Phosphorylation of the Receptor for PTH and PTHrP Is Required for Internalization and Regulates Receptor Signaling

Hesham A. W. Tawfeek, Fang Qian and Abdul B. Abou-Samra

Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have previously shown that agonist-dependent phosphorylation of the PTH/PTHrP receptor occurs on its carboxyl-terminal tail. Using site- directed mutagenesis, phosphopeptide mapping, and direct sequencing of cyanogen bromide-cleaved fragments of phosphoreceptors, we report here that PTH-dependent phosphorylation occurs on the serine residues at positions 491, 492, 493, 495, 501, and 504, and that the serine residue at position 489 is required for phosphorylation. When these seven sites were mutated to alanine residues, the mutant receptor was no longer phosphorylated after PTH stimulation. The phosphorylation-deficient receptor, stably expressed in LLCPK-1 cells, was impaired in PTH-dependent internalization and showed an increased sensitivity to PTH stimulation; the EC50 for PTH-stimulated cAMP accumulation was decreased by 7-fold. Furthermore, PTH stimulation of the phosphorylation-deficient PTH/PTHrP receptor caused a sustained elevation in intracellular cAMP levels. These data indicate that agonist-dependent phosphorylation of the PTH/PTHrP receptor plays an important role in receptor function.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PTH MAINTAINS calcium ion homeostasis by binding to a specific G protein-coupled receptor located on bone and kidney cells, which also binds PTHrP (1, 2). PTHrP regulates bone development and differentiation by interacting with the PTH/PTHrP receptor found on chondrocytes and bone stromal cells (3, 4, 5, 6). Binding of PTH or PTHrP to the PTH/PTHrP receptor stimulates the accumulation of several intracellular second messengers, such as cAMP and IP3 (2, 7), increases the phosphorylation of the PTH/PTHrP receptor on several serine residues within its carboxyl-terminal tail (8, 9), stimulates receptor internalization (10, 11, 12), and causes desensitization of the PTH/PTHrP receptor-second messenger system (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)

It has been shown that agonist-stimulated phosphorylation of the PTH/PTHrP receptor is not dependent on PKA or PKC (9, 24). Similarly, activation of PKA and PKC is neither necessary nor required for agonist-stimulated receptor internalization (12, 25). However, ligand-stimulated internalization of the PTH/PTHrP receptor requires occupancy with an agonist (12, 25). These data, showing similarities in the cellular mechanisms involved in receptor phosphorylation and receptor internalization, suggest that phosphorylation of the PTH/PTHrP receptor may play a role in receptor internalization.

The role of PTH/PTHrP receptor phosphorylation in internalization and signaling has been challenged by two recent studies. Malecz et al. (8) reported that a phosphorylation-deficient opossum PTH/PTHrP receptor stably expressed in HEK 293 cells internalizes normally. Overexpression of G protein receptor kinases (GRKs) together with a C-terminally truncated PTH/PTHrP receptor in COS-1 cells inhibited signaling through mechanisms other than receptor phosphorylation (26).

Altogether, the available data suggest that phosphorylation of the PTH/PTHrP receptor is neither required for receptor internalization (8) nor involved in regulating PTH/PTHrP receptor signaling (26).

In a preliminary report we have shown that mutating the potential phosphorylation sites for the PTH/PTHrP receptor impairs its internalization (27). This study was therefore performed to fully investigate the role of receptor phosphorylation in internalization and PTH-stimulated cAMP accumulation. Here we mapped the PTH-dependent phosphorylation sites and demonstrated that receptor phosphorylation is required for efficient internalization and that it influences PTH-stimulated cAMP accumulation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Fine Mapping of the PTH-Dependent Phosphorylation Sites
We have previously mapped the phosphorylation sites within the PTH/PTHrP receptor to two CNBr-cleaved fragments, F1 and F2, which were resolved on tricine SDS-PAGE as heterogeneous bands with apparent molecular masses of 8 and 10 kDa, respectively. These two bands correspond to the fragments flanked by the methionine residues at positions 445 and 499, and 499 and 553, respectively (9). To characterize the phosphorylated residues, we performed clustered and single residue mutations using site-directed mutagenesis. The potential phosphorylation sites in the F1 fragment are predicted to occur after position 480 (9); these sites are 489, 491, 492, 493, and 495. F2 contains 13 potential phosphorylation sites occurring at positions 501, 503, 504, 506, 515, 519, 526, 527, 531, 544, 546, 548, and 552. Single and cluster mutations were performed on each of the potential phosphorylation sites; the mutant receptors were expressed in COS-7 cells and challenged with PTH in presence of [32P]orthophosphate. The phosphorylated receptor was immunoprecipitated, cleaved with CNBr, and analyzed on tricine-SDS-PAGE. None of the single mutations showed any detectable change in the phosphorylation of the intact uncleaved receptor (data not shown). In contrast, after CNBr cleavage, some single and combined mutations resulted in decreased phosphorylation of the F1 or F2 fragment. Data from 20 receptor constructs bearing single or combined mutations indicated that receptor phosphorylation was decreased when any of the following residues was mutated: S489, S491, S492, S493, S495, S501, and S504. These results suggest that these sites are either phosphorylated or required for phosphorylation.

To distinguish between the two possibilities, we performed radiosequencing of the carboxyl-terminal tail of the PTH/PTHrP receptor. To facilitate sequencing of CNBr-cleaved phosphopeptides, we mutated the alanine residue at position 480 to methionine; this mutation places the first potential phosphorylation site at the eighth sequencing cycle. Additionally, we mutated the methionine residue at position 499 to alanine; this mutation resulted in a single phosphorylated CNBr-cleaved fragment (instead of the two fragments, F1 and F2). The A480M/M499A mutant receptor had normal expression, ligand binding, and PTH-stimulated cAMP accumulation (data not shown). Phosphorylation of the A480M/M499A receptor resulted in a single CNBr-cleaved fragment as predicted (Fig. 1AGo, lane 3). The CNBr-cleaved fragment was eluted from SDS-PAGE and subjected to amino-terminal microsequencing, and the radioactivity released during each cycle was counted and plotted (Fig. 1BGo, circles). Residue S489 was associated with the release of an insignificant amount of radioactivity, suggesting that S489 is not phosphorylated. In contrast, amino acid S491 was associated with the release of the highest amount of radioactivity; this indicated that this residue is frequently phosphorylated upon PTH challenge. The radioactivity decreased only slightly at amino acid S492 sequencing and then increased slightly at S493, suggesting either that both of these residues are phosphorylated or that only S493 is phosphorylated. The residues S495, S501, and S504 had a small, but consistent, increase in radioactivity, suggesting that these sites are phosphorylated. The high radioactivity released with S491 might have overshadowed the radioactivity released in the subsequent sequencing cycles. Therefore, the sequencing was repeated in an A480M/M499A receptor mutant in which the S491 site was mutated to alanine. Functional characterization of the A480M/M499A/S491A mutant revealed normal expression, ligand binding, and PTH stimulation of cAMP accumulation (data not shown). Phosphorylation of the A480M/M499A/S491A mutant resulted in a single CNBr-cleaved fragment similar to that produced with the A480M/M499A double mutant (Fig. 1AGo, lane 2). The sequencing of the phosphorylated CNBr-cleaved fragment of the A480M/M499A/S491A mutant indicated that both S492 and S493 are phosphorylated. The released radioactivity increased with S492 and was further increased with S493 (Fig. 1BGo, triangles). Furthermore, the S501 and S504 cycles showed clear peaks of radioactivity that were released with each of these sequencing cycles (Fig. 1BGo, triangles).



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Figure 1. Radiosequencing of the Phosphorylated PTH/PTHrP Receptor Fragments

A, Mutant PTH/PTHrP receptor constructs carrying double mutations, A480M/M499A, or triple mutations, S491A/A480M/M499A, were expressed in COS-7 cells, 32P-phosphorylated in the presence of PTH, immunoprecipitated with the PTH/PTHrP receptor antiserum beads, CNBr-cleaved, desalted, lyophilized, analyzed on SDS-PAGE, and autoradiographed for 1 h. B, The radioactive band was cut from the gel, and the phosphorylated peptide was electroeluted, desalted, lyophilized, and sequenced on a pulsed liquid-gas phase sequencer (PE Applied Biosystems, Foster City, CA, model 477A) as indicated in Materials and Methods. The amount of radioactivity released was counted for 20 min and plotted as counts per min. The experiment was repeated twice (for each construct) with similar results.

 
Recently, it was reported that phosphorylation of the recombinant opossum PTH/PTHrP receptor expressed in HEK 293 cells occurs on six serine residues (8). Therefore, the corresponding six serine residues in the rat PTH/PTHrP receptor sequence, S489, S491, S492, S493, S495, and S504, were mutated in a single construct (6SA). We also constructed another mutant in which S489, S491, S492, S493, S495, S501, and S504 were mutated to alanine residues (7SA). The 6SA and 7SA mutant receptors were expressed in COS-7 cells to the same level as the wild-type (WT) receptor (data not shown). Mutant 6SA had a dramatic decrease in PTH-stimulated phosphorylation; however, a slight, but detectable, phosphorylation was observed after PTH challenge (Fig. 2Go). In contrast, the mutant 7SA had no detectable basal or ligand-dependent phosphorylation (Fig. 2Go).



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Figure 2. Phosphorylation of the PTH/PTHrP Receptor Mutants Bearing Six or Seven Serine to Alanine Mutations (6SA or 7SA)

WT or mutant receptors were expressed in COS-7 cells. The cells were labeled with 32P for 2 h, and NlePTH (200 nM) or vehicle was added during the last 40 min of the labeling period. The cells were lysed with RIPA buffer, and the lysates were immunoprecipitated with PTH/PTHrP receptor antiserum beads as indicated in Materials and Methods, immunoprecipitated (no CNBr treatment), analyzed on SDS-PAGE, and autoradiographed for 48 h. The experiment was repeated three times with similar results.

 
The Phosphorylation-Deficient (PD) PTH/PTHrP Receptor Functions Normally in COS-7 Cells
Ligand binding and cAMP stimulation properties of the PD mutant receptor (7SA) were examined in COS-7 cells and compared with those of the WT receptor (Fig. 3Go). The PD mutant receptor bound PTH normally (Fig. 3AGo) and increased cAMP accumulation to the same levels as the WT receptor (Fig. 3BGo). Cell surface receptor expression of the PD mutant receptor was similar to that of the WT receptor (Fig. 3CGo). As shown above, stimulation of the PD receptor did not result in receptor phosphorylation (Fig. 3DGo), and Western blot analysis of the immunoprecipitates revealed a similar amount of immunoreactivity in both PD and WT receptors (Fig. 3EGo).



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Figure 3. Characterization of the PD PTH/PTHrP Receptor Transiently Expressed in COS-7 Cells

WT or PD PTH/PTHrP receptors were transiently expressed in COS-7 cells. A, PTH binding. Cells grown in 24-well plates were rinsed (twice) with 1 ml binding buffer (50 mM Tris-HCl, 100 mM NaCl, 5 mM KCl, 2 mM CaCl2, 5% heat-inactivated horse serum, and 0.5% FBS, pH 7.7) and incubated with [125I]NlePTH in binding buffer at 15 C for 2 h in the presence of nonradioactive NlePTH (0–1000 nM). Cells were then washed (three times) with cold PBS and solubilized with 1 N NaOH, and the radioactivity in the whole lysate was determined using a gamma-counter (model 6/400 Plus, Micromedic Systems, Inc., Horsham, PA). B, PTH-stimulated cAMP accumulation. The cells were challenged with increasing concentrations of NlePTH in presence of IBMX (2 mM) for 20 min at 37 C. Intracellular cAMP was measured using a specific RIA. C, Cell surface PTH/PTHrP receptor immunoreactivity was measured using an antibody binding assay. The data are the mean ± SD of three experiments. D, Basal and PTH-stimulated (200 nM) receptor phosphorylation was determined as described above. E, Western blot analysis of the immunoprecipitate to control for the amount of immunoprecipitated receptors. The experiments were repeated three times with similar results.

 
Phosphorylation Deficiency of the PTH/PTHrP Receptor Impairs Internalization and Increases Sensitivity for PTH Stimulation
To understand the role of receptor phosphorylation in receptor internalization, we developed LLCPK-1 cell lines stably expressing WT or PD PTH/PTHrP receptors. The cell lines were screened for ligand binding and PTH-stimulated cAMP accumulation and selected as previously described (7). WT6 and PD7 cell lines expressing similar numbers of WT and PD PTH/PTHrP receptors, respectively, were selected (Fig. 4Go). Radioligand binding was similar in both cell lines (Fig. 4AGo). PD7 cells had an increased potency for PTH-stimulated cAMP accumulation compared with WT6 cells (Fig. 4BGo); the EC50 for PTH-stimulated cAMP accumulation decreased by 7-fold (P < 0.05; n = 4).



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Figure 4. Characterization of the PD PTH/PTHrP Receptor Stably Expressed in LLCPK-1 Cells

LLCPK-1 cell lines stably expressing the WT (WT6) or the PD (PD7) PTH/PTHrP receptors were developed as explained in Materials and Methods. A, Binding of [125I]NlePTH (100,000 cpm/well) to confluent cells in 24-well plates was performed as described in Fig. 3Go. Specific binding (mean ± SD of binding in the presence of nonradioactive PTH, B/maximum binding, B0) of triplicate determinations was plotted on the y-axis. The B0 for WT6 and PD7 cells was 20% and 18% of the total added radioactivity, respectively. The data are the mean ± SD of three experiments. B, PTH-stimulated cAMP accumulation. The cells were challenged with increasing concentrations of NlePTH in presence of IBMX (2 mM) for 20 min at 37 C. Intracellular cAMP was measured using a specific RIA. The basal values for both WT6 and PD7 cell lines were less than 8 pmol/well, and the maximal stimulation levels were 294 ± 12 and 132 ± 6 pmol/well, respectively. The data are the mean ± SD of three experiments.

 
To test whether phosphorylation is required for internalization, we examined agonist-dependent internalization of the PD PTH/PTHrP receptor. PTH challenge (100 nM, 37 C, 5–100 min) significantly (P < 0.01) decreased cell surface PTH/PTHrP receptor immunoreactivity in WT6 cells (Fig. 5Go). In contrast, a similar treatment did not decrease cell surface receptor immunoreactivity in PD7 cells (Fig. 5Go).



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Figure 5. Internalization of the PD PTH/PTHrP Receptor

Effects of PTH on cell surface PTH/PTHrP receptor immunoreactivity. LLCPK-1 cells stably expressing WT (WT6) or PD (PD7) PTH/PTHrP receptors were incubated with NlePTH (100 nM, 37 C) for different time periods. Cells were then rinsed with ice-cold PBS and incubated with the PTH/PTHrP receptor antiserum (G48) for 120 min at 4 C, with a rabbit antisheep IgG antiserum for 90 min, then with an 125I-labeled goat antirabbit IgG (200,000 cpm/well) for another 90 min. After each incubation the cells were rinsed (three times). The amount of cell-associated radioactivity was measured and is reported on the y-axis. The antibody binding to the control (cells treated with vehicle) for WT6 and PD7 cells was 6% and 5% of the total added radioactivity, respectively. The data are the mean ± SD of three experiments.

 
To study real-time internalization of the PD PTH/PTHrP receptor, we constructed a green fluorescent protein (GFP)-tagged PD PTH/PTHrP receptor by inserting the epitope tag in the middle of the amino-terminal sequence of the receptor at a site that was shown previously not to disturb receptor functions (28). The GFP-tagged PD receptor was stably expressed in LLCPK-1 cells. Three cell lines, PD-GFP2, PD-GFP8, and PD-GFP11, were selected. PTH binding in the three cell lines was not different from that observed in the cell lines expressing the GFP-tagged WT receptor (Fig. 6AGo). The EC50 for PTH-stimulated cAMP accumulation in the PD-GFP cell lines decreased by an average of 7-fold compared with those expressing the GFP-tagged WT receptors (Fig. 6BGo); this observation is similar to that obtained in cell lines expressing non-GFP-tagged receptors. In some PD receptor cell lines maximal cAMP stimulation was increased; however, this increase was not consistent among all PD receptor cell lines. On the other hand, all PD receptor cell lines showed decreased EC50. We compared the effects of PTH on cell surface receptor immunoreactivity in these cells expressing GFP-tagged WT and GFP-tagged PD PTH/PTHrP receptors. All cell lines expressing the PD PTH/PTHrP receptor showed an impaired internalization of the PTH/PTHrP receptor after PTH challenge (Fig. 7Go, upper panel).



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Figure 6. Characterization of the GFP-Tagged PD PTH/PTHrP Receptor Stably Expressed in LLCPK-1 Cells

LLCPK-1 cell lines stably expressing GFP-tagged WT (WT-GFP27 and WT-GFP36) or GFP-tagged PD (PD-GFP2, PD-GFP8, and PD-GFP11) PTH/PTHrP receptors were developed as explained in Materials and Methods. A, Binding of [125I]NlePTH (100,000 cpm/well) to confluent cells in 24-well plates was competed off by increasing concentrations of unlabeled NlePTH. Specific binding (mean ± SD of the binding in the presence of nonradioactive PTH, B/maximum binding, B0) of triplicate determinations was plotted on the y-axis. The B0 for WT-GFP27, WT-GFP36, PD-GFP11, PD-GFP8, and PD-GFP2 cells was 21%, 19%, 18%, 10%, and 13% of the total added radioactivity, respectively. The data are the mean ± SD of three experiments. B, PTH-stimulated cAMP accumulation. The cells were challenged with increasing concentrations of NlePTH in the presence of IBMX (2 mM) for 20 min at 37 C. Intracellular cAMP (mean ± SD of triplicate determinations) was measured using a specific RIA. The basal values for all cell lines were less than 8 pmol/well. The maximal stimulation levels for WT-GFP27, WT-GFP36, PD-GFP11, PD-GFP8, and PD-GFP2 cells were 244 ± 5, 433 ± 26, 471 ± 30, 162 ± 7, and 421 ± 24 pmol/well, respectively. The data are the mean ± SD of three experiments.

 


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Figure 7. Internalization of PTH/PTHrP Receptor and Its Ligand in LLCPK-1 Cells Stably Expressing GFP-Tagged WT or GFP-Tagged PD PTH/PTHrP Receptor

Upper panel, Effects of PTH on cell surface PTH/PTHrP receptor immunoreactivity. LLCPK-1 cells stably expressing GFP-tagged WT (WT-GFP27) or GFP-tagged PD (PD-GFP2 and PD-GFP11) PTH/PTHrP receptors were incubated with NlePTH (1–1000 nM, 40 min, 37 C), then cell surface receptor immunoreactivity was measured as described above. The antibody binding to the control (cells treated with vehicle) for WT-GFP27, PD-GFP2, and PD-GFP11 cells was 7%, 4%, and 6% of the total added radioactivity (200,000 cpm/well), respectively. The data are the mean ± SD of three experiments. Middle panel, Effects of PTH (100 nM, 40 min) on the subcellular localization of GFP-tagged WT and GFP-tagged phosphorylation-deficient PTH/PTHrP receptors. LLCPK-1 cells stably expressing GFP-tagged WT (WT-GFP27) or GFP-tagged PD (PD-GFP11) PTH/PTHrP receptors were incubated with NlePTH (100 nM, 40 min, 37 C), then fixed and examined using a confocal microscope. The experiment was repeated three times with similar results. Lower panel, Effects of phosphorylation deficiency on ligand internalization. LLCPK-1 cells stably expressing GFP-tagged WT (WT-GFP27) or GFP-tagged PD (PD-GFP11) PTH/PTHrP receptors were incubated with radioiodinated PTH-(1–34) (100,000 cpm/well) for 4 h on ice. At the end of the incubation, the unbound ligand was washed twice using ice-cold PBS and replaced with medium. The cells were then incubated at 37 C for 0, 10, 30, 40, 60, 120, or 240 min. For each incubation, the free, the surface-bound or acid-sensitive (collected using acid wash buffer), and the internalized or acid-resistant (collected using 1 M NaOH) fractions were collected and counted. The data are the percentage ± SD of the acid-resistant/total radioactivity ratio in the three fractions, determined three times. Total binding at time zero was 6300 ± 300 and 6000 ± 250 cpm for WT-GFP27 and PD-GFP11, respectively. Nonspecific binding was less than 0.5% of the total added radioactivity.

 
As previously reported (12), cells stably expressing a GFP-tagged WT PTH/PTHrP receptor (WT-GFP27) showed green fluorescence at the periphery of the cells (Fig. 7Go, middle panel). Treatment with PTH (100 nM, 40 min, 37 C) caused a rapid redistribution of the green fluorescence with the appearance of dense vesicles in the cytoplasm (Fig. 7Go, middle panel). In nontreated PD-GFP11 cells, the receptors were also localized to the periphery of the cells (Fig. 7Go, middle panel). However, treatment with PTH had a slight effect on receptor internalization and resulted in the appearance of only few dense cytoplasmic vesicles (Fig. 7Go, middle panel).

To further study the role of receptor phosphorylation in internalization, we examined internalization of radioiodinated PTH in WT-GFP27 and PD-GFP11 cells. Cells expressing the PD receptor showed impaired radioligand internalization (Fig. 7Go, lower panel). The radioactivity in the acid-resistant fraction (internalized) was significantly higher in the WT than in the PD receptor cell lines, was maximum at 60 min of incubation (Fig. 7Go, lower panel), and then decreased at 120 and 240 min. The decrease in the radioactivity in the acid-resistant fraction for the WT receptor cells at 120 and 240 min was accompanied by increased free radioactivity in the medium (data not shown); this indicates the release of the internalized radioligand in the medium.

The PD PTH/PTHrP Receptor Shows a Sustained cAMP Elevation after PTH Stimulation
To examine whether phosphorylation of the PTH/PTHrP receptor plays a role in the termination of signaling and recovery of PTH-induced cAMP responses, cell lines stably expressing PD PTH/PTHrP receptors were challenged with PTH (10 nM) for 10 min; PTH was then removed, and the cells were allowed to recover for 0–60 min at 37 C in the absence of PTH. Accumulation of intracellular cAMP was measured in the presence of isobutylmethylxanthine (IBMX; 2 mM) for 10 min at the end of each recovery period. The levels of cAMP accumulation in the cells expressing WT receptors rapidly decreased toward the basal level observed in nontreated cells (Fig. 8AGo). In contrast, the cell lines expressing PD PTH/PTHrP receptors had sustained cAMP elevation; intracellular cAMP levels remained higher than those found in the cells expressing WT receptors (Fig. 8AGo).



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Figure 8. Recovery of PTH-Induced cAMP Responses in the PD PTH/PTHrP Receptor Cell Lines

A, Sustained elevation of intracellular cAMP levels in the phosphorylation-deficient PTH/PTHrP receptor cell lines. LLCPK-1 cell lines stably expressing WT (WT6), PD (PD7), GFP-tagged WT (WT-GFP27 and WT-GFP36), or GFP-tagged PD (PD-GFP2 and PD-GFP11) PTH/PTHrP receptors were treated with 10 nM PTH for 10 min at 37 C. PTH was then removed, and the cells were rinsed with ice-cold PBS and allowed to recover for different periods of time (0–60 min) at 37 C. At the end of each recovery period, intracellular cAMP accumulation was measured for 10 min in the presence of IBMX (2 mM). The data are presented as a percentage of the control value (cAMP response to 10 nM PTH in the presence of 2 mM IBMX for 10 min in control cells). The basal cAMP levels for all cell lines were less than 8 pmol/well. The stimulated values in control cells were 147 ± 3, 139 ± 13, 234 ± 24, 186 ± 4, 295 ± 13, and 217 ± 18 pmol/well for WT6, WT-GFP27, WT-GFP36, PD7, PD-GFP11, and PD-GFP2 cells, respectively. The data are the mean ± SD of three experiments. B and C, Desensitization in WT and phosphorylation-deficient PTH/PTHrP receptor cell lines. LLCPK-1 cell lines stably expressing WT (B) or PD (C) PTH/PTHrP receptors were treated with vehicle (control) or 100 nM PTH (pretreated) for 60 min at 37 C. The cells were placed on ice and washed initially by ice-cold PBS, followed by a 2-min acid wash buffer (50 mM glycine and 150 mM NaCl, pH 3 using acetic acid), and finally by ice-cold PBS. Cyclic AMP accumulation was measured in response to a second challenge with different doses of PTH in the presence of IBMX (2 mM) for 30 min at 37 C. The data are the mean ± SD of three experiments performed in four cell lines, two expressing WT receptors (one GFP tagged and one non-GFP tagged) and two expressing PD receptors (one GFP tagged and one non-GFP tagged).

 
We also examined in classic desensitization experiments whether PTH-stimulated cAMP accumulation desensitizes after a second PTH challenge. LLCPK-1 cells stably expressing WT (WT6 and WT-GFP27) or PD PTH/PTHrP (PD7 and PD-GFP11) receptors were treated with PTH (100 nM) or vehicle for 60 min at 37 C (Fig. 8Go). The cells were placed on ice, and PTH was removed by an initial ice-cold PBS wash, followed by a 2-min acid wash (50 mM glycine and 150 mM NaCl, pH 3, using acetic acid) and a final ice-cold PBS wash. Cyclic AMP accumulation was measured in response to a second challenge with different doses of PTH in the presence of IBMX. In cells expressing WT receptors, cAMP levels decreased toward basal levels (Fig. 8BGo, pretreated 0), and a second PTH challenge resulted in a robust cAMP response similar to that of control cells (Fig. 8BGo, pretreated). In contrast, cells expressing PD receptors showed sustained elevation of cAMP levels (Fig. 8BGo, pretreated 0). Similar results were obtained in cells pretreated with PTH for shorter time periods (5, 20, and 40 min; data not shown). This sustained elevation did not increase in response to a second PTH challenge (Fig. 8BGo, pretreated). Although acid wash decreased basal cAMP in PTH-pretreated WT cells, cAMP levels in PTH-pretreated PD receptor cells remained markedly elevated. As acid wash removed all bound radioligand from the cell surface receptors of both WT and PD receptor cells (data not shown), the sustained cAMP levels in PTH-pretreated PD receptor cells is not secondary to residual ligand occupancy.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Stimulation of G protein-coupled receptors results in the activation of second messenger systems, receptor phosphorylation and internalization, and desensitization of the receptor-effector responsiveness. It has been reported that the PTH/PTHrP receptor is phosphorylated after challenge with PTH (8, 9, 24), that the phosphorylation acceptor sites occur in the cytoplasmic tail (8, 9), and that PTH/PTHrP receptor is a substrate for GRK2, -3, and -5 (8, 26) but not for PKA and PKC (9, 24). Mapping of the phosphorylation sites using CNBr cleavage of WT receptor identified two phosphopeptides from the cytoplasmic tail to be phosphate acceptor sites (9). Fine mapping of the phosphorylation acceptor sites of the opossum PTH/PTHrP receptor, using serine to threonine mutations and TLC, revealed that six serine residues at positions 483, 485, 486, 489, 495, and 498 are the sites for PTH-stimulated receptor phosphorylation (8). These residues correspond to S489, S491, S492, S495, 501, and S504 of the rat PTH/PTHrP receptor. Mutagenesis data for the rat PTH/PTHrP receptor confirmed that mutations of S489, S491, S492, S493, and S495 markedly reduced phosphorylation of the F1 fragment, whereas mutations of S501 and S504 reduced phosphorylation of the F2 fragment. Radiosequencing, showing the release of radioactivity at each sequencing cycle, confirmed the mutagenesis data for all residues except S489. Taken together, the mutagenesis and radiosequencing data suggest that residues S491, S492, S493, S495, S501, and S504 are the phosphorylation acceptor sites and that S489 is required for the phosphorylation of nearby serine residues within F1.

The role of receptor phosphorylation in internalization has been well documented for several G protein-coupled receptors (29, 30, 31, 32, 33, 34, 35, 36). Previous data using an opossum receptor stably expressed in HEK293 cells indicated that phosphorylation is not required for internalization (8). The current study, however, demonstrates that internalization of the PD PTH/PTHrP receptor, examined using three independent approaches, was severely impaired. The discrepancy between our data and those of Malecz et al. (8) may arise from differences in cell lines and/or receptor species. In fact, a recent report from the same group of investigators using HEK293 cells demonstrated a 30% impairment in internalization of opossum PTH/PTHrP receptors with mutations in the potential phosphorylation sites (37). Collectively, these data indicate that phosphorylation of the PTH/PTHrP receptor is important for its internalization.

Expression of inactive GRK2 and truncated PTH/PTHrP receptor, which lacks the phosphorylation sites, decreased PTH/PTHrP receptor-stimulated GTPase activity; this indicated that GRK2 binding and uncoupling of PTH/PTHrP receptor from the G protein do not require phosphorylation (26). However, in these experiments the PTH/PTHrP receptor was examined in a pharmacological system in which some individual components were overexpressed. In this regard, overexpression of ß-arrestin 1 rescued sequestration of a ß-adrenergic receptor mutant that otherwise was defective in agonist-induced phosphorylation and internalization (32).

It is interesting that LLCPK-1 cell lines expressing the PD receptor mutant had a higher sensitivity for PTH stimulation than those expressing WT receptor. The decreased EC50 for PTH-stimulated cAMP accumulation implies an important role for receptor phosphorylation in the responsiveness to PTH. The PD receptor may have a decreased affinity for binding ß-arrestin(s), and therefore results in enhanced Gs coupling and adenylate cyclase stimulation. Alternatively, the increased sensitivity may be secondary to the defect in internalization, i.e. the PD receptor remains on the cell surface longer than the WT receptor. The EC50 values of WT and PD receptors were similar in transiently transfected COS-7 cells. Indeed, COS-7 cells express the large T antigen that induces the expression of a high copy number of transiently transfected genes cloned in a vector containing the simian virus 40 promoter. This results in the expression of several million PTH/PTHrP receptors with decreased binding affinity (7), which makes the COS-7 cell system inappropriate for studying high affinity signaling.

G protein-coupled receptor phosphorylation and internalization subserve several biological functions. Yu et al. (38) reported that a carboxyl-terminally truncated TRH receptor exhibited sustained signaling properties; this implies a role for the cytoplasmic tail in preventing sustained stimulation. Our finding that PD-PTH/PTHrP receptor cells had a sustained response to PTH stimulation is in agreement with the study by Yu et al. (38) and suggests that sustained stimulation is due to lack of phosphorylation of the carboxyl-terminal tail. The fact that cAMP levels return to control values faster in WT cells than in PD-PTH/PTHrP receptor cells indicates that phosphorylation of the PTH/PTHrP receptor is essential for the process of recovery from stimulation. Interestingly, the sustained stimulation in the PD receptor cells persisted despite the complete removal of PTH by acid wash, suggesting the involvement of other cellular mechanisms in this response, e.g. impaired ß-arrestin(s) binding to the nonphosphorylated receptor.

Desensitization, classically described as a decreased response to a second or persistent stimulus, is an important process, which prevents excessive stimulation. A role for receptor phosphorylation in desensitization has been reported in other G protein-coupled receptors (30, 31, 36, 39, 40, 41, 42, 43, 44, 45, 46). The current study investigates the relationship among the early cellular events, receptor phosphorylation (9), internalization (12), and short-term desensitization. No short-term desensitization in PTH/PTHrP receptor was observed in LLCPK-1 cells. Although classic desensitization of PTH/PTHrP receptor has been reported previously (13, 14, 15, 16, 17, 18, 19, 20, 21, 22), it required long treatment with PTH. Additionally, it was reported that different levels of expression of ß-adrenergic receptor kinase in lung cell lines resulted in a remarkable difference in short-term desensitization of the ß2-adrenergic receptor (47). It is possible that the LLCPK-1 cell line does not express optimal levels of some intracellular molecule(s) involved in rapid desensitization of the PTH/PTHrP receptor. Previous studies have demonstrated that LLCPK-1 cells expressing PTH/PTHrP receptor in a range of 20,000–400,000 receptors/cell had the same maximal PTH-stimulated cAMP accumulation (48). Therefore, it is not surprising that the 35–40% decrease in PTH/PTHrP receptor number in LLCPK-1 cells (expressing ~200,000 receptors/cell), due to PTH-stimulated internalization, did not result in a parallel decrease in cAMP response.

In conclusion, we have identified six serine residues within the carboxyl-terminal tail of the PTH/PTHrP receptor as being agonist-dependent phosphate acceptor sites and the serine residue at position 489 as being required for phosphorylation. Further, we demonstrate that phosphorylation plays an important role in PTH/PTHrP receptor internalization and that agonist- dependent phosphorylation of the PTH/PTHrP receptor is important for regulating receptor signaling through the adenylate cyclase/cAMP pathway.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
[Nle8,18,Tyr34]Bovine PTH-(1–34)NH2 (NlePTH) was synthesized by a solid phase method (Endocrine Unit, Massachusetts General Hospital, Boston, MA). All chemicals were of the highest grade available and were obtained from either Sigma (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA). Na125I (2125 Ci/mmol), [32P]orthophosphate (8500–9120 Ci/mmol), and chemiluminescence kit were purchased from NEN Life Science Products (Boston, MA). Restriction enzymes were purchased from U.S. Biochemical Corp. (Cleveland, OH), New England Biolabs (Beverly, MA), Promega Corp. (Madison, WI), or Life Technologies, Inc. (Gaithersburg, MD). FBS, normal sheep serum, peroxidase-conjugated antisheep IgG, IBMX, and forskolin were obtained from Sigma; streptomycin, penicillin, and lipofectin were purchased from Life Technologies, Inc.; CNBr-activated Sepharose 4B was obtained from Pharmacia Biotech (Uppsala, Sweden); tissue culture media were prepared by the Massachusetts General Hospital media facility; flasks, plates, and other tissues cultures supplies were obtained from Corning, Inc. (Oneonta, NY); Vectashield was obtained from Vector Laboratories, Inc. (Burlingame, CA); and Immobilon membranes were purchased from Millipore Corp. (Bedford, MA).

Tissue Culture
COS-7 and LLCPK-1 cells were cultured in DMEM supplemented with 10% FBS. All media contained 1 µg/ml streptomycin and 100 U/ml penicillin. The cells were incubated in a humidified atmosphere containing 95% air and 5% CO2 at 37 C. Media were replaced every other day. When the cells became confluent, they were lifted by trypsinization, diluted, and passaged to new flasks.

Site-Directed Mutagenesis and Construction of Phosphorylation-Deficient Receptor Mutants
The WT PTH/PTHrP receptor cDNA cloned in pcDNA1, R15B (2), was used for single-strand plasmid preparation. All receptor mutations were then created on the R15B backbone by site-directed mutagenesis according to the method of Kunkel et al. (49). The veracity of all mutations was confirmed by sequence analysis. A PD PTH/PTHrP receptor (PD-PTH/PTHrP receptor or 7SA) was constructed by mutating seven serine residues to alanine residues at positions 489, 491, 492, 493, 495, 501, and 504. Another receptor mutant, 6SA, in which six serine residues were mutated to alanine residues at positions 489, 491, 492, 493, 495, and 504 was also constructed.

Construction of GFP-Tagged Receptors
The sequence of GFP was introduced in the WT or PD-PTH/PTHrP receptor cDNAs by creating a unique restriction enzyme site using site-directed mutagenesis and/or PCR. The sequence encoding full-length GFP was digested from the expressed GFP plasmid (CLONTECH Laboratories, Inc., Palo Alto, CA) and was ligated in-frame in the middle of exon E2, an extracellular domain whose deletion or mutation does not disturb ligand binding or signal transduction (28). The 69- to 73-amino acid sequence (Trp-Thr-Pro-Ala-Ser) from the E2 exon of the rat PTH/PTHrP receptor was replaced with 251 amino acids encoding the GFP sequence. The location of the GFP insertion within exon E2 was selected so that the epitopes recognized by the antirat PTH/PTHrP receptor antibody, G48, are not interrupted.

Cell Transfection
For transient transfection experiments, COS-7 cells in 10-cm plates were transfected with 5 µg plasmid DNA using the diethylaminoethyl-dextran method. One day after transfection, the cells were trypsinized and replated in 24-well plates. Two days later, radioligand binding, PTH-stimulated cAMP accumulation, and antibody binding assays were performed. To study PTH/PTHrP receptor internalization, LLCPK-1 cell lines stably expressing WT, GFP-tagged WT, PD, or GFP-tagged PD PTH/PTHrP receptors were developed. LLCPK-1 cells were cotransfected with cDNA encoding the WT or the mutant receptor sequence and the psv2Neo plasmid using the lipofectin method of transfection. The cells were grown in the presence of G418. Cell colonies that survived selection were expanded and examined by fluorescent microscopy. Cell lines that had radioligand binding greater than 10,000 cpm/well of total added radioactivity (100,000 cpm/well) were expanded and examined for receptor expression by Western blot analysis. All cell lines expressed a single immunoreactive band (~80 or ~107 kDa), which corresponded to the molecular mass of the WT receptor or the fusion protein, respectively. Cell lines expressing the WT (WT6), GFP-tagged WT (WT-GFP27 and WT-GFP36), PD (PD7), or GFP-tagged PD PTH/PTHrP receptor (PD-GFP2, PD-GFP8 and PD-GFP11) were selected for this study.

Phosphorylation of PTH/PTHrP Receptor
Confluent cells, in 6-cm tissue culture dishes, were washed once with phosphate- and serum-free DMEM and incubated with the same medium for 40 min at 37 C. [32P]Orthophosphate (0.5 mCi/3.5 ml) in fresh phosphate- and serum-free DMEM was then added, and incubation was continued for additional 2 h at 37 C (labeling period). The cells were treated with PTH (0–40 min) during the last 0–40 min of the 2-h labeling period. At the end of the labeling period, the cells were rinsed (three times) with ice-cold PBS and lysed with 0.8 ml/dish of RIPA buffer [140 mM NaCl, 50 mM Tris (pH 8), 1% Triton X-100, 0.5% deoxycholic acid, and 0.1% SDS] containing phosphatase inhibitors (300 nM okadaic acid, 10 mM tetrasodium pyrophosphate, 0.1 mM sodium orthovanadate, and 10 mM NaF) and proteinase inhibitors (1 mM phenylmethylsulfonylfluoride and 20 µg/ml aprotinin). The cell lysate was immunoprecipitated with the PTH/PTHrP receptor antiserum beads (9).

Immunoprecipitation and Western Blot
The anti-PTH/PTHrP receptor antiserum, G48 (9), was used for immunoprecipitation and Western blots. This antiserum was raised in sheep against a synthetic rat PTH/PTHrP receptor peptide, which corresponded to residues 88–108. The animal was subsequently boosted with eight other synthetic receptor fragments that represented different domains from the extracellular loops, the cytoplasmic loops, and the carboxyl-terminal tail. The crude Igs were precipitated with saturated ammonium sulfate, dissolved in PBS, dialyzed against coupling buffer (0.5 M NaCl and 0.1 M NaHCO3, pH 8.3), and then coupled to CNBr-activated Sepharose 4B beads following the manufacturer’s recommendations. Normal sheep serum was processed in an identical manner to construct normal IgG-Sepharose beads. The cell lysate (0.5 ml) was first incubated with 50 µl normal sheep IgG beads for 1 h. The supernatant was then collected and further incubated with 30 µl PTH/PTHrP receptor antiserum beads for 1 h at 4 C. The beads were rinsed (six times) with 0.8 ml ice-cold RIPA buffer. The receptor protein was then eluted from the beads by adding 35 µl SDS-sample buffer and incubating for 10 min at room temperature. The eluted receptor was subjected to 5–20% gradient SDS-PAGE and analyzed by autoradiography for 16–48 h.

For Western blots, the receptor protein that had been resolved on 5–20% SDS-PAGE was electrotransferred onto an Immobilon-P membrane; the membrane was blocked with 5% nonfat dry milk and 0.2% Tween 20 in PBS and then incubated with the PTH/PTHrP receptor antiserum, G48, at a dilution of 1:2000 for 2 h at room temperature. The membrane was rinsed (three times) with 0.2% Tween 20 in PBS, and a peroxidase-conjugated rabbit antisheep antiserum was added for 1 h at room temperature. The excess second antiserum was removed, the blots were rinsed as described above, and the bands were developed using a chemiluminescence kit.

CNBr Digestion of the Receptor Protein
The receptor protein, adsorbed on the PTH/PTHrP receptor antiserum beads, was directly subjected to the CNBr cleavage. CNBr (0.5 ml 100 mM in 70% formic acid) was added to the drained beads at room temperature for 16 h on a rotator. The supernatant was collected, air-dried, dissolved in ddH2O, and lyophilized. The cleaved receptor fragments were then resolved on 16.5% tricine-SDS-PAGE. The gel was dried and autoradiographed for 48 h.

Sequencing of the CNBr-Cleaved Phosphorylated Receptor Fragments
To facilitate N-terminal sequencing of the CNBr-cleaved phosphorylated receptor fragments, a methionine residue was introduced at position 480; this places the first potential phosphorylation site closer to the amino terminus of the cleaved fragment. Additionally, to prevent the formation of two CNBr-cleaved phosphorylated fragments, the methionine residue at position 499 was mutated to alanine; this resulted in a single CNBr-cleaved fragment that carries all of the potential phosphorylation sites. The resulting double mutant, A480M/M499A was expressed in COS-7 cells and was shown to function normally. As predicted, CNBr cleavage of the A480M/M499A double mutant resulted in a single phosphorylated band that was resolved as an approximately 15-kDa band on the tricine-SDS-PAGE. The phosphorylated band was identified by autoradiography of the wet gel for 1 h and was then cut from the gel. The phosphorylated CNBr-cleaved peptide was eluted from the gel fragment, desalted on a Sephadex G-25 column, lyophilized, and reconstituted in 50 µl ddH2O. The sample was sequenced on a pulsed liquid-gas phase sequencer (model 477A, PE Applied Biosystems) in the peptide core facility of the Endocrine Unit (Ashok Khatri, Endocrine Unit, Massachusetts General Hospital). In brief, the purified CNBr fragment was covalently coupled to an aryl-amine-derived polyvinylidene difluoride membrane, which was placed in the sequencer. After each sequencing cycle, the membrane was rinsed with methanol, followed by heptane/ethyl acetate (1:1, vol/vol), to ensure full recovery of the phosphorylated residues from the membrane. At each cycle, the released residue was collected and analyzed on a ß-spectrophotometer to determine its radioactivity content.

Cell Surface Receptor Quantification
Cell surface expression of the PTH/PTHrP receptor was assessed using G48 antibody. Cells grown in 24-well plates were rinsed (three times) with PBS, pH 7.4, and incubated at room temperature for 120 min with the G48 antibody (at a 1:2000 dilution in PBS and heat-inactivated FBS) or nonimmune IgG. The cells were then rinsed (three times) with PBS, incubated at room temperature for an additional 90 min with a rabbit antisheep antibody (Kirkegaard & Perry Laboratories, Gaithersburg MD; at a 1:500 dilution in PBS and heat-inactivated FBS), and rinsed with PBS (three times), and an 125I-labeled goat antirabbit IgG (NEN Life Science Products; 200,000 cpm/well) was added for 90 min. The incubation was terminated by removing the supernatant and rinsing the cells with PBS (three times). The cells were then solubilized in 1 N NaOH, and the radioactivity was counted. For internalization experiments, the cells were always placed on ice after treatment and washed with ice-cold PBS, and the G48 antibody incubation was performed at 4 C.

Internalization of Radioiodinated PTH
LLCPK-1 cells stably expressing the GFP-tagged WT (WT-GFP27) or the GFP-tagged PD (PD-GFP11) PTH/PTHrP receptors grown in 24-well plates were incubated with radioiodinated PTH-(1–34) (100,000 cpm/well in DMEM containing 20 mM HEPES buffer and 0.1% BSA) for 4 h on ice. The unbound ligand was removed using ice-cold PBS wash once and was replaced with control medium. The cells were transferred to 37 C incubator for 0, 10, 30, 40, 60, 120, or 240 min. After each incubation, three fractions of radioactivity were collected and counted. The first fraction collected was the free ligand in the medium and represents the dissociated and/or released ligand in the medium. The second fraction collected was the result of acid wash buffer (50 mM glycine and 150 mM NaCl, pH 3, using acetic acid) and represents the surface-bound ligand; it is called the acid-sensitive fraction. The third fraction was collected after lysing the cells in 1 M NaOH and represents the internalized ligand; it is called the acid-resistant fraction. Ligand internalization was calculated and is presented as the percentage of radioactivity in the acid-resistant fraction/the total radioactivity in the three fractions.


    FOOTNOTES
 
Address requests for reprints to: Dr. Abdul B. Abou-Samra, Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114. E-mail: samra{at}helix.mgh.harvard.edu

This work was supported by the NIH (NIDDK Grant DK-11794-26 and NRSA Grant DK-10087-01).

Abbreviations: CNBr, Cyanogen bromide; GFP, green fluorescent protein; GRK, G protein receptor kinase; IBMX, isobutylmethylxanthine; NlePTH, [Nle8,1,Tyr34]bovine PTH-(1–34)NH2; PD, phosphorylation deficient; RIPA buffer, 140 mM NaCl, 50 mM Tris (pH 8), 1% Triton X-100, 0.5% deoxycholic acid, and 0.1% SDS; WT, wild type.

Received for publication January 5, 2001. Accepted for publication September 26, 2001.


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