Department of Pharmacology (C.G., L.M., M.K., M.A.), The University of Iowa, Iowa City, Iowa 52242-1109; and Department of Molecular and Cellular Biology (R.N., N.L.W.), Baylor College of Medicine, Houston, Texas 77030
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, Iowa 52242-1109. E-mail: mario-ascoli{at}uiowa.edu.
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ABSTRACT |
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Using a number of mutations of the GTALL motif, we now show that only the first two residues (GT) of this motif are necessary and sufficient to induce recycling of the internalized agonist-rLHR complex. Phosphoamino acid analysis and mutations of the GT motif show that phosphorylation of the threonine residue is not necessary for recycling. Lastly, we show that addition of portions of the C-terminal tail of the hLHR that include the GT motif to the C-terminal tails of the rat follitropin or murine -opioid receptors promotes the post-endocytotic recycling of these G protein-coupled receptors.
We conclude that the GT motif present in the C-terminal tail of the hLHR is a transferable motif that promotes the postendocytotic recycling of several G protein-coupled receptors and that the GT-induced recycling does not require the phosphorylation of the threonine residue.
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INTRODUCTION |
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The rat LHR (rLHR) and human LHR (hLHR) provide a rather unique tool to understand the structural features of GPCRs that determine their intracellular fates because they share a high degree of amino acid sequence homology (18), yet they follow a divergent fate once internalized (10). 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 (10). Using chimeras and exchange mutants of these two receptors, we showed previously that, when grafted into the C-terminal tail of the rLHR, a five-amino-acid residue motif (GTALL) present in the C-terminal tail of the hLHR can redirect the internalized agonist-rLHR complex from a degradation to a recycling pathway (10). The GTALL motif is interesting because it shares two structural features (a phosphate acceptor and a dileucine sequence) with the C-terminal tetrapeptide (DSLL) of the ß2-adrenergic receptor (ß2-AR), a motif that is known to be important for the recycling of the internalized ß2-AR (19, 20). Like the GTALL motif, the DSLL motif is also transferable in that it can reroute the internalized rLHR (10) and the internalized mDOR (14) from a degradation to a recycling pathway. Because serine/threonine phosphorylation (19, 21) and dileucine motifs (19, 20) have been implicated as important determinants of the fate of internalized receptors, the experiments described here were designed to better define the structural features of the GTALL motif that induce the recycling of the internalized agonist-rLHR complex.
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RESULTS |
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As shown in Table 1, the routing of the hLHR or rLHR to a recycling or degradation pathways is not complete. Whereas the majority of the rLHR is degraded, the internalized hLHR is distributed to the degradation and recycling pathways in roughly equal proportions. We believe that this is due to the experimental conditions used. Because sorting to a recycling pathway is not 100% efficient and because the recycled hCG remains bound to the receptor (see Table 1
and Refs. 6, 7, 10), the recycled hormone undergoes additional rounds of internalization leading to the eventual degradation of substantial amounts of the internalized hormone. Importantly, however, the extent of recycling and degradation of 125I-hCG is basically the same in cells expressing the hLHR-wild type (wt) or rLHR-GT (Table 1
).
The fate of the internalized rLHR-wt, hLHR-wt, and rLHR-GT were also directly ascertained using confocal microscopy in cells cotransfected with the myc-tagged receptors and Rab5a-GFP (an endosomal marker, see Ref. 22) or procathepsin D-GFP (a lysosomal marker, see Ref. 23). The results presented in Fig. 2 show that in cells incubated without hCG the rLHR-wt and rLHR-GT are localized mostly to intracellular compartments, whereas the hLHR-wt is localized mostly at the plasma membrane. The large intracellular pools of rLHR-wt and rLHR-GT do not colocalize with either Rab5a-GFP or procathepsin D-GFP and are likely to represent the immature 68-kDa LHR precursor, which is localized in the endoplasmic reticulum (reviewed in Ref. 18). This interpretation is in agreement with results obtained by Western blots which show that the intracellular (i.e. 68 kDa) precursor of the LHR is much more prevalent in cells transfected with the rLHR-wt and rLHR-GT than in cells transfected with the hLHR (see Fig. 3
and Refs. 18, 24). Although the presence of the large intracellular pool of rLHR precursor poses a problem in visualizing the surface rLHR that becomes internalized after hCG stimulation, colocalization experiments with the endosomal and lysosomal markers clearly show that such internalization does takes place (Fig. 2
). After addition of hCG, a portion of the rLHR-wt redistributes into intracellular compartments that are rich in Rab5a (i.e. endosomes) as well as intracellular compartments that are rich in procathepsin D (i.e. lysosomes). In contrast, after addition of hCG, the rLHR-GT redistributes into endosomes but not into lysosomes (Fig. 2
). The data presented in Fig. 2
also show that the internalization of the cell surface hLHR-wt is easier to visualize because the relative abundance of the intracellular hLHR precursor is low compared with that of the mature cell surface hLHR. After addition of hCG, the internalized hLHR-wt redistributes into endosomes but not into lysosomes.
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The Addition of the GT Motif Prevents the Down-Regulation of the rLHR
An agonist-induced decrease in the cell surface LHR is another assay that can be used to discern the fate of the LHR after agonist stimulation. Because the extent of agonist-induced decrease of the cell surface LHR (down-regulation) is dictated by the rate of internalization of the receptor vs. the rate of recycling of the internalized receptor (6, 25, 26), we predicted that the hCG-induced down-regulation of the cell surface rLHR-GT mutant would be less than that of the rLHR-wt if the rates of internalization of hCG mediated by the rLHR-wt and by rLHR-GT are comparable.
To test this prediction, we used an immunoprecipitation/immunoblotting approach to measure the levels of the cell surface LHR after stimulation with hCG. Because the immunoprecipitation is done using whole cell lysates, three species of the LHR can be detected (reviewed in Ref. 18). The 68-kDa band is much more prevalent in cells expressing the rLHR (see left and middle panels of Fig. 3) than in cells expressing the hLHR (see right panel of Fig. 3
) and it represents an intracellular, immature precursor of the LHR that is thought to be localized in the endoplasmic reticulum (also see Fig. 2
above). The 85-kDa band is much more prevalent in cells expressing the hLHR (see right panel of Fig. 3
) than in cells expressing the rLHR (see left and middle panels of Fig. 3
), and it represents the mature cell surface LHR. The 165-kDa LHR is mostly an aggregate of the 68-kDa immature receptor, which is again more prevalent in cells expressing the rLHR than in cells expressing the hLHR.
As expected, incubation of the cells with hCG resulted in a decrease in the density of the 85-kDa band (i.e. the mature cell surface LHR) but had no effect on the intensity of the intracellular LHR precursors. The intensity of the 85-kDa band was thus measured in several experiments before and after agonist stimulation, and a summary of the quantitative data obtained is presented in Table 2. This table also displays the rates of internalization of hCG mediated by each of the three receptors. These results show that, in spite of a slightly faster rate of internalization, a property that enhances down-regulation (26), the extent of hCG-induced down-regulation of the cell surface rLHR-GT is much lower than that of the cell surface rLHR-wt as predicted by the divergent fates of the internalized receptors. Also, as shown previously (27), the half-time of internalization of the hLHR-wt is much shorter than that of the rLHR-wt.
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The Rerouting of the rLHR to a Recycling Pathway by the Addition of the GT Sequence Does Not Require Threonine Phosphorylation
Because the hCG-induced activation of the rLHR and the hLHR results in the phosphorylation of several sites present in their C-terminal tails (28, 29, 30), and the GT sequence contains a phosphate acceptor (threonine), we considered the possibility that the phosphorylation of the threonine present in the GT sequence is necessary for recycling. This hypothesis was tested by performing phosphoamino acid analysis on rLHR-wt, hLHR-wt, and the rLHR-GT mutant as well as by mutagenesis of the GT sequence.
Because previous phosphoamino acid analysis and mutagenesis studies of the rLHR have shown that this receptor is phosphorylated mostly on serine residues (28, 29, 31), we reasoned that if the threonine present in the GT motif is phosphorylated we should observe an increase in the phosphothreonine content of the rLHR-GT when compared with the rLRH-wt. The results summarized in Fig. 4 show that this is not the case. The amount of phosphothreonine detectable in the rLHR-wt is minimal, and there is no increase in the phosphothreonine content of the rLHR-GT mutant when compared with rLHR-wt. In addition, and in agreement with previous mutagenesis studies (30) the amount of phosphothreonine detected in the hLHR-wt, a recycling receptor that contains the GT sequence as part of its wild-type C-terminal tail is also minimal (Fig. 4
).
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Lastly, previously characterized mutants of the rLHR or the hLHR that were rendered phosphorylation-deficient by simultaneous mutation or removal of several C-terminal tail serine residues that become phosphorylated upon agonist activation (shown in red in Fig. 1, also see Refs. 10, 29, 30, 33) were shown to follow the same fate as their wild-type counterparts (data not shown and Ref. 10).
Taken together, these data allow us to conclude that the phosphorylation of the threonine residue of the GT motif, or the phosphorylation of other serine residues present in the C-terminal tail of the rLHR or hLHR, are not involved in determining the fate of the internalized receptors.
The Recycling Properties of the GT Motif Are Transferable to Other GPCRs
Because the sorting of internalized GPCRs is dictated mostly by their C-terminal tails (10, 12, 14, 16, 20, 34, 35, 36), one would predict that the portion of the C-terminal tail of the hLHR that contains the GT motif should be able to redirect another internalized GPCR from a degradation to a recycling pathway. This possibility was tested by examining the properties of mutants of the mDOR and the rat FSH receptor (rFSHR) modified to express the GT sequence at their C-terminal tails.
The mDOR is another GPCR that is routed to a lysosomal degradation pathway upon agonist-induced internalization (13, 14). This GPCR was chosen as a model to further ascertain the recycling properties of the GT motif because it is only distantly related to the LHR (see alignment in Fig. 1) and because recent studies have shown that replacing its last 6 residues with the last 10 residues of the ß2-AR redirect the internalized mDOR from a degradation to a recycling pathway (14). Thus, we prepared a similar construct (designated mDOR/hLHR) in which the last 6 residues of the C-terminal tail of the mDOR were replaced with the last 17 residues of the C-terminal tail of the hLHR. The fates of HA-tagged versions of the mDOR-wt and the mDOR/hLHR constructs transiently expressed in 293T cells were compared by confocal imaging. The confocal micrographs shown in Fig. 5
show that, as expected (13, 14), most of the influenza hemagglutinin epitope (HA)-mDOR-wt is localized at the surface of transiently transfected 293T and that agonist stimulation results in a redistribution of the mDOR from the cell surface to Rab5a-rich compartments (i.e. endosomes) and procathepsin D-rich compartments (i.e. lysosomes). The HA-mDOR/hLHR is also localized mostly at the cell surface, but agonist stimulation results in a redistribution of this receptor from the cell surface to endosomes and not to lysosomes. These data clearly show that a portion of the C-terminal tail of the hLHR that encompasses the GT motif promotes the postendocytotic recycling of another GPCR (the mDOR) that is normally routed to a lysosomal degradation pathway.
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DISCUSSION |
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Although all investigators agree that the DSLL motif is essential for the recycling of the internalized ß2-AR (19, 20) and that it can induce the recycling of nonrecycling GPCRs when added to their C-terminal tails (10, 14), there is no agreement on the identity of the cellular protein(s) that mediate the DSLL-dependent GPCR recycling. Cao et al. (19) implicated a PDZ domain protein known as ezrin binding protein (EBP)50 or sodium-hydrogen exchange regulatory factor as being responsible for this event, whereas we (10) and Cong et al. (20) have presented evidence that exclude EBP50/NHERF from being involved in this process. Lastly, Cong et al. (20) have presented evidence that implicates a protein that participates in membrane fusion (N-ethylmaleimide-sensitive factor) instead of EBP50/NHERF as being responsible for the DSLL-dependent recycling of the internalized ß2-AR. A general role for the DSLL motif in the recycling of internalized GPCRs is unlikely, however, because most internalized GPCRs are routed to a recycling pathway (3, 4, 5), but there is only one other GPCR (the P2Y1 purinergic receptor, see Ref. 37) that has a similar C-terminal sequence (D-S/T-x-L). Based on these considerations, it appears likely that there may be other motifs present in the C-terminal tails of GPCRs that are involved in determining the fate of the internalized receptors.
Structure function studies on the fates of the internalized hLHR and rLHR can provide important data about this issue because, in spite of a high degree of amino acid sequence identity, the agonist-activated hLHR and rLHR follow a different fate once internalized. The internalized hCG-rLHR complex is routed mostly to a lysosomal degradation pathway, whereas the internalized hCG-hLHR complex is routed mostly to a recycling pathway (this paper and Ref. 10). We have previously concluded (10) that the lysosomal routing of the internalized rLHR is due to the lack of sorting motif(s) needed for recycling rather than the presence of sorting motif(s) needed for degradation for three reasons. First, progressive deletions of the C-terminal tail of the rLHR fail to reroute the internalized hCG-rLHR complex from a degradation to a recycling pathway (9). Second, grafting the C-terminal tail of the rLHR into the hLHR does not reroute the internalized hCG-hLHR complex from a recycling to a degradation pathway (10). Third, addition of the DSLL C-terminal tetrapeptide of the ß2-AR to the extreme C-terminus of the rLHR, grafting the C-terminal tail of the hLHR into the rLHR, or grafting short sequences, GTALL or GT, present in the C-terminal tail of the hLHR into the corresponding position of the C-terminal tail of the rLHR reroutes the internalized agonist-rLHR complex from a degradation pathway to a recycling pathway (this paper and Ref. 10).
Because of the obvious structural similarities between the GTALL and the DSLL motifs (they both have a phosphate acceptor and a leucine dimer), it was reasonable to hypothesize that one or both of these features may play a ubiquitous role in determining the fate of internalized GPCRs. The data presented here show that the leucine dimer of the GTALL motif is not needed for recycling and identify the GT sequence as being necessary and sufficient to induce the recycling of the internalized agonist-rLHR complex. Our results also show that the threonine residue present in the GT motif does not need to be phosphorylated for recycling to occur. We show that there is no increase in threonine phosphorylation of the rLHR-GT when compared with the rLHR-wt and that there is little or no threonine phosphorylation in the hLHR. Moreover, substitution of the threonine of the GT motif with another phosphate acceptor (serine) or with two acidic amino acids that mimic the negative charge of phosphothreonine or phosphoserine do not support recycling. These results stand in contrast with the DSLL-dependent recycling of the ß2-AR or the DSLL-induced recycling of the rLHR both of which are disrupted by mutations of the leucine dimer (10, 20). In addition, the phosphate acceptor (serine) of the DSLL motif of the ß2-AR can be phosphorylated by GRK5 in vitro (38) and overexpression of GRK5 reportedly inhibits the recycling of the internalized ß2-AR (19). Moreover, because mutation of the serine of the DSLL motif to a residue that cannot be phosphorylated (alanine) or to one that mimics the charge of the phosphorylated serine (aspartic acid) disrupted recycling of the internalized ß2-AR and the in vitro association of the ß2-AR with EBP50/NHERF it was proposed that the GRK-5 catalyzed phosphorylation of the serine of the DSLL motif disrupted recycling by preventing the association of the ß2-AR with EBP50/NHERF (19). The effects of a serine to threonine exchange were not examined, however. When considered together, the data on the differential effects of mutations of the leucine dimer and the phosphate acceptor imply that the mechanisms by which the GT and the DSLL motifs induce GPCR recycling are rather different.
Another important difference between the DSLL and GT motifs is the role that they play in their native context (i.e. the ß2-AR and the hLHR, respectively). Whereas mutation or removal of the DSLL motif disrupt the recycling of the internalized ß2-AR (see above and Refs. 19, 20), mutation or removal of the GT motif of the hLHR does not reroute the internalized agonist-hLHR complex from a recycling to a degradation pathway (10). As already discussed elsewhere (10), the apparent lack of involvement of the GT motif in the recycling of the internalized hLHR may be explained by the presence of redundant sorting motifs in the hLHR. Because the C-terminal tail of the rLHR does not appear to have any sorting motifs that promote recycling or degradation (as discussed above), then the grafting of recycling motifs, such as the GT or the DSLL motifs, on the C-terminal tail of the rLHR would be sufficient to reroute most of the internalized rLHR to a recycling pathway.
An important similarity of the DSLL and GT motifs is that their recycling properties are transferable to other GPCRs. As shown here for the GT motif and elsewhere for the DSLL motif (10, 14), both of these motifs induce the recycling of the rLHR and the mDOR, two distantly related GPCRs that are normally sorted to a lysosomal degradation pathway. Moreover, we also show here that grafting the C-terminal tail of the rLHR into another recycling GPCR (the rFSHR) reroutes portion of the internalized rFSHR to a degradation pathway unless a GT motif is included in the C-terminal tail of the rLHR. When the C-terminal tail of the rLHR grafted into the rFSHR is modified to contain a GT motif, however, the recycling properties of the rFSHR are preserved.
As already outlined above, a general role for the DSLL motif in GPCR recycling is unlikely because most internalized GPCRs recycle but do not terminate with a DSLL or homologous sequence, and the GT motif identified here is too short to be used as a query of GPCR databases to determine if its presence correlates with GPCR recycling. Fortuitously, however, we noticed that this motif is present in the C-terminal tail of the type A endothelin receptor and it is absent in the type B endothelin receptor and the presence of this motif correlates with the fate of the internalized endothelin receptors (see Fig. 1 of Ref. 16). Thus, the agonist-activated endothelin B receptor is targeted to a lysosomal degradation pathway, whereas the agonist-activated endothelin A receptor is targeted to a recycling pathway (15, 16, 17, 39). Interestingly, switching the C-terminal tails of these two receptors was also shown to influence their fate, but the specific residues responsible for this phenomenon were not identified (16).
The identify of the proteins that mediate the postendocytotic sorting of GPCRs are just beginning to be revealed. EBP50/NHERF and N-ethylmaleimide-sensitive factor have both been implicated as being involved in the DSLL-dependent recycling of GPCRs (19, 20), and recent experiments conducted with the mDOR and the thrombin receptor have identified two proteins, sorting nexin 1 (40) and GPCR-associated sorting protein (41) as proteins that participate in the sorting of internalized GPCRs to the lysosomes. An important future challenge for us will be to identify the proteins that sort the internalized LHR to a degradation or a recycling pathway. This question is actively being investigated by searching for cellular proteins that show preferential binding to the C-terminal tails of the rLHR and hLHR in a GT-dependent manner.
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MATERIALS AND METHODS |
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Human embryonic kidney 293 cells 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 of plasmid DNA, using the calcium phosphate method of Chen and Okayama (44), when 7080% confluent. After an overnight incubation with the transfection mixture, the cells were washed and used 24 h later. The preparation and properties of 293L(wt-1) cells, a clonal line of 293 cells stably expressing the rLHR-wt at a high density, have been described (45, 46, 47). Additional clonal lines of 293 cells stably expressing the myc-rLHR-GT mutant, designated 293Lmyc(GT-1), were obtained by selection of the transfected cells with 700 µg/ml of G418 as described elsewhere (33, 45).
Rate of Internalization of hCG
Transiently transfected cells were incubated with a subsaturating concentration (0.5 nM) of 125I-hCG at 37 C and the surface bound and internalized radioactivity were measured as a function of time after hormone addition. The surface bound and internalized radioactivity were measured using a brief exposure of the cells to an isotonic pH 3 buffer (48), and the half-times of internalization were calculated from the slope of the line obtained by plotting the internalized radioactivity against the integral of the surface-bound radioactivity using at least five different data points collected at 10-min intervals after the addition of 125I-hCG (43, 49).
Fate of the Internalized hCG-LHR Complex
Transiently transfected cells were allowed to internalize 125I-hCG during a 2-h incubation at 37 C with a saturating concentration of hormone (50 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 (10, 48, 49). This was defined as t = 0, and the cells (which now contain only internalized 125I-hormone) were incubated for an additional 2 h at 37 C. At this time the medium was saved and the cells were washed with cold medium, and they were 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 (10, 48, 49).
Confocal Microscopy
Cells were plated in eight-chamber coverslip culture vessels coated with polylysine (BioCoat from Becton Dickinson, Franklin Lakes, NJ). They were cotransfected (in a total volume of 400 µl) with 100 ng of the appropriate myc-LHR or HA-mDOR constructs, and 8 ng of Rab5-GFP or cathepsin D-GFP using the methods described above. Two days after transfection, the cells expressing the LHR constructs were incubated with or without hCG (52 nM) for 2 h at 37 C and the cells expressing the DOR constructs were incubated with DADLE (10 µM) for 30 min at 37 C. The medium was removed, and the cells were washed twice with PBS (137 mM NaCl; 2.7 mM KCl; 1.4 mM NaH2PO4; 4.3 mM Na2HPO4, pH 7.4) and fixed during a 30-min incubation at room temperature with 4% paraformaldehyde (dissolved in PBS). The fixed cells were washed twice again and then incubated for 1 h at room temperature with PBS containing 50 mg/ml BSA. This solution was removed, and the cells were incubated for another hour at room temperature with a 1:100 dilution of the anti-myc monoclonal antibody (9E10) or an anti-HA monoclonal antibody (12CA5) dissolved in PBS containing 5 mg/ml BSA. After washing four times with PBS, the cells were incubated for another hour at room temperature with a 1:2000 dilution of CY5-labeled antimouse IgG. Finally, they were washed three to four times with PBS, dried, and mounted in Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA). The CY5-labeled LHR and the arrestin-3-GFP were visualized with a Bio-Rad Laboratories, Inc. (Richmond, CA) confocal microscope at the Central Microscopy Facility of The University of Iowa.
Phosphoaminoacid Analysis
Phosphorylated receptors were immunoprecipitated from 293L(wt-1), 293Lmyc(GT-1) cells or from 293T cells transiently expressing the myc-hLHR-wt. The choice of stably transfected or transiently transfected cells as a source of rLHR and hLHR was based entirely on receptor abundance (18). Cells were metabolically labeled with 32P (400 µCi/ml) for 3 h and stimulated with a saturating concentration of agonist (50 nM) for 15 min (cells expressing the rLHR-wt or rLHR-GT) or 60 min (cells expressing the hLHR-wt) as described elsewhere (28, 29, 30, 33, 47). The rLHR-wt expressed in 293L(wt-1) cells is not myc-tagged and was immunoprecipitated with a polyclonal antibody to the rLHR as described elsewhere (33, 50). Because the rLHR-GT expressed in 293Lmyc(GT-1) cells and the hLHR expressed in transiently transfected cells are both tagged with the myc epitope (see above), these receptors were immunoprecipitated with the 9E10 antibody as described before (30).
The immunoprecipitates were bound to either protein A agarose (for the polyclonal antibody) or to protein G-agarose for the monoclonal antibody, the beads were washed extensively (30, 33, 50), and the immune complexes were eluted during a 15-min incubation with 1 M acetic acid. The eluates were dried under a stream of nitrogen and phosphoamino acid analysis of the immunoprecipitated and eluted proteins was carried out using a HTLE-7000 electrophoresis system as described by Van der Geer and Hunter (51). Briefly, the eluted proteins were hydrolyzed in 6 N HCl for 1 h at 110 C and dried using a Speed Vac with 4 µg of phosphoamino acid standards (phosphoserine, phosphothreonine, and phosphotyrosine). The dried samples were resuspended in 20 µl of 2.5% (vol/vol) formic acid, 7.8% (vol/vol) acetic acid (pH 1.9) and spotted on a thin-layer chromatography plate (EM Science, Gibbstown, NJ). The samples were electrophoresed in the first dimension in the same pH 1.9 buffer at 1500 V for 20 min. The plate was dried, turned 90 degrees counterclockwise, and electrophoresed in the second dimension in 5% (vol/vol) acetic acid, 0.5% (vol/vol) pyridine (pH 3.5) at 1300 V for 16 min. The plate was dried for 45 min, sprayed with 0.5% ninhydrin, and heated in an oven at 65 C for 10 min. The 32P-labeled phosphoamino acids were detected by autoradiography and identified by matching with the standards.
Receptor Down-Regulation
293T cells that had been transiently transfected with the myc-rLHR-wt, myc-rLHR-GT, or myc-hLHR-wt were washed twice with assay medium (Waymouths 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 a saturating concentration (50 nM) of hCG for 6 h at 37 C. At the desired time, the cells were placed on ice and lysed (52). The myc-tagged LHR were immunoprecipitated with the 9E10 antibody and the immunoprecipitates were resolved on sodium dodecyl sulfate gels and electrophoretically transferred to polyvinylidene difluoride membranes as described elsewhere (52, 53). The antibody 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. (Rockford, IL) and a Kodak (Rochester, NY) digital imaging system as described elsewhere (52). This digital image capture system is set up to alert us when image saturation occurs and to prevent us from measuring the intensity of such images.
The immunoprecipitation/immunoblot analysis described above and illustrated in Fig. 3 results in the identification of the mature LHR present at the cell surface (
85 kDa), an intracellular LHR precursor with a molecular mass of approximately 68 kDa, and an approximately 165-kDa aggregate of the 68-kDa precursor (18). Because only the cell surface LHR is expected to change as a result of agonist activation, we used the intensity of the 68-kDa band to correct for loading and variability in receptor expression. Thus, to calculate down-regulation (see Table 2
) the amount of cell surface receptor (i.e. 85 kDa) was divided by the amount of receptor precursor (i.e. 68 kDa) and the corrected data from the cells incubated with hCG for 6 h were expressed as the percentage of the corrected data from the cells that had hCG added but were processed immediately after hormone addition.
Hormones and Supplies
Human kidney 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 9E10 antibody coupled to horseradish peroxidase was purchased from Roche Molecular Biochemicals and the 12CA5 monoclonal antibody to the HA epitope was from Roche. The CY5-labeled secondary antibody was from The Jackson Laboratory (Bar Harbor, ME). DADLE was from Sigma (St. Louis, MO). Human kidney 293T cells are a derivative of 293 cells that express the Simian virus 40 T antigen (54) and were provided to us by Dr. Marlene Hosey (Northwestern University, Chicago, IL). Purified hCG (CR-127, 13,000 IU/mg) and hFSH (AFP-5720D, prepared from human pituitaries) were purchased from the National Hormone and Pituitary Agency (NIDDK, NIH, Bethesda, MD) and purified recombinant hCG and hFSH were provided by Ares-Serono (Randolph, MA).1 125I-hCG was prepared as described elsewhere (55). Partially purified hCG (
3000 IU/mg) was purchased from Sigma, and it was used only for the determination of nonspecific binding. Partially purified equine FSH was kindly donated by Dr. George Bousfield (Wichita State University, Wichita, KS). Cell culture medium was obtained from the Media and Cell Production Core of the Diabetes and Endocrinology Research Center of the University of Iowa. Other cells culture supplies and reagents were obtained from Corning, Inc. (Corning, NY) and Invitrogen (Carlsbad, CA), respectively. All other chemicals were obtained from commonly used suppliers.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: ß2-AR, ß2-Adrenergic receptor; EBP, ezrin binding protein; FSHR, FSH receptor; GPCRs, G protein-coupled receptors; HA, influenza hemagglutinin epitope; hCG, human chorionic gonadotropin; hLHR, human LHR; LHR, lutropin receptor; mDOR, murine -opioid receptors; NHERF, sodium-hydrogen exchange regulatory factor; rLHR, rat LHR; rFSHR, rat FSHR; wt, wild-type.
1 Results obtained with the two hormone preparations were indistinguishable.
Received for publication April 30, 2002. Accepted for publication November 20, 2002.
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REFERENCES |
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