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J Biol Chem, Vol. 274, Issue 42, 29968-29975, October 15, 1999
From the Division of Bone and Mineral Metabolism, Harvard-Thorndike
and Charles A. Dana Research Laboratories, Department of Medicine, Beth
Israel Deaconess Medical Center and Harvard Medical School, Boston,
Massachusetts 02215
Endocytosis and intracellular trafficking of the
human parathyroid hormone receptor subtype 1 (hPTH1-Rc) and its ligands
was monitored independently by real-time fluorescence microscopy in stably transfected HEK-293 cells. Complexes of fluorescence-labeled parathyroid hormone (PTH)-(1-34) agonist bound to the hPTH1-Rc internalized rapidly at 37 °C via clathrin-coated vesicles, whereas fluorescent PTH-(7-34) antagonist-hPTH1Rc complexes did not. A functional C terminus epitope-tagged receptor (C-Tag-hPTH1-Rc) was
immunolocalized to the cell membrane and, to a lesser extent, the
cytoplasm. PTH and PTH-related protein agonists stimulated C-Tag-hPTH1-Rc internalization. Relocalization to the cell membrane occurred 1 h after removal of the ligand. Endocytosis of
fluorescent PTH agonist-hPTH1-Rc complexes was blocked by the protein
kinase C (PKC) inhibitor staurosporine but not by the specific protein kinase A inhibitor
N-(2-(methylamino)ethyl)-5-isoquinoline-sulfonamide. Fluorescent PTH antagonist-hPTH1-Rc complexes were rapidly internalized after PKC activation by phorbol 12-myristate 13-acetate or thrombin, but not after stimulation of the cAMP/protein kinase A pathway by
forskolin. In cells co-expressing the hPTH1-Rc and a green fluorescent
protein- G protein-coupled receptors
(GPCRs)1 represent a major
class of membrane-bound proteins that mediate a wide variety of
biological functions, including expression of the biological actions of
various hormones. The responsiveness of GPCRs to extracellular stimuli, and particularly to their natural ligands, is regulated by several mechanisms, including receptor phosphorylation, coupling and uncoupling from G proteins, receptor internalization (endocytosis), and regulation of receptor gene transcription (1-3). In particular, a model for
agonist-activated GPCR endocytosis, which involves mobilization of
Little is known about the molecular mechanisms regulating
internalization and intracellular trafficking of a structurally distinct class of GPCRs, designated Class II (15). The natural ligands
of this GPCR subfamily, which includes receptors for parathyroid hormone (PTH) and PTH-related protein (PTHrP), glucagon, calcitonin, secretin, vasoactive intestinal peptide, and others, are medium-sized polypeptides. Understanding of the mechanisms underlying
ligand-receptor bimolecular recognition and interactions within the
GPCR Class II subfamily is advancing rapidly (16-20). Nevertheless,
important questions remain regarding the relationship among receptor
occupancy, signaling, and endocytosis. These issues are particularly
interesting in light of recent models that propose multiple
conformational states for GPCRs (21-23), which may be stabilized by
different classes of ligands (i.e. agonists, partial
agonists, antagonists, and inverse agonists) and may mediate distinct
molecular and cellular events.
PTH is the major regulator of calcium and phosphate homeostasis and
plays a central role in bone metabolism (24). Activation of the
parathyroid hormone receptor subtype 1 (PTH1-Rc) by PTH stimulates both
adenylyl cyclase/cAMP/protein kinase A (PKA) and the phospholipase
C/inositol 1,4,5-trisphosphate/cytosolic Ca2+ and
diacylglycerol/PKC pathways (25-27). In addition, PTH activates G
protein-coupled receptor kinases, such as GPCR kinase 2 (28-30). Which, if either, of these second messenger pathways is linked to
receptor endocytosis has not been determined definitely. Previous studies have provided indirect evidence suggesting that PTH1-Rc internalization occurs upon agonist stimulation, possibly via clathrin-coated vesicles (31, 32). Recovery of receptor function following blunted response to PTH stimulation has been attributed to
receptor recycling to the cell surface (33). More recently, both
positive and negative endocytic signals have been identified as
originating in the cytoplasmic tail of the opossum PTH1-Rc, transiently
expressed in COS-7 cells (32). These studies suggest that structural
changes in the receptor might contribute to the internalization
process. However, neither receptor phosphorylation nor protein kinase
activation seemed to play a role in agonist-induced internalization of
the opossum PTH1-Rc when expressed in a human cell background (30, 34).
Therefore, the relationship among ligand-receptor interactions,
receptor activation, signaling, and endocytosis in the PTH system
remains to be elucidated.
Real-time monitoring of biomolecules by fluorescence microscopy is a
powerful methodology for the study of internalization and trafficking
of both hormones and receptors in living cells. In the present study,
we designed and synthesized fluorescence-labeled PTH agonists and
antagonists, as well as an epitope-tagged human PTH1-Rc, in order to
examine independently the trafficking of both receptor and ligand and
to determine directly the relationships between receptor occupancy,
activation of specific intracellular signaling pathways, and
endocytosis of ligand-hPTH1-Rc complexes. Furthermore, by using a
GFP- Materials
Boc-protected amino acids, N-hydroxybenzotriazole
N,N'-dicyclohexylcarbodiimide, and
p-methylbenzydrylamine resin were purchased from Applied
Biosystems (Foster City, CA). Fmoc-protected amino acids were from
Bachem (Torrence, CA). B&J brand dichloromethane, acetonitrile,
methanol, N-methylpyrrolidone and
N,N'-dimethylformamide were obtained from Baxter (McGraw
Park, IL). 5-Carboxyfluorescein succinimidyl ester,
5-carboxymethylrhodamine succinimidyl ester and fluorescein-labeled
transferrin were purchased from Molecular Probes (Eugene, OR).
Dulbecco's modified Eagle's medium, fetal bovine serum, EDTA
(Versene®), trypsin, and phosphate-buffered saline (PBS) were obtained
from Life Technologies (Life Technologies, Inc.). Tissue culture
disposables and plasticware (Costar®) were purchased from Corning
(Corning, NJ). All other reagents, including staurosporine, forskolin,
cycloheximide, phorbol 12-myristate 13-acetate (PMA), and
N-(2-(methylamino)ethyl)-5-isoquinoline-sulfonamide (H8)
were from Sigma.
Peptide Synthesis
[Nle8,18,Lys13(N [Nle8,18,D-2-Nal12,Lys13(N
PTH-(1-34), PTHrP-(1-34), PTH-(7-34), and
Bpa1-PTHrP-(1-36) were synthesized and purified as
previously reported (35). The pure products were characterized by
analytical HPLC, electron spray mass spectrometry, and amino acid analysis.
Cell Culture
Native human embryonic kidney cells (HEK-293) and HEK-293 cells
stably transfected with the hPTH1-Rc (C-21 and C-20 clones expressing
~400,000 and ~40,000 receptors/cell, respectively) or with a
C-Tag-hPTH1-Rc (HPTD8 clone, see below) were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% (v/v) fetal bovine
serum (27, 36).
PTH1-Rc Binding
Stably transfected HEK-293 cells (clones C-21, C-20, and HPTD8)
were subcultured in 24-well plates and grown to confluence. Radioreceptor assays were carried out as described (35) using HPLC-purified
125I-[Nle8,18,Tyr34]bPTH-(1-34)NH2
as radioligand (37). Curves were fitted by CA-Cricket Graph III
(version 1.0), and Scatchard analysis was performed as described
previously (36).
Adenylyl Cyclase Assay
Stably transfected HEK-293 cells (clones C-21, C-20, and HPTD8)
were subcultured in 24-well plates and grown to confluence. Adenylyl
cyclase activation by PTH agonists and inhibition of PTH-(1-34)-stimulated adenylyl cyclase activation by PTH antagonists was determined in the presence of 1 mM
isobutylmethylxanthine as described (35). Cyclic AMP was isolated by
the two-column chromatographic method (38). In experiments evaluating
the effects of forskolin (10-100 µM), PMA (1-10
µM), or staurosporine (1-5 µM) on adenylyl
cyclase activity, cells were incubated with these agents for 30 min at
37 °C in the presence of isobutylmethylxanthine prior to PTH stimulation.
Intracellular Calcium Determinations
The stimulation of increases in intracellular calcium levels by
PTH-(1-34) and Bpa1-PTHrP-(1-36) was assessed
spectroscopically in Fura-2-loaded HEK-293 cells stably transfected
with hPTH1-Rc (clone C-21) as described (39).
Detection of Fluorescent PTH Agonists and Antagonists in
Living Cells
In order to perform real-time microscopy with
fluorescence-labeled PTH ligands, C-21 cells were plated on 25-mm glass
coverslips (1.5 × 105 cells/coverslip) and cultured
for 48 h. Coverslips were then rinsed twice in cold PBS and
mounted in an open-air chamber. After 10 min in cold PBS/1% bovine
serum albumin (blocking buffer), coverslips were incubated with
fluorescence-labeled PTH agonists or antagonists for 5 min on ice,
unless otherwise indicated, and the unbound ligand was subsequently
removed by washing three times with cold PBS. Coverslips were then
covered with 1 ml of PBS/0.1% bovine serum albumin, and the cell
chamber was placed at 37 °C in a temperature-controlled block for
the duration of the experiment. Competition of fluorescent ligands
binding was performed by preincubating the coverslips 30 min at
37 °C with unlabeled PTH ligands, using concentrations as indicated
in the figure legends. When co-localization of fluorescent PTH ligands
with fluorescein-conjugated transferrin was investigated, coverslips
were incubated for 5 min on ice with both reagents simultaneously;
subsequent treatment was carried out as described above.
In some experiments, cells were treated with sucrose or with activators
or inhibitors of PKA (forskolin or H8, respectively) and of PKC (PMA or
staurosporine, respectively). In this case, cells on coverslips were
preincubated with the pharmacological agents for 30 min at 37 °C,
using concentrations as detailed in the figure legends. Subsequently,
these reagents were washed and cells were incubated with the
fluorescent ligands as described above. Coverslips were then covered
with 0.1% PBS/bovine serum albumin containing the pharmacological
agents and maintained at 37 °C for the duration of the observation.
When thrombin was used as an activator of PKC, there was no
preincubation, but coverslips were first incubated with the fluorescent
ligands as described above and then treated with thrombin (1 µM) at 37 °C for the duration of the observation.
Fluorescence was viewed on a Nikon epifluorescence microscope (Nikon
Diaphot 300®) with filters selective for rhodamine and fluorescein
labeling. Micrographs were taken using a Sensys CCD® digital camera
(Photometrics, Tucson, AZ) and the Image-pro Plus® software (media
Cybernetics, Silver Spring, MD). Z-mode (confocal-like) analysis, in
which sequential images were taken at 0.1-µm intervals throughout the
cell body, was used for co-localization of two differentially
fluorescence-labeled entities.
In order to perform real-time fluorescence microscopy of
Generation of a C-terminal Epitope-tagged hPTH1-Rc
(C-Tag-hPTH1-Rc)
The full-length hPTH1-Rc cDNA, previously cloned into
pcDNA1 (40), was amplified by polymerase chain reaction using
primers designed to delete the stop codon (GenBankTM
accession number L04308; nucleotides 26-42, forward, and 1790-1807, reverse). The 2.0-kb polymerase chain reaction product was sub-cloned into the TOPO® ligation site of the mammalian expression vector pCDNA3.1/V5/His (Invitrogen, Carlsbad, CA), in frame with the 3'-V5
and 3'-His6 tag sequences followed by a stop codon.
Integrity and orientation of the final construct were verified by
dideoxynucleotide sequencing. Plasmid pCDNA3-C-Tag-hPTH1-Rc was
transfected into HEK-293 cells using Fugene® (Roche Molecular
Biochemicals), and stable transfectants were selected with G418 (800 µg/ml). Because preliminary experiments in COS-7 cells transiently
transfected with this plasmid demonstrated a 4-6-fold increase of cAMP
levels in response to 100 nM PTH-(1-34), G418-resistant
clones were screened for expression of the C-Tag-hPTH1-Rc by adenylyl
cyclase assay. Stable expression of the C-Tag-hPTH1-Rc was further
confirmed by Western blot using mouse anti-V5 or anti-His6
monoclonal antibodies (diluted 1:100 to 1:1000, Invitrogen, Carlsbad,
CA) and horseradish peroxidase-conjugated secondary antibody (Roche
Molecular Biochemicals). The specificity of both antibodies for
the epitope-tagged receptor was assessed by using as controls PTH1-Rc
negative HEK-293 and C-21 cells, which express ~400,000
hPTH1-Rc/cell.
Localization of the C-Tag-hPTH1-Rc in Fixed Cells by Indirect
Immunofluorescence
HEK-293 cells stably expressing the C-Tag-hPTH1-Rc (HPTD8 cells)
were grown to 50-60% confluency on glass coverslips as described above. Cells were then treated as detailed in the figure legends, transferred to ice, rinsed in PBS, and fixed and permeabilized in neat
methanol for 15 min at In some experiments, in order to prevent de novo protein
synthesis, cells were preincubated 2 h with cycloheximide (25 µg/ml) at 37 °C and maintained in the presence of cycloheximide
during and after treatment with PTH.
Characterization of Fluorescent PTH
Analogs--
Fluorescence-labeled agonists (Rho-PTH-(1-34) and
Fluo-PTH-(1-34)) and antagonists (Rho-PTH-(7-34) and
Fluo-PTH-(7-34)) derived from the sequence of bovine parathyroid
hormone (bPTH) were synthesized by solid phase methodology followed by
postsynthetic site-selective modification of the Cellular Distribution of Fluorescent PTH Agonist and
Antagonist--
Fig. 1 illustrates
real-time monitoring of the cellular distribution of the agonist
Rho-PTH-(1-34) or the antagonist Rho-PTH-(7-34) in living C-21 cells
by fluorescence microscopy. In cells incubated with either ligand
(10-100 nM, 5 min on ice to prevent spontaneous internalization) and extensively washed to remove unbound ligand, fluorescence was initially well delineated on the cell membrane (Fig.
1, a and e). The ligand distribution was hPTH1-Rc
specific: it was inhibited by preincubation (30 min at 37 °C) with
nonfluorescent PTH-(7-34) (Fig. 1i) or PTH-(1-34) (not
shown) and was undetectable in nontransfected HEK-293 cells (Fig.
1j). No change in ligand distribution occurred in cells
maintained at 4 °C (data not shown). However, within minutes after
raising the temperature to 37 °C, the agonist Rho-PTH-(1-34) formed
clusters on the cell membrane and subsequently was internalized (Fig.
1, b and c). After 50 min, there was coalescence
of the intracellular fluorescence, with complete disappearance of the
ligand from the cell surface (Fig. 1d). Analogous results
were obtained with Fluo-PTH-(1-34) (data not shown).
Following a 5-min preincubation at 4 °C, receptor-bound PTH
antagonist Rho-PTH-(7-34) was observed to be evenly distributed on the
surface of cells (Fig. 1, e-h). In contrast to the findings with the agonist, increasing the temperature to 37 °C produced no
internalization and the pattern of fluorescence remained essentially unchanged. Analogous results were obtained with Fluo-PTH-(7-34) (data
not shown). Only a few isolated fluorescent dots, representing internalized Rho-PTH-(7-34), were detectable in the immediate submembranal region after 50 min at 37 °C (Fig. 1h).
After prolonged observation (2 h), there was no evidence of further
internalization or intracellular coalescence of the fluorescence (data
not shown), suggesting that the observed small fraction of internalized
Rho-PTH-(7-34) probably represents a slow, constitutive cycling of
hPTH1-Rc. Increasing the concentration of Rho-PTH-(7-34) (from 100 to
1000 nM), the preincubation temperature (from 4 to
25 °C) and duration (from 5 to 10 min) had no further effects on the
cellular distribution of the antagonist.
Characterization and Immunolocalization of C-Tag-hPTH1-Rc--
In
order to directly visualize trafficking of the hPTH1-Rc, a modified
hPTH1-Rc containing both His6 and V5 epitope tags at the C
terminus (C-Tag-hPTH1-Rc) was designed. PTH-(1-34) binding
(IC50 = 3 nM) and stimulation of adenylyl
cyclase activity (EC50 = 20 nM) in HEK-293
cells stably transfected with the C-Tag-hPTH1-Rc (clone HPTD8,
~70,000 Rcs/cells) demonstrated receptor function comparable to
unmodified hPTH1-Rc expressed at a similar level in HEK-293 cells
(clone C-20) (27, 36).
HPTD8 cells fixed and permeabilized with methanol were used to follow
the receptor by indirect fluorescence using a monoclonal antibody
directed against the epitope-Tag of the C-Tag-hPTH1-Rc and
rhodamine-labeled anti-mouse IgG as secondary antibody (Fig. 2). In the absence of agonist ligand, the
C-Tag-hPTH1-Rc was localized to the cell surface, and, less
extensively, to the cytoplasm (Fig. 2a). One hour of
incubation with 100 nM PTH-(1-34) (Fig. 2b) or PTHrP (1-34) (not shown) induced extensive internalization of the
receptor. After removal of the ligand from the medium, a progressive relocalization of receptors to the cell membrane was visualized and
completed within 1-2 h (Fig. 2c). Identical results were
obtained in HPTD8 cells fixed and permeabilized with
paraformaldehyde/Triton X-100 and treated as described above (not
shown). In cells preincubated for 2 h in the presence of
cycloheximide (25 µg/ml) in order to inhibit de novo
protein synthesis, C-Tag-hPTH1-Rcs were initially expressed on the cell
membrane and less markedly in the cytoplasm, and they were internalized
after PTH treatment (Fig. 2, d and e,
respectively). However, under these conditions, relocalization to the
cell membrane was delayed and incomplete even after 2 h (Fig. 2,
f and g).
These changes in membranal receptor population were in full accordance
with the total binding capacity for the radioiodinated PTH-(1-34)
analog. Time-dependent recovery of radiolabeled PTH-(1-34) binding capacity (almost back to the initial value) was observed 2 h after wash-out of the ligand (Fig. 3)
in HPTD8 cells. In contrast, the recovery of radioligand binding in
cycloheximide-treated HPTD8 cells (2 h at 25 µg/ml) was reduced and
incomplete even after 2 h following wash-out of the ligand (Fig.
3).
Clathrin-mediated Internalization of PTH with the hPTH1-Rc--
In
order to assess whether endocytosis of fluorescent agonist associated
with the hPTH1-Rc was specifically mediated by clathrin-coated vesicles, C-21 cells were incubated with Rho-PTH-(1-34) in hypertonic medium (0.45 M sucrose). These conditions have been
reported to selectively disrupt clathrin-coating of endosomes (41).
Under these conditions, internalization of Rho-PTH-(1-34) was
completely prevented (data not shown). Furthermore, in HPTD8 cells
(expressing the C-Tag-hPTH1-Rc) co-incubated with Rho-PTH-(1-34) and
fluorescein-conjugated transferrin (a marker of the clathrin-coated
pit-mediated internalization pathway), intracellular co-localization of
the fluorescence of both ligands was observed after 15 min at 37 °C
(Fig. 4). These data indicate that
fluorescent PTH arrived at its intracellular locale via clathrin-coated
vesicles.
Effect of PKA and PKC on Endocytosis--
The following
experiments were carried out in order to examine the role of PKA and
PKC activation on internalization of the hPTH1-Rc and its ligands.
Endocytosis of the agonist Rho-PTH-(1-34) in C-21 cells (Fig.
5a) was completely blocked by
the PKC inhibitor staurosporine (5 µM) (Fig.
5b). Under the same experimental conditions, the PKA
inhibitor H8 (50 µM) was ineffective (Fig.
5c). These results are independent of the number of
hPTH1-Rcs on cell surface, because identical findings were obtained in
C-20 cells, which stably express a 10-fold lower level of hPTH1-Rc
(data not shown). Importantly, staurosporine (5 µM)
significantly increases basal cAMP levels in C-21 cells and markedly
potentiates PTH-stimulated cAMP production (EC50 = 0.28 versus 3.5 nM with and without staurosporine, respectively). In addition, the magnitude of intracellular calcium transients stimulated by 100 nM PTH-(1-34) was similar
(150 ± 20 nM) in C-21 cells in the presence and
absence of staurosporine (5 µM). These observations
therefore strongly suggest that neither activation of the cAMP/PKA
pathway nor increases in intracellular calcium levels are directly
involved in the internalization of agonist-occupied hPTH1-Rc, whereas
PKC may play a critical role in this process.
To support this hypothesis, C-21 cells incubated with the antagonist
Rho-PTH-(7-34) were treated with the PKC activator PMA (1 µM) or thrombin (1 µM). Endocytosis of the
otherwise noninternalized Rho-PTH-(7-34) (Fig. 5d) was
markedly induced by both PMA (Fig. 5e) and thrombin (Fig.
5f). As was seen for the agonist Rho-PTH-(1-34), internalization of the antagonist was completely inhibited by staurosporine (5 µM) (Fig. 5g). Moreover,
internalized Rho-PTH-(7-34) co-localized with fluorescein-transferrin,
indicating that PKC-activated endocytosis of the PTH antagonist also
occurred through clathrin-coated vesicles (data not shown). In
contrast, treatment of C-21 cells with forskolin (10-100
µM) did not produce Rho-PTH-(7-34) internalization (Fig.
5h), despite increasing cAMP 20-fold.
HPTD8 cells expressing the C-Tag-hPTH1-Rc also were incubated with PMA
(1 µM) but without any hPTH1-Rc ligand. In this case, the
receptor remained mostly localized on the cell surface, suggesting that
no significant internalization occurs in the absence of ligand (data
not shown).
The objective of the present study was to investigate the
relationship among receptor occupancy, activation of PKA- and
PKC-dependent second messenger pathways, and endocytosis of
the hPTH1-Rc and its ligands. Direct evidence of GCPR internalization
has been reported previously (Refs. 1-14 and references therein).
However, data concerning endocytosis of the hPTH1-Rc are sparse and
mostly indirect (30-34). In particular, the molecular and biochemical mechanisms responsible for hPTH1-Rc internalization remain
undetermined. Our approach to studying trafficking in the PTH1-Rc
system was to use three independent lines of investigation based on
fluorescence microscopy methodologies. One avenue is based on
monitoring the intracellular trafficking of PTH ligands, both agonists
and antagonists, that have been labeled with a fluorescent moiety. The
second avenue examines the distribution of the receptor by
immunofluorescence microscopy using a newly created C-terminal
epitope-tagged hPTH1-Rc (C-Tag-hPTH1-Rc). The third avenue is to
monitor changes in the cellular distribution of a Immunostaining of C-Tag-hPTH1-Rc in stably transfected HEK-293 cells
indicates that unoccupied receptors are present predominantly on the
cell surface and to a lesser extent intracellularly. Agonist occupancy
and activation of the receptor causes a rapid and
temperature-dependent clustering of fluorescent PTH-hPTH1Rc
complexes on the cell membrane, followed by endocytosis. Inhibition of
agonist-occupied receptor internalization by hypertonic medium and
co-localization of internalized PTH agonist-hPTH1-Rc complexes with
transferrin confirms that the process of receptor endocytosis is
mediated by clathrin-coated pits, as previously suggested (31, 32). We
found no evidence to indicate that the internalized fluorescent
Rho-PTH-(1-34) agonist, which accumulates intracellularly, is recycled
to the membrane. It is likely that, similar to other ligands for GPCRs
(1), endosomes target PTH to lysosomes for degradation.
In contrast, following removal of PTH-(1-34) from the medium, the
internalized hPTH1-Rc relocalizes to the cell membrane within 1 h,
as assessed by both immunostaining and radioligand binding. Recycling
of the internalized hPTH1-Rc still occurs, even in the presence of
cycloheximide, an inhibitor of protein synthesis, in agreement with
previous observations (33). These data suggest that the receptors
originally monitored are returned to the cell surface. However, the
slower rate and lower extent of receptor relocalization to the cell
membrane in the presence of cycloheximide suggests some role for
protein biosynthesis during recycling. Whether this observation
reflects a reduction in de novo synthesis of hPTH1-Rc or a
depletion of particular trafficking-adaptor molecules remains to be elucidated.
Agonist stimulation of hPTH1-Rc-expressing cells causes a very rapid
mobilization to and clustering of Although PTH-(7-34) antagonists do not induce rapid internalization of
the hPTH1-Rc nor mobilize Because agonist stimulation of the hPTH1-Rc activates both the protein
kinase A and C pathways (25-27), the role of these intracellular signaling events in ligand and receptor endocytosis was investigated. Our data indicate that PKA activity is neither sufficient nor required
for internalization of ligand-occupied hPTH1-Rc: inhibition of PKA by
the agent H8 does not prevent endocytosis of the fluorescent Rho-PTH-(1-34) agonist, and maximal exogenous stimulation of the cAMP/PKA pathway by forskolin does not alter the membrane distribution of fluorescent Rho-PTH-(7-34) antagonist. In contrast, exogenous PKC
activation by PMA or, in a more specific manner, heterologous PKC
activation by thrombin, markedly increases internalization of
antagonist-occupied hPTH1-Rc. However, PKC activation per se is not sufficient to promote receptor internalization: pretreatment with PMA did not significantly affect either binding of radiolabeled PTH-(1-34) or the cell surface distribution of unoccupied
C-Tag-hPTH1-Rc. Furthermore, inhibition by staurosporine of either
agonist-, PMA-, or thrombin-induced PKC activity completely blocked
ligand-receptor complex internalization. Moreover, the essential role
of the PKC pathway in hPTH1-Rc endocytosis was independently validated
by the inability of Bpa1-PTHrP-(1-36), which selectively
activates the cAMP/PKA pathway without producing intracellular
calcium transients, to induce In contrast to our observations, Nissenson and co-workers (30, 34)
recently reported that protein kinases activity, including PKA, GPCR
kinase 2, and, notably, PKC, is only minimally involved in endocytosis
of the agonist-activated opossum PTH1-Rc receptor. Although the reasons
for such an apparent discrepancy need to be further investigated, they
may originate from species-related differences in the receptor or the
cell systems studied (43, 44).
The lack of internalization of unoccupied hPTH1-Rc and the ability of
PTH antagonist-occupied receptor to be internalized suggests that
occupancy of the receptor induces conformational changes that may be a
prerequisite for endocytosis. The conformational features required for
internalization clearly are distinct from those leading to receptor
activation and signaling. Antagonist-promoted internalization in the
absence of receptor signaling has been observed for CCK (45) and
endothelin subtype A (46) receptors, even in the absence of either
exogenous or heterologous protein kinase activation. In addition, in
staurosporine-treated cells, Rho-PTH-(1-34)-occupied hPTH1-Rcs do not
necessarily internalize, despite efficient activation of both
Gs and Gq by the PTH agonist, leading to
increased cAMP and cytosolic calcium levels. These observations also
indicate that receptor activation and G protein coupling are neither
sufficient nor required for ligand-receptor endocytosis.
In conclusion, we have generated a series of novel molecular reagents
that enable us to study directly and independently endocytosis and
intracellular trafficking of both the human PTH1-Rc and PTH ligands in
living cells using fluorescence microscopy. Our results provide new
insights into the relationship among hPTH1-Rc occupancy, activation,
and signaling (particularly through the PKC pathway), and endocytosis,
as well as We thank Dr. Paola Marignani for invaluable
help with the fluorescence microscopy and Dr. Joseph Alexander for
helpful discussions and critical reading of the manuscript. We also
thank Dr. Marc Caron for the *
This work was supported in part by Grant RO1-DK47940 from
the NIDDK, National Institutes Health (to M. R.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Beth Israel Deaconess
Medical Center, Division of Bone and Mineral Metabolism (HIM 944), 330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-0904; Fax:
617-667-4432; E-mail: abisello@caregroup.harvard.edu.
2
V. Behar, A. Bisello, M. Rosenblatt, and M. Chorev, unpublished data.
The abbreviations used are:
GPCR, G
protein-coupled receptor;
PTH, parathyroid hormone;
bPTH, bovine PTH;
PTH-(1-34), [Nle8,18,Tyr34]bPTH-(1-34)NH2;
PTH-(7-34), [Nle8,18,D-Trp12,Tyr34]bPTH-(7-34)NH2;
PTHrP, PTH-related protein;
hPTH, human PTH;
hPTH1-Rc, hPTH receptor
subtype 1;
Endocytosis of Ligand-Human Parathyroid Hormone Receptor 1 Complexes Is Protein Kinase C-dependent and Involves
-Arrestin2
REAL-TIME MONITORING BY FLUORESCENCE MICROSCOPY*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-arrestin2 fusion protein (-Arr2-GFP), PTH agonists
stimulated -Arr2-GFP mobilization to the cell membrane. Subsequently, fluorescent PTH-(1-34)-hPTH1Rc complexes and
-Arr2-GFP co-localized intracellularly. In conclusion,
agonist-activated hPTH1-Rc internalization involves -arrestin
mobilization and targeting to clathrin-coated vesicles. Our results
also indicate that receptor occupancy, rather than receptor-mediated
signaling, is necessary, although not sufficient, for endocytosis of
the hPTH1-Rc. Activation of PKC, however, is absolutely required.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-arrestins followed by agonist-receptor complex internalization through clathrin-coated pits, has been well characterized for the
2-adrenergic receptor (4-6). Some of these key features are common
to other GPCRs (7-9), but notable exceptions exist. For example,
although the angiotensin receptor AT1 is rapidly internalized in
response to agonist stimulation, the homologous AT2 receptor is not
(10). Although the µ-opioid receptor agonist etorphin promotes
-arrestin mobilization and receptor internalization, morphin does
not (11). Moreover, in contrast to the widely observed requirement for
receptor occupancy to enable endocytosis, heterologous protein kinase C
(PKC) activation triggers internalization of the
1b-adrenoreceptor even in the absence of ligand (12).
Also, whereas endocytosis of many agonist-occupied GPCR involves
clathrin-coated vesicles (1), internalization of -adrenergic
receptor and cholecystokinin receptors may occur independently of
clathrin-coated pits (13, 14).
-arrestin2 fusion protein (-Arr2-GFP) (7), we gained insights
into the involvement of this adaptor molecule in both endocytosis and
trafficking of the hPTH1-Rc.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-5-carboxymethylrhodamine),L-2-Nal23,Arg26,27,Tyr34]bPTH-(1--
34)NH2
(Rho-PTH-(1-34)) and
[Nle8,18,Lys13(N
-5-carboxymethylfluorescein),L-2-Nal23,Arg26,27,Tyr34]bPTH-(1-34)NH2
(Fluo-PTH-(1-34))
The synthesis of
N
-Fmoc-[Nle8,18,L-2-Nal23,Arg26,27,Tyr34]bPTH-(1-34)NH2
was carried out by the solid phase methodology using
Boc/N-hydroxybenzotriazole/N-methylpyrrolidone
chemistry, liquid hydrogen fluoride cleavage, and HPLC
purification as reported previously (17). Five mg (8 × 10
7 mol; peptide content, 70%) of
N-Fmoc-[Nle8,18,L-2-Nal23,Arg26,27,Tyr34]bPTH-(1-34)NH2
were dissolved in 1 ml of N,N'-dimethylformamide in the
presence of 2.6 equivalents of either 5-carboxyfluorescein succinimidyl
ester or 5-carboxymethylrhodamine succinimidyl ester and 5 µl of
N,N-diisopropylethylamine. Reactions were followed by
HPLC/ES-MS. After completion of the reaction (approximately 4 h)
the mixture was treated for 30 min with 300 µl of piperidine to
remove the N-Fmoc protecting group. The
reaction mixture was then diluted with 30 ml of water and lyophilized.
The crude Rho-PTH-(1-34) and Fluo-PTH-(1-34) were purified by HPLC as
described (17).
-5-carboxymethylrhodamine),L-2-Nal23,Arg26,27,Tyr34]bPTH-(7--
34)NH2
(Rho-PTH-(7-34)) and
[Nle8,18,D- 2-Nal12,Lys13(N-5-carboxymethylfluorescein),L-2-Nal23,Arg26,27,Tyr34]bPTH-(7-34)NH2
(Fluo-PTH-(7-34))The synthesis of Rho-PTH-(7-34) and
Fluo-PTH-(7-34) were carried out using same protocol used for the
PTH-(1-34) analogs with
N-Fmoc-[Nle8,18,D-2-Nal12,L-2-Nal23, Arg26,27,Tyr34]bPTH-(7-34)NH2
as peptide precursor.
-Arrestin2-GFP Mobilization and Trafficking in Living
Cells
-arrestin mobilization and trafficking, C-21 cells grown on glass coverslips (as described above) were transfected with 0.3 µg of -Arr2-GFP-p(S65T)-N3 plasmid (7) (a generous gift of Dr. Marc Caron,
Duke University). Twenty-four hours after transfection, coverslips were
prepared (as described above) and incubated with ligands at 37 °C
using concentrations and durations as detailed in the figure legends.
Fluorescence was viewed as described above.
20 °C. Alternatively, fixation and
permeabilization were performed using a 2% paraformaldehyde/0.2% Triton X-100 solution for 30 min on ice. Blocking was performed by
incubating the cells for 10 min at room temperature in 4% goat serum.
Primary antibody (mouse anti-V5 mAb), diluted 1:100 in PBS/1% bovine
serum albumin, was applied to the specimens for 1 h at room
temperature, followed by three washes with the same buffer.
Rhodamine-tagged secondary antibody (goat anti-mouse (H+L) IgG,
preadsorbed against human serum (Jackson Immunoresearch, West Grove,
PA)) was diluted 1:300 and applied in the same conditions as the
primary antibody. Coverslips were then rinsed in PBS and mounted for
immunofluorescence microscopy, which was performed as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino group of
Lys13 with N-hydroxysuccinimide esters of either
5-carboxyrhodamine or 5-carboxyfluorescein. The structural integrity of
these peptides was established by amino acid analysis and electron
spray mass spectrometry. The purity, established by analytical
reverse-phase HPLC, exceeded 98%. The characterization of the
biological properties of these novel PTH analogs in C-21 cells is
reported in Table I. Both Rho-PTH-(1-34)
and Fluo-PTH-(1-34) are agonists for the hPTH1-Rc. Their binding
affinities and cyclase activities are 5-15-fold lower than those of
PTH-(1-34) (Table I). Rho-PTH-(7-34) and Fluo-PTH-(7-34) also bind
to the hPTH1-Rc and inhibit PTH-(1-34)-induced cAMP accumulation.
Similar results were obtained in HPTD8 cells.
In vitro activities of the fluorescence-labeled PTH analogs in HEK-293
cells stably expressing the hPTH1-Rc (clone C-21)
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Fig. 1.
Internalization of the fluorescence-labeled
agonist Rho-PTH-(1-34) and lack of internalization of the antagonist
Rho-PTH-(7-34). C-21 cells, stably expressing the hPTH1-Rc, were
incubated 5 min at 4 °C with the agonist Rho-PTH-(1-34) (100 nM) (a-d) or the antagonist Rho-PTH-(7-34)
(100 nM) (e-h), washed, and
maintained at 37 °C. Micrographs (magnification, × 100) were taken
immediately (a and e) and after 5 min
(b and f), 15 min (c and
g), and 50 min (d and h). Panels
a-d show binding (a), clustering (b), and
time-dependent endocytosis of Rho-PTH-(1-34) (c
and d); panels e-h show predominant localization
of Rho-PTH-(7-34) on the cell membrane at all time points. Controls
were C-21 cells preincubated for 30 min at 37 °C with unlabeled
PTH-(7-34) (100 nM) before incubation with Rho-PTH-(1-34)
(100 nM) (i) and nontransfected HEK-293 cells
incubated for 5 min at 4 °C with Rho-PTH-(7-34) (100 nM) (j). The figure is representative of six
separate experiments.
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Fig. 2.
PTH agonist-induced endocytosis and recycling
of epitope-tagged hPTH1-Rc (C-Tag-hPTH1-Rc). HPTD8 cells, stably
transfected with C-Tag-hPTH1-Rc and fixed with methanol, were
immunostained using a mouse anti-V5-epitope-Tag monoclonal antibody and
rhodamine-conjugated goat anti-mouse IgG. Micrographs (magnification, × 60) show C-Tag-hPTH1-Rc immunolocalization on the cell membrane and
partly in the cytoplasm in untreated HPTD8 cells (a),
internalization after 1 h incubation with PTH-(1-34) (100 nM) (b), and relocalization to the membrane
1 h after removal of the ligand (c). In HPTD8 cells
pretreated and maintained in the presence of cycloheximide (25 µg/ml), cell membrane localization of the C-Tag-hPTH1-Rc
(d) and internalization after 1 h PTH-(1-34) treatment
(100 nM) (e) occurred normally,
whereas relocalization to the cell surface after 1 h
(f) and 2 h (g) following removal of the
ligand was impaired. Controls were HPTD8 cells incubated with secondary
antibody in the absence of primary antibody (h) and
nontransfected (native) HEK-293 cells immunostained as described above
(i). The figure is representative of four separate
experiments.
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Fig. 3.
Recycling of C-Tag-hPTH1-Rc in HPTD8
cells. HPTD8 cells stably expressing C-Tag-hPTH1-Rc (white
bars) and HPTD8 cells pretreated for 2 h with cycloheximide
(25 µg/ml) (black bars) were treated for 15 min with 100 nM PTH-(1-34) at 37 °C in Dulbecco's minimal essential
medium/fetal bovine serum (10%) and thoroughly washed with PBS. Cells
were then maintained in Dulbecco's modified Eagle's medium/fetal
bovine serum (10%) with (white bars) or without
(black bars) cycloheximide (25 µg/ml) at 37 °C for the
indicated time. Competitive binding of
125I-[Nle8,18,Tyr34]bPTH-(1-34)NH2
was determined at 4 °C as described under "Experimental
Procedures." Total binding was measured in nontreated HPTD8 cells,
and nonspecific binding was assessed in the presence of competing
10 6 M PTH-(1-34). Data are expressed as
percentage of specific binding ± S.E. of three independent
determinations. Similar results were obtained in two additional
experiments.
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Fig. 4.
Clathrin-coated pits mediate internalization
of PTH agonist-hPTH1-Rc complexes: co-localization of internalized
Rho-PTH-(1-34) with fluorescein-conjugated transferrin. HPTD8
cells stably expressing C-Tag-hPTH1-Rc were incubated for 5 min at
4 °C with Rho-PTH-(1-34) (10 nM) and
fluorescein-conjugated transferrin (25 µg/ml), washed, and maintained
for 15 min at 37 °C. Rho-PTH-(1-34) fluorescence is shown in
red (a), and fluorescein-conjugated transferrin
fluorescence is shown in green (b). An overlay
image (c) is shown in which orange-yellow spots
represent co-localization of Rho-PTH-(1-34) and fluorescein-conjugated
transferrin. The figure is representative of three separate
experiments.
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Fig. 5.
Role of PKC or PKA on internalization of PTH
agonist- and antagonist-occupied hPTH1-Rc: PKC activity is required for
hPTH1-Rc endocytosis. C-21 cells were incubated for 5 min with the
agonist Rho-PTH-(1-34) (100 nM) (a-c) or the
antagonist Rho-PTH-(7-34) (100 nM) (d-h) and
maintained for 30 min at 37 °C. Internalization of Rho-PTH-(1-34)
(a) was inhibited by staurosporine (5 µM)
(b) but not by the PKA inhibitor H8 (50 µM)
(c). The antagonist Rho-PTH-(7-34) was internalized by
treatment with PMA (1 µM) (e) and thrombin (1 µM) (f); staurosporine (5 µM)
prevented thrombin (1 µM)-induced internalization of
Rho-PTH-(7-34) (g). Forskolin (10 µM) did not
affect the distribution of Rho-PTH-(7-34) (h). The figure
is representative of four separate experiments.
-Arrestin2 Mobilization and Trafficking--
Because
-Arrestins have been shown to be involved in the internalization
process of several GCPRs (Refs. 3, 6, and 42 and references therein),
we investigated whether or not the cellular distribution of
-Arr2-GFP changed in response to hPTH1-Rc stimulation. In resting
C-21 cells transiently expressing -Arr2-GFP, the GFP fluorescence
was evenly distributed throughout the cytoplasm (Fig.
6A, a). Upon receptor
activation by full agonists, such as PTH-(1-34) and PTHrP-(1-34) (100 nM), -Arr2-GFP was rapidly (2 min) mobilized to the cell
membrane (Fig. 6A, b). Within 15 min in the presence of
agonist, -Arr2-GFP clusters translocated into the cytoplasm (Fig.
6A, c). During this process, -Arr2-GFP co-localized with
Rho-PTH-(1-34) (50 nM), at first on the cell membrane and
later intracellularly (Fig. 6B). Fifteen min after removal
of the agonist, -Arr2-GFP redistributed evenly in the cytoplasm
(data not shown). In contrast, neither the antagonist PTH-(7-34) (Fig.
6A, d-f), nor Bpa1-PTHrP-(1-36) (Fig.
6A, g-i), an "agonist" that selectively activates adenylyl cyclase (EC50 = 4 nM) without
eliciting intracellular calcium
transients,2 stimulated
translocation of -Arr2-GFP.
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Fig. 6.
A, fluorescence microscopy of
-Arr2-GFP mobilization. C-21 cells transiently expressing
-Arr2-GFP were incubated at 37 °C with the agonist PTHrP-(1-34)
(100 nM) (a-c), the antagonist PTH-(7-34) (1 µM) (d-f), or the selective adenylyl
cyclase-activating agonist Bpa1-PTHrP-(1-36) (100 nM) (g-i). Micrographs (magnification, × 100)
at times 0 (a, d, and g), 2 min
(b, e, and h), and 15 min (c, f, and
i) show that only the full agonist PTHrP-(1-34) stimulates
-Arr2-GFP mobilization to the cell membrane (b) followed
by retranslocation to the cytoplasm (c). B,
co-localization of -Arr2-GFP with Rho-PTH-(1-34)-hPTH1Rc complexes.
C-21 cells transiently expressing -Arr2-GFP were incubated for 10 min at 4 °C with Rho-PTH-(1-34) (50 nM), washed, and
maintained at 37 °C. Micrographs were taken after 2 min
(a-c) and 15 min (d-f). Monitoring
Rho-PTH-(1-34) fluorescence (in red) (a and
d) and -Arr2-GFP fluorescence (in green)
(b and e), it was observed that both appear at
first to cluster on the cell membrane (a and b)
and then in the cytoplasm (d and e). In the
overlay images (c and f), orange-yellow
spots represent co-localization of Rho-PTH-(1-34) and
-Arr2-GFP. Images in both panels are representative of three
separate experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-arrestin2-GFP
fusion protein (7) in relation to PTH1-Rc activation and the movement
of PTH-PTH1-Rc complexes.
-arrestin2 to the cell membrane,
followed later by return to the cytoplasm. The time course and pattern
of -Arr2-GFP redistribution following receptor activation coincide
with those of PTH-hPTH1Rc complex movement, indicating that
-arrestins are involved not only at the time of initiation of the
endocytic process but also in the intracellular trafficking of
hPTH1-Rc. These observations, analogous to those reported for the
angiotensin AT1A and neurotensin receptors, but different from
2-adrenergic and other receptors (42), provide further evidence that
the cellular trafficking of -arrestins themselves is differentially
regulated by the activation of distinct GPCRs (42). The observation
that -Arr2-GFP rapidly redistributes evenly in the cytoplasm after
removal of PTH agonist suggests that arrestins are not involved in the
recycling of the internalized hPTH1-Rc to the cell membrane.
-Arr2-GFP, a minor fraction of the
fluorescent antagonist accumulates intracellularly over time. These
findings are consistent with the existence of a different pathway
responsible for slow agonist- and arrestin-independent receptor
cycling. Importantly, the availability of fluorescent antagonists that
bind to the receptor with high affinity but that are not actively
internalized provides a unique reagent for the study of the functional
relationship among receptor occupancy, signaling, and ligand-receptor
complex endocytosis.
-arrestin2 mobilization. Taking these
data together, we conclude that hPTH1-Rc internalization requires
occupancy by ligand and concomitant activation (not necessarily
homologous) of the PKC signaling pathway.
-arrestin trafficking. These reagents may prove useful in
elucidating the molecular mechanisms underlying hPTH1-Rc receptor
desensitization and their relevance to the wide spectrum of metabolic
outcomes produced by various regimens of PTH treatment.
ACKNOWLEDGEMENTS
-Arr2-GFP cDNA.
FOOTNOTES
Supported by a postdoctoral fellowship grant from the Swiss
National Science Foundation and the Foundation des Bourses de Recherche
en Medecine et Biologie.
ABBREVIATIONS
-Arr2, -Arrestin2;
Bpa1-PTHrP-(1-36), [p-benzoylphenylalanine1,Ile5,Arg11,13,Tyr36]PTHrP-(1-36)NH2;
Fluo-PTH-(1-34), [Nle8,18,Lys13(N-5-carboxymethylfluorescein),L-2-Nal23, Arg26,27,Tyr34]bPTH-(1-34)NH2;
Fluo-PTH-(7-34), [Nle8,18,D-2-Nal12,Lys13(N-5-carboxymethylfluorescein),L-2-Nal23,Arg26,27,Tyr34]bPTH- (7-34)NH2;
GFP, green fluorescent protein;
H8, N-(2-(methylamino)-ethyl)-5-isoquinoline-sulfonamide;
HEK, human embryonic kidney;
HPLC, high performance liquid
chromatography;
Nal, naphtylalanine;
Nle, norleucine;
PBS, phosphate-buffered saline;
PKA, protein kinase A;
PKC, protein kinase
C;
PMA, phorbol 12-myristate 13-acetate;
PTH1-Rc, PTH receptor subtype
1;
Rho-PTH-(1-34), [Nle8,18,Lys13(N-5-carboxymethylrhodamine),L-2-Nal23,Arg26,27,Tyr34]bPTH-(1-34)NH2;
Rho-PTH-(7-34), [Nle8,18,D-2-Nal12,Lys13(N-5-carboxymethylrhoda-mine),L-2-Nal23,Arg26,27,Tyr34]bPTH-(7-34)NH2.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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