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Molecular Endocrinology, doi:10.1210/me.2003-0293
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Molecular Endocrinology 18 (2): 434-446
Copyright © 2004 by The Endocrine Society

The Postendocytotic Trafficking of the Human Lutropin Receptor Is Mediated by a Transferable Motif Consisting of the C-Terminal Cysteine and an Upstream Leucine

Colette Galet, Takashi Hirakawa and Mario Ascoli

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

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutants of the human (h) lutropin receptor (LHR) were analyzed using a combination of biochemical and imaging approaches to define motifs that participate in the postendocytotic sorting of this G protein-coupled receptor (GPCR).

We show that a substantial portion of the human chorionic gonadotropin internalized by the hLHR sorts to a recycling pathway, and the internalized hLHR accumulates in endosomes because of the C-terminal cysteine (Cys699) and an upstream Leu683 present in the hLHR. The removal or simultaneous mutation of these two residues reroutes the internalized human chorionic gonadotropin to a degradation pathway and the internalized hLHR to lysosomes. We also show that grafting the 17 C-terminal residues of the hLHR into the C-terminal tail of two GPCRs that are routed to a lysosomal/degradation pathway (the rat LHR or the murine {delta} opioid receptor) reroutes them to an endosomal/recycling pathway. This is due to the Leu683 and Cys699 combination and another recycling motif (Gly687Thr688) that was previously identified in the hLHR. The importance of both motifs can be readily ascertained in the context of a murine {delta} opioid receptor/hLHR chimera. The importance of the Gly687Thr688 motif is revealed mostly in the context of a rat LHR/hLHR chimera.

These studies define a novel, noncontiguous, transferable motif that participates in the sorting of internalized GPCRs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ALTHOUGH MUCH HAS been learned recently about the pathways by which G protein-coupled receptors (GPCRs) are internalized (reviewed in Refs. 1, 2, 3, 4, 5, 6), there is still a paucity of information regarding the structural features of GPCRs that are involved in the postendocytotic sorting of the receptors and on the intracellular proteins that mediate sorting (reviewed in Refs. 3, 5, 6, 7, 8, 9).

Understanding the processes involved in the sorting of internalized GPCRs is particularly important because their fate has important functional implications. Most internalized GPCRs are sorted into endosomes and quickly recycled back to the plasma membrane (4, 5, 7) but a few, such as the rodent or porcine lutropin receptors (LHRs) (10, 11, 12, 13, 14), the human thrombin receptor (15, 16, 17), the murine {delta}-opioid receptor (18, 19, 20, 21), and the human endothelin B receptor (22, 23, 24), are instead directed to lysosomes where they are degraded. Recycling of internalized GPCRs is thought to be involved in the resensitization of GPCRs and is important in preserving cellular responsiveness by maintaining a relatively constant density of receptors at the cell surface (4, 5, 7). Conversely, lysosomal degradation of internalized GPCRs is thought to be involved in the acute termination of signaling (16, 24, 25), and it also contributes to a more prolonged attenuation of signaling because it results in a net loss of cell surface receptors (10, 26).

The rat (r) LHR and the human (h) LHR provide us with a unique opportunity to understand the structural features of GPCRs that determine their postendocytotic sorting because they share a high degree of amino acid sequence homology (27), yet they have divergent fates after internalization (14, 28). The internalized agonist-hLHR complex is routed mostly to a recycling pathway, whereas the internalized agonist-rLHR complex is routed mostly to a degradation pathway (13, 14, 28, 29). Using chimeras and more discrete mutations that exchanged divergent residues, we previously showed that, when grafted into the C-terminal tail of the rLHR, a two-amino acid residue sequence (Gly687Thr688) present in the extreme C-terminal tail of the hLHR can redirect the internalized agonist-rLHR complex from a degradation to a recycling pathway (14, 28). Curiously, however, removal of the Gly687Thr688 sequence of the hLHR by truncation of its C-terminal tail had little or no effect on the recycling of the internalized agonist-hLHR complex (14), thus suggesting the presence of additional recycling motifs. The experiments presented here were designed to identify these additional motifs.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Truncations of the C-Terminal Tail of the hLHR Reroute the Internalized [125I]hCG (Human Chorionic Gonadotropin) from a Recycling to a Degradation Pathway and Promote the Lysosomal Accumulation of the Internalized hLHR
Our initial attempt to define additional residues of the C-terminal tail of the hLHR that may participate in recycling involved analyzing the fate of the [125I]hCG internalized by 293T cells transiently expressing progressive C-terminal truncations of the hLHR. As expected from previous results (14, 28), the data presented in Table 1Go show that during a 2-h postendocytotic sorting assay, equivalent proportions of the [125I]hCG internalized by cells expressing the hLHR-wt are recycled to the cell surface or degraded and released back into the medium. The new data presented in Table 1Go show that progressive truncations of the C-terminal tail of the hLHR changed this sorting pattern in two distinguishable ways, as highlighted by the two boxes. Removal of the C-terminal Cys699 (i.e. the hLHR-t698 mutant in Table 1Go) and additional truncations that removed residues up to His684 (i.e. the hLHR-t683 mutant in Table 1Go) induced a slight increase in the amount of degraded hormone released and a slight decrease in the amount of recycled hormone. A truncation that removed Leu683 (see hLHR-t682 in Table 1Go) and additional truncations that removed residues up to Ser674 (see hLHR-t673 in Table 1Go) induced a further increase in the amount of degraded hormone released and a further decrease in the amount of recycled hormone. The sorting of the [125I]hCG internalized by this latter set of truncated mutants is, in fact, very similar to that of the rLHR-wt (also shown in Table 1Go), which was previously shown to route most of the internalized [125I]hCG to a lysosomal degradation pathway (10, 11, 13, 14, 28).


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  		Table 1. Effect of Progressive hLHR Truncations on the Fate of the Internalized [125I]hCG

 
In previous publications (14, 28) we have shown that, when grafted into the equivalent position of the rLHR, the Gly687Thr688 motif of the hLHR redirects a substantial portion of the [125I]hCG internalized by the rLHR from a degradation to a recycling pathway. These same studies (14, 28) have also shown that the removal of this motif does not affect the recycling of the hLHR, and several of the mutants shown in Table 1Go confirm this prior observation. First, three truncations that remove the Gly687Thr688 motif (i.e. hLHR-t686, -t685, and -t684) fall into the group of truncations that do not have major effects on the recycling or degradation of the internalized [125I]hCG. Second, a mutation of the Gly687Thr688Ile689 motif to the corresponding sequence of the rLHR (see hLHR-QPI in Table 1Go) does not reroute the internalized [125I]hCG to a degradation pathway.1

The functional properties of the two types of hLHR truncations described above are better illustrated in Fig. 1AGo, where a more complete time course of the postendocytotic sorting of hCG mediated by two prototypical truncations (hLHR-t685 and hLHR-t682) expressed in 293T cells is illustrated. Both truncations decrease the recycling and enhance the degradation of the internalized [125I]hCG, but these effects are clearly more pronounced for hLHR-t682 than for hLHR-t685. The data presented in Fig. 1AGo also show that the postendocytotic sorting of [125I]hCG mediated by hLHR-t682 is indistinguishable from that of the rLHR-wt, which, as already noted above, is targeted mostly to a degradation pathway (10, 11, 13, 14, 28).



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  		Fig. 1. Truncations of the hLHR Reroute the Internalized [125I]hCG to a Degradation Pathway

A, 293T cells were transiently transfected with the indicated constructs. The fate of the internalized [125I]hCG was measured as described in Materials and Methods. Results are expressed as a percent of the total intracellular [125I]hCG radioactivity present at t = 0 (10,000–20,000 cpm/well in individual experiments) and they represent the mean ± SEM of three independent transfections. B, MA-10 cells were transiently transfected with the indicated constructs. The fate of the internalized [125I]hCG was measured as described in Materials and Methods. Results are expressed as a percent of the total intracellular [125I]hCG radioactivity present at t = 0 (5,000 to 10,000 cpm/well in individual experiments) and they represent the mean ± SEM of three independent transfections.

 
To ensure that the results obtained with these hLHR mutants are not dependent on the cell type, we also compared their behavior when expressed in MA-10 cells, a mouse Leydig tumor cell line that expresses small amounts of endogenous LHR (30, 31). This is an appropriate model to address this question for two reasons. First, the sorting of the hCG internalized by the transfected hLHR constructs can be readily compared with the sorting of the hCG internalized by the endogenous mouse (m) LHR which, like the rLHR, also routes most of the internalized hormone to a lysosomal degradation pathway (10, 13, 14, 29). Second, studies with the transfected hLHR constructs are minimally affected by the presence of endogenous murine (m) LHR because the transfected hLHR constructs are expressed at a density of approximately 100,000 receptors per cell whereas the endogenous mLHR is expressed at a density of about 1,000 receptors per cell (31). Note, however, that under our experimental conditions, receptor density has no effect on the sorting of the internalized hCG (data not shown, and Ref. 32).

The data presented in Fig. 1BGo show that the sorting of the hCG internalized by MA-10 cells expressing the hLHR-wt, -t685, and -t682 is similar to that displayed by 293T cells expressing the same constructs. Moreover, these data show that the behavior of hLHR-t682 is similar to that of the endogenous mLHR, which as noted above, has been previously shown to route most of the internalized hCG to a lysosomal degradation pathway (10). Further studies were conducted using only 293T cells because these can be transfected with the economical calcium phosphate method.

The fates of the hLHR-wt and hLHR-t682 were ascertained using two different approaches. The first approach involved the visualization of receptors by confocal imaging in cells cotransfected with Rab5a-green fluorescent protein (GFP) (an endosomal marker) and pro-cathepsin D-GFP (a lysosomal marker). The results presented in Fig. 2Go, A and B, show that the hLHR-wt and hLHR-t682 localize mostly to the cell surface in cells that were not treated with hCG. An hCG-induced internalization of the hLHR-wt and hLHR-t682 are also clearly demonstrated, but the internalized hLHR-wt localizes mostly to endosomes (Fig. 2AGo) whereas the internalized hLHR-t682 localizes to endosomes and lysosomes (Fig. 2BGo). Because Table 1Go and Fig. 1AGo show that 20–30% of the [125I]hCG internalized by the hLHR-wt is degraded, these data predict that some of the hLHR-wt would also localize to the lysosomes. Our inability to detect the hLHR-wt in this compartment by confocal imaging (Fig. 2AGo) is likely due to the less quantitative nature of this experimental approach and the low levels of intact (i.e. immunoreactive) hLHR-wt that are likely to be present in the lysosomes at the 2-h time point used in the confocal analysis.



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  		Fig. 2. Subcellular Localization of Several hLHR Constructs

293T cells were transiently cotransfected with the indicated myc-hLHR constructs and Rab5a-GFP (left panels) or the indicated myc-hLHR constructs and procathepsin D-GFP (right panels). The transfected cells were washed and incubated with (52 nM) or without hCG at 37 C for 2 h as indicated. The cells were fixed and the receptors (in red) were visualized using an anti-myc monoclonal antibody (9E10) and a CY5-conjugated antimouse antibody. Rab5a-GFP and procathepsin D-GFP are shown in green, and colocalized components are shown in yellow. The cells were observed and analyzed using a Bio-Rad confocal microscope at the Central Microscopy Facility of The University of Iowa.

 
The second approach used to determine the fate of the hLHR-wt and hLHR-t682 is a more quantitative approach in which the density of the hLHR at the cell surface was measured by immunoprecipitation before and after addition of hCG to surface-biotinylated cells (33). The results of these experiments are presented in Fig. 3Go and show that during a 6-h incubation with hCG there is an approximately 25% and 85% loss, respectively, in the density of the hLHR-wt and hLHR-t682 at the cell surface.2



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  		Fig. 3. Agonist-Induced Down-Regulation of the Cell Surface hLHR-wt and hLHR-t682

Transiently transfected 293T cells were biotinylated, stimulated with a saturating concentration of hCG (52 nM), and lysed immediately or after a 6-h incubation at 37 C. Lysates were immunoprecipitated with the 9E10 antibody, and the amount of surface receptor was visualized and quantitated on Western blots using streptavidin covalently coupled to horseradish peroxidase as described in Materials and Methods. The relevant portions of a representative blot are shown. The numbers under each lane represent the quantitation of receptor levels (mean ± SEM of three to five independent transfections) expressed as percent of the amount of cell surface receptors present at t = 0.

 
Identification of Leu683 and Cys699 as the Two C-Terminal Tail Residues Required for Recycling of the Internalized [125I]hCG and for Preventing the Lysosomal Accumulation of the Internalized hLHR
Based on the functional properties of the C-terminal truncations shown in Table 1Go, we hypothesized that two residues, Cys699 (the C terminus) and Leu683, were needed for the recycling of the [125I]hCG internalized by the hLHR. This hypothesis was tested by analyzing the behavior of individual or combined mutations of Leu683 and Cys699. Two mutations of Leu683 were examined, a traditional L683A mutation and a L683V mutation because Leu683 is replaced by a Val in the rLHR, a homologous receptor that does not recycle. The results of these mutagenesis experiments are presented in Table 2Go and show that, in the context of the full-length hLHR, the two different mutations of Leu683 have only minor effects on the routing of the internalized [125I]hCG. Table 2Go also shows that a point mutation of the C-terminal Cys (see hLHR-C699A) had little or no effect on the routing of the internalized [125I]hCG. In fact, the behavior of hLHR-C699A is very similar to that of mutants that remove the C-terminal Cys699 (see hLHR-t698), or the C-terminal tetrapeptide (see hLHR-t695) (see Tables 1Go and 2Go).


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  		Table 2. Effect of Selected hLHR Mutations on the Fate of the Internalized [125I]hCG

 
We next examined the behavior of a construct harboring combined mutations of Leu683 and Cys699 (hLHR-L683A/C699A). Table 2Go shows that this mutant reroutes most of the internalized [125I]hCG to a degradation pathway. Because the cell surface expression of hLHR-C699A and hLHR-L683A/C699A was relatively poor compared with that of other mutants, we also tested additional constructs that harbor a mutation of Leu683 and deletions of small portions of the C-terminal tail. Table 2Go shows that a hLHR construct containing an L683A mutation and a deletion of Cys699 (see hLHR-L683A/t698) reroutes most of the internalized [125I]hCG to a degradation pathway. The same is true when two different point mutations of Leu683 are introduced in the context of a truncation that removes the last four residues of the hLHR (see hLHR-L683A/t695 and hLHR-L683V/t695). In fact, the sorting of the internalized [125I]hCG mediated by hLHR-L683A/t698 or hLHR-L683A/t695 is very similar to that mediated by the rLHR (Table 2Go) or by truncations of the hLHR that remove the Leu683-Cys699 region (compare the data in Tables 1Go and 2Go). The rerouting of [125I]hCG mediated by the hLHR-L683V/t695 mutant is not as pronounced as that of the other combination mutants examined. The reasons for this apparent difference were not investigated.

The postendocytotic sorting of one of these receptor mutants (hLHR-L683A/t698) was also analyzed by confocal microscopy. Figure 2CGo shows that addition of hCG to cells expressing the internalized hLHR-L683A/t698 resulted in the accumulation of the receptor in endosomes and lysosomes. The subcellular localization of the internalized hLHR-L683A/t698 was, in fact, indistinguishable from hLHR-t682, a truncation that removes the C-terminal Cys and Leu683 (compare Fig. 2Go, B and C).

Collectively, these data show that the recycling of the internalized [125I]hCG and the lack of accumulation of the internalized hLHR in lysosomes is dependent on both Leu683 and the C-terminal Cys699.

C-Terminal Sequences Encoding the Leu683 and Cys699 Combination Influence the Postendocytotic Fate of Other Internalized GPCRs
To determine whether the Leu683 and Cys699 combination described above influences the postendocytotic fate of other GPCRs, we took advantage of the finding that grafting the Leu683-Cys699 fragment of the hLHR into the C-terminal end of two distinct GPCRs that are normally routed to lysosomes (i.e. the rLHR and the murine opioid receptor, mDOR) reroutes them to endosomes (14, 28). This fragment contains two motifs that could potentially promote recycling, i.e. the Leu683 and Cys699 combination characterized here and the Gly687Thr688 sequence characterized previously (14, 28).

To determine whether the sorting of the mDOR/hLHR chimera to endosomes is due to the Leu683 and Cys699 combination or the Gly687Thr688 sequence, we prepared two mutants of this chimera (Fig. 4AGo). In the mDOR/hLHR-1 chimera the transferred Gly687Thr688 sequence was mutated to Gln-Pro, the corresponding sequence of the rLHR that does not promote recycling. In the mDOR/hLHR-2 chimera the last four residues of the grafted hLHR tail were removed, and the grafted Leu683 of the hLHR was mutated to Ala. In this and other experiments we opted to test the involvement of Cys699 of the hLHR by removing the C-terminal tetrapeptide of the hLHR instead of mutating or removing the C-terminal Cys699 for two reasons. First, constructs with point mutations of Cys699 or lacking Cys699 are not expressed as robustly as constructs lacking the C-terminal tetrapeptide; and second, because the role of the C-terminal Cys699 in recycling may be context dependent, we reasoned that it was safer to remove and/or add (see below) a small peptide containing this residue rather than to remove and/or add the single Cys residue.



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  		Fig. 4. Fate of Several mDOR Constructs

A, Amino acid sequences of the C-terminal tails of the hLHR and mDOR and chimeras thereof. The amino acid sequences of the hLHR and mDOR are shown in red and blue, respectively. The partial box at the left of the sequences marks the cytoplasmic end of transmembrane 7 (TM7). Identical residues are highlighted in gray, and dots indicate gaps introduced for optimal alignment. The red arrow on the hLHR sequence highlights the start of the 17-residue sequence that was grafted into the mDOR when constructing the mDOR/hLHR chimera. The blue arrow on the mDOR sequence highlights the start of the six-residue sequence that was substituted by the hLHR sequence when constructing the mDOR/hLHR chimera. The boxes in the amino acid sequences of the mDOR/hLHR-1 and mDOR-hLHR-2 chimeras highlight the mutated and/or deleted residues. B–E, 293T cells were transiently cotransfected with the indicated HA-mDOR constructs and Rab5a-GFP (left panels) or the indicated HA-mDOR constructs and procathepsin D-GFP (right panels). The transfected cells were washed and incubated with (10 µM) or without DADLE [(D-Ala2, D-Leu5 enkephalin), an mDOR agonist] at 37 C for 30 min as indicated. The cells were fixed and the receptors (in red) were visualized using an anti-HA monoclonal antibody (12CA5) and a CY5-conjugated antimouse antibody. Rab5a-GFP and cathepsin D-GFP are shown in green, and colocalized components are shown in yellow. The cells were observed and analyzed using a Bio-Rad confocal microscope at the Central Microscopy Facility of The University of Iowa. F, Stably transfected 293 cells expressing the indicated HA-mDOR constructs were biotinylated, stimulated with DADLE (20 µM), and lysed immediately or after a 6-h incubation at 37 C. Lysates were immunoprecipitated with the 3F10 antibody, and the amount of surface receptor was visualized and quantitated on Western blots using streptavidin covalently coupled to horseradish peroxidase as described in Materials and Methods. The relevant portions of a representative blot are shown. The numbers under each lane represent the quantitation of receptor levels (mean ± SEM of three to five experiments) expressed as percent of the amount of cell surface receptors present at t = 0.

 
The postendocytotic trafficking of the wild-type mDOR, the original mDOR/hLHR chimera, and the two mutant chimeras described above was ascertained by confocal imaging (Fig. 4Go, B–E), and the fate of the cell surface receptors was measured by immunoprecipitation from surface-biotinylated cells (Fig. 4FGo). As expected from previous data (18, 19, 28), the internalized wild-type mDOR is routed to lysosomes (Fig. 4BGo), but the mDOR/hLHR chimera is routed mostly to endosomes (Fig. 4CGo). An mDOR/hLHR chimera with a mutated GlyThr motif (see mDOR/hLHR-1 mutant chimera) is routed mostly to endosomes (Fig. 4DGo), but a chimera with a truncation of the last four grafted residues and mutation of the grafted Leu (the mDOR/hLHR-2 mutant chimera) is routed to lysosomes (Fig. 4EGo). The two constructs that can be detected in lysosomes in agonist-stimulated cells (i.e. the mDOR and the mDOR/hLHR-2 chimera; see Fig. 4Go, B and E) display more than a 50% reduction in the levels of cell surface receptor upon agonist stimulation (Fig. 4FGo), and one of the constructs that cannot be detected in lysosomes (i.e. the mDOR/hLHR chimera; see Fig. 4CGo) displays no reduction in the levels of cell surface receptor upon agonist stimulation (Fig. 4FGo). There is a slight discrepancy between the imaging data and the cell surface receptor assay with the mDOR/hLHR-1 chimera, however. Because the imaging data indicated that this chimera does not accumulate in the lysosomes (Fig. 4DGo), we expected it to behave like the original mDOR/hLRH chimera and show no reduction in the levels of cell surface receptor. As shown in Fig. 4FGo, however, the density of cell surface receptors in cells expressing this chimera declined approximately 35% after agonist stimulation. This could be a reflection of differences in the sensitivity or the quantitative vs. qualitative nature of the two methods employed. Alternatively, the agonist-induced reduction in the levels of cell surface mDOR/hLHR-1 chimera may occur by a pathway that is unrelated to agonist-induced internalization and postendocytotic trafficking. This is a likely possibility because a proteasome-dependent pathway has also been implicated in the agonist-induced down-regulation of the mDOR (34).

Collectively, these data show that the rerouting of the internalized mDOR that is induced by the Leu683-Cys699 fragment of the hLHR is mediated mostly by Leu683 and the C-terminal tetrapeptide of the transferred hLHR sequence. Although we cannot completely exclude the involvement of the Gly687Thr688 motif that is also present in the transferred hLHR sequence, we can safely conclude that this motif is not as important as the Leu683 and C-terminal tetrapeptide combination.

A similar series of experiments was conducted by following the fate of the [125I]hCG internalized by the equivalent chimera of the rLHR (designated rLHR/hLRH; see Table 3Go). In this case the fate of the internalized hormone reflects the fate of the receptor because the internalized hCG-LHR complex does not dissociate after endocytosis and subsequent sorting (10, 11, 13, 14, 28). Similar to the results obtained with the mDOR, we previously showed that grafting the Leu683-Cys699 fragment of the hLHR unto the C-terminal tail of the rLHR reroutes the internalized [125I]hCG from a degradation to a recycling pathway (see Table 3Go and Refs. 14 and 28). Several mutations of this chimera were then examined to determine whether the rerouting effect was due to the grafted Gly687Thr688 motif or to the grafted Leu683 and C-terminal tetrapeptide combination. The removal of the last four residues and the mutation of Leu683 of the grafted hLHR sequence induced a small increase in the amount of degraded [125I]hCG and a small decrease in the amount of recycled [125I]hCG when compared with the original rLHR/hLHR chimera (compare rLHR/hLHR-1 with rLHR/hLHR in Table 3Go). Likewise, mutation of the grafted Gly687Thr688 to GlnPro, the corresponding sequence of the rLHR had small effects on the routing of the [125I]hCG internalized by the original chimera (compare rLHR/hLHR-2 with rLHR/hLHR in Table 3Go). The only mutant chimera that more fully reversed the rerouting of the internalized [125I]hCG back to that observed with the rLHR is a chimera in which all potential recycling motifs of the hLHR were mutated (compare rLHR/hLHR-3 with rLHR/hLHR and rLHR in Table 3Go). Thus, the rerouting of the internalized [125I]hCG that is induced when the Leu683-Cys699 fragment of the hLHR is grafted into the rLHR is mediated by Leu683 and the C-terminal tetrapeptide as well as by the Gly687Thr688 motif present in the transferred hLHR sequence.


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  		Table 3. Fate of the [125I]hCG Internalized by Cells Expressing Chimeras of the rLHR and hLHR and Other Selected Mutants of the rLHR

 
In a complementary set of experiments done with the rLHR (a receptor that is routed to a lysosomal degradation pathway), we mutated Val661 (a residue that corresponds to Leu683 of the hLHR; see Table 3Go) to a Leu and substituted the last two residues of the rLHR with the C-terminal pentapeptide of the hLHR (RYTEC, see Table 3Go). We opted to do this instead of simply adding a C-terminal Cys to the rLHR to mimic the overall length of the hLHR and because the role of the C-terminal Cys in recycling may be context dependent. The results summarized in Table 3Go show that neither of these two modifications, when introduced alone or in combination, had an effect on the routing of the [125I]hCG internalized by the rLHR. In agreement with previous results, however (see Refs. 14 and 28), a QP to GT mutation of the rLHR reroutes the internalized [125I]hCG to a recycling pathway (Table 3Go). The rerouting of [125I]hCG detected in the rLHR(QP/GT) mutant is in fact very similar or identical to that detected in an rLHR construct containing a V to L mutation, a QP to GT mutation, and an added RYTEC sequence (see Table 3Go). These results show that the ability of the Leu683-C-terminal tetrapeptide combination to promote recycling is context dependent. The functional properties of this motif are readily detectable in the context of the hLHR-wt (Table 2Go) and a chimera of the rLHR or mDOR that contains the last 17 residues of the hLHR (Table 3Go and Fig. 4Go) but are not detectable by more discrete substitutions done in the context of the rLHR as shown in Table 3Go.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Our analysis of truncations of the hLHR and more defined point mutations show that the accumulation of the internalized hLHR in endosomes, the recycling of the internalized ligand (hCG) in an intact form, and the maintenance of a relatively constant level of cell surface hLHR during endocytosis of the ligand are all dependent on two distant residues present in the C-terminal tail, the C-terminal Cys699 and an upstream Leu (Leu 683). Both of these residues are required for all of these processes as shown here by the behavior of hLHR constructs harboring the individual and simultaneous mutations or deletions. Importantly, our data also show that, when transferred to the C-terminal tail of two GPCRs that are normally routed to lysosomes (the rLHR and the mDOR), a fragment of the hLHR that contains these two residues prevents the lysosomal accumulation of these receptors and/or the agonist-induced loss of surface receptors that accompanies endocytosis. Therefore, the Leu683 and Cys699 combination identified here is necessary not only for the recycling of the hLHR but it is also sufficient to induce the recycling of two other GPCRS when introduced in the right context.

Our previous studies of chimeras of the hLHR and rLHR had identified a simple linear amino acid sequence of the hLHR (Gly687Thr688) that is sufficient to promote the recycling of the closely related rLHR when grafted into its C-terminal tail but is not necessary for the recycling of the hLHR (Table 1Go and Refs. 14 and 28). The new data presented here show that the ability of the Gly687Thr688 to promote recycling is detected mostly in the context of the C-terminal tail of the rLHR and the rLHR/hLHR chimeras (Table 3Go). The Gly687Thr688 motif of the hLHR is involved only secondarily in the recycling of the internalized mDOR induced by the C-terminal tail of the hLHR (Fig. 4Go). Because of these results we can now conclude that the Gly687Thr688 motif is unlikely to be directly involved in recycling. It is more likely that, when transferred into the C-terminal tail of the rLHR, the Gly687Thr688 motif disrupts the conformation of this tail, thus exposing other motifs that more directly promote the recycling of the rLHR. The location and identity of these putative recycling motifs are not known but they are presumably unique to the rLHR because the Gly687Thr688 motif has only a small effect on the trafficking of the mDOR/hLHR chimera. In contrast, the motif defined here by the Leu683 and Cys699 combination of the hLHR is more likely to directly affect recycling because this motif is necessary for the recycling of the hLHR, and it is sufficient to promote the recycling of the mDOR and the rLHR as shown by the mDOR/hLHR and rLHR/hLHR chimeras.

When internalized by cells expressing the rLHR, most of the [125I]hCG is degraded and released into the medium, whereas a small percentage is quickly recycled to the medium. We have previously concluded that this is the default pathway because progressive deletions of the C-terminal tail of the rLHR do not reroute the internalized [125I]hCG to a recycling pathway (13). Some of the [125I]hCG internalized by the rLHR can be rerouted to a recycling pathway, however, by specific amino acid sequences present in recycling GPCRs as shown here and elsewhere. These include adding the DSLL [amino acid of the C-terminal tetrapeptide of the ß2-adrenergic receptor (AR)] to the extreme C terminus of the rLHR, grafting the Leu683-Cys699 fragment of the hLHR into the corresponding position of the rLHR, or grafting a shorter sequence (such as Gly687Thr688) present in the C-terminal tail of the hLHR into the corresponding position of the C-terminal tail of the rLHR (present paper and Refs. 14 and 28). A similar conclusion can be made from studies conducted on the mDOR. This GPCR also appears to be routed to the lysosomes by default (19), but it can be induced to recycle by the DSLL C-terminal tetrapeptide of the ß2-AR (19), the Leu683-Cys699 fragment of the hLHR (current paper and Ref. 28) or a newly described recycling signal present in the C-terminal tail of the µ-opioid receptor (20). Therefore, these data argue that the recycling of internalized GPCRs is a regulated process rather than a default pathway followed after internalization. Moreover, these results indicate that there is a diversity of endocytic recycling motifs present in GPCRs.

The recycling of the internalized [125I]hCG that is observed in cells expressing the hLHR-wt and the rerouting of the internalized [125I]hCG to a recycling pathway that is observed by addition of recycling motifs to the rLHR is not complete. Some of the internalized hormone still escapes recycling and is routed to a degradation pathway (present paper and Refs. 14 and 28). This is likely to be due to the high affinity of the hCG-LHR complex (14, 32). Because the recycled hormone remains bound to the receptor, the hormone-receptor complex can undergo many rounds of internalization. Even if only a small percentage of the internalized hormone is routed to a degradation pathway with each round of endocytosis, this futile cycle could ultimately result in the degradation of a substantial amount of the internalized hormone.

The routing of internalized GPCRs from endosomes back to the plasma membrane or to the lysosomes may be mediated by posttranslational modifications such as phosphorylation or ubiquitination (35, 36, 37, 38) and/or by association of the internalized GPCRs with cellular proteins such as the nonvisual arrestins (39, 40), N-ethylmaleimide sensitive factor (NSF, see Ref. 41), ezrin-radixin-moesin-binding phosphoprotein-50/sodium-hydrogen exchange regulatory factor (EBP50/NHERF, see Ref. 38), sorting nexins (17), or a newly identified GPCR-associated sorting protein (see Ref. 42). Whereas significant progress has been made in understanding these processes, the identity of the GPCR residues that mediate sorting and/or participate in the binding of the aforementioned cellular proteins are still not fully defined. The data presented here make an important contribution to this area by identifying a new GPCR motif that is necessary for the recycling of the hLHR and is sufficient to promote recycling of other GPCRs. One fully transferable motif that is necessary for the recycling of the ß2-AR, the C-terminal tetrapeptide (DSLL), has been previously identified (14, 19, 38, 41). Another fully transferable GPCR (comprised of ~17 residues) that is present in the C-terminal tail of the µ-opioid receptor was identified while this paper was under review (20). Although these three motifs are located in the C-terminal tails of GPCRs, only two, the DSLL motif of the ß2-AR and the Leu683 and Cys699 combination of the hLHR, involve the extreme C terminus. All three motifs, however, contain at least one Leu, but the DSLL motif of the ß2-AR and the recycling motif of the µ-opioid receptor are linear sequences, whereas the Leu683 and Cys699 combination of the hLHR is not. Interestingly, leucine residues present in the third intracellular loop of the mDOR have also been shown to participate in the sorting of this GPCR (21).

Because neither of the two residues of the Leu683 and Cys699 combination are susceptible to posttranslational modifications, they may promote recycling by directly mediating the association of the hLHR with one or more cellular proteins or by modulating the association of the hLHR with enzymes that catalyze the phosphorylation and/or ubiquitination of other hLHR residues. It is not known whether the hLHR is ubiquitinated, but the phosphorylation of the hLHR does not appear to participate in sorting because the phosphorylation sites of the rLHR and the hLHR are highly conserved (33, 43, 44). Moreover, the postendocytotic fate of a hLHR mutant in which all phosphorylation sites were mutated to alanine is identical to that of the hLHR-wt (data not shown), and truncations of the rLHR that remove all phosphorylation sites do not reroute the internalized rLHR to a recycling pathway (13). Because there are two proteins, NSF (41) and EBP50/NHERF (38), that have been implicated in the DSLL-dependent recycling of the ß2-AR, we have examined their ability to bind to the hLHR and to participate in the recycling of this receptor. Although weak binding of the hLHR to EBP50/NHERF can be detected in vitro (14, 45) we have been unable to demonstrate the involvement of either EBP50/NHERF or NSF using either dominant-negative or overexpression approaches (our unpublished data and Ref. 45) in the recycling of the hLHR. We have recently shown, however, that the recycling of the hLHR is regulated by its association with a previously identified protein known as GAIP-interacting protein C terminus, GIPC (45). The formation of this complex occurs through the single PDZ type I domain of GIPC and it requires Cys699, but not Leu683, of the hLHR (45). Although the association of GIPC with the hLHR provides a molecular explanation for the involvement of Cys699 in recycling, it is possible that Leu683 may bind additional protein(s) that also participate in recycling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids and Cells
The preparation and characterization of expression vectors for the myc-hLHR-wt (in pcDNA3.1) and the myc-rLHR-wt (in pcDNA1Neo) have been described (33, 47). The expression vectors for human Rab5a-GFP and human pro-cathepsin D-GFP were kindly provided by Dr. P. Stahl (Washington University, St. Louis, MO) and Dr. J. Backer (Albert Einstein College of Medicine, New York, NY), respectively. A vector for a hemagglutinin (HA)-tagged form of mDOR was kindly provided by Dr. Ping Yee Law (University of Minnesota, Minneapolis, MN), and it was subcloned into pcDNA3.1 for expression. The mDOR/hLHR and rLHR/hLHR chimeras were prepared as described previously (14, 28). The different mutants of the hLHR, mDOR/hLHR, and rLHR/hLHR chimeras used here were constructed using standard PCR strategies.

Human embryonic kidney 293 and 293T cells were maintained in DMEM containing 10 mM HEPES, 10% newborn calf serum, and 50 µg/ml gentamicin, pH 7.4. Cells were plated in gelatin-coated 35-mm wells and transiently transfected with 0.5 µg plasmid DNA, using the calcium phosphate methods of Chen and Okayama (48), when 70–80% confluent. After an overnight incubation with the transfection mixture, the cells were washed and used 24 h later. Nonclonal stably transfected 293 cells expressing the different mDOR constructs were prepared by selection with 700 µg/ml G418. MA-10 cells were maintained and transfected as described previously (30, 31).

Fate of the Internalized hCG
Transiently transfected cells were allowed to internalize [125I]hCG during a 2-h incubation at 37 C with a saturating concentration of hormone (52 nM). After washing to remove the free hormone, the surface-bound [125I]hCG was released by a brief exposure of the cells to an isotonic pH 3 buffer (14, 49, 50). This was defined as t = 0, and the cells (which now contain only internalized [125I]hCG) were incubated for an additional 2 h at 37 C. At the end of this second incubation the medium was saved, after which the cells were washed with cold medium and briefly exposed again to the isotonic pH 3 buffer to release and measure any of the internalized hormone that had recycled back to the surface. The acid-stripped cells were solubilized with NaOH to measure residual radioactivity that remained internalized. Finally, the saved medium was precipitated with 10% trichloroacetic acid to determine the amount of degraded and undegraded [125I]hCG released (14, 49, 50).

Down-Regulation of the Cell Surface LHR
293T cells transiently transfected with the myc-hLHR-wt or mutants thereof were washed twice with PBS (137 mM NaCl, 2.7 mM KCl, 1.4 mM KH2PO4, 4.3 mM Na2HPO4) pH 8, and then biotinylated for 30 min at room temperature by incubation with a freshly prepared solution (0.5 mg/ml) of sulfo-NHS-LC-Biotin, (Pierce Chemical Co., Rockford, IL) as described previously (33). After biotinylation, the cells were washed once with DMEM containing 10 mM HEPES and 10% newborn calf serum and incubated for 5 min in the same medium to quench the excess unreacted biotin. Cells were then washed once with cold assay medium (Waymouth’s MB752/1 supplemented with 1 mg/ml BSA, 20 mM HEPES, and 50 µg/ml gentamicin, pH 7.4). Some cells were saved on ice and processed immediately (t = 0 samples) whereas others were incubated in 1 ml of warm assay medium containing 52 nM hCG for 6 h at 37 C. At the indicated times, the cells were placed on ice and lysed as described previously (33). The receptors were immunoprecipitated with 9E10 antibody (33), and the immunoprecipitates were resolved on sodium dodecyl sulfate gels and electrophoretically transferred to polyvinylidene difluoride membranes as described elsewhere (51). The blots were revealed using streptavidin conjugated to horseradish peroxidase, and the proteins were finally visualized and quantitated using the Super Signal West FEMTO Maximum Sensitivity system of detection from Pierce Chemical Co. and a Kodak digital imaging system (Eastman Kodak, Rochester, NY) as described elsewhere (33). This image capture system is set up to alert us when image saturation occurs and to prevent us from measuring the intensity of such images.

Nonclonal populations of 293 cells stably expressing the HA-mDOR or mutants thereof were biotinylated as described above and lysed immediately or after a 4-h incubation at 37 C with D-Ala2, D-Leu5 enkephalin (DADLE) (20 µM) as described above. The HA-tagged receptors were immunoprecipitated using an anti-HA monoclonal antibody (3F10) as described elsewhere (33), and the immunoprecipitates were analyzed and quantitated as described above.

Confocal Microscopy
Confocal microscopy experiments were accomplished as recently described (28, 52). Briefly, 293T cells were plated in eight-chamber coverslip culture vessels coated with polylysine (Biocoat from Becton Dickinson and Co., Franklin Lakes, NJ). They were cotransfected (in a total volume of 400 µl) with 100 ng of expression vectors for the appropriate receptors (myc-hLHR-wt or mutants thereof or HA-mDOR/hLHR chimeras) and 10 ng of expression vectors for Rab5a-GFP or procathepsin D-GFP. Two days after the transfection, the cells expressing the myc-hLHR-wt (or mutants thereof) were incubated with or without hCG (52 nM) for 2 h, whereas the cells expressing the HA-mDOR/hLHR chimeras were incubated with or without DADLE (10 µM) for 30 min. The cells were then washed and treated as previously described (28, 52). The myc-hLHR was visualized by incubating the cells (1 h at room temperature) with a 1:100 dilution of an anti-myc monoclonal antibody (9E10) or an anti-HA monoclonal antibody (12CA5) dissolved in PBS containing 5 mg/ml BSA. After washing three times, the cells were incubated for another hour at room temperature with a 1:1000 dilution of Cy5-conjugated sheep antimouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Finally, the cells were washed three or four times, dried, and mounted as previously described (28, 52). The Cy5-labeled receptors and the Rab5a or cathepsin D-GFP were visualized with a Bio-Rad confocal microscope (Bio-Rad Laboratories, Inc., Hercules, CA) at the Central Microscopy Facility of the University of Iowa. An oil x60 objective was used; the iris opening was 2 to 2.2 for each color filter.

Hormones and Supplies
The 293 cells and the 9E10 hybridoma cell line were obtained from the American Type Culture Collection (Manassas, VA). The 9E10 cells were used by the Hybridoma Facility of the Cancer Center of the University of Iowa to prepare a concentrated supernatant containing the 9E10 antibody. The two anti-HA monoclonal antibodies (12CA5 and 3F10) were purchased from Roche Clinical Laboratories (Indianapolis, IN) and the peroxidase-conjugated streptavidin was from Vector Laboratories, Inc. (Burlingame, CA). Human kidney 293T cells are a derivative of 293 cells that express the SV40T antigen (53) and were provided to us by Dr. Marlene Hosey (Northwestern University, Chicago, IL). Purified hCG (CR-127, ~13,000 IU/mg) was purchased from Dr. A. Parlow and the National Hormone and Pituitary Agency (NIDDK, National Institute of Health, Bethesda, MD), and purified recombinant hCG was kindly provided by Ares-Serono (Randolph, MA). [125I]hCG was prepared as described elsewhere (54). Partially purified hCG (~3000 IU/mg) and DADLE were purchased from Sigma Chemical Co. (St. Louis, MO). The partially purified hCG was used only for the determination of nonspecific binding. Cell culture medium and other cell culture supplies and reagents were obtained from Corning, Inc. (Corning, NY) and Invitrogen (San Diego, CA), respectively. All other chemicals were obtained from commonly used suppliers.


   FOOTNOTES
 
This work was supported by NIH Grant CA-40629 (to M.A.). T.H. was partially supported by a fellowship from the Lalor Foundation, and C.G. was partially supported by a fellowship from the American Heart Association.

1 Many other exchange mutants in which progressively smaller sequences of the rLHR were substituted with the corresponding sequences of the hLHR showed that the Gly687Thr688 motif is the minimal sequence of the hLHR that can induced recycling of the [125I]hCG internalized by rLHR.28 Back

2 A longer incubation time with hCG was used for these experiments because the loss of cell surface receptors occurs with a slower time course than the recycling and/or degradation of the internalized hormone. Back

Abbreviations: AR, Adrenergic receptor; CG, chorionic gonadotropin; DADLE, D-Ala2, D-Leu5 enkephalin; DSLL, the amino acid of the C-terminal tetrapeptide of the ß2-AR; EBP50/NHERF, ezrin-radixin-moesin-binding phosphoprotein-50/sodium-hydrogen exchange regulatory factor; GFP, green fluorescent protein; GIPC, GAIP-interacting protein C terminus; GPCR, G protein-coupled receptor; HA, hemagglutinin; LHR, lutropin receptor; LHR-wt, wild-type LHR; mDOR, murine {delta}-opioid receptor; NSF, N-ethylmaleimide-sensitive factor.

Received for publication July 24, 2003. Accepted for publication October 27, 2003.


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 MATERIALS AND METHODS
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