Evidence that Palmitoylation of Carboxyl Terminus Cysteine Residues of the Human Luteinizing Hormone Receptor Regulates Postendocytic Processing

Utpal M. Munshi1, Christine L. Clouser1, Helle Peegel and K. M. J. Menon

Departments of Biological Chemistry and Obstetrics/Gynecology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0617

Address all correspondence and requests for reprints to: K. M. J. Menon, 6428 Medical Sciences Building I, 1301 Catherine Street, Ann Arbor, Michigan 48109-0617. E-mail: kmjmenon{at}umich.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Palmitoylation is a well-conserved posttranslational modification among members of the G protein-coupled receptor superfamily. The present study examined the role of palmitoylation in endocytosis and postendocytic trafficking of the human LH receptor (LHR). Palmitoylation of the LHR was determined by incorporation of [3H]palmitic acid into wild-type (WT) or mutant receptor in which the potential palmitoylation sites, C643 and C644, were mutated to glycine residues. The WT receptor showed incorporation of [3H]palmitic acid into the mature 90-kDa form of the receptor whereas mutation of the two Cys residues abrogated this incorporation, indicating that Cys 643 and C644 are the sites of palmitoylation. The role of palmitoylation on endocytosis and postendocytic processing was examined by testing the ability of the WT and mutant receptor to undergo internalization, recycling, and lysosomal degradation. Compared with the WT receptor, the mutant receptor showed increased internalization and decreased recycling, suggesting that retention of palmitic acid residues at Cys 643 and 644 promotes LHR recycling. The role of palmitoylation on receptor recycling was substantiated by demonstrating that a different mutant, D578H LHR, which is deficient in palmitoylation, also recycled less efficiently. Furthermore, the data show that palmitoylation, not the rate of internalization, determines the efficiency of recycling. The present study shows that palmitoylation of cysteine residues 643 and 644 of the human LHR is a determinant of recycling.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE LH RECEPTOR (LHR) is a G protein-coupled receptor (GPCR) that mediates steroidogenesis in gonadal cells through its interaction with LH/human chorionic gonadotropin (hCG) and subsequent activation of the Gs-cAMP signaling pathway (1, 2). The extracellular domain of the LHR is posttranslationally modified by N-linked glycosylation (3, 4, 5), whereas the C-terminal tail of the rat receptor has been shown to undergo palmitoylation at two adjacent cysteine residues (6) and LH/hCG-stimulated phosphorylation at four serine residues (7). Although palmitoylation is a well-conserved posttranslational modification of several members of the GPCR family, its function varies from one receptor to another (8). For example, abrogation of the palmitoylation sites of the vasopressin V2 receptor or the {delta}-opioid receptor hinders receptor processing (8), whereas mutation of the palmitoylation site in the ß2-adrenergic receptor significantly reduces ligand-mediated stimulation of cAMP production (9). Similar mutations in the CCR5 receptor decrease ligand-induced internalization (10).

Previous studies have reported that two cysteine (C621/622) residues in the C-terminal tail of the rat LHR are palmitoylated (6, 11). We further showed that mutation of these cysteine residues did not affect ligand binding, cAMP production (6, 12), or stimulation of the inositol phosphate signaling pathway (13), but resulted in an increased rate of hCG-induced receptor internalization (6), which was attributed to enhanced interaction of the mutant receptor with the arrestin-mediated internalization pathway (13). Furthermore, the palmitoylation-deficient mutant receptors were found to be more prone to phorbol 12-myristate 13 acetate-stimulated phosphorylation (13). It is not known whether palmitoylation might play an additional role in trafficking of the internalized LHR.

After internalization, a portion of the human LHR is recycled to the cell surface to undergo a new round of agonist binding and subsequent G protein activation (14). Specific structural elements, including a GT motif and a Cys and Leu in the C-terminal tail of LHR, have been shown to determine the fate of the internalized receptor (14, 15, 16, 17). Interestingly, the portion of receptor that is recycled is thought to undergo dephosphorylation before trafficking to the cell surface. Because phosphorylation of the rat LHR is enhanced by mutations of the two palmitoylation sites (13), the present study examines whether palmitoylation of the carboxyl-terminal cysteine residues has any role in the postendocytic routing of the hLHR. Our results indicate that the palmitoylation status of the hLHR plays a role in the postendocytic processing of the internalized receptor.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Palmitoylation of the hLHR/hCG Receptor
Before testing the effect of the palmitoylation state of the receptor on postendocytic routing, we sought first to examine whether the hLHR is palmitoylated. To test this, 293T cells transiently transfected with hLHR cDNA constructs were examined for the ability to incorporate [3H]palmitic acid into the hLHR. The LHR cDNA construct encoded a FLAG epitope at the carboxyl terminus of the receptor to allow receptor isolation by immunoprecipitation before separation by SDS-PAGE. Figure 1AGo shows that [3H]palmitic acid was incorporated into a prominent 90-kDa band corresponding to the mature LHR, demonstrating that the hLHR is palmitoylated. Sequence homology to other palmitoylated GPCRs suggests that two adjacent cysteine residues, both Cys 643 and Cys 644 in the C-terminal tail of the hLHR, represent potential sites for palmitoylation. To determine whether the hLHR/hCG receptor undergoes palmitoylation at these Cys residues, Cys 643 and 644 in hLHR were mutated to glycine residues, and the ability of the mutant receptor to incorporate [3H]palmitic acid was examined. The results showed that, in contrast to the wild-type (WT) hLHR (Fig. 1AGo, lane 1), little or no [3H]palmitic acid was incorporated into the mutant hLHR (Fig. 1AGo, lane 2). The possibility that lack of [3H]palmitic acid incorporation into the mutant hLHR was due to differences in receptor expression was then determined by Western blot analysis. Results showed a prominent band at 90 kDa from cells transfected with WT and mutant hLHR, indicating that mutation of the cysteine residues did not adversely affect receptor expression (Fig. 1BGo). These results show that the absence of a prominent 90-kDa band in [3H]palmitic acid-labeled cells expressing the mutant receptor (Fig. 1AGo) is not due to lack of receptor expression, but due to the loss of the receptor palmitoylation site. Because the Cys643/644Gly mutant failed to incorporate [3H]palmitic acid, other cysteine residues were not considered as potential sites of palmitoylation.



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Fig. 1. Palmitoylation of hLHR

A, Incorporation of [3H]palmitic acid into WT or C643/644G hLHR in transiently transfected 293 T cells. Transfected cells were metabolically labeled with [3H]palmitic acid for 6 h. Cells were solubilized with PBS containing 1% Triton X-100, and the receptor was immunoprecipitated with FLAG antibody. Bound proteins were eluted with sodium dodecyl sulfate loading buffer as described in Materials and Methods and analyzed by SDS-PAGE under reducing conditions followed by fluorography and autoradiography. B, Western blot analysis of WT and C643/44G hLHR. LHR from transfected cells was immunoprecipitated and separated by SDS-PAGE before Western blot analysis was performed as described in Materials and Methods. C, Scatchard analysis of WT LHR. 293T cells were transiently transfected with WT or C643/644G LHR. Approximately 40 h after transfection, the intact cells were subjected to [125I]hCG binding assay as described in Materials and Methods. D, Scatchard analysis of C643/644G LHR.

 
Effect of Mutation of Cys 643/644 on Ligand Binding
To determine whether the C643/644G mutant receptor is functionally similar to the WT hLHR, Scatchard analysis was performed at 4 C to compare the equilibrium binding parameters of the WT with the mutant receptor. Cells expressing mutant hLHR or WT hLHR were incubated with increasing concentrations of [125I]hCG at 4 C, and Scatchard analysis was performed as described in Materials and Methods. The analysis showed identical affinities (0.3 nM) for the receptors expressed in cells transfected with the WT and C643/644G LHR (Fig. 1Go, C and D). In the subsequent experiments, the amount of plasmid used in transfections was adjusted to obtain comparable levels of expression of WT and mutant LHR at the cell surface.

Effect of Mutation of C643 and C644 to Glycine on hCG-Induced Recycling of LHR
Before determining the effect of palmitoylation on recycling of the hLHR, hCG-induced internalization was examined in cells expressing WT or mutant hLHR as described in Materials and Methods. The results presented in Fig. 2Go show that the internalization of the mutant receptor was higher than the WT hLHR (Fig. 2AGo). Furthermore, the increased internalization occurs largely through an arrestin-mediated pathway as expression of a dominant-negative form of arrestin (319–418) significantly reduces internalization of the mutant LHR as well as the WT LHR (Fig. 2BGo). As expected, WT arrestin increased internalization of both WT and mutant receptor.



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Fig. 2. Effect of Mutation of Cys643 and Cys644 to Glycines on Ligand-Induced Internalization of hLHR

A, Internalization of WT and C643/644G LHR. 293 T cells transiently transfected with plasmids encoding for WT or C643/644G LHR were incubated with 100 ng/ml [125I]hCG for the indicated times. Cells were then washed to remove free [125I]hCG and treated with acidic buffer to release surface-bound [125I]hCG as described in Materials and Methods. Radioactivity associated with cells after treatment with acidic buffer represents the internalized [125I]hCG. Each point represents the mean ± SEM of triplicate determinations. B, Internalization of WT and C643/644G (indicated as GG) when coexpressed with WT or dominant-negative arrestin (319–418). The results in this figure are representative of two separate experiments and show the average of triplicate measurements ± SEM.

 
After internalization, most GPCRs are either recycled to the plasma membrane or targeted for degradation in the lysosomes. We next examined whether palmitoylation has a role in the postendocytic trafficking of the hLHR. Specifically, cells transiently transfected with cDNA encoding WT or mutant receptor were incubated with [125I]hCG for 2 h at 37 C to facilitate internalization. Cell surface-bound hormone was subsequently removed by acid wash, and the trafficking fate of the remaining cell-associated radioactivity was measured at the indicated times as described in Materials and Methods. As expected, Fig. 3Go shows that surface-bound radioactivity representing recycled receptor increased during the incubation time for both the WT and mutant receptor, but the fraction of WT LHR recycled to the cell surface was higher than that seen for cells expressing the mutant receptor (panel A). For both the WT and mutant receptor, secreted radioactivity also increased during the incubation time (panel B), whereas cell-associated radioactivity decreased (panel C). Whether the recycling property of the receptor is related to the cell surface receptor concentration was then tested. The results presented in Table 1Go show that the percent recycling of the WT LHR and C643/644G LHR was not related to the initial cell surface receptor concentration. Taken together, these results suggest that lack of palmitoylation reduces recycling of the internalized receptor.



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Fig. 3. Time Course and Fate of Internalized WT or C643/644G hLHR

Cells were transiently transfected with 0.68 µg of WT LHR or 1.4 µg of C643/644G LHR encoding plasmids in 35-mm wells. Approximately 40 h later, cells were preincubated in 1 ml of Waymouth’s MB752/1 media containing 1 mg/ml BSA and 20 mM HEPES (pH 7.4) for 30–60 min at 37 C. Cells were then incubated with 21 ng/ml of [125I]hCG for 2 h at 37 C. After washing three times to remove the free hormone, the surface-bound hormone was released from cells by exposure to an isotonic buffer (pH 3). This washing procedure was repeated once. Cells were then incubated for the indicated times in media containing 100 ng/ml of nonlabeled hCG as described in Materials and Methods. At the times indicated, the media were removed and saved. The cells were washed with cold medium, and this wash, together with the saved media, was counted to determine the secreted radioactivity (panel B). The cells were then exposed to the isotonic buffer (pH 3) two times to release internalized hormone that had recycled back to the surface (panel A). The acid-stripped cells were solubilized with NaOH, and the radioactivity associated with the cells is shown in panel C. Six 35-mm wells were used for each time point. Four wells contained [125I]hCG only to determine total binding, and two wells additionally contained an excess of nonlabeled hCG to determine nonspecific binding. The radioactivity associated with these two wells was used to correct for nonspecific binding. Results are shown from one experiment (average of four measurements ± SEM). % Recycled = [cell surface cpm at t = x/(cell surface cpm at t = x + cell-associated cpm at t = x + secreted cpm at t = x)] x 100. % secreted = [secreted cpm at t = x/(cell surface cpm at t = x + cell-associated cpm at t = x + secreted cpm at t = x)] x 100. % Cell associated = [cell-associated cpm at t = x/(cell surface cpm at t = x + cell-associated cpm at t = x + secreted cpm at t = x)] x 100.

 

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Table 1. Relationship between Cell Surface Receptor Expression and Receptor Recycling

 
We then tested whether other palmitoylation-deficient mutant receptors also undergo decreased recycling. Because our previous studies showed that the constitutively active mutant rat LHR receptor (D556G) undergoes reduced levels of palmitoylation (18), we first tested whether the constitutively active hLHR (D578H) is also resistant to palmitoylation by examining its ability to incorporate [3H]palmitic acid. Figure 4AGo shows that D578H incorporated significantly less [3H]palmitic acid compared with WT LHR. The reduced level of [3H]palmitic acid incorporation in the mutant was not due to decreased expression as determined by Western blot analysis (Fig. 4BGo). Scatchard analysis (Fig. 4Go, C and D) showed that the WT and mutant receptor had comparable binding affinity (0.3 nM and 0.2 nM, respectively). The ability of the palmitoylation-deficient mutant hLHR to undergo recycling was tested by transiently transfecting 293T cells with WT or D578H hLHR cDNA and measuring internalization and recycling. Consistent with previous observations (19, 20), the D578H mutant receptor underwent increased internalization as compared with the WT receptor after the initial 2-h incubation (data not shown). Examination of recycling of D578H hLHR showed that the mutant receptor underwent decreased recycling as compared with the WT receptor (Fig. 5Go, panel A). The results also showed that secreted radioactivity was increased in cells expressing (panel B) both WT and mutant receptor whereas cell-associated radioactivity was decreased (panel C). These results show that, like C643/644G LHR, another mutant receptor that is defective in palmitoylation (D578H LHR) also undergoes decreased recycling.



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Fig. 4. Palmitoylation of D578H LHR

A, Incorporation of [3H]palmitic acid into WT or D578H LHR expressed in 293 T cells. Transiently transfected cells were metabolically labeled with [3H]palmitic acid for 6 h. The cells were solubilized and the receptor analyzed as described in Materials and Methods. B, Western blot analysis of WT and D578H hLHR. LHR from transfected cells was immunoprecipitated and separated by SDS-PAGE before Western blot analysis was performed as described in Materials and Methods. C, Scatchard analysis of WT LHR. 293T cells were transiently transfected with WT LHR. Approximately 40 h after transfection, the intact cells were subjected to [125I]hCG binding assay as described in Materials and Methods. D, Scatchard analysis of D578H LHR.

 


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Fig. 5. Fate of Internalized WT and D578H LHR

Cells transfected with 1.7 µg of WT or D578H LHR encoding plasmid were incubated with 21 ng/ml [125I]hCG for 2 h to allow for internalization. Surface-associated radioactivity was then released by exposure of cells to an isotonic buffer (pH 3). The fate of the internalized receptor was then examined as described in Materials and Methods and in the legend of Fig. 3Go. Results shown are from one experiment (average of four measurements ± SEM). A, % Recycled = [cell surface cpm at t = x/(cell surface cpm at t = x + cell-associated cpm at t = x + secreted cpm at t = x)] x 100. B, % Secreted = [secreted cpm at t = x/(cell surface cpm at t = x + cell-associated cpm at t = x + secreted cpm at t = x)] x 100. C, % Cell associated = [cell-associated cpm at t = x/(cell surface cpm at t = x + cell-associated cpm at t = x + secreted cpm at t = x)] x 100.

 
The possibility that the reduced ability of palmitoylation-deficient mutants to undergo recycling (Figs. 3Go and 5Go) is a function of their tendency to undergo rapid internalization was then tested. To examine this, the cDNA concentrations of the WT and the mutant hLHR were altered to obtain equal amounts of internalized radioactivity after a 2-h incubation at 37 C (Fig. 6AGo). Following this incubation, recycling of WT LHR was still greater than C643/644G 2 h after internalization (Fig. 6BGo). Thus, the differing efficiencies of recycling of the WT and C643/644G hLHR are independent of the rate of internalization.



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Fig. 6. Recycling of Equivalent Amounts of Internalized WT and C643/644G LHR

A, Internalized radioactivity. Cells were transfected with 1.36 µg of WT or 0.68 µg of C643/644G LHR and incubated with [125I]hCG for 2 h at 37 C to facilitate internalization. The surface-bound [125I]hCG was removed by acid wash, and the amount of internalized radioactivity was determined. B, Recycled radioactivity. The cells described in panel A were then incubated at 37 C for an additional 2 h to allow for receptor recycling. The amount of recycled radioactivity was then determined by measuring surface-bound [125I]hCG.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GPCRs have been shown to undergo internalization after ligand binding, but the postendocytic processing of the receptors shows differences among members of the family (21). It is believed that upon agonist binding, GPCRs undergo phosphorylation, and the phosphorylated receptor interacts with arrestin, which in turn interacts with the heavy chain of clathrin as well as ß2-adaptin subunit of the clathrin adapter protein AP-2 to target the receptor to clathrin-coated pits at the cell surface (22). These pits are then removed from the cell surface by the action of dynamin, resulting in the formation of receptor containing endosomes (23). Sorting from the endosomes leads to either a recycling pathway, which is believed to require dephosphorylation before the receptor is targeted back to the cell surface, or the receptor may be targeted from the endosomes to the lysosomes for degradation (24). In the case of LHR, it has been previously reported that two motifs, a GT and a Cys and upstream Leu in the carboxyl terminus, play a role in recycling (14, 15, 16, 17). In the present studies we have shown that the palmitoylation state of the LHR also plays an important role in the postendocytic processing of the receptor. This is based on the observation that two palmitoylation-deficient hLHRs undergo a lower level of recycling compared with the WT receptor.

Accumulating evidence shows that palmitoylation is a dynamic process and that agonist and other cellular factors can result in depalmitoylation (reviewed in Ref. 25). In fact, the palmitoylation state of the ß2-adrenergic receptor has been shown to be regulated by agonist binding (26). Previous studies from our laboratory have shown that palmitoylation-deficient rat LHR is more prone to phosphorylation in response to phorbol 12-myristate 13 acetate (13). This is in agreement with previous reports that palmitoylation-deficient GPCR may be hyperphosphorylated (8, 27), which may result in increased accessibility of the C-terminal tail of GPCRs to the endocytic machinery, causing an increase in the rate of internalization.

The studies presented here show that palmitoylation promotes recycling of the hLHR as abrogation of palmitoylation significantly decreases recycling of this receptor. The results also show that palmitoylation is not the only determinant of recycling as the palmitoylation-deficient mutant was still capable of recycling although at a considerably reduced level. This is consistent with previous findings that other elements are also involved in LHR recycling (14, 15, 16, 17). The mechanism by which palmitoylation state of the receptor affects postendocytic processing of the hLHR is not understood. However, because recycling from endosomes to the cell surface is believed to require dephosphorylation, it would appear that the reduced level of recycling of the internalized depalmitoylated receptor might be related to its hyperphosphorylated state (Fig. 7Go) (13). Thus, it is possible that palmitoylation-deficient hyperphosphorylated receptors may be more resistant to dephosphorylation, which in turn would prevent efficient recycling leading to increased lysosomal degradation. The minimally phosphorylated, palmitoylated receptors may be more susceptible for dephosphorylation, and this pool is therefore efficiently recycled to the cell surface. This notion is consistent with a recent report on the neurokinin 1 receptor that showed inefficient recycling of the receptor when stimulated with agonist concentrations known to promote extensive phosphorylation (28). In contrast, recycling of the neurokinin 1 receptor was rapid after stimulation with agonist at concentrations known to promote minimal phosphorylation (28).



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Fig. 7. Schematic of Postendocytic Trafficking of WT and Palmitoylation-Deficient hLHR

Palmitoylation of the receptor is shown as two wavy lines attached to the intracellular, C-terminal tail of the receptor whereas phosphorylation is shown as filled circles attached to the C-terminal tail. Upon activation, the palmitoylated WT receptor is phosphorylated at the C-terminal tail, internalized, and targeted to the endosome where the receptor is dephosphorylated. After dephosphorylation, the receptor recycles back to the cell surface. The palmitoylation-deficient receptor is also internalized to the endosome where, because of hyperphosphorylation, it may be more resistant to dephosphorylation, thereby targeting it for degradation by the lysosome.

 
Interestingly, D578H, a naturally occurring mutant of the hLHR was shown to incorporate less [3H]palmitic acid compared with WT LHR. Similar to the palmitoylation-deficient C643/644G hLHR, recycling of D578H hLHR was also defective. That both mutants incorporate less palmitic acid and undergo decreased recycling strongly suggests that lack of palmitoylation reduces recycling of internalized receptor back to the cell surface. Although both mutants underwent increased internalization, our data (Fig. 6Go) suggest that palmitoylation, not the rate of internalization, regulates the extent of receptor recycling.

The results presented here suggest that the ligand-mediated transient down-regulation of the LHR may be partially regulated by depalmitoylation, which increases receptor internalization. Because the depalmitoylated receptor is likely to be hyperphosphorylated, the internalized receptor may be targeted for degradation rather than recycled to the cell surface. In conclusion, we have established that the hLHR is palmitoylated in the C-terminal tail and that this posttranslational modification promotes receptor recycling.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DMEM, Waymouth’s MB 752/1 media, and Hanks’ buffered saline solution (HBSS) were purchased from Life Technologies (Gaithersburg, MD). FLAG antibodies and hCG were obtained from Sigma Chemical Co. (St. Louis, MO). Enhanced chemiluminescence detection system was purchased from Amersham Pharmacia Biotech (Arlington Heights, IL). Na125I was purchased from NEN Life Science Products (Boston, MA). [3H]palmitic acid was obtained from PerkinElmer Corp. (Boston, MA). The QuikChange XL site-directed mutagenesis kit was purchased from Stratagene (La Jolla, CA). cDNA constructs for the hLHR and D578H LHR were kindly provided by Andrew Shenker (Northwestern University, Evanston, IL). cDNA constructs for WT and dominant-negative arrestin were generously provided by Jeffrey L. Benovic (Thomas Jefferson University, Philadelphia, PA).

Site-Directed Mutagenesis
C643/644 LHR was prepared by site-directed mutagenesis using Stratagene’s QuikChange XL kit according to the manufacturer’s instructions using template LHR cDNA constructs. The following oligonucleotide and its complementary strand were used for mutagenesis: 5'-CTGAGCAAATTTGGCGGCGGTAAACGTCGGGCTG-3'.

Transient Expression of hLHR in 293T Cells
Human embryonic kidney cells (293T cells) were grown in DMEM as previously described (13). Cells in exponential growth phase were plated 5–9 h before transfection at a density of 6 x 105 per 35-mm well, 2 x 106 for 6-cm plates, and 4–5 x 106 for 10-cm plates. Cells were transfected by the calcium phosphate coprecipitation method with 5 µg WT cDNA, 11 µg C643/644G, 4.3 µg D578H, and 10 µg of vector (pFLAG-CMV-1) per 10-cm plate for [3H]palmitic acid incorporation and Western blot analysis. For Scatchard analysis, cells were transfected with 5 µg WT cDNA or 11 µg of either C643/644G or D578H cDNA.

Immunoprecipitation and Western Blot Analysis
Transfected cells were harvested approximately 40 h after transfection and lysed in PBS 1% Triton X-100 containing protease inhibitors. The cell lysate was then precleared with protein A agarose and then centrifuged at 14,000 x g for 5 min. The precleared lysate was incubated with M2 FLAG antibody conjugated to agarose beads as per the manufacturer’s instructions (Sigma). Protein A agarose was then added, and the samples were incubated for an additional hour at 4 C. After extensive washes, the proteins were eluted from the beads with sodium dodecyl sulfate loading buffer containing 50 mM dithiothreitol and 5% ß-mercaptoethanol. Proteins were then separated by SDS-PAGE and transferred to nitrocellulose. Western blot analysis was performed using M2 FLAG antibody as the primary antibody (1:1,000) and a horseradish peroxidase-conjugated secondary antibody (1:10,000). The receptor was detected using enhanced chemiluminescence according to the manufacturer’s instructions (Amersham).

[3H]Palmitic Acid Incorporation
Cells expressing WT or mutant LHR were labeled with [3H]palmitic acid as previously described (6). Briefly, transfected cells were preincubated for 20 min with serum-free DMEM containing 10 mM HEPES. After preincubation, media were replaced with serum-free media containing 285 µCi/ml [3H]palmitic acid and incubated at 37 C for 6 h under 5% CO2. Before use, [3H]palmitic acid was treated with 0.3 ml of serum-free medium containing 0.2% fatty acid-free BSA and diluted with 3.2 ml of the serum-free medium to a final concentration of 0.017% fatty acid-free BSA. LHR was then immunoprecipitated as described above and separated by SDS-PAGE under reducing conditions. Fluorography was performed with Enlightening (NEN Life Sciences Products) according to the manufacturer’s protocol. The gels were dried at 80 C for 2 h and exposed to Kodak X-Omat AR film (Eastman Kodak Co., Rochester, NY) at –70 C for 5–16 d.

[125I]hCG Binding to Transfected Cells
Scatchard analysis was performed as previously described by our laboratory with slight modification (6). Briefly, cells were incubated with increasing concentrations of [125I]hCG (5–300 ng/ml) at 4 C for 20 h with or without a 1000-fold excess of unlabeled hCG to determine nonspecific binding. The cells were then washed twice at 4 C with Waymouth’s media. The radioactivity remaining with the cell pellet was then counted in a Gamma Trac 1290-counter (Tm Analytic, Elk Grove Village, IL). Specific binding was determined by subtracting nonspecific binding from total binding.

Receptor-Mediated [125I]hCG Internalization Assay
Internalization was performed as previously described from our laboratory (6, 29). Briefly, 4–5 x 106 cells were transiently transfected in 10-cm cell culture dishes with plasmids containing WT (2.24 µg) or C643/644G (7.7 µg) hLHR. Forty hours after transfection, cells were harvested and resuspended in Waymouth’s medium containing 0.1% BSA. Cells were then incubated with 100 ng/ml of [125I]hCG for the indicated times at 37 C. Nonspecific binding was measured by adding excess nonlabeled hCG to designated tubes. The internalization reactions were terminated by the addition of 2 ml of cold Waymouth’s medium, and the cells were pelleted at 250 x g for 5 min at 4 C to remove unbound [125I]hCG. The supernatant was removed, and this wash was repeated. To remove surface-bound [125I]hCG, the cell pellets were incubated with a low pH buffer (100 mM NaCl; 50 mM glycine, pH 3.0) at 4 C for 5 min. This procedure was repeated, and both acid washes were pooled and assayed for 125I using a {gamma}-counter. The radioactivity associated with the pellet was then determined. The radioactivity associated with the acid washes represents the surface-bound hormone whereas the radioactivity associated with the pellet after acid wash represents internalized hormone. Nonspecific binding was subtracted from total binding to calculate specific binding.

Fate of Internalized Hormone
The fate of internalized hormone was examined to determine recycling using a previously published procedure (30) with slight modification. Briefly, cells expressing WT or mutant LHR were preincubated in 1 ml of Waymouth’s MB752/1 medium containing 1 mg/ml BSA and 20 mM HEPES (pH 7.4) for 30–60 min at 37 C. Cells were then incubated with 21 ng/ml [125I]hCG for 2 h at 37 C to allow for internalization to occur. To account for nonspecific binding, 25 µg of the unlabeled hCG were added to designated plates, and [125I]hCG binding detected in cells from these plates was subtracted from total binding to obtain the specific binding. After the 2 h, cells were washed three times with cold HBSS containing 0.1% BSA to remove excess ligand associated with the cells. Surface receptor-bound ligand was then removed with acid washes performed at 4 C for 2 min as described earlier. The washing was repeated once. The acid washed cells were then washed once with Waymouth’s containing BSA. All washes were counted separately to determine the total amount of surface-bound radioactivity. To follow the fate of the internalized receptor, cells were incubated for the indicated times in Waymouth’s/BSA containing 100 ng/ml of nonlabeled hCG to prevent reassociation of any undegraded [125I]hCG secreted into the media. After the indicated times, medium was removed and saved. Cells were washed once with cold HBSS/BSA, and this buffer as well as the saved media were counted. The nonspecific radioactivity in the wash media was found to be equal to the background level, showing that all of the nonspecifically bound [125I]hCG had been removed. The total radioactivity in the saved media and the wash represents total secreted radioactivity. The [125I]hCG reappearing on the cell surface represents recycled [125I]hCG. To determine the amount of recycled receptor, surface-bound [125I]hCG was removed with two acid washes, 2 min each, which were counted to determine the amount of recycled hormone. Finally, cells were solubilized with 0.5 N NaOH, and the radioactivity associated with the cells was collected and assayed to determine the amount of cell-associated hormone. For a typical 4-h recycling experiment shown in Fig. 3Go, specific [125I]hCG binding for the WT LHR was 43,133 ± 1062 cpm. Of the original radioactivity, at the 2-h time interval, 12,712 ± 524 cpm were secreted into the medium, 11,313 ± 341 cpm were recycled, and 19,107 ± 360 cpm were cell associated.


    FOOTNOTES
 
This work was supported by National Institutes of Health (NIH) Grant HD R37 06656. U.M.M. is a predoctoral fellow supported by NIH Training Grant 5 T32 HD 07048. C.L.C. is a predoctoral fellow supported by NIH Training Grants 5 T32 HD 07048 and T3 GM 08353.

First Published Online November 11, 2004

1 Both U.M.M. and C.L.C. contributed equally to this work and should both be considered first authors. Back

Abbreviations: CG, Chorionic gonadotropin; GPCR, G protein-coupled receptor; HBSS, Hanks’ balanced saline solution; LHR, LH receptor; WT, wild-type.

Received for publication August 25, 2004. Accepted for publication November 1, 2004.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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