expression vectors encoding for GST fusion proteins of the full-length EBP50 or derivatives thereof were prepared as described (15, 28). GST fusion proteins were also prepared as described elsewhere (15, 28). Expression vectors (pcDNA3, Invitrogen) encoding the C-terminally HA-tagged EBP50-wt and EBP50{Delta} (an EBP50 mutant lacking the last 61 amino acids) are described in Ref. 11 and were kindly provided by Tracy Cao and Mark von Zastrow (University of California at San Francisco). An additional expression vector (pcDNA3.1, Invitrogen) encoding for EBP50{Delta} without an epitope tag was also constructed from the full-length expression vector encoding for the EBP50 GST fusion protein using standard PCR strategies. An expression vector for an N-terminally FLAG-tagged construct encoding for the ERM binding domain of EBP50 (designated ERM-BD) was prepared by subcloning a portion of EBP50 (coding for residues 298–358) into the pFLAG-CMV2 vector (Sigma, St. Louis, MO).

Human embryonic kidney (293) cells were obtained from the American Type Culture Collection (Manassas, VA; CRL 1573) and maintained in DMEM containing 10 mM HEPES, 10% newborn calf-serum, and 50 µg/ml gentamicin, pH 7.4. Cells were plated in 35-mm wells or 100-mm dishes and transfected with not more than 2 or 10 µg of plasmid DNA, respectively (42), when 70–80% confluent. After an overnight incubation with the transfection mixture, the cells were washed and used 24 h later. Clonal lines of 293 cells stably expressing the HA-tagged version of EBP50{Delta} were obtained by selection of the transfected cells with 700 µg/ml of G418 (43). Resistant colonies were then tested for the expression of EBP50{Delta} using an antibody to the HA epitope as described below.

Fate of the Internalized Hormone
Transiently transfected cells were incubated with 125I-hCG (0.5–2 nM) for 2 h at 37 C. After washing to remove the free hormone, the surface-bound hormone was released by a brief exposure of the cells to an isotonic pH 3 buffer (21). Warm, hormone-free medium was then added back (t = 0), and the cells were returned to the incubator. At the times indicated the medium was removed and saved. The cells were washed with cold medium, and they were briefly exposed again to the isotonic pH 3 buffer, thus releasing any internalized hormone that had recycled back to the surface. The acid-stripped cells were solubilized with NaOH. The radioactivity that remained associated with the cells after the acid elution and the radioactivity released by the acid treatment were subsequently quantitated in a {gamma} counter. The saved medium was precipitated with 10% trichloroacetic acid to determine the amount of degraded and undegraded 125I-hCG released (21).

hCG Binding Assays
The equilibrium binding parameters for 125I-hCG were measured using intact cells that had been cotransfected with dynamin-K44A to prevent internalization (see Ref. 19). Binding parameters were measured during a 1-h incubation of intact cells (plated in 35-mm wells) with seven different concentrations (0.3–90 nM) of 125I-hCG at room temperature. All binding assays were corrected for nonspecific binding (measured in the presence of an excess of partially purified hCG). The binding data were simply fitted to a sigmoidal equation (44) using DeltaGraph software (Delta Point, Monterey, CA), and this equation was used to calculate the maximal amount of cell-associated hormone and the apparent dissociation constant (Kd).

Other Methods
The interaction between the myc-tagged rLHR-wt, rLHR-DSLL, or rLHR-DSLA with different EBP50 derivatives was determined by measuring the ability of detergent lysates prepared from transiently transfected cells to bind to the indicated GST fusion proteins. Lysates of cells expressing the myc-tagged rLHR constructs were prepared and partially purified on a wheat germ agglutinin agarose column as described elsewhere (25, 26), except that the lysis buffer contained 1% NP-40 and 60 mM octylglucoside. Equal amounts of partially purified lysate protein were incubated with 25 µg of the appropriate GST fusion proteins bound to glutathione agarose, washed, and eluted exactly as described in Ref. 15 . The eluted samples were resolved on SDS gels and electrophoretically blotted as described elsewhere (25). Blots were visualized using a monoclonal antibody to the myc epitope (9E10) and the enhanced chemiluminescence (ECL) detection system.

The expression of the endogenous EBP50 or the different EBP50 constructs transfected was ascertained on Western blots of cell lysates prepared as described above but without lectin purification. Depending on the construct transfected, blots were visualized using a monoclonal antibody to residues 128–249 of human EBP50 (Transduction Laboratories, Inc., Lexington, KY), a monoclonal antibody to the HA epitope (12CA5 from Roche Molecular Biochemicals, Indianapolis, IN), or a monoclonal antibody to the FLAG epitope (M2 from Sigma, St. Louis, MO) and the ECL detection system.

Hormones and Supplies
Purified hCG (CR-127, ~13,000 U/mg) was kindly provided by Dr. A. Parlow and 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, and it was used only to correct for nonspecific binding. 125I-hCG was prepared as previously described (45). Cell culture supplies and reagents were obtained from Corning, Inc. (Corning, NY) and Life Technologies, Inc., Gaithersburg, MD), respectively. All other chemicals were obtained from commonly used suppliers.


    ACKNOWLEDGMENTS
 
We wish to thank Tracy Cao and Mark von Zastrow (University of California at San Francisco, San Francisco, CA) for the expression vectors encoding for the HA-tagged forms of EBP50 and EBP50{Delta}. We also thank Deborah Segaloff (University of Iowa, Iowa City, IA) for her comments on this manuscript and Professor Masatomo Mori (First Department of Internal Medicine, Gunma University, Gunma, Japan) for his support. The initial hLHR plasmid used in these experiments was kindly provided to us by Ares Serono (Randolph, MA).


    FOOTNOTES
 
This work was supported by grants from the NIH: CA-40629 to MA and GM-36652 to AB. The services and facilities provided by the Diabetes and Endocrinology Research Center of the University of Iowa (supported by NIH grant DK-2529 5) are also gratefully acknowledged.

Abbreviations: ß2AR, ß2-adrenergic receptor; ECL, enhanced chemiluminescence; ERM, ezrin-radixin-moesin; GPCR, G protein-coupled receptor; GST, glutathione-S-transferase; HA, hemagglutinin; LHR, LH receptor; mLHR, mouse LHR; pLHR, porcine LH; rLHR, rat LHR.

1 This construct could not be detected with the commercially available antibody to EBP50 because this antibody recognizes an epitope (residues 128–249) that was removed from ERM-BD. Back

2 The same results were also obtained using transient co-transfections of 293 cells with myc-rLHR-DSLL and a different construct of EBP50{Delta} that was not tagged with the HA-epitope or by transient expression of myc-rLHR-DSLL in 293 cells stably expressing the HA-tagged version of EBP50{Delta} (data not shown). Back

Received for publication January 23, 2001. Accepted for publication June 5, 2001.


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