From the Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599
Received for publication, October 31, 2000, and in revised form, December 11, 2000
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ABSTRACT |
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Agonist-promoted regulation of the uridine
nucleotide-activated human P2Y4 receptor (P2Y4-R) and P2Y6 receptor
(P2Y6-R) was studied. Incubation of P2Y4-R-expressing 1321N1 human
astrocytoma cells with the cognate agonist UTP resulted in rapid
desensitization of the inositol phosphate response and a 50% loss of
cell surface receptors. In contrast, incubation of P2Y6-R-expressing
cells with the cognate agonist UDP caused neither rapid desensitization nor rapid loss of cell surface receptors. Removal of UTP from the
medium of UTP-pretreated cells resulted in rapid and complete recovery
of surface P2Y4-R even after 12 h of agonist treatment. Although
extended incubation with UDP also caused a loss of surface P2Y6-R,
rapid recovery of surface P2Y6-R did not occur following removal of
agonist. Pharmacological studies indicated that neither protein kinase
C nor other Ca2+-activated kinases were involved in
agonist-promoted desensitization or loss of surface P2Y4-R or P2Y6-R.
Mutational analyses were carried out to identify domains involved in
agonist-dependent regulation of P2Y4-R. Sequential
truncation of the carboxyl-terminal domain revealed that sequence
between amino acids 332 and 343 was necessary for UTP-promoted
desensitization and internalization. Further mutational analyses of the
three serines in this domain confirmed that Ser-333 and Ser-334 play a
major role in these agonist-promoted changes in P2Y4-R. Experiments
were carried out with [32P]Pi-labeled cells
to ascertain the role of phosphorylation in regulation of P2Y4-R.
Incubation with UTP for 2 min caused a marked increase in
phosphorylation of both the wild-type P2Y4-R and the P2Y4-343
truncation mutant. In contrast, no UTP-promoted phosphorylation of the
P2Y4-332 truncation mutant was observed. Taken together, these results
demonstrate differential regulation of uridine nucleotide-activated P2Y4-R and P2Y6-R and indicate that Ser-333 and Ser-334 in the carboxyl
terminus of P2Y4-R are important for UTP-dependent
phosphorylation, desensitization, and loss of surface receptors.
Adenine nucleotides function as neurotransmitters in the
peripheral and central nervous systems and as autocrine or paracrine signaling molecules in most other tissues (1-3). Two large classes of
receptors, consisting of the ligand-gated P2X receptors and the
G-protein-coupled P2Y receptors (4-6), are broadly distributed and
promote a myriad of physiological responses to ATP and ADP, including
neurotransmission, muscle contraction, immunological responses, and
platelet aggregation (2, 7). A complex family of ecto-enzymes rapidly
hydrolyze or interconvert extracellular nucleotides thereby either
terminating their signaling action or producing an active metabolite of
altered P2 receptor selectivity (8).
The existence of extracellular pyrimidinergic signaling, first
suggested over a decade ago (9-11), was brought into focus in studies
of UTP-promoted second messenger responses (12-16). UTP was confirmed
as an extracellular signaling molecule by demonstration of regulated
release of endogenous UTP (17, 18) and by cloning of uridine
nucleotide-activated G-protein-coupled receptors (19-22). The P2Y2-R,
which is equipotently activated by ATP and UTP, was the first
molecularly identified uridine nucleotide-activated receptor (19).
However, the demonstration (23), and then cloning (20-22) of uridine
nucleotide-specific members of the P2Y receptor family placed the
pyrimidinergic signaling hypothesis on firm ground. The human
P2Y4-R1 is specifically
activated by UTP (20, 21, 24) and competitively antagonized by ATP
(25); nucleoside diphosphates are inactive. In contrast, the human
P2Y6-R is specifically activated by UDP and is insensitive to UTP or
other triphosphates (22, 24).
The biological significance of P2Y receptors that exhibit strict
specificity for either UTP or UDP is not yet understood, and evidence
for physiological release of UDP in concentrations that activate P2Y6-R
is not available. Thus, the extracellular breakdown of UTP to UDP may
provide an important source of activating agonist for P2Y6-R (26). If
metabolic formation of UDP is important, then temporal differences
might be expected in both the rapidity by which these receptors are
activated upon release of nucleotide and in the length of their
activation. The relative capacity of P2Y4-R and P2Y6-R to undergo
agonist-induced desensitization also would interplay with possible
differences in their rapidity and longevity of activation due to
differences in the extracellular presence of their cognate agonists.
Little is known about the regulation of P2Y-R in general and of the
uridine nucleotide activated P2Y-R in particular. Moreover, P2Y4-R and
P2Y6-R differ markedly in potential sites for phosphorylation by second
messenger-regulated kinases and GRKs. Thus, we initiated a comparative
examination of the agonist-promoted changes in the activities of these
receptors and of the mechanisms that underlie these changes. Major
differences in the mechanisms of regulation of P2Y4-R versus
P2Y6-R are described below. We also report identification of two
adjacent serines in the carboxyl terminus of the P2Y4-R receptor that
play a major role in agonist-dependent phosphorylation, desensitization, and internalization of this receptor.
Construction of Mutant P2Y4-R and P2Y6-R cDNAs--
Mutant
receptor cDNA constructs were generated by polymerase chain
reaction using cloned Pfu polymerase (Stratagene) and
primers that incorporated an EcoRI or MluI restriction site
at the amino terminus and a XhoI site at the carboxyl
terminus. All receptor cDNAs were digested and ligated into a pLXSN
retroviral expression vector that incorporated an HA epitope tag
(YPYDVPDYAS) after the initial methionine residue. Truncation mutations
were made of the P2Y4-R carboxyl terminus using primers that engineered a stop codon directly downstream of Ser-332, Ser-343, and Ser-355. PCR
products were generated using the upstream primer
5'-CCGCCTCGATCCTCCCTT-3' and the following downstream primers:
5'-GAGACTCGAGTCAGTCCTGGGGGGTGGCC-3' for P2Y4/355,
5'-GAGACTCGAGTCAATCCTCAGGCAGGGACACTA-3' for P2Y4/343, and
5'-GAGACTCGAGTCAGGCAGCCGTGCGGGGCT-3' for P2Y4/332. Point mutations were
made in the P2Y4-R carboxyl terminus and P2Y4-R and P2Y6-R using
four-primer PCR. The upstream primer employed for all reactions was 5'- CCGCCTCGATCCTCCCTT-3', the downstream primer for mutations in
the third intracellular loop was 5'-CCTGGGGACTTTCCACAC-3', and the
downstream primer for mutations in the carboxyl terminus was
5'-GACTATGGTTGCTGACTAATTG-3'. The following internal primers were used:
5'-TCTCGCCTCCGCGCTCTCCGCACCA-3' and 5'-TGGTGCGGAGAGCGCGGAGGCGAGA-3' for
P2Y4(S243A), 5'-CGTGGCAAGTCGGCCCGCAT-3' and
5'-ATGCGGGCCGACTTGCCACG-3' for P2Y6(A237S), and
5'-CACTAGTGCCAGGGCAGCGGCAGCCGTGCGGGGCTG-3' and
5'-GCTGCCCTGGCACTAGTGGCCCTGCCTGAGGATAGCAG-3' for P2Y4(S333A, S334A,
S339A). P2Y4 S/T-A, a full-length P2Y4-R construct with 10 serine and
threonine residues mutated to alanine, was engineered in a stepwise
fashion by first using four-primer PCR with 5'-CCGCCTCGATCCTCCCTT-3' as the upstream primer, 5'-GACTATGGTTGCTGACTAATTG-3' as the downstream primer, and 5'-GGCGGCCGCCCACCTGCAGGCGGCATCCTCAGGCAGGGACAC-3' and 5'-TGCAGGTGGGCGGCCGCCCCCCAGGACAGT-3' as internal primers. The product
from this reaction was amplified with four-primer PCR, the same
upstream and downstream primers, and
5'-CACTAGTGCCAGGGCAGCGGCAGCCGTGCGGGGCTG-3' and
5'-GCTGCCCTGGCACTAGTGGCCCTGCCTGAGGATGCCG-3' as internal primers. The
product from this reaction was amplified with the following two
primers: 5'-CCGCCTCGATCCTCCCTT-3' and
5'-TAGGAGCAGCACAAGCAGCGTCCTGGGGGGCGGCC-3' and the final,
full-length construct was generated using 5'-CCGCCTCGATCCTCCCTT-3' and
5'-GAGACTCGAGTTACAATCTATCTGCTCTAGGAGCAGCACAAGCAGC-3'. Additional point
mutant constructs were made using four-primer PCR and P2Y4/343 as
a template. 5'-CCGCCTCGATCCTCCCTT-3' and 5'-TAGGGGCGGGACTATGGTTG-3' were employed as upstream and downstream primers, respectively, and the
following internal primers were used:
5'-CACTAGTGCCAGGGAAGCGGCAGCCGTGC-3' and
5'-GCTTCCCTGGCACTAGTGTC-3' for P2Y4/343(S333A),
5'-CACTAGTGCCAGGGCAGAGGCAGCCGTGC-3' and 5'-TCTGCCCTGGCACTAGTGTC-3' for
P2Y4/343(S334A), and 5'-CACTAGTGCCAGGGCAGCGGCAGCCGTGC-3' and
5'-GCTGCCCTGGCACTAGTGTC-3' for P2Y4/343(S333A,S334A).
P2Y4/343(S339A) was generated using full-length P2Y4-R sequence as
the template and 5'-CCGCCTCGATCCTCCCTT-3' and
5'-GAGACTCGAGTCAATCCTCAGGCAGGGCCACTAGTG-3' as upstream and
downstream primers, respectively. P2Y4/343(S333A,S334A,S339A) was
constructed with P2Y4(S333A,S334A,S339A) as the template and 5'-CCGCCTCGATCCTCCCTT-3' and
5'-GAGACTCGAGTCAATCCTCAGGCAGGGCCACTA-3' as upstream and downstream
primers, respectively. The chimera P2Y6/310-Y4 was generated with
four-primer PCR using 5'-CCGCCTCGATCCTCCCTT-3' and
5'-CCTGGGGACTTTCCACAC-3' as upstream and downstream primers, respectively, and 5'-CCGGCGGAACTTCTTCTGG-3' and
5'-CCAGAAGAAGTTCCGCCGGCAGCTCCGTCAGCTCTGTG-3' as internal primers.
Cell Culture and Expression of Receptor
Constructs--
P2Y-R-expressing 1321N1 human astrocytoma cells were
grown in DMEM with 4.5 g/liter glucose (Life Technologies, Inc.)
supplemented with 5% FBS (Hyclone) in a 37 °C humidified atmosphere
with 5% CO2 and 95% air. Retrovirus packaging and cell
infections were performed as previously described (27). Following
infection, receptor-expressing 1321N1 cells were selected in medium
containing 1 mg/ml G418 (Life Technologies, Inc.). Stable cell lines
were maintained in medium containing 0.6 mg/ml G418.
Radioimmunoassay for Detection of Surface
P2Y-R--
P2Y-R-expressing 1321N1 cells were seeded at 3 × 105 cells/well in 12-well plates (Corning/Costar) coated
with 10 µg/ml fibronectin (Collaborative Bioproducts). Assays were
performed on confluent cells 2 days after plating. For most
experiments, assays were initiated by replacing the medium with DMEM
containing 50 mM HEPES (pH 7.5), and equilibrating for
1 h at 37 °C prior to assay of agonist-promoted responses. Drug
incubations of longer than 1-h duration were performed in a humidified,
37 °C/5% CO2 incubator without a medium change. Drug
treatments were terminated by placing plates in an ice bath, aspirating
the medium, adding 2% cold paraformaldehyde (Sigma), and incubating at
room temperature for 10 min. Following a wash with HBSS containing
Ca2+ and Mg 2+, HEPES/DMEM with 10%
heat-inactivated FBS was added to each well for 30 min. Anti-HA.11 raw
ascites fluid (BabCo/Covance) was added without changing the medium to
a final concentration of 1:1000 for 1 h. Cells were washed two
times with HBSS containing Ca2+ and Mg2+,
followed by addition (typically 100,000 cpm/assay) of
125I-rabbit anti-mouse antibody (PerkinElmer Life Sciences)
diluted in HEPES/DMEM with 10% heat-inactivated FBS to a concentration of 1:500. Following a 2-h incubation at room temperature, the cells
were washed twice with HBSS containing Ca2+ and
Mg2+. Cells were solubilized with 1 M NaOH and
transferred to glass tubes for quantitation of radioactivity by gamma counting.
In Vivo Labeling and Immunoprecipitation of
P2Y4-R--
Confluent 100-mm dishes of P2Y4-R-expressing cells were
washed with phosphate-free DMEM and incubated in phosphate-free DMEM for 1 h in a 37 °C humidified incubator with 5%
CO2 and 95% air. Cells were labeled with 500 µCi of
[32P]orthophosphate for 3 h. Following labeling,
cells were treated with UTP for 2 min, transferred to an ice bath, and
washed with ice-cold PBS. Ice-cold lysis buffer containing 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 5 mM EDTA, 50 mM NaF, 10 mM sodium
pyrophosphate, 200 µM Na3VO4, 1%
Triton X-100, 200 mM phenylmethylsulfonyl fluoride, 10 µg/ml benzamidine A, 1 mM pepstatin A, 4.3 mg/ml
leupeptin, and 10 mg/ml TPCK was added to each dish for 5 min. The cell
extracts were transferred to screw cap tubes, rocked for 1.5 h at
4 °C, and centrifuged at 13,000 × g for 30 min at
4 °C. The resultant supernatants were transferred to fresh tubes
containing 30 µl of protein A/G PLUS-agarose beads (Santa Cruz
Biotechnology, Inc.) that were preblocked with 1% BSA for 30 min.
Following rocking for 45 min at 4 °C, the samples were centrifuged
for 15 s at 13,000 × g, and the supernatants were
transferred to tubes containing 2 µl of anti-HA.11 raw ascites fluid
(BabCo/Covance) and 50 µl of preblocked protein A/G beads. Tubes were
rocked for 1.5 h at 4 °C followed by a quick centrifugation.
The pelleted beads were washed twice with the Triton X-100-containing
lysis buffer and resuspended in 40 µl of 2× Laemmli sample buffer.
Samples were loaded onto a 9% polyacrylamide gel, electrophoresed, and
transferred to nitrocellulose. Radioactivity associated with the
immunoprecipitated P2Y4-R was measured with a phosphorimaging
screen. Immunoblots were generated by blocking with 3% BSA/TBST for
1 h or overnight and incubating with 1:1000 anti-HA-purified
monoclonal antibody (Babco/Covance) in 3% BSA/TBST for 2 h. Blots
were washed four times with TBST followed by a 2.5-h incubation with
1:10,000 anti-mouse horseradish peroxidase-conjugated antibody in 3%
BSA/TBST. Following four washes with TBST, protein bands were
visualized with ECL reagent.
Inositol Phosphate Assay--
P2Y-R-expressing 1321N1 human
astrocytoma cells were seeded in 24-well plates at 1 × 105 cells/well 2 days prior to assay. The evening before
each assay, the medium was removed and replaced with 200 µl of
inositol-free DMEM containing 2 µCi/ml [3H]inositol.
Drug incubations were performed in a 37 °C water bath in the
presence of 10 mM LiCl and terminated by addition of 0.9 ml
of 50 mM ice-cold formic acid. After a 20-min incubation on ice, 0.3 ml of 150 mM ammonium formate was added to each
well. The supernatant from each well was transferred to Dowex
AG1-X8 columns, and total [3H]inositol phosphates were
quantitated as previously described (23, 24).
Data Analysis--
All experiments were repeated at least three
times in triplicate assay except where indicated in the figure legends.
Results are presented as the mean ± S.E.
Agonist-induced Desensitization and Loss of Cell Surface P2Y4-R and
P2Y6-R--
Agonist-induced phosphorylation of serine and threonine
residues in the third intracellular loop and carboxyl terminus has been
implicated in desensitization and subsequent internalization of a broad
range of G-protein-coupled receptors (28, 29). Although the P2Y4-R has
multiple putative phosphorylation sites in these domains, the P2Y6-R
contains only a single threonine residue in the carboxyl terminus,
suggesting that these two uridine nucleotide-activated receptors differ
significantly in their regulatory mechanisms. To test this hypothesis
we examined agonist-induced desensitization by quantitating the rate of
accumulation of total inositol phosphates in the presence of LiCl and
examined agonist-dependent loss of cell surface P2Y-R
utilizing a "surface binding assay" that employs HA-antibody
followed by a 125I-labeled secondary antibody (see
"Experimental Procedures").
Human 1321N1 astrocytoma cells stably expressing either P2Y4-R or
P2Y6-R were labeled with [3H]inositol, and
agonist-induced desensitization was assessed. The rate of inositol
phosphate accumulation in P2Y4-R-expressing cells rapidly declined in
the presence of UTP, reaching a near steady state within 10 min (Fig.
1). In contrast, incubation of P2Y6-R-expressing 1321N1 cells with UDP resulted in a linear
accumulation of inositol phosphates for at least 30 min (Fig. 1 and
data not shown). Thus, as was reported previously by Robaye et
al. (30), P2Y4-R undergoes rapid agonist-induced desensitization,
whereas P2Y6-R fails to desensitize, even after 30 min of agonist
incubation.
The surface binding assay was employed to determine whether the marked
differences in capacity of P2Y4-R and P2Y6-R to undergo desensitization
were reflected in agonist-dependent changes in surface
receptors. Incubation of P2Y4-R-expressing cells with UTP for 20 min
resulted in an ~50% decrease in the amount of surface P2Y4-R (Fig.
2). The EC50 (886 ± 300 nM) of UTP for inducing loss of surface P2Y4-R during a
20-min incubation (data not shown) was similar to the EC50
(541 ± 200 nM) of UTP for promotion of inositol
phosphate accumulation and the EC50 of UTP (780 ± 60 nM (25)) for stimulation of Ca2+ mobilization.
In contrast to the changes observed in surface P2Y4-R, a 20-min
incubation of P2Y6-R-expressing cells with UDP had essentially no
effect on surface receptor levels (Fig. 2). Thus, differences in the
capacity of these receptors to undergo rapid agonist-induced
desensitization were paralleled by differences in agonist-induced loss
of surface receptors.
Extended time courses for agonist-induced loss of surface receptors
were generated to further compare P2Y4-R and P2Y6-R. P2Y4-R levels
decreased rapidly during agonist incubation reaching a near steady
state within 10 min that decreased very slowly if at all over the
ensuing 12 h (Fig. 3). Although
agonist-promoted changes in surface P2Y6-R occurred more slowly, with
extended incubation (>1 h), decreases in the surface levels of this
uridine nucleotide-activated receptor also were observed (Fig. 3).
To study the reversibility of agonist-induced loss of surface
receptors, we took advantage of the nucleotide-hydrolyzing activity of
the enzyme apyrase. This approach was necessary, because antagonists of
P2Y4-R or P2Y6-R are not available, and mechanical stimulation caused
by medium changes of cultured cells results in release of large amounts
of cellular UTP into the medium (17, 18). The reversibility of the loss
of surface P2Y4-R or P2Y6-R was examined after various times of
incubation with UTP or UDP followed by addition of apyrase at a
concentration that completely hydrolyzed nucleotide within 1 min (data
not shown). Surprisingly, surface P2Y4-R returned to control levels
within 30 min of addition of apyrase, irrespective of the time (up to
12 h) of preincubation with agonist (Fig.
4A). The agonist-promoted loss
of cell surface P2Y4-R and the complete recovery of P2Y4-R to the
surface after hydrolysis of agonist in the medium suggest efficient
recycling of internalized receptors. This possibility was supported by
the fact that cycloheximide at a concentration (10 µg/ml) that
inhibited protein synthesis had no effect on the rapid recovery of
P2Y4-R after removal of agonist from the medium (data not shown).
Direct evidence for recycling was provided by experiments in which
P2Y4-R were prelabeled with primary antibody prior to addition of
agonist. Cell surface immunoreactivity moved to an intracellular
compartment following addition of agonist (40 ± 15% of control
after 20 min in the presence of 100 µM UTP).
Antibody-bound receptors then returned to the cell surface after
agonist removal by addition of apyrase (95 ± 2% of control
levels after removal of agonist for 60 min). The reversibility of
UDP-induced changes in the cell surface levels of P2Y6-R also was
studied after long-term agonist treatment. In marked contrast to
P2Y4-R, removal of UDP from the medium with apyrase after 3-h
preincubation did not result in rapid recovery of P2Y6-R to the cell
surface (Fig. 4B). Thus, in the presence of UTP P2Y4-R are
internalized and sequestered in an intracellular pool from which
receptors rapidly recycle to the cell surface once agonist is removed.
In contrast, P2Y6-R are more slowly lost from the cell surface in the
presence of agonist and fail to rapidly recycle once agonist is removed
from the medium.
Potential Regulation of P2Y4-R and P2Y6-R by Second
Messenger-regulated Kinases--
P2Y4-R and P2Y6-R are both coupled to
Gq and phospholipase C (6), and PKC has been broadly implicated in
feed-back regulation of the inositol lipid signaling pathway (31).
Moreover, PKC has been shown to phosphorylate a number of
Gq/phospholipase C-linked GPCR (32-34). Because the P2Y4-R contains a
strong PKC consensus sequence in its third intracellular loop and
several weaker sequences in its carboxyl terminus, we investigated the
potential involvement of PKC in agonist-promoted loss of surface P2Y4-R
and P2Y6-R. Incubation of P2Y4-R-expressing 1321N1 cells with the
phorbol ester activator of PKC, PMA, caused only a small loss of
surface P2Y4-R compared with that observed during a parallel incubation with UTP (Fig. 5A). Although
the PKC inhibitor, bisindolemaleimide, blocked the small
PMA-induced changes, it had no effect on UTP-induced loss of surface
P2Y4-R (Fig. 5B). Down-regulation of PKC by overnight incubation with 1 µM PMA also had no effect on the time
course of occurrence or extent of UTP-promoted loss of cell surface
P2Y4-R (Fig. 5C). In contrast, down-regulation of PKC
blocked the acute effects of PMA on cell surface P2Y4-R (Fig.
5C). As was observed with short-term incubation with UDP,
incubation of P2Y6-R-expressing cells with PMA had no effect on cell
surface receptors (Fig. 5A).
The potential role of the PKC-consensus site (RLRS) in the
third intracellular loop of the P2Y4-R also was examined. Mutation of
this serine to alanine (P2Y4(S243A); see Fig.
6) failed to inhibit agonist-induced
internalization of P2Y4-R (data not shown). Interestingly, although
this serine is lacking in the third cytoplasmic loop of the P2Y6-R, the
adjoining residues are conserved. Thus, we mutated the corresponding
residue of the P2Y6-R to serine (P2Y6/A237S; see Fig. 6) to
recapitulate the sequence found in the P2Y4-R. This mutation failed to
confer in the P2Y6-R a capacity to undergo rapid
agonist-dependent loss of surface receptors (data not
shown). Therefore, this potential site for PKC phosphorylation is not involved in agonist-induced changes in surface levels of P2Y4-R, and
engineering this site into the P2Y6-R also did not uncover UDP-induced
loss of surface receptors. Taken together, these results support the
idea that mechanisms largely unrelated to PKC mediate agonist-promoted
P2Y4-R sequestration. No effects on agonist-promoted loss of surface
P2Y4-R were observed after elevation of Ca2+ levels with
ionomycin or after increasing cyclic AMP levels with forskolin (data
not shown).
Identification of Regulatory Sites in the Carboxyl-terminal Domain
of the P2Y4-R by Mutational Analysis--
A series of receptor mutants
was constructed with the goal of identifying the regions and residues
important for regulation of P2Y4-R signaling (Fig. 6). Each mutant
P2Y4-R was stably expressed in 1321N1 cells, and its capacity to
promote inositol lipid hydrolysis was compared with that of the
wild-type P2Y4-R. Concentration effect curves for UTP at wild type and
two mutant P2Y4-R are presented in Fig.
7. The EC50 values for
stimulation (5-min assay) of inositol phosphate accumulation by UTP
ranged from 200 to 2000 nM for all of the mutant receptors
reported in this study (data not shown). The EC50 for UTP
(in a 5-min assay) at the wild type P2Y4-R stably expressed in parallel
with these various mutants was 440 ± 250 nM. The
maximal effect of 100 µM UTP for stimulation of inositol phosphate accumulation in a 5-min assay (0.4-2.5% conversion of [3H]inositol lipids into [3H]inositol
phosphates) with the mutant receptors also was within the range of
maximal effect (0.99 ± 0.42% conversion) observed with 100 µM UTP at the wild type P2Y4-R in a 5-min assay. Thus, each mutant receptor promoted inositol lipid hydrolysis with
characteristics similar to that of the wild type receptor. The enhanced
maximal response of the P2Y4/332 truncation mutant illustrated in Fig. 7 apparently occurs due to the fact that this receptor does not desensitize (see data and discussion below).
The carboxyl-terminal region of the P2Y4-R is ~55 amino acids in
length and contains 11 serines and threonines as potential sites for
phosphorylation. To determine whether the carboxyl terminus is
important in P2Y4-R desensitization and trafficking, a series of
truncations was made after residues 355, 343, and 332 (Fig. 6).
Truncations of the carboxyl terminus that removed the final 11 amino
acids (and four serines/threonines), i.e. P2Y4/355 (data not
shown), or final 23 amino acids (and seven serines/threonines), i.e. P2Y4/343, had no effect on agonist-induced loss of cell
surface P2Y4-R (Fig. 8) or
desensitization (Fig. 9). In contrast,
removal of an additional 11 residues (and three serines),
i.e. P2Y4/332, markedly inhibited agonist-induced loss of
cell surface P2Y4-R (Fig. 8) and prevented agonist-induced
desensitization (Fig. 9; also see Fig. 7). Therefore, residues between
332 and 343 in the carboxyl terminus of P2Y4-R are required for
agonist-induced desensitization and loss of surface receptors. Although
these data establish the P2Y4-R carboxyl terminus as a critical region
for agonist-induced regulation of this receptor, chimeric addition of
the P2Y4-R carboxyl terminus to P2Y6-R, i.e. P2Y6/310-Y4
(Fig. 6), failed to confer UDP-induced loss of surface receptors to the
chimeric P2Y6-R (data not shown).
The data from the carboxyl terminus truncation studies suggest that
amino acids within the region that differs between the two mutants,
P2Y4/343 and P2Y4/332, are crucial for regulation of P2Y4-R signaling.
This region contains three putative phosphorylation sites,
i.e. Ser-333, Ser-334, and Ser-339. To evaluate their role in regulation of P2Y4-R these serines were mutated to alanines (P2Y4/S333A,S334A,S339A). The capacity of this mutant P2Y4-R to undergo
agonist-induced desensitization (Fig. 9) or agonist-promoted loss of
surface receptors (Fig. 10) was greatly
reduced but not completely inhibited. Because alternative sites in the
carboxyl terminus, i.e. the seven serines/threonines
carboxyl-terminal to Ser-339, could be phosphorylated after mutation of
Ser-333, Ser-334, and S339, the truncation mutant P2Y4/343 was used as the "wild type" sequence to assess more directly the role(s) of amino acids between positions 332 and 343. All three of the serines were mutated individually in the truncated receptor, all three serines
were mutated simultaneously in a triple mutant, and a double mutant of
the serines at positions 333 and 334 also was constructed. Mutation of
individual serines did not inhibit agonist-induced loss of surface
receptors. In contrast, simultaneous mutation of Ser-333 and Ser-334 or
of Ser-333, Ser-334, and S339 reduced the capacity of UTP to induce
loss of surface receptors by ~80% (Fig.
11). The inhibition observed with these
two mutants was similar to that seen with a full-length construct,
designated S/T-A (see Fig. 6), containing mutations to alanine at all
potential phosphorylation sites carboxyl-terminal to residue 332 (Fig.
11). Taken together, these data illustrate that serines 333 and 334 are
important sites for agonist-induced desensitization and loss of
surface P2Y4-R.
Agonist-promoted Phosphorylation of P2Y4-R--
Experiments were
carried out to demonstrate directly that the P2Y4-R is phosphorylated
in an agonist-dependent fashion and to establish whether
the regulatory domain identified by mutational analysis also
contributes to the residue(s) responsible for this phosphorylation.
Cells expressing HA-tagged P2Y4-R were prelabeled with
[32P]Pi and then incubated with UTP for 2 min. The extent of [32P]phosphorylation of P2Y4-R was
established as described under "Experimental Procedures." A small
amount of 32P-phosphorylation of P2Y4-R was detected in the
absence of added agonist (Fig. 12).
This phosphorylation was not observed in cells not expressing P2Y4-R,
and therefore, either represents basal P2Y4-R phosphorylation unrelated
to P2Y4-R activation or occurs due to the presence of constitutively
released extracellular UTP as we have recently described (26). Addition
of UTP for 2 min resulted in a marked increase in P2Y4-R
phosphorylation (Fig. 12). This agonist-promoted phosphorylation
apparently does not occur in the final seven serines/threonines of the
carboxyl terminus, because a similar amount of
agonist-dependent phosphorylation was observed with the
P2Y4/343-R truncation mutant. In contrast, the capacity of UTP to
induce phosphorylation was completely lost with the P2Y4/332 truncation
mutant (Fig. 12). These results indicate that phosphorylation occurs on
one or more of the three serines present between residues 332 and 343 in the carboxyl terminus. Together with the mutational analyses
presented above, these results indicate that Ser-333 and Ser-334 are
key regulatory residues in the events of agonist-induced
phosphorylation, desensitization, and loss of cell surface P2Y4-R.
The observation of a broad range of uridine nucleotide-activated
physiological responses (2), the cloning of two receptors that are
specifically activated by uridine nucleotides (20-22), and the
demonstration of regulated release of cellular UTP (17, 18) establish
pyrimidinergic signaling as a physiologically important regulatory
pathway. The UTP versus UDP specificity of P2Y4-R
versus P2Y6-R adds potential complexity to pyrimidinergic signaling, and we have demonstrated here that these two P2Y-R exhibit
very different regulatory properties. The P2Y4-R undergoes rapid
agonist-induced desensitization but does not down-regulate. In
contrast, the P2Y6-R does not undergo rapid agonist-induced desensitization but does down-regulate. Delineation of the molecular basis of the very different modes of regulation of these two uridine nucleotide-activated receptors will be important to establish. In the
current study we have shown that agonist-dependent
phosphorylation of either of two adjacent serines in the carboxyl
terminus of P2Y4-R contributes an initiating step in both
desensitization and internalization of this receptor.
The rapidly occurring desensitization of P2Y4-R is a predictable
regulatory response of a GPCR and confirms results previously reported
by Robaye et al. (30) for P2Y4-R. Our kinetic analyses were
of insufficient detail to resolve the time course of occurrence of
desensitization from that of loss of surface receptors, but desensitization occurred at least as rapidly as did the receptor trafficking response. Agonist-induced desensitization of phospholipase C-linked GPCR has been exceptionally difficult to quantitate (35), because activity cannot be assessed in membranes prepared from agonist-preincubated cells as is the case with studies of adenylyl cyclase-linked receptors. Measurement of intact cell
Ins(1,4,5)P3 levels (35-39) may have certain advantages
over quantitation of total inositol phosphates in the presence of LiCl
as in the current study. Desensitization of the
Ins(1,4,5)P3 response almost certainly occurs faster than
that revealed here in a measurement that includes not only
Ins(1,4,5)P3 but also all of its downstream metabolites. Thus, we only can conclude that agonist-induced desensitization of
P2Y4-R is very rapid and likely occurs prior to loss of surface receptors. That agonist-stimulated accumulation of total inositol phosphates is essentially linear during activation of P2Y6-R expressed in the same cells, provides validation of the qualitative, if not
quantitative, aspects of our analyses.
The P2Y4/343 carboxyl-terminal truncation mutant retained all of the
phenotypical responses of the wild type receptor. In contrast, although
UTP activated the P2Y4/332 mutant with properties similar to the wild
type receptor, this truncated receptor essentially lost its capacity to
undergo UTP-induced desensitization or loss of surface receptors.
Although other residues in the 332 to 343 domain could provide these
regulatory properties, the serines at positions 333, 334, and 339 are
potential targets for kinase-promoted phosphorylation. Our studies
showed for the first time that agonist-dependent phosphorylation occurs in a P2Y-R (Fig. 12). Moreover, the occurrence of UTP-stimulated phosphorylation in the P2Y4/343 truncation mutant but
not in the P2Y4/332 truncation mutant confirms that the domain between
residues 332 and 343 provides the principle sites of
agonist-dependent phosphorylation. Mutational analyses of
individual or combinations of serines in this domain indicate that
Ser-333 and Ser-334 are the important sites of regulation and strongly
suggest that phosphorylation of either of these two serines is a key
step in agonist-dependent desensitization and loss of
surface P2Y4-R. However, our results do not rule out an additional
contributing role of phosphorylation at residues elsewhere in
P2Y4-R.
Although the P2Y4-R exhibits a commonly described phenotype of rapid
desensitization and loss of surface receptors, it is unusual in that
agonist-promoted down-regulation of this receptor apparently does not
occur. That is, following agonist removal from the medium (after times
of preincubation up to 12 h) surface receptors were replenished
within 30-60 min of incubation. Such results could be a vagary of a
cell line engineered to express a recombinant receptor. However, P2Y6-R
and P2Y2-R (40) expressed under the same conditions in 1321N1 cells
(and apparently to similar levels as P2Y4-R) both down-regulate. We
also have extensively studied down-regulation of the endogenous
The P2Y6-R falls into a relatively small group of GPCR that do not
undergo rapid agonist-induced desensitization. This observation is not
entirely surprising, because the P2Y6-R lacks serines and threonines in
its third cytoplasmic loop and only a single threonine occurs in the
carboxyl-terminal domain. Two serines and two threonines are present in
the first cytoplasmic loop, but this domain has not been predictably
important in GPCR coupling to heterotrimeric G-proteins or in their
agonist-dependent regulation. Simple replacement of the
carboxyl-terminal domain of P2Y6-R with that of P2Y4-R did not confer
to P2Y6-R a capacity to undergo rapid agonist-induced loss of surface
receptors. It is uncertain whether such results reflect a
regulation-resistant contribution of noncarboxyl-terminal sequence of
P2Y6-R, or whether they simply reflect lack of proper structural
context in which the phosphorylation of Ser-333 and Ser-334 apparently
produce signals for desensitization/internalization in the P2Y4-R protein.
The overriding view of agonist-induced regulatory changes in GPCR
signaling has followed from studies of the adenylyl cyclase-coupled As with receptor-regulated adenylyl cyclase, much of the early thinking
on regulation of receptor-promoted inositol lipid signaling included
models involving protein kinase C-mediated feed-back regulation of
responsiveness. This view was supported by large inhibitory effects of
phorbol ester activators of PKC on receptor-stimulated inositol
phosphate accumulation (50-52), although it could be argued that the
extent of PKC activation by PMA greatly exceeds that normally occurring
through receptor-promoted activation of phospholipase C. Experiments
utilizing pharmacological inhibition or down-regulation of PKC clearly
confirmed a role for this second messenger-regulated kinase in
agonist-promoted desensitization of inositol lipid signaling (31, 52).
However, very few of these studies established that the effects of PKC were at the level of the involved GPCR, and PKC could act at multiple levels in the inositol lipid signaling pathway. For example,
phospholipase C The presence of a potential consensus site (S243) for PKC
phosphorylation in the carboxyl-terminal portion of the third
cytoplasmic loop of P2Y4-R suggested that PKC might play a role in the
marked agonist-induced desensitization and loss of cell surface
receptors that occurs upon activation of this receptor. However,
incubation of P2Y4-R expressing cells with PMA did not mimic these
UTP-promoted effects, and neither down-regulation of PKC nor
pharmacological inhibition of its activity altered the time course or
extent of agonist promoted loss of surface P2Y4-R. Moreover, serine to
alanine mutation of S243 did not modify agonist-dependent
regulatory effects. Thus, although UTP induces profound changes in
P2Y4-R, little or none of these effects follow from a feed-back
involvement from its most proximally activated protein kinase. Because
ionomycin also failed to induce changes in UTP responsiveness or modify those occurring during incubation with UTP,
Ca2+/calmodulin-regulated kinases also are not involved.
GRKs recently have been implicated in regulation of Gq/phospholipase
C-coupled GPCR (29, 55-57), and our experiments that exclude PKC in
regulation of P2Y4-R indirectly suggest that GRKs also regulate P2Y4-R.
However, coexpression of GRK2 with P2Y4-R in Cos-7 cells had no effect on UTP-dependent desensitization or loss of surface
receptors whereas in parallel experiments GRK2 expression markedly
augmented desensitization and internalization of the
P2Y2-R.2 Perhaps another GRK
family kinase, e.g. GRK5, is involved in regulation of
P2Y4-R. Alternatively, casein kinase 1 The P2Y-R family is comprised of a group of five Gq/phospholipase
C-linked GPCR that exhibit novel selectivity for extracellular adenine
and uridine di- and triphosphates. The ATP- and UTP-activated P2Y2-R
was shown previously to undergo agonist-induced internalization (40,
60), and Garrad et al. (60) reported that the capacity of
UTP to induce P2Y2-R sequestration was reduced by truncation of the
carboxyl terminus. The current work reveals that the subfamily of two
pyrimidinergic P2Y-R exhibit very different properties of regulation
and that each of these receptors exhibits a property of regulation not
common among GPCR. Although our work has not established the identity
of the kinase involved in regulation of P2Y4-R, it identifies in P2Y-R
for the first time sites of phosphorylation important for
agonist-dependent regulation of receptor responsiveness and
cellular translocation. It will be important to establish whether
phosphorylation per se is sufficient to functionally
uncouple P2Y4-R from Gq and to establish the role this phosphorylation
plays, for example, in interaction with arrestin, in internalization,
and in a recycling response that rapidly and completely replenishes
surface P2Y4-R even during extended activation of the receptor.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Agonist-promoted inositol phosphate
accumulation in P2Y4-R- and P2Y6-R-expressing cells. P2Y4-R- and
P2Y6-R-expressing 1321N1 cells were prelabeled with
[3H]inositol and then incubated in the presence of 10 mM LiCl and 100 µM UTP (P2Y4-R), or 100 µM UDP (P2Y6-R). Total [3H]inositol
phosphate accumulation was quantitated at the indicated times.
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Fig. 2.
Agonist-promoted effects on cell surface
P2Y4-R and P2Y6-R. P2Y4-R and P2Y6-R-expressing 1321N1 cells were
incubated in the absence (open bars) or presence
(filled bars) of 100 µM UTP and 100 µM UDP, respectively, for 10 min. Surface P2Y-R were
quantitated using anti-HA antibody followed by a
125I-labeled secondary antibody.
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Fig. 3.
Time course of agonist-promoted decrease in
cell surface P2Y4-R and P2Y6-R. P2Y4-R- and P2Y6-R-expressing
1321N1 cells were incubated with 100 µM UTP ( ) and 100 µM UDP (
), respectively, for the indicated times.
Surface P2Y-R were quantitated using anti-HA antibody followed by a
125I-labeled secondary antibody.
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Fig. 4.
Recovery of surface P2Y4-R and P2Y6-R.
A, P2Y4-R-expressing 1321N1 cells were incubated for various
times with 100 µM UTP. At the indicated times, apyrase
(at a concentration that immediately hydrolyzed the UTP) was added to
the medium, and cell surface P2Y4-R were measured 30 or 60 min later
using anti-HA antibody followed by a 125I-labeled secondary
antibody. The data are from triplicate assays in an experiment that was
representative of two similar experiments. B,
P2Y6-R-expressing 1321N1 cells were incubated in the absence (control)
or presence of 100 µM UDP for 1 or 3 h. Apyrase (at
a concentration that immediately hydrolyzed UDP) then was added to the
medium, and cell surface P2Y6-R were measured 30 or 60 min later using
anti-HA antibody followed by 125I-labeled secondary
antibody. The data are the mean ± S.E. and are representative of
results from three experiments.
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Fig. 5.
Pharmacological analyses of the potential
role of PKC in regulation of cell surface P2Y4-R and P2Y6-R.
A-C, surface levels of P2Y-R were measured using an anti-HA
antibody followed by a 125I-labeled secondary antibody.
A, P2Y4-R (light bar) or P2Y6-R (dark
bar)-expressing 1321N1 cells were incubated with vehicle
(DMSO) or 10 µM PMA for 10 or 20 min or with
100 µM UTP (agonist) or 100 µM
UDP (agonist) for 20 min. B, P2Y4-R-expressing
1321N1 cells were incubated for 20 min with 100 µM UTP,
10 µM PMA, vehicle + 100 µM UTP
(D+U), 10 µM bisindolmaleimide + 100 µM UTP (B+U), or 10 µM
bisindolemaleimide + 10 µM PMA (B+P).
C, P2Y4-R-expressing 1321N1 cells were incubated in the
absence (light bar) or presence (dark bar) of 1 µM PMA overnight to down-regulate PKC. Cells then were
incubated with 100 µM UTP for the indicated times or with
10 µM PMA for 30 min.
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Fig. 6.
Mutations of P2Y4-R and P2Y6-R. A series
of deletions and mutations was made in the carboxyl terminus and third
intracellular loop of the human P2Y4-R and human P2Y6-R.
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Fig. 7.
Agonist-promoted inositol phosphate
accumulation in P2Y4-, P2Y4/343-, and P2Y4/332-R-expressing cells.
P2Y4-, P2Y4/343-, and P2Y4/332-R-expressing 1321N1 cells were
prelabeled with [3H]inositol and then incubated in the
presence of 10 mM LiCl and the indicated concentrations of
UTP for 5 min. Total [3H]inositol phosphate accumulation
was measured and divided by the total labeling of [3H]-
inositol lipids to determine the percent conversion to
[3H]inositol phosphates.
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Fig. 8.
Truncation of the carboxyl terminus prevents
UTP-promoted loss of cell surface P2Y4-R. 1321N1 Cells stably
expressing wild type P2Y4-R ( ), P2Y4-R truncated at amino acid 343 (
), or P2Y4-R truncated at amino acid 332 (
) were incubated for
the indicated times with 100 µM UTP. Cell surface
receptors were detected using anti-HA antibody followed by
125I-labeled secondary antibody.
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Fig. 9.
Agonist-promoted inositol phosphate
accumulation in cells expressing mutant P2Y4-R. 1321N1 cells
stably expressing wild type P2Y4-R ( ), P2Y4-R truncated at amino
acid 343 (
), P2Y4-R truncated at amino acid 332 (
), or P2Y4-R
with serine to alanine mutations at residues 333, 334, and 339 (
)
were prelabeled with [3H]inositol. The cells were then
challenged with 100 µM UTP in the presence of 10 mM LiCl for the indicated times, and total
[3H]inositol phosphates were quantitated.
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Fig. 10.
Agonist-promoted loss of cell surface
P2Y4-R. 1321N1 cells stably expressing either wild type P2Y4-R
( ) or a P2Y4-R with serine to alanine mutations at residues 333, 334, and 339 (
) were incubated with 100 µM UTP for the
indicated times. Surface P2Y4-R were quantitated using an anti-HA
antibody followed by 125I-labeled secondary antibody.
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Fig. 11.
Agonist-promoted loss of surface receptors
in cells expressing truncated/mutated P2Y4-R. 1321N1 cells stably
expressing mutant P2Y4-R were incubated with 100 µM UTP
for 20 min. Surface P2Y4-R were quantitated using an anti-HA antibody
followed by 125I-labeled secondary antibody.
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Fig. 12.
Agonist-promoted phosphorylation of wild
type P2Y4 and mutant receptors. Wild type 1321N1 cells
(WT) or 1321N1 cells stably expressing P2Y4-, P2Y4/343-, and
P2Y4/332-R were labeled with 500 µCi of
[32P] orthophosphate and incubated with 100 µM UTP for 2 min. Cells were lysed, and receptors were
immunoprecipitated, resolved on SDS-polyacrylamide gel electrophoresis,
and transferred to nitrocellulose. A PhosphorImager was used to measure
receptor phosphorylation. The data are presented as a densitometric
scan of gels from a single experiment and as bar graphs of
densitometric quantitation averaged from two experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenergic receptor (41, 42) and M3 muscarinic receptor (43, 44)
in this same cell line. Thus, the P2Y4-R is a GPCR that both rapidly
desensitizes and rapidly recovers from desensitization irrespective of
the length of time of activation. It will be important to determine both the physiological significance and structural basis for this unusual property of P2Y4-R.
2-adrenergic receptor (45, 46). The intuitive model of regulation is
one of second messenger-regulated kinases immediately downstream of
GPCR feeding back to regulate the activities of cohort proteins of the
pathway. This model has been substantiated in studies of the
2-adrenergic receptor by demonstrating that cyclic
AMP-dependent protein kinase is in part responsible for
agonist-induced desensitization (47-49). However, the dominant concept
is that members of the GRK family of kinases catalyze receptor
phosphorylation to provide the major initiating event in
agonist-dependent receptor desensitization and
internalization (28, 29).
is phosphorylated by PKC, and its activity is
inhibited (53, 54). Nonetheless, several phospholipase C-linked GPCR
are phosphorylated in response to activation of protein kinase C
(32-34).
catalyzes agonist-dependent phosphorylation and regulation of the
Gq/phospholipase C-coupled M3-muscarinic receptor, and potentially
other Gq-linked receptors (58, 59).
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ACKNOWLEDGEMENTS |
---|
We are indebted to Suzanne Delaney, Gary Waldo, Jose Boyer, JoAnn Trejo, and Rob Nicholas for many helpful discussions and suggestions, to JoAnn Trejo for her insightful comments on the manuscript, and to David Rinker for his outstanding help in producing the manuscript.
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FOOTNOTES |
---|
* This work was supported by United States Public Health Services Grants GM38213 and HL34322.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: Dept. of Pharmacology,
CB#7365, University of North Carolina School of Medicine, Chapel Hill,
NC 27599. Tel.: 919-966-4816; Fax: 919-966-5640; E-mail:
tkh@med.unc.edu.
Published, JBC Papers in Press, December 12, 2000, DOI 10.1074/jbc.M009909200
2 S. Delaney and T. K. Harden, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: P2Y-R, P2Y receptor; GPCR, G-protein-coupled receptor; GRK, G-protein-coupled receptor kinase; PCR, polymerase chain reaction; HA, hemagglutinin A; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; HBSS, Hanks' buffered saline solution; BSA, bovine serum albumin; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; Ins(1, 4,5)P3, inositol 1,4,5-trisphosphate.
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REFERENCES |
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