Identification of Phosphorylation Sites in the G Protein-Coupled Receptor for Parathyroid Hormone. Receptor Phosphorylation Is Not Required for Agonist-Induced Internalization
Nicole Malecz,
Tom Bambino,
Margaret Bencsik and
Robert A. Nissenson
Endocrine Research Unit Veterans Administration Medical Center
and the Departments of Medicine and Physiology University of
California San Francisco, California 94121
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ABSTRACT
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In some G protein-coupled receptors (GPCRs),
agonist-dependent phosphorylation by specific GPCR kinases (GRKs)
is an important mediator of receptor desensitization and endocytosis.
Phosphorylation and the subsequent events that it triggers, such as
arrestin binding, have been suggested to be regulatory mechanisms for a
wide variety of GPCRs. In the present study, we investigated whether
agonist-induced phosphorylation of the PTH receptor, a class II GPCR,
also regulates receptor internalization. Upon agonist stimulation, the
PTH receptor was exclusively phosphorylated on serine residues.
Phosphoamino acid analysis of a number of receptor mutants in which
individual serine residues had been replaced by threonine identified
serine residues in positions 485, 486, and 489 of the cytoplasmic tail
as sites of phosphorylation after agonist treatment. When serine
residues at positions 483, 485, 486, 489, 495, and 498 were
simultaneously replaced by alanine residues, the PTH receptor was no
longer phosphorylated either basally or in response to PTH. The
substitution of these serine residues by alanine affected neither the
number of receptors expressed on the cell surface nor the ability of
the receptor to signal via Gs. Overexpression of GRK2, but not GRK3,
enhanced PTH-stimulated receptor phosphorylation, and this
phosphorylation was abolished by alanine mutagenesis of residues 483,
485, 486, 489, 495, and 498. Thus, phosphorylation of the PTH receptor
by the endogenous kinase in HEK-293 cells occurs on the same residues
targeted by overexpressed GRK2. Strikingly, the rate and extent of
PTH-stimulated internalization of mutated PTH receptors lacking
phosphorylation sites were identical to that observed for the wild-type
PTH receptor. Moreover, overexpressed GRK2, while enhancing the
phosphorylation of the wild-type PTH receptor, had no affect on the
rate or extent of receptor internalization in response to PTH. Thus,
the agonist-occupied PTH receptor is phosphorylated by a kinase similar
or identical to GRK2 in HEK-293 cells, but this phosphorylation is not
requisite for efficient receptor endocytosis.
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INTRODUCTION
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G protein-coupled receptors (GPCRs) comprise a large group
of intrinsic membrane proteins that are responsible for the
transduction of extracellular signals to the interior of the cell.
After agonist binding, the magnitude and duration of receptor signaling
are tightly controlled by acute regulatory processes that include
uncoupling of the receptor from its cognate G protein (desensitization)
and translocation of the receptor from the plasma membrane to endocytic
vesicles (internalization). The mechanisms underlying these regulatory
processes have been most extensively studied for rhodopsin and the
ß-adrenergic receptor (ß-AR). In both cases, perturbation of
receptor regulation is associated with pathological effects in
vivo, highlighting the physiological importance of these processes
(1, 2, 3, 4). It is almost certain that regulation of other GPCRs is likewise
physiologically significant, and it is therefore crucial to evaluate
the extent to which mechanistic models based on studies with rhodopsin
and the ß-AR are broadly applicable to the GPCR superfamily.
Agonist-induced receptor phosphorylation appears to be a common feature
of the regulation of a number of GPCRs. In the case of the ß-AR and
rhodopsin, agonist binding leads to rapid phosphorylation of the
cytoplasmic domain of the receptor by a GPCR kinase (GRK) (5). This
phosphorylation facilitates the interaction between the receptor and an
arrestin protein (6), a process that physically uncouples the receptor
from its cognate G protein (7, 8). In the case of the ß-AR, arrestin
binding to the phosphorylated receptor also serves to target the
receptor to clathrin-coated pits and thereby to promote receptor
internalization (9, 10). If the phosphorylation of the ß2-AR is
reduced [either by mutagenesis of the receptor or by overexpressing a
dominant negative GRK2 (11)], or if a dominant negative arrestin is
overexpressed (7), agonist-induced receptor sequestration is
significantly reduced. This phenotype can be rescued by overexpressing
GRK2 and ß-arrestin, showing that they work synergistically and that
they are both crucial for ß2-AR internalization (11, 12). A similar
dependence of internalization on phosphorylation has been described for
a number of other GPCRs (13, 14, 15, 16). Barak et al. (17) have
recently tested a variety of GPCRs for their ability to bind a
ß-arrestin-GFP fusion protein in response to agonist stimulation.
They found translocation to the plasma membrane of the arrestin-green
fluorescent protein (GFP) in response to 16 different ligand-activated
GPCRs, suggesting that recruitment of arrestins may be a widespread
mechanism for the regulation of GPCRs.
Little is known about the role of phosphorylation in the regulation of
class B GPCRs, a structurally distinct GPCR subfamily that includes
receptors for PTH/PTH-related peptide (PTHrP), secretin, glucagon and
related peptides, calcitonin, and others. The opossum PTH receptor is
rapidly phosphorylated upon agonist stimulation (18), and this
phosphorylation was not evident in a C-terminally truncated receptor
(termed T474) that lacks all but the membrane-proximal 16 amino acids
of the lengthy cytoplasmic tail (19). This truncated, nonphosphorylated
receptor was found to be well expressed at the cell surface and
resembled the wild-type (wt) PTH receptor in its ability to signal to
adenylyl cyclase, but displayed a marked (50%) reduction in
agonist-stimulated receptor internalization (20). It is unclear whether
the endocytic defect seen with this truncated receptor is related to
the lack of phosphorylation, since the truncation not only prevented
phosphorylation but also deleted a positive endocytic signal from the
cytoplasmic tail (20). The purpose of the present study was to localize
the sites of PTH receptor phosphorylation, and to express a minimally
mutated, phosphorylation-deficient receptor to evaluate the role of
phosphorylation in PTH receptor internalization.
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RESULTS
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Localization of Sites of Agonist-Stimulated Phosphorylation in the
PTH Receptor
Initial 32P-labeling studies were carried out in human
embryonic kidney (HEK)-293 cells expressing the wt PTH receptor. As
previously described (18), basal phosphorylation of the receptor was
detectable, and the level of phosphorylation was increased
approximately 3-fold by treatment of cells with PTH. Phosphoamino
analysis of the immunoprecipitated receptor revealed that
phosphorylation occurred exclusively on serine residues, both basally
and after treatment with PTH (Fig. 1
).

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Figure 1. Phosphoamino Acid Analysis of
32P-Labeled wt PTH Receptor
The receptor was metabolically labeled with
[32P]orthophosphoric acid, immunoprecipitated, and
processed as described in Materials and Methods. The
cellulose TLC plates were exposed to Kodak X-ray film at -80 C. The
upper panels show receptor phosphoserine residues from
cells treated with 1 µM PTH for 10 min before
immunoprecipitation. The lower panels show receptor
phosphoserine residues from untreated cells. The autoradiograms on the
right were exposed for a shorter time; Cerenkov counting
indicated that PTH treatment produced approximately a 3-fold increase
in receptor phosphoserine content. The autoradiograms on the
left were overexposed to emphasize the absence of
phosphotyrosine and phosphothreonine. The positions of the phosphoamino
acid markers are indicated.
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These results suggested a possible mutagenesis strategy for identifying
sites of receptor phosphorylation. Some serine/threonine kinases are
capable of phosphorylating either serine or threonine in a given
sequence context. If a mutated PTH receptor, bearing a serine to
threonine transversion, contains phosphothreonine upon phosphoamino
acid analysis, this would strongly suggest that the corresponding
serine residue is a phosphoacceptor site in the wt receptor. Such an
approach has been successfully used by Giannini et al. (21)
to identify phosphorylation sites in the C5a receptor. As a guide to
choosing the appropriate serine residues for mutagenesis, we took
advantage of previous results from our laboratory concerning
the effects of C-terminal truncation on receptor phosphorylation.
Deletion of all but the proximal 16 amino acids from the cytoplasmic
tail produced a receptor (T474) that was not phosphorylated either
basally or in response to PTH (19), suggesting that the phosphorylated
residues lie distal to residue 474 in the cytoplasmic tail. However,
two other truncation mutants of the receptor, T494 and T507, did show
phosphorylation. In an attempt to identify the sites of
phosphorylation, serine residues proximal to position 507 were targeted
for mutagenesis (Fig. 2
). Five of the
first six serine residues in the proximal portion of the cytoplasmic
tail were individually mutated to threonine, and the mutant receptors
were expressed in HEK-293 cells and subjected to phosphoamino acid
analysis. Only mutant receptors with substitutions S485T, S486T, or
S489T, but not receptors with S469T or S467T mutations showed the
appearance of phosphothreonine (Fig. 3
).
Serine 483 was not targeted individually, but a mutant receptor was
tested in which serine residues at positions 483, 485, 486, and 489
were simultaneously mutated to threonine. This mutant receptor
displayed phosphothreonine, as expected, but also contained
phosphoserine, indicating that serine residues distal to S489 were also
phosphoacceptor sites.

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Figure 2. Schematic Drawing of the wt PTH Receptor
The proximal portion of the wt PTH receptors cytoplasmic tail with
all the potential phosphorylation sites are shown.
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Figure 3. Phosphoamino Acid Analysis of
32P-Labeled PTH Receptor Mutants
Individual serine residues in the cytoplasmic tail of the PTH receptor
were replaced by threonine residues, and each mutant receptor was
expressed in HEK-293 cells. In the S(483489)T mutant all serine
residues between position 483 and 489 were changed to threonine. Cells
were metabolically labeled with [32P]orthophosphoric acid
for 2 h and incubated for 10 min either without (basal) or with 1
µM PTH (+PTH). After immunoprecipitation the receptor was
subjected to phosphoamino acid analysis as described in
Materials and Methods. The phosphoserine and
phosphothreonine signals are shown.
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In an attempt to create a phosphorylation-deficient receptor, two
alanine mutants were studied, one in which all serine residues between
position 467 and 498 [S(467498)A] and another in which all serine
residues between positions 483 and 498 [S(483498)A] were converted
to alanine. When expressed in HEK-293 cells, both of these mutant
receptors lacked detectable phosphorylation both basally and in
response to PTH (Fig. 4
). To determine
whether this lack of phosphorylation was due to low receptor
expression, competitive ligand-binding analysis was carried out and
receptor numbers were determined by Scatchard analysis (Fig. 5A
). The wt receptor (990,000 receptors
per cell) was found to be expressed at a level only slightly greater
than that of the S(467498)A mutant (724,000 receptors per cell) and
the S(483498)A mutant (799,000 receptors per cell). Dose-response
studies for PTH-stimulated cAMP production were carried out with each
of these cell lines, and they were similarly sensitive to PTH
[EC50 values of 0.6 nM for the wt PTH
receptor, 0.4 nM for S(467498)A, and 0.3 nM
for S(483498)A] and displayed similar maximal cAMP responses (Fig. 5B
). The results obtained indicate that both basal and PTH-stimulated
phosphorylation of the PTH receptor occur on serine residues between
positions 483 and 498 in the cytoplasmic tail. Moreover,
phosphorylation of these residues is not absolutely required either for
achieving high levels of receptor expression or for initiation of
receptor signaling to adenylyl cyclase. However, it is possible that
differences in signaling properties between the wt and the
phosphorylation-deficient PTH receptor would become evident under
conditions of lower levels of receptor expression.

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Figure 4. Phosphorylation of the wt and Mutant PTH Receptors
Expressed in HEK-293 Cells
The mutants tested were S(467498)A, a mutant in which all serine
residues between positions 467 and 498 were converted to alanine, and
S(483498)A, a mutant in which all serine residues between positions
483 and 498 were converted to alanine. Phosphorylation was carried out
in cells treated without (basal) or with 1 µM PTH, as
described in Materials and Methods. After labeling, the
receptors were immunoprecipitated and evaluated by SDS-PAGE. The mol wt
of the PTH receptor is indicated.
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Figure 5. Comparison of wt and Phosphorylation-Deficient PTH
Receptors in HEK-293 Cells
A, Estimates of functional levels of receptor expression and of
receptor affinity determined by Scatchard analysis of competitive
ligand binding data, as described in Materials and
Methods. B, Dose-response curves for bPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 )-stimulated
cAMP production, and the resulting EC50 values. Data are
expressed as the mean ± SEM of triplicate samples.
The results are representative of three independent experiments.
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Investigation of the Involvement of Known GRKs in PTH Receptor
Phosphorylation
Two approaches were taken to explore the possible role in PTH
receptor phosphorylation of GRKs known to be important for other GPCRs
(8). First, GRKs 2 or 3 were overexpressed in HEK-293 cells stably
expressing the wt PTH receptor. The cDNAs encoding the GRKs were
subcloned into an episomal vector (pCEP4) and transfected into HEK-293
cells; successfully transfected cells were selected in the presence of
hygromycin, as described in Materials and Methods. Under
these conditions, overexpression of GRK2 was readily apparent by
Western blotting (Fig. 6A
, inset). GRK3 was overexpressed to a similar degree as GRK2
(data not shown). Overexpression of GRK2 resulted in a 3- to 4-fold
increase in agonist-induced phosphorylation of the wt PTH receptor
(Fig. 6A
). To evaluate whether overexpressed GRK2 phosphorylated the
same sites as the endogenous kinase, we established HEK-293 cell lines
stably overexpressing GRK2 and the phosphorylation-deficient mutants
S(467498)A or S(483498)A. If overexpressed GRK2 phosphorylated the
receptor at sites different from those used by the endogenous kinase,
there should be retention of some phosphorylation in the mutant
receptors. However, neither mutant S(467498)A nor mutant
S(483498)A exhibited detectable phosphorylation despite the
overexpression of GRK2 (Fig. 6B
). These results suggest that GRK2 and
the endogenous GRK in HEK-293 cells utilize the same phosphoacceptor
sites in the PTH receptor. Overexpression of GRK3 produced a slight
(<2-fold) increase in phosphorylation of the wt PTH receptor (data not
shown).

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Figure 6. Effects of GRK2 on PTH Receptor Phosphorylation in
HEK-293 Cells
A, Cells stably transfected with the wt PTH receptor alone or with the
wt PTH receptor and either GRK2 or a DN-GRK2 were metabolically labeled
with [32P]orthophosphoric acid either without (basal) or
in the presence of 1 µM PTH. Receptors were
immunoprecipitated and then separated by SDS-PAGE, and the respective
bands were excised and phosphorylation was quantified by Cerenkov
counting. The inset shows a Western blot performed with
a polyclonal GRK2 antibody in untransfected cells (lane 1), and in
cells transfected with a plasmid-encoding GRK2 (lane 2) or DN-GRK2
(lane 3). B, Phosphorylation of wt and mutant PTH receptors in cells
overexpressing GRK2. The cells were metabolically labeled with
[32P]orthophosphoric acid, after which the PTH receptor
was immunoprecipitated and separated by SDS-PAGE, and autoradiograms
were developed. The mol wt of the PTH receptor is indicated.
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The results of the above studies suggest a possible role for GRK2 in
PTH receptor phosphorylation. In the second approach, we used a
C-terminal fragment of GRK2 that has previously been shown to function
as a dominant inhibitor of GRK2 catalytic activity (22). A cDNA
encoding this dominant-negative (DN) GRK2 was subcloned into pCEP4 and
was transfected into HEK-293 cells stably expressing the wt PTH
receptor. Western blotting confirmed successful expression of the
DN-GRK2 (Fig. 6
, inset). As shown in Fig. 6A
, agonist-induced phosphorylation was not suppressed despite the presence
of high levels of DN-GRK2. Overexpression of another DN-GRK2 (K220R)
(23) likewise failed to suppress agonist-induced phosphorylation of the
PTH receptor (data not shown).
Relationship between Receptor Phosphorylation and Endocytosis
In HEK-293 cells expressing the wt PTH receptor,
internalization of the receptor was detected as early as 2.5 min
after the addition of agonist, and reached a maximum (4050% of bound
radioligand) after 1015 min (Fig. 7A
).
The kinetics and extent of internalization of the
phosphorylation-deficient S(467498)A and S(483498)A PTH receptors
were indistinguishable from those of wt, indicating that
phosphorylation of the receptor is not required for efficient
agonist-stimulated endocytosis.

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Figure 7. Internalization of PTH Receptors in HEK-293 Cells
A, Time course for agonist-stimulated internalization of the wt
PTH receptor and phosphorylation-deficient PTH receptor mutants. B,
Effect of hypertonic sucrose on PTH receptor internalization. HEK-293
cells expressing wt PTH receptors or phosphorylation-deficient PTH
receptor mutants were incubated in the absence or presence of 0.45
M sucrose (to disrupt clathrin lattices), and
internalization was measured after 10 min of exposure to
[125I]PTHrP. C, Lack of effect of overexpression of GRK2
on agonist-stimulated internalization of the wt PTH receptor.
Internalization was assessed as non-acid-extractable radioligand uptake
(see Materials and Methods). Similar results were
obtained in three independent experiments each with triplicate data
points.
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Disruption of clathrin lattices by treatment of HEK-293 cells
with hypertonic sucrose inhibited the internalization of both the wt
and phosphorylation-deficient PTH receptors by 6070% (Fig. 7B
). This
result is similar to a previous report for the wt PTH receptor in COS-7
cells (20) and indicates that the majority of the endocytosed receptor
is internalized via a clathrin-coated pit mechanism regardless of
whether the receptor is phosphorylated. It is unclear whether the
non-sucrose-inhibitive component reflects an alternative
mechanism of internalization (e.g. caveolae) or is due to
incomplete disruption of the clathrin lattice.
To determine whether hyperphosphorylation of the wt PTH receptor
would influence receptor endocytosis, we evaluated agonist-dependent
receptor internalization in HEK-293 cells overexpressing GRK2 (Fig. 7C
). Although the PTH receptor in GRK2 overexpressing cells was
hyperphosphorylated (Fig. 6A
), the initial rate and extent of receptor
internalization were unaffected.
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DISCUSSION
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Endocytosis of GPCRs in response to agonist binding is a
widespread phenomenon that is important in the down-regulation of
responsiveness that accompanies chronic exposure to agonist. For most
GPCRs, endocytosis appears to occur through a classic clathrin-coated
pit mechanism (24, 25), although a few exceptions have been noted
(e.g. 26). At least two structural features in the
cytoplasmic domain of GPCRs directly contribute to receptor
endocytosis. First, the cytoplasmic domain of some GPCRs contains
sequences (e.g. Y-X-X-hydrophobic; L-L) that mediate the
interaction of the receptor with clathrin-associated adaptor proteins
(27, 28). Second, agonist-stimulated phosphorylation of the cytoplasmic
domain of GPCRs can facilitate their association with clathrin-coated
pits. In the case of the ß-AR, agonist binding leads to
phosphorylation of the receptor by a GRK, which in turn facilitates the
association of an arrestin protein with the receptor. Evidence suggests
that the arrestin-phosphoreceptor complex then associates with
clathrin-coated pits through the direct binding of arrestin to clathrin
(9). However, not all GPCRs depend on agonist-induced phosphorylation
for their internalization: a cholecystokinin receptor with a truncated
cytoplasmic tail internalized normally (29), as did a secretin receptor
with a truncation that eliminated phosphorylation (30). A limitation of
these studies is the use of truncation to eliminate the sites of
phosphorylation, since truncation may also induce conformational
changes in the receptor, thereby altering its accessibility or its
affinity for the endocytic machinery.
In light of the above, an important objective of the present
study was to identify the sites of phosphorylation in the PTH receptor,
allowing the expression of a form of the receptor that is minimally
mutated but lacks all of the phosphorylation sites. Previous studies
suggested that phosphorylation occurs in the proximal portion of the
cytoplasmic tail. A PTH receptor C-terminally truncated at position 474
(and thus lacking all but the proximal 16 amino acids in the
cytoplasmic tail) was found to be well expressed but was not
phosphorylated either basally or in response to PTH (19),
whereas truncation mutants T494 and T507 were
phosphorylated (E. Blind, P. Turner, and R. A. Nissenson,
unpublished observations). The results of the present study demonstrate
that, in HEK-293 cells, the opossum PTH receptor is phosphorylated
solely on serine residues in the cytoplasmic tail despite the presence
of six threonine residues. To identify these serine residues, we
adopted the approach used for the C5a anaphylatoxin receptor in which
candidate serine residues were mutated to threonine, and the receptor
was then scanned for the presence of phosphothreonine after stimulation
by agonist (21). Five of the six proximal serine residues in the
cytoplasmic tail were individually converted to threonine and subjected
to phosphoamino acid analysis. The serine residues identified in this
way were then mutated to alanine together with two downstream serine
residues to create a phosphorylation-deficient receptor. These
approaches allowed us to make the following conclusions about PTH
receptor phosphorylation: neither serine 467 nor serine 469 was
phosphorylated, a result in accordance with the observation that T474
was not phosphorylated, since no phosphothreonine appeared if threonine
was substituted for either of the two serine residues. Although this
result could be explained by the inability of the kinase to recognize a
threonine in these positions, the mutant receptor S(483498)A with
serine residues 467 and 469 left intact also was not phosphorylated,
indicating that S467 and S469 are not sites of phosphorylation. Serine
residues 485, 486, or 489 were phosphorylated in response to agonist,
as shown by the presence of phosphothreonine after the threonine
substitutions in those sites, identifying them as targets for a kinase
activated after ligand treatment; S489 was also phosphorylated basally.
A phosphorylation-deficient receptor for both the basal and the
agonist-stimulated phosphorylation was obtained when all six serine
residues between residues 483 and 498 were mutated to alanine,
indicating that serine residues 483, 495, and 498 were also potential
phosphorylation sites, although it is not yet clear whether all three
of these sites are phosphorylated. The proximal part of the cytoplasmic
tail is highly homologous in sequence between different species and,
interestingly, these six serine residues between positions 483 and 498
are conserved in the human, rat, and opossum PTH receptor, suggesting
evolutionary conservation of the phosphoacceptor sites.
Previous studies demonstrated that purified protein kinase
A and protein kinase C were each capable of phosphorylating the
proximal portion of the cytoplasmic tail of the PTH receptor in
vitro (19), and these kinases were implicated in basal
phosphorylation of the PTH receptor in intact HEK-293 cells (18).
However, neither of these kinases was found to be responsible for
agonist-stimulated phosphorylation of the receptor in intact HEK-293
cells (18). GRK2 was also shown to be effective in phosphorylating the
cytoplasmic tail of the PTH receptor in vitro, and it was
suggested that GRK2 or a related kinase was likely to mediate
agonist-dependent phosphoryation in vivo (19). Since the
importance of agonist-induced phosphorylation as a control mechanism
for GPCR trafficking has mainly been shown for receptors phosphorylated
by GRK2, and because GRK2 has been shown to be expressed in
osteoblastic cells (31), it was of particular interest in the present
study to assess whether the PTH receptor was a substrate for GRK2
in vivo. Comparison of the sequence surrounding the sites of
phosphorylation by GRK2 in the C5a receptor (21) and the ß2-AR (5)
with the sites phosphorylated in the PTH receptor does not yield an
obvious consensus sequence. However, overexpression of GRK2 led to a 3-
to 4-fold increase in receptor phosphorylation, indicating that GRK2 is
capable of utilizing the PTH receptor as a substrate in
vivo.
Two approaches were taken to assess whether GRK2 is likely to be
the endogenous kinase responsible for agonist-stimulated
phosphorylation of the PTH receptor in HEK-293 cells. First, we
evaluated whether the sites of augmented receptor phosphorylation
observed in the presence of overexpressed GRK2 were the same as those
used by the endogenous kinase. That this is the case was demonstrated
by the inability of the phosphorylation-deficient mutated PTH receptor
to be phosphorylated even in the presence of overexpressed GRK2. The
second approach tested whether the endogenous kinase could be inhibited
by altered forms of GRK2 previously shown to display dominant-negative
activity in certain cell systems. However, suppression of
agonist-stimulated PTH receptor phosphorylation was not observed with
overexpression of either the K220R mutant, which is no longer
catalytically active, or the C-terminal GRK2 fragment, which binds the
ß
-subunits of the G-protein. It is not clear whether this
result represents a failure of the dominant-negative constructs to
effectively suppress endogenous GRK2 activity, or whether the relevant
endogenous kinase is an enzyme distinct from GRK2.
Identifying the sites of agonist-stimulated PTH receptor
phosphorylation allowed us to take multiple approaches to address the
role of phosphorylation in receptor endocytosis. Overexpression of
GRK2, which enhanced PTH-stimulated receptor phosphorylation, had no
influence on the rate or extent of receptor internalization.
Conversely, expression of dominant-negative forms of GRK2 did not
influence receptor internalization although, as discussed above, we
have not evaluated directly whether GRK2 activity is suppressed in
these cell lines. Most importantly, targeted mutations of serine
residues in the proximal portion of the cytoplasmic tail of the PTH
receptor abolished PTH-stimulated receptor phosphorylation but had no
effect on the kinetics of receptor endocytosis. When mutations are
introduced, there is always the possibility that the effects seen are
caused by the altered amino acid sequence; i.e. an
unphosphorylated wt PTH receptor may behave differently than a mutant
receptor with alanines in place of serines. However, by narrowing the
potential phosphorylation sites down to six, we kept the necessary
mutations to a minimum, and there was no evidence that introduction of
alanine residues at those sites produced any alterations in receptor
expression or signaling.
These results demonstrate that, in HEK-293 cells,
agonist-stimulated PTH receptor internalization occurs by a mechanism
that is not dependent on receptor phosphorylation. The mechanism
underlying PTH receptor internalization, therefore, is distinct from
that proposed for the ß-AR and certain other class I GPCRs, which
involves phosphorylation of the receptor by a GRK followed by arrestin
binding to the phosphorylated receptor (6, 7). It is likely that the
structural determinants of PTH receptor endocytosis include sequences
(e.g. Y-X-X-hydrophobic; L-L) known to mediate the
association of other membrane proteins with components of
clathrin-coated pits (32). Indeed, the sequence Y-G-P-M lies within a
region of the cytoplasmic tail of the PTH receptor that has previously
been shown to be important for optimal PTH-stimulated internalization
(20). Our findings appear to differ from those obtained by Fukayama
et al. (33), who reported that expression of a
dominant-negative form of GRK2 (K220R) reduced acute agonist-induced
down-regulation of the endogenous PTH receptor in SaOS-2 human
osteosarcoma cells. It is conceivable that phosphorylation of the PTH
receptor by GRK2 is involved in PTH-stimulated receptor internalization
in SaOS-2 cells, but not in HEK-293 cells. Alternatively, the high
level of overexpression of the PTH receptor in HEK-293 cells may have
masked an effect of K220R GRK2 in the present study. It is also
possible that K220R GRK2 may influence acute PTH receptor endocytosis
in SaOS-2 cells independently from any effect it may have on receptor
phosphorylation. The availability of phosphorylation-deficient, mutated
forms of the PTH receptor, such as those described in the present
studies, will be useful in more clearly defining the role of
phosphorylation in receptor trafficking and signaling.
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MATERIALS AND METHODS
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Materials
Bovine PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) and PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) were obtained from Bachem
(Torrance, CA). Human embryonic kidney (HEK) 293 cells and the
expression vector pCEP4 were obtained from Invitrogen (San Diego, CA).
Carrier-free [32P] orthophosphoric acid was obtained from
New England Nuclear Corp. (Boston, MA). All oligonucleotides were
synthesized and purified at University of California San Francisco
Biomolecular Resource Facility.
Site-Directed Mutagenesis
Oligonucleotide-mediated mutagenesis was performed using the
site-directed mutagenesis kit (CLONTECH, Palo Alto, CA) according to
the instructions of the manufacturer using a
BglII/AflIII selection primer. Mutagenic primers
were designed to introduce the mutation of choice and either added or
removed a restriction site, to facilitate identification of mutant
clones.
Generation of PTH Receptor and PTH Receptor Mutant-Expressing
Cell Lines
Opossum PTH/PTHrP receptor cDNA in pcDNA1 (kindly provided by
Drs. H. Jueppner and G. V. Segre; Endocrine Unit, Massachusetts
General Hospital, Boston, MA), and the respective receptor mutants
obtained by site-directed mutagenesis were subcloned into the
expression vector pCEP4 or pcDNA31, amplified in Escherichia
coli HB101 and column purified (Plasmid maxi Kit, QIAGEN,
Chatsworth,CA) for transfection. HEK-293 cells were maintained in DMEM
containing 10% FCS and antibiotics (100 U/ml penicillin and 100
µg/ml streptomycin) in 5% CO2 at 37 C. Calcium
phosphate-mediated transfection was performed as previously described
(34). After an overnight incubation at 37 C, cells were washed twice
with calcium-magnesium-free PBS, grown for another day in DMEM, and
then subjected to selection with 200 µg/ml hygromycin for at least 3
weeks. Pooled hygromycin-resistant clones were used for all the
experiments using pCEP4 vectors. HEK cell lines stably expressing the
wt PTH receptor were isolated as follows. Cells were transfected (as
described above) with cDNA encoding the wt PTH receptor cloned into
pcDNA31, and selection was carried out in the presence of 400 µg/ml
G418 until distinct colonies appeared. Clonal lines were isolated by
limiting dilution, and a line displaying high levels of PTH receptor
expression (assessed by ligand binding assays and by Western blotting)
was chosen for further studies.
Generation of GRK2- and -3 Expressing HEK-293 Cell Lines
cDNAs for GRK2 and -3 (kindly provided by Dr. J. Benovic) were
subcloned into pCEP4 or, in the case of GRK2, also into pcDNA31.
Transfection was carried out as described above; individual colonies
were lifted individually from the culture dish and allowed to expand
further in the presence of G418; the selection of positive clones was
carried out as described above. Hygromycin-resistant pools of cells
(pCEP4) or G418-resistant clonal cell lines (pcDNA31) were evaluated
by Western blotting for GRK expression. Studies were carried out with
cells estimated to express GRKs at a level exceeding endogenous levels
by at least 10-fold.
Ligand Binding and Internalization
PTH receptor binding and internalization studies were carried
out as previously described (20). In brief, HEK 293 cells were grown
for 3 days in 35-mm wells to confluence and incubated in 1 ml of DMEM
containing 20 mM HEPES, 0.1% BSA (DHB), 60,000 cpm of
125I-labeled human (h)PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) and varying
concentrations of unlabeled bovine (b)PTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). Under these
conditions, the concentration of labeled hPTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) added was
approximately 0.1 nM. After a 1-h incubation at room
temperature, cells were washed, collected in 1 ml of 0.8 M
NaOH, and bound [125I]hPTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) was assessed. In
studies for Scatchard analysis these conditions were modified in that
incubations were carried out at 4 C for at least 2 h in 12-well
plates in a volume of 0.5 ml. Receptor affinity and number were
calculated using the GraphPad Prism program. For internalization
studies, the cells were incubated at room temperature for varying times
with [125I]hPTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) amide and washed twice in
ice-cold PBS, and surface-bound ligand was extracted by two 5-min
incubations on ice with 50 mM glycine buffer, pH 3.0,
containing 0.1 M NaCl. The internalized radioligand was
then extracted by exposing cells to 0.8 M NaOH. Receptor
internalization is expressed as the percent of cell-associated
radioligand remaining after acid washing. To disrupt clathrin lattices,
cells were treated with 0.45 M sucrose in DHB for 1 h
at 37 C before binding and internalization studies.
cAMP Assay
For the assay of cAMP levels, cells prepared as described above
were incubated in 1 ml DHB, 1 mM isobutylmethylxanthine,
and various concentrations of bovine (b)PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) for 10 min at room
temperature. Cells were then washed twice in ice-cold PBS, and the
cellular cAMP was extracted with 1.5 ml 95% ethanol and quantified by
RIA (35).
32P Labeling and Immunoprecipitation
Labeling and immunoprecipitation were carried out as previously
described (18). Briefly, HEK-293 cells were grown to confluence in
35-mm wells, washed in phosphate- and serum free DMEM, and incubated in
the same media for 30 min. To label the intracellular ATP pools, cells
were incubated in 0.7 ml phosphate and serum-free DMEM, 20
mM HEPES, and 200 µCi [32P]orthophosphoric
acid (5 mCi/ml) for 2 h. PTH was added to the appropriate wells 10
min before the end of the 2-h period. Cells were lysed by adding 170
µl RIPA buffer/well (150 mM NaCl, 50 mM
Tris-HCl, pH 7.5, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS)
supplemented with phosphatase inhibitors: 300 nM okadaic
acid, 10 mM tetrasodium pyrophosphate, 0.1 mM
sodium orthovanadate, and 10 mM sodium fluoride, and rocked
intermittently for 1 h at 4 C. The PTH receptor was
immunoprecipitated with a monoclonal anti-PTH receptor antibody
immobilized on Sepharose beads. Immunoprecipitates were subjected to
SDS-PAGE, and the radiolabeled receptor was visualized by
autoradiography and quantitated by Cerenkov counting. Gel loading was
normalized to levels of receptor expression, determined by Scatchard
analysis and/or by Western blotting.
Phosphoamino Acid Analysis
The 32P-labeled receptor was excised from the dried
SDS-polyacrylamide gel, and protein hydrolysis was carried out in 200
µl of 6 M HCl/band at 110 C for 90 min. The hydrolyzed
protein was then transferred to a new tube and dried in a Speed Vac
centrifuge. The dried residue was resolubilized in 5 µl of buffer 1
at pH 1.9 [formic acid-acetic acid-H2O; 22:78:900
(vol/vol/vol)], spotted on a thin layer cellulose plate (Merck) and
separated by electrophoresis for 40 min at 1300 V followed by a second
electrophoresis in buffer 2 at pH 3.5 [pyridine-acetic
acid-H2O; 5:50:945 (vol/vol/vol)] for 15 min at 1000 V in
the orthogonal direction. Phosphoserine, phosphothreonine, and
phosphotyrosine (0.5 mg each) were included in the sample and made
visible after the separation by ninhydrin staining. The dried thin
layer plates were subjected to autoradiography.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Jeffrey Benovic for cDNAs for GRK2 and 3 and Dr.
Robert J. Lefkowitz for the minigene of the carboxyl terminus of GRK
2.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Robert A. Nissenson, Endocrine Research Unit, Department of Medicine, University of California, San Francisco, Veterans Affairs Medical Center, 4150 Clement Street (111N), San Francisco, California 94121-1598. E-mail:
Chicago{at}itsa.ucsf.edu
This work was supported by the Medical Research Service of the
Department of Veterans Affairs and by NIH Grant DK-35323 (to R.A.N.).
Dr. Nissenson is a Research Career Scientist of the Department of
Veterans Affairs.
Received for publication June 15, 1998.
Revision received August 25, 1998.
Accepted for publication August 28, 1998.
 |
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