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
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ABSTRACT
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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.
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INTRODUCTION
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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.
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RESULTS
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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. 1A
, 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. 1B
, 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. 1A
, 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. 1B
, triangles). Furthermore, the S501 and S504 cycles showed
clear peaks of radioactivity that were released with each of these
sequencing cycles (Fig. 1B
, 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.
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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. 2
). In contrast, the mutant 7SA had no
detectable basal or ligand-dependent phosphorylation (Fig. 2
).

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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.
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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. 3
). The PD mutant
receptor bound PTH normally (Fig. 3A
) and increased cAMP accumulation
to the same levels as the WT receptor (Fig. 3B
). Cell surface receptor
expression of the PD mutant receptor was similar to that of the WT
receptor (Fig. 3C
). As shown above, stimulation of the PD receptor did
not result in receptor phosphorylation (Fig. 3D
), and Western blot
analysis of the immunoprecipitates revealed a similar amount of
immunoreactivity in both PD and WT receptors (Fig. 3E
).

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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 (01000 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.
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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. 4
).
Radioligand binding was similar in both cell lines (Fig. 4A
). PD7 cells
had an increased potency for PTH-stimulated cAMP accumulation compared
with WT6 cells (Fig. 4B
); 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. 3 . 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.
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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, 5100 min)
significantly (P < 0.01) decreased cell surface
PTH/PTHrP receptor immunoreactivity in WT6 cells (Fig. 5
). In contrast, a similar treatment did
not decrease cell surface receptor immunoreactivity in PD7 cells (Fig. 5
).

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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.
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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. 6A
). 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. 6B
); 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. 7
, 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 (11000 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-(134) (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.
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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. 7
, 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. 7
, middle panel). In
nontreated PD-GFP11 cells, the receptors were also localized to the
periphery of the cells (Fig. 7
, 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. 7
, 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. 7
, 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. 7
, 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 060 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. 8A
). 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. 8A
).

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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 (060 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).
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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. 8
). 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. 8B
, pretreated 0), and a second PTH challenge resulted in a
robust cAMP response similar to that of control cells (Fig. 8B
, pretreated). In contrast, cells expressing PD receptors showed
sustained elevation of cAMP levels (Fig. 8B
, 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. 8B
, 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.
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DISCUSSION
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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 20,000400,000
receptors/cell had the same maximal PTH-stimulated cAMP accumulation
(48). Therefore, it is not surprising that the 3540%
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
|
---|
Materials
[Nle8,18,Tyr34]Bovine
PTH-(134)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 (85009120
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 (040 min) during the last
040 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 88108. 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 manufacturers
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 520% gradient SDS-PAGE and analyzed by
autoradiography for 1648 h.
For Western blots, the receptor protein that had been resolved on
520% 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-(134) (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-(134)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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