ADP-ribosylation Factor-dependent Phospholipase D2
Activation Is Required for Agonist-induced µ-Opioid Receptor
Endocytosis*
Thomas
Koch
,
Lars-Ove
Brandenburg,
Stefan
Schulz,
Yingjian
Liang,
Jochen
Klein§, and
Volker
Höllt
From the Department of Pharmacology and Toxicology,
Otto-von-Guericke University, 39120 Magdeburg, Germany and the
§ Department of Pharmacology, Johannes Gutenberg-University,
55101 Mainz, Germany
Received for publication, July 7, 2002, and in revised form, November 14, 2002
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ABSTRACT |
Agonist exposure of many G protein-coupled
receptors induces a rapid receptor phosphorylation and uncoupling from
G proteins. Resensitization of these desensitized receptors requires
endocytosis and subsequent dephosphorylation. Using a yeast two-hybrid
screen, the rat µ-opioid receptor (MOR1, also termed MOP) was found
to be associated with phospholipase D2 (PLD2), a phospholipid-specific phosphodiesterase located in the plasma membrane, which has been implicated in the formation of endocytotic vesicles.
Coimmunoprecipitation experiments in HEK293 cells coexpressing MOR1 and
PLD2 confirmed that MOR1 constitutively interacts with PLD2. Treatment
with the µ receptor agonist DAMGO
([D-Ala2, Me Phe4,
Glyol5]enkephalin) led to an increase in PLD2 activity,
whereas morphine, which does not induce MOR1 receptor internalization,
failed to induce PLD2 activation. The DAMGO-mediated PLD2 activation
was inhibited by brefeldin A, an inhibitor of ADP-ribosylation factor (ARF) but not by the protein kinase C (PKC) inhibitor calphostin C
indicating that opioid receptor-mediated activation of PLD2 is ARF- but
not PKC-dependent. Furthermore, heterologous stimulation of
PLD2 by phorbol ester led to an accelerated internalization of the
µ-opioid receptor after both DAMGO and morphine exposure. Conversely
the inhibition of PLD2-mediated phosphatidic acid formation by
1-butanol or overexpression of a negative mutant of PLD2 prevented agonist-mediated endocytosis of MOR1. Together, these data
suggest that PLD2 play a key role in the regulation of agonist-induced endocytosis of the µ-opioid receptor.
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INTRODUCTION |
Phospholipase D (PLD)1
is a widely distributed phospholipid-specific diesterase that
hydrolyzes phosphatidylcholine (PC) to phosphatidic acid (PA) and
choline and is assumed to play an important function in cell regulation
(1, 2). Signal-dependent activation of PLD was demonstrated
in numerous cell types stimulated by various hormones, growth factors,
cytokines, neurotransmitters, adhesion molecules, drugs, and physical
stimuli (reviewed in Ref. 3). Pathways leading to PLD activation
include protein serine/threonine kinases, e.g. protein
kinase C, small GTPases, e.g. ADP-ribosylation factor (ARF),
RhoA and Ral, phosphatidylinositol 4,5-bisphosphate (PIP2), and
tyrosine kinases (4-6). Recently two mammalian PLDs (PLD1 and PLD2)
have been identified (7-10). Subcellular fractionation studies have
demonstrated the presence of PLD1 in intracellular membranes,
e.g. ER, Golgi, and vesicular compartment (9, 11), whereas
PLD2 was largely associated with the plasma membrane (9). After
stimulation with serum, redistribution of PLD2 from the plasma membrane
into submembraneous endocytotic vesicles (early endosomes) was observed
(9). Another study revealed that PLD2 is associated with the EGF
receptor (12). Interestingly, EGF receptor endocytosis is impaired when
PLD activity is inhibited (13) suggesting a role for PLD2 in receptor trafficking.
Using the yeast two-hybrid system to identify proteins that interact
with the µ-opioid receptor, we isolated a rat cDNA encoding for
the NH2 terminus of PLD2. We therefore investigated the
potential role of PLD2 in the process of agonist-mediated endocytosis
of the µ-opioid receptor. We show that PLD2 is constitutively
associated with the µ-opioid receptor. Furthermore, we provide
evidence that the opioid receptor-mediated activation is
ARF-dependent and essential for receptor endocytosis.
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MATERIALS AND METHODS |
Yeast Two-hybrid Studies--
The µ-opioid receptor was
subcloned into pGBKT7 vector (Clontech, Heidelberg,
Germany) and used as bait to screen a rat brain MATCHMAKER cDNA
library (Clontech). The yeast two-hybrid screen was
carried out according to the protocol of the manufacturer. 280 clones
were positive on plates lacking leucine, tryptophan, histidine, and
adenine, 6 of which were further confirmed by yeast mating and
filterlift assays for
-galactosidase: one encoding for PLD2, two
clones encoding for known proteins, and three clones encoding for novel proteins.
Epitope Tagging and Cloning of cDNA--
The rat µ-opioid
receptor (MOR1) was tagged at the NH2 terminus with the HA
epitope tag sequence MYPYNVPNYA using polymerase chain reaction and
then subcloned into the pEAK10 expression vector (Edge Bio Systems,
Gaithersburg, MD). Dr. S. Ryu (Pohang, South Korea) kindly provided the
human PLD1b and PLD2 cDNAs subcloned into pcDNA3.1 expression
vector. The PLD2 truncation mutant (nPLD2) expressing only the
NH2-terminal amino acids 1-235 of PLD2 lacking the active
site motif of the enzyme was constructed by PCR mutagenesis. To
introduce a HindIII restriction site, the forward primer
5'-GCG GCC GCG AAG CTT ATG ACG GCG ACC CCT GAG-3'was
synthesized. The sequence for the reverse nPLD2-mutagenesis primer
introducing a XbaI restriction site and an amber stop codon
in amino acid position 235 of the PLD2 gene was 5'-CCA GCC
ACC TCT AGA ACC AGC GAT AAC AAA CTT-3'. The amplified 695-bp
NH2-terminal fragment of PLD2 (nPLD2) was subcloned in the
HindIII and XbaI sites of pcDNA3.1.
Generation of Cell Lines Coexpressing µ-Opioid Receptor and
PLD1b, PLD2, or the NH2-terminal Fragment of PLD2
(nPLD2)--
Cells were first transfected with peak10:MOR1 plasmid
containing puromycin resistance using the calcium phosphate
precipitation method (14). Stable transfectants were selected in the
presence of 1 µg/ml puromycin (Sigma). To generate cell lines
coexpressing HAMOR1 and PLD1b, PLD2, or nPLD2, cells were subjected to
a second round of transfection using Effectene (Qiagen, Hilden,
Germany) and selected in the presence of 1 µg/ml puromycin and 500 µg/ml G418 (Invitrogen). The whole pool of resistant cells was used without selection of individual clones. Receptor expression and PLD1b
or PLD2 expression was monitored using receptor ligand binding assays,
PLD activity assays, Western blot analysis, and confocal microscopy as
described below.
Radioligand Binding Assays--
Binding studies were performed
on membranes prepared from stably transfected cells. The dissociation
constant (KD) and number of [3H]DAMGO
binding sites (Bmax) were calculated by Scatchard analysis using at least six concentrations of [3H]DAMGO in a range
from 0.3 to 9 nM as previously described (15). Nonspecific
binding was determined as radioactivity bound in the presence of 1 µM unlabeled DAMGO.
Immunoprecipitation and Western Blot Analysis--
Cells were
plated onto poly-L-lysine-coated 150-mm dishes and grown to
80% confluence. Cells were then lysed, and the resulting lysate was
subjected to Western blot analysis or immunoprecipitation as described
(15-17). Briefly, receptor proteins were immunoprecipitated with 100 µl of protein A-agarose beads preloaded with 10 µg of anti-HA
antibodies. After SDS-polyacrylamide gel electrophoresis and
electroblotting, membranes were incubated with 1 µg/ml rabbit anti-human PLD antibody (kindly provided by Dr. S. Ryu, Pohang, South
Korea) or 1 µg/ml mouse anti-human ARF1 (Dianova, Hamburg, Germany)
for 12 h at 4 °C. Immunoreactive bands were visualized by using
an enhanced chemiluminescence detection system (Amersham Biosciences).
PLD Assays--
PLD activity was determined using a
transphosphatidylation assay (18). HEK293 cells coexpressing MOR1 and
PLD1b or PLD2 were kept in serum-free OPTIMEM containing
[1,2,3-3H]glycerol (1 µCi/ml; specific activity 40 Ci/mmol; American Radiolabeled Chemicals, St. Louis, MO) for 24 h
in order to label phospholipids. Cells were then exposed to serum-free
medium containing drugs and 2% ethanol. The following drugs were added
as aqueous solutions resulting in final concentrations of 1 µM DAMGO, 1 µM morphine, 1 µM
phorbol-12-myristate-13-acetate (PMA), 1 µM calphostin C, or 25 µg/ml brefeldin A (BFA) (all from Sigma). During the assay, BFA
was added 15 min before PMA or agonists. After 30 min of drug treatment, cells were extracted in 2.5 ml of ice-cold methanol/water (3:2, v/v). Subsequently, 1.5 ml chloroform and 0.35 ml H2O
were added, and the lipid phase was separated by thin layer
chromatography using the lower phase of methanol/chloroform/water
(10:10:9, v/v/v). Individual phospholipids were stained with iodine,
identified by standards, and spots corresponding to phosphatidylethanol
(PtdEtOH), PA, and PC were isolated and subjected to liquid
scintillation counting. PLD activity was expressed as percent
[3H]PtdEtOH of the total cellular PC concentration.
Confocal Microscopy--
HEK293 cells stable expressing MOR1 or
coexpressing MOR1 and PLD2 were grown onto
poly-L-lysine-coated coverslips overnight. Cells were then
exposed to 1 µM DAMGO or 1 µM morphine for
30 min at 37 °C to induce receptor endocytosis with or without 1 µM PMA stimulation. Cell fixation, antibody incubation,
and confocal analysis were carried out as previously described
(15-17).
Quantitative Internalization Assay--
Cells were seeded at a
density of 2 × 105 per well and grown onto
poly-L-lysine-treated 24-well plates overnight. After
washing, cells were preincubated with 1 µg of affinity-purified
rabbit anti-HA antibody for 2 h in OPTIMEM 1 (Invitrogen) at
4 °C. Cells were then treated as indicated with 1 µM
DAMGO, 1 µM morphine, and/or 1 µM PMA, 1%
butanol, or 1% isobutyl alcohol in OPTIMEM for 60 min. To prevent
receptors from recycling back to the plasma membrane, the culture
medium was supplemented with 50 µM monensin. Subsequently, cells were incubated with peroxidase-conjugated anti-rabbit antibody (1:1000, Amersham Biosciences) for 2 h at room temperature. Plates were developed with 250 µl of ABTS solution (Roche Molecular Biochemicals). After 10-30 min, 200 µl of the substrate solution from each well were transferred to a 96-well plate
and analyzed at 405 nm using a microplate reader.
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RESULTS |
Identification of PLD2 as a µ-Opioid Receptor-interacting Protein
by Yeast Two-hybrid Screen--
To identify tail-interacting proteins
that could modulate endocytosis of rat µ-opioid receptor we used the
yeast two-hybrid system (MATCHMAKER GAL4 Two-Hybrid-System 3);
(Clontech). A yeast two-hybrid screen of a rat
cDNA library using the rat MOR1-tail domain (COOH-terminal amino
acids 340-398) as bait led to the finding of a cDNA encoding only
the NH2-terminal amino acids 116-226 (representing exons
4-8) of PLD2. This NH2-terminal fragment of the PLD2
harbors a major part of the phosphoinositide-binding Phox homologous
(PX) domain (amino acids 63-192) of the enzyme. The interaction
between the COOH terminus of MOR1 (fused with Gal4 binding domain as
bait protein) and the NH2-terminal domain of PLD2 (fused
with the Gal4-activating domain) was verified in a yeast mating and
-galactosidase assay according to the protocol of the manufacturer.
As negative control empty Gal4-BD with Gal4-AD-PLD2 and empty Gal4-AD
vector with Gal4-BD-MOR1, as well as a fusion of Gal4-BD with human
lamin C, which neither forms complexes nor interacts with most other
proteins, were used.
MOR1 Stimulates PLD2 Activity in HEK293 Cells--
Since PLD2
activation has been previously described for various G protein-coupled
receptors, the association of MOR1 and PLD2 indicated that agonist
stimulation of the MOR1 might activate the PLD2. Therefore, we stable
expressed MOR1 and full-length human PLD2 in HEK293 cells. MOR1 and
PLD2 expression was monitored by ligand binding experiments, Western
blot, and immunocytochemical analyses. Saturation binding experiments
(n = 3-5) revealed no substantial differences between
MOR1 and MOR1-PLD2 expressing cells with respect to their affinities
(KD) to [3H]DAMGO (1.4 ± 0.3 nM and 1.4 ± 0.4 nM for MOR1 and
MOR1-PLD2, respectively) and their numbers of binding sites
(Bmax) (1329 ± 399 fmol/mg protein and 938 ± 107 fmol/mg protein for MOR1 and MOR1-PLD2, respectively). We then
incubated MOR1-PLD2 expressing HEK293 cells for 2, 5, 10, 20, and 30 min with the µ-agonist DAMGO and determined the relative PLD
activity. As shown in Fig. 1A, treatment with the µ-receptor selective agonist DAMGO led to a time-dependent increase in the PLD2 activity with a maximum
(3-fold increase in activity) after 30 min, whereas an incubation for 30 min with morphine failed to induce activation of PLD2. The observed
DAMGO-induced PLD2 activation was opioid receptor-mediated, because it
could be completely blocked by the opioid antagonist naloxone.
Activation of PKC by PMA also promoted a 4.5-fold increase in PLD2
activity, which was not blocked by naloxone (data not shown).
Furthermore, DAMGO-mediated activation of PLD2 could not be blocked by
the PKC inhibitor calphostin C (Fig. 1B), indicating that
activation of PKC seems not to play a major role in the opioid receptor-induced PLD2 activation in these cells. We therefore examined
whether activation of PLD2 by the opioid receptor involves ARF-GTP
proteins. Fig. 1B shows that DAMGO-mediated activation of
PLD2 was completely blocked by the ARF inhibitor brefeldin A indicating
that PLD2 activation by the µ-opioid receptor is ARF-dependent. BFA had no effect on the PMA-induced PLD2
activation (data not shown). In MOR1 and human PLD1b coexpressing
HEK293 cells, PLD1b activity was stimulated by PMA treatment, whereas DAMGO incubation did not lead to an increase in the PLD1b activity (Fig. 1C) indicating that the opioid receptor specifically
activates PLD2.

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Fig. 1.
Opioid receptor-mediated PLD2 activation in
HEK293 cells. A, HEK293 cells stable coexpressing MOR1 and
PLD2 were incubated in serum-free medium containing 2% ethanol and
exposed or not exposed to either 1 µM DAMGO, 1 µM morphine, or 1 µM PMA in the presence or
absence of 1 µM naloxone for the indicated time periods.
B, HEK293 cells stable coexpressing MOR1 and PLD2 were
incubated in serum-free medium containing 2% ethanol and exposed or
not exposed to 1 µM DAMGO in the presence or absence of 1 µM calphostin C or 25 µg/ml brefeldin A for 30 min.
C, HEK293 cells stable coexpressing MOR1 and PLD1b were
incubated in serum-free medium containing 2% ethanol and exposed or
not exposed to 1 µM DAMGO or 1 µM PMA for
30 min. PLD activity was determined as described under "Materials and
Methods." The control condition is assayed with 2% ethanol for 30 min. Note that (i) DAMGO and PMA but not morphine promoted a robust
increase in PLD2 activity, (ii) the DAMGO-mediated stimulation of PLD2
activity was time-dependent and completely blocked by
naloxone, (iii) the DAMGO-mediated PLD2 activation was ARF- but not
PKC-dependent, and (iv) PLD1b was activated by PMA but not
after DAMGO treatment. Values represent means ± S.E. of
triplicate determinations from three independent experiments.
Asterisks indicate significant difference (p < 0.05) compared with ethanol-treated control cells as determined
using ANOVA followed by Bonferroni test.
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MOR1 Interacts with PLD2 and ARF in HEK293 Cells--
To analyze
an interaction between MOR1, PLD1b, PLD2, and ARF1 in HEK293 cells, we
carried out coimmunoprecipitation studies. Expression of HAMOR1, PLD1b,
PLD2, and ARF was examined by directly immunoblotting lysates from
these cells with specific antibodies against HA tag, PLD1b, PLD2, or
ARF1 (Fig. 2, lysate). For
coimmunoprecipitation, HAMOR1 receptors were precipitated from lysates
of HAMOR1-expressing cells and cells coexpressing HAMOR1 and PLD1b or
PLD2 using anti-HA antibodies. The resulting precipitates were
immunoblotted with antibodies directed against PLD1b, PLD2, or ARF1. As
shown in Fig. 2A (lanes 2 and 3), PLD2
was detected in immunoprecipitates from cells coexpressing HAMOR1 and
PLD2, suggesting that MOR1 is physically associated with PLD2 in
vivo. Surprisingly, after agonist treatment we observed a decrease
in the amount of coimmunoprecipitated PLD2 in HAMOR1-PLD2-expressing
cells (Fig. 2A, lane 3). In immunoprecipitates from HAMOR1-expressing control cells no PLD2 was detected, which might
be because of the low basal PLD2 expression levels (Fig. 2A, lane 4).

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Fig. 2.
Interaction of MOR1, PLD2, and ARF.
Membrane proteins from HEK293 cells stable coexpressing HAMOR1 and
PLD1b (HAMOR1-PLD1b) or PLD2 (HAMOR1-PLD2) were
extracted and either immunoblotted directly (A and
B, lane 1, lysate) or
immunoprecipiated using anti-HA antibodies (A and
B, lane 2, IP HA). Membrane proteins
from HAMOR1- and PLD2-expressing HEK293 cells pretreated with DAMGO for
30 min (HAMOR1-PLD2/DAMGO) were extracted and
immunoprecipitated using anti-HA antibodies (A, lane
3, IP HA). Membrane proteins from HEK293 cells
expressing HAMOR1 alone (HAMOR1) were extracted and
immunoprecipiated using anti-HA antibodies (A, lane
4, IP HA). Membrane proteins from HEK293 cells
expressing PLD2 alone (PLD2) were extracted and either
immunoblotted directly (A, lane 5,
lysate) or immunoprecipitated using anti-HA antibodies
(A, lane 6, IP HA). The resulting
immunoprecipitates were electrophoretically separated, transferred to
nitrocellulose and detected with anti-PLD, anti-ARF1, and anti-HA
antibodies. Note that PLD2 and ARF1 were coimmunoprecipitated with the
µ-opioid receptor only from cells coexpressing HAMOR1 and PLD2 but
not from cells expressing HAMOR1 or PLD2 alone. The positions of
molecular mass markers are indicated on the left (in kDa).
Arrows point to PLD1b, PLD2, ARF1, and MOR1. Two additional
experiments gave similar results. Densitometric measurements revealed
that in HAMOR1-PLD2 coexpressing HEK293 cells 68 ± 8% of the
expressed PLD2 protein and 4 ± 1% of the expressed ARF protein
were coimmunoprecipitated with the µ-opioid receptor, whereas, after
agonist stimulation 35 ± 6% of the expressed PLD2 protein and
8 ± 2% of the expressed ARF protein were coimmunoprecipitated
with the µ-opioid receptor.
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In addition, ARF was coimmunoprecipitated from HAMOR1-PLD2-coexpressing
cells but not from cells stable expressing HAMOR1 alone (Fig.
2A, lanes 2-4), indicating that ARF binds to the
HAMOR1-PLD2 signaling complex and not directly to the MOR1 receptor.
After DAMGO treatment an increase in the amount of coimmunoprecipitated ARF protein was detected in HAMOR1-PLD2 cells (Fig. 2A,
lane 3). PLD2 and ARF were not nonspecifically
immunoprecipitated with anti-HA antibodies, because in PLD2-expressing
control cells no PLD2 and ARF were detected after precipitation with
anti-HA antibodies (Fig. 2A, lane 6).
Furthermore, from HAMOR1-PLD1b-coexpressing control cells no PLD1b or
ARF was coimmunoprecipitated with MOR1 (Fig. 2B, lane
2) indicating that MOR1 is not associated with PLD1b in
vivo.
Heterologous Activation of PLD2 by Phorbol Ester Influences the
Agonist Selectivity of µ-Opioid Receptor Endocytosis--
First, we
examined the subcellular distribution of HAMOR1 and PLD2 using double
immunofluorescence and confocal microscopy. As shown in Fig.
3, A and A', both
MOR1 and PLD2 were confined to the plasma membrane in HEK293 cells. To
test the possibility that physical association would promote
cointernalization of PLD2 and MOR1 after agonist-exposure,
MOR1-PLD2-expressing cells were incubated in the presence of 1 µM DAMGO for 30 min. The results in Fig. 3, B
and B' reveal that the MOR1 receptor is rapidly internalized while PLD2 remained at the plasma membrane.

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Fig. 3.
Subcellular distribution of MOR1 and PLD2 in
HEK293 cells. HEK293 cells stable coexpressing HAMOR1 and PLD2
were either not treated (A and A') or treated
with 1 µM DAMGO for 30 min (B and
B'). Cells were subsequently fixed, subjected to double
immunofluorescent staining using a mixture of rat anti-HA and rabbit
anti-PLD2 antibodies, and examined by confocal microscopy. Note that in
untreated cells both HAMOR1 and PLD2 were confined to the plasma
membrane. In contrast, after treatment with DAMGO only the µ-opioid
receptor but not PLD2 was internalized into the vesicular endocytotic
compartment. Shown are representative results from one of three
independent experiments performed in duplicate. Scale bar,
20 µM.
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We next examined whether the activation of PLD2 by the µ-opioid
receptor influences the agonist-induced receptor endocytosis. Therefore, HEK293 cells stable expressing HAMOR1 or HAMOR1 and PLD2
were incubated with anti-HA antibodies at 4 °C to label cell surface
receptors. The cells were then treated with DAMGO, morphine in the
presence or absence of the PKC activator PMA for 30 min at 37 °C.
During the experiment, the culture medium was supplemented with 50 µM monensin to prevent internalized receptors from
recycling back to the plasma membrane. Cells were subsequently fixed,
permeabilized, and bound anti-HA antibodies were immunofluorescently
detected. The subcellular distribution of the receptor proteins was
then analyzed by confocal microscopy. Fig.
4 shows that without agonist incubation
(control), µ-opioid receptors were almost exclusively confined to the
plasma membrane. After 30 min of DAMGO incubation at 37 °C, the
µ-opioid receptor exhibited robust receptor endocytosis. In contrast,
after incubation with morphine the MOR1 receptor was highly resistant
to agonist-mediated endocytosis. Preincubation with PKC activator PMA
alone did not increase the endocytosis of MOR1. In the presence of
overexpressed PLD2, however, the combination of morphine and PMA
resulted in a robust receptor internalization suggesting that both the
agonist-induced conformational change and stimulation of PLD2 activity
were required for receptor endocytosis under these conditions.

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Fig. 4.
PLD2 modulates agonist-selectivity of
µ-opioid receptor endocytosis. HEK293 cells
expressing HAMOR1 alone (upper panel) or coexpressing HAMOR1
and PLD2 (lower panel) were either not treated
(control) or treated with 1 µM DAMGO, 1 µM morphine, and/or 1 µM PMA for 30 min at
37 °C as indicated. Cells were subsequently fixed, fluorescently
labeled with anti-HA antibody and the subcellular distribution of
receptor protein was examined by confocal microscopy. Note that DAMGO
treatment resulted in robust receptor internalization for both HAMOR1
and HAMOR1-PLD2 expressing cells, whereas morphine failed to induce
receptor internalization. In HAMOR1-PLD2-coexpressing cells; however,
coactivation of PLD2 by the PKC activator PMA led to the induction of
morphine-induced receptor internalization. Shown are representative
results from one of four independent experiments performed in
duplicate. Scale bar, 20 µM.
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Quantitative analysis of receptor endocytosis by ELISA confirmed that
in HAMOR1-PLD2 coexpressing HEK293 cells, PMA treatment enhances the
rate of receptor endocytosis after both DAMGO and morphine in
HAMOR1-PLD2 coexpressing HEK293 cells (Fig.
5). In contrast, in cells expressing the
MOR1 receptor alone, PMA treatment did not significantly affect the
rate of agonist-mediated receptor endocytosis indicating that the
PMA-induced facilitation of receptor endocytosis depends on PLD2 and is
not simply caused by increased PKC activity in these cells.

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Fig. 5.
Quantitative analysis of agonist-induced
endocytosis of MOR1. HEK293 cells expressing HAMOR1 or HAMOR1-PLD2
were either not treated or treated with 1 µM DAMGO or 1 µM morphine in the presence or absence of 1 µM PMA for 60 min. Cell surface receptors were labeled
with affinity-purified rabbit anti-HA antibody followed by a
peroxidase-conjugated anti-rabbit antibody. Receptor internalization,
quantified as the percent loss of cell surface receptors in
agonist-treated cells, was measured by ELISA as described under
"Materials and Methods." Data are presented as means ± S.E.
of five independent experiments performed in duplicate.
Asterisk indicates a significant difference
(p < 0.05) and double asterisks indicate
significant difference (p < 0.001) between HAMOR1- and
HAMOR1-PLD2-expressing cells (ANOVA followed by Bonferroni test).
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Agonist-induced MOR1 Endocytosis Is Inhibited by Primary but Not
Secondary Alcohol--
Next, we examined whether the production of PA
by PLD2 is required for the induction of opioid receptor endocytosis in
HEK293 cells. For inhibition of PA production the primary alcohol
1-butanol was used, which is known to be preferentially used over water by PLD2 in the transphosphatidylation reaction to generate
phosphatidylalcohol instead of phosphatidic acid. This reaction is
highly specific for primary alcohols, whereas secondary alcohols (such
as isobutyl alcohol) are not utilized by PLD2 (1). Since 1.5% of
butanol was found to inhibit the PLD activity but also to induce toxic effects (19), we used 1% of butanol for inhibition of PA production. Fig. 6A shows that
DAMGO-mediated endocytosis of HAMOR1 was strongly inhibited by the
primary alcohol (1-butanol) but not the secondary alcohol (isobutyl
alcohol). This inhibitory effect of primary alcohol (1-butanol) on the
agonist-induced endocytosis of the µ-opioid receptor was confirmed by
quantitative analysis (Fig. 6B). These data strongly suggest
that PA production by PLD2 is required for the induction of opioid
receptor endocytosis.

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Fig. 6.
Inhibition of agonist-induced endocytosis of
MOR1 by the primary alcohol (1-butanol). A, HAMOR1- or
HAMOR1-PLD2-expressing HEK293 cells were either not subjected or
subjected to 1 µM DAMGO with or without 1% 1-butanol or
1% isobutyl alcohol for 30 min at 37 °C as indicated. Cells were
subsequently fixed, fluorescently labeled with anti-HA antibody, and
the subcellular distribution of receptor protein was examined by
confocal microscopy. DAMGO treatment resulted in robust receptor
internalization for both HAMOR1 and HAMOR1-PLD2 cells, which was
inhibited in the presence of primary alcohol 1-butanol. Note that
secondary alcohol isobutyl alcohol has no effect on the agonist-induced
internalization of the µ-opioid receptor (A). Shown are
representative results from one of four independent experiments
performed in duplicate. Scale bar, 20 µM.
B, HEK293 cells expressing HAMOR1 or HAMOR1-PLD2 were
subjected to 1 µM DAMGO with or without 1% 1-butanol or
1% isobutyl alcohol for 60 min (B). Cell surface receptors
were labeled with affinity-purified rabbit anti-HA antibody followed by
a peroxidase-conjugated anti-rabbit antibody. Receptor internalization,
quantified as the percent loss of cell surface receptors in
agonist-treated cells, was measured by ELISA as described under
"Materials and Methods." Data are presented as means ± S.E.
of five independent experiments performed in duplicate. The
double asterisks indicate significant difference
(p < 0.001) between cells treated only with DAMGO and
cells treated with DAMGO plus 1-butanol or isobutyl alcohol (ANOVA
followed by Bonferroni test).
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Agonist-induced MOR1 Endocytosis Is Decreased by Coexpression of an
NH2-terminal Fragment of PLD2--
In a yeast two-hybrid
screen we identified the NH2 terminus of the PLD2 as the
important MOR1 interacting site of the enzyme. This region harbors the
phosphoinositide binding domain of the PLD2 but lacks the PLD2 active
site motif. After overexpression of the NH2 terminus (amino
acids 1-235) of the PLD2 in HAMOR1 cells (HAMOR1-nPLD2), the
DAMGO-induced MOR1-internalization was reduced by nearly 50% compared
with control cells expressing HAMOR1 alone (Fig.
7), suggesting that nPLD2 competes for
binding of full-length endogenous PLD2 to MOR1 and impairs opioid
receptor-mediated PLD2 activation and subsequent receptor
internalization. Furthermore, costimulation of HAMOR1-nPLD2-expressing
cells with DAMGO and PMA resulted in a restoration of receptor
endocytosis to the same extent as in HAMOR1-expressing cells (Fig. 7),
indicating that PMA stimulation of PLD2 bypasses the nPLD2-mediated
impairment of opioid receptor induced PLD2 activation. This effect is
not simply due to increased PKC activity in these cells, because PMA treatment alone did not induce the endocytosis of MOR1 in both HAMOR1-
and HAMOR1-nPLD2-expressing cells.

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Fig. 7.
Reduction of agonist-induced endocytosis of
MOR1 by overexpression of an NH2-terminal fragment of
PLD2. HAMOR1- or HAMOR1-nPLD2-expressing HEK293 cells were
subjected to 1 µM DAMGO and/or 1 µM PMA for
60 min at 37 °C as indicated. Cell surface receptors were labeled
with affinity-purified rabbit anti-HA antibody followed by a
peroxidase-conjugated anti-rabbit antibody. Receptor internalization,
quantified as the percent loss of cell surface receptors in
agonist-treated cells, was measured by ELISA as described under
"Materials and Methods." Note that in HAMOR1-nPLD2 expressing
cells DAMGO-treatment resulted in a 50% reduced receptor endocytosis
compared with HAMOR1 control cells, whereas after combined stimulation
with DAMGO and PMA both cell lines showed similar endocytosis rates.
Data are presented as means ± S.E. of five independent
experiments performed in duplicate. The double asterisks
indicate significant difference (p < 0.001) between
HAMOR1- and HAMOR1-nPLD2-expressing cells (ANOVA followed by Bonferroni
test).
|
|
 |
DISCUSSION |
Stimulation of PLD activity has been observed for numerous G
protein-coupled receptors including the VPAC 1 and 2 (for vasoactive intestinal polypeptide) receptors, PAC1 (for pituitary
adenylate cyclase-activating peptide) receptor (20), metabotropic
glutamate receptors (21, 22), m1-m4 muscarinic receptors (23), the endothelin receptor (24), the
2-adrenergic receptor (25), and the D2
dopamine receptor (26). However, the mechanisms of PLD activation by G
protein-coupled receptors as well as the cellular functions of
activated PLD are still incompletely understood. For the first time,
the present study provides evidence for an essential role of PLD2 in
agonist-induced endocytosis of a G protein-coupled receptor namely the
µ-opioid receptor.
The present study demonstrates that the µ-opioid receptor interacts
specifically with PLD2 and not with PLD1b. Using a yeast two-hybrid
technique we identified the Phox homologous (PX) domain in the
NH2 terminus of the PLD2 to be the important site for the interaction with the COOH terminus of the µ-opioid receptor. PX domains are known to be phosphoinositide-binding motifs found in a
number of signaling and adapter proteins (Ref. 27 for review), but
their precise function is still poorly defined. Recently, PX domains in
proteins mediating the protein trafficking were shown to be required
not only for the association with cellular membranes but also for the
association with various receptors (e.g. epidermal growth
factor, platelet-derived growth factor) (28, 29). The fact that PX
domains are present in the NH2 terminus of both PLD1b and
PLD2, whereas only PLD2 was found to be associated with the µ-opioid
receptor might be due mainly to sequence differences in the
NH2 termini of PLD1b and PLD2.
The specific interaction between MOR1 and PLD2 was confirmed by
coimmunoprecipitation experiments in HEK293 cells stable expressing MOR1 and PLD2. The interaction between MOR1 and PLD2 was shown to be
constitutive and not inducible by agonist-treatment. In HAMOR1-PLD2
cells ~68% of the expressed PLD2 was coimmunoprecipitated with the
MOR1, whereas agonist-stimulation resulted in only ~35% coimmunoprecipitated PLD2. This reduction might be due to the separation of MOR1 and membranal localized PLD2 after agonist-induced receptor internalization. It was further shown that stimulating the
µ-opioid receptor with DAMGO activates PLD2 and that this DAMGO-mediated elevation of PLD2 activity was completely blocked by the
opioid antagonist naloxone. The DAMGO-mediated PLD2 activation was
ARF-dependent because it was completely blocked by the ARF inhibitor brefeldin A, which inhibits ARF-specific guanine nucleotide exchange proteins by locking them in an abortive complex with ARF-GDP.
In addition, after DAMGO stimulation a 50% increase in the amount of
coimmunoprecipitated ARF1 with MOR1 was detected. Thus, it is possible
that the opioid-mediated PLD2 stimulation may involve a direct
interaction of the µ-opoid receptor with small G proteins,
e.g. ARF. The NPXXY motif, which is highly
conserved within the seventh transmembrane domain of many G
protein-coupled receptors including the µ-opioid receptor, was
previously demonstrated to represent a specific ARF binding site
implicated in receptor-mediated PLD activation (30). However, for
coimmunoprecipitation of MOR1 and ARF the presence of PLD2 seemed to be
required, because ARF was only detected in coimmunoprecipitates from
MOR1-PLD2- but not from MOR1-expressing cells. Therefore, it is
reasonable to assume that ARF binds directly to PLD2 rather than to
MOR1, but it cannot be excluded that a conformational change of MOR1 in the MOR1-PLD2 signaling complex is necessary to facilitate ARF binding
to MOR1. However, after agonist-stimulation we observed an increase in
the coimmunoprecipitation of ARF (from ~4 to ~8% of total ARF)
from MOR1-PLD2-expressing cells supporting the hypothesis of an
ARF-dependent activation pathway of PLD2 by MOR1.
We further demonstrate that PLD2 can be activated by heterologous
stimulation of PKC with PMA. However, the finding that DAMGO-induced PLD2 stimulation was not blocked by calphostin C suggests that PKC is
not required for opioid receptor-mediated activation of PLD2. Similar
to phosphoinositide-specific phospholipase C, phospholipase A2, and sphingomyelinase, PLD2 is a signal- and receptor-activated phospholipase; however, little is known about the cellular effects of
PLD2 and its primary metabolite PA. It has been demonstrated that the
second messenger PA can activate phosphatidylinositol 4-phosphate-5-kinase leading to the production of PIP2 (31, 32), which
can further activate PLD in a positive feedback. In addition, an
increase in the level of PA after PLD activation results in a change of
the physical properties, e.g. charge and pH, of cellular
membranes, thereby facilitating vesicle formation. Whereas PLD1 seems
to play a role for the vesicle formation from the Golgi apparatus (33,
34), PLD2 has been suggested to be involved in vesicle formation from
the plasma membrane (9).
Thus, it is reasonable to speculate that local activation of PLD2 by
the µ-opioid receptor might play a key role in the stimulation of
vesicle formation during µ-opioid receptor endocytosis. Because PLD2
levels in wild-type HEK293 cells were below the detection limit in
Western blot and immunocytochemical assays, we stably coexpressed PLD2
and MOR1. Immunocytochemical studies revealed that both proteins were
localized at the plasma membrane and that coexpression of PLD2 led to a
significant increase in the agonist-induced endocytosis of the
µ-opioid receptor. However, cointernalization of PLD2 and MOR1 was
not observed in agonist-treated HAMOR1-PLD2-expressing cells. On the
other hand, preventing the PLD2-mediated production of PA by treatment
with the primary alcohol (1-butanol) led to an ~80% reduction of
agonist-induced receptor endocytosis. Furthermore, overexpression
of an inactive nPLD2 mutant which competes for binding of full-length
PLD2 to the MOR1 resulted in a ~50% reduction of agonist-induced
receptor endocytosis. Heterologous activation of endogenous PLD2 by PMA
together with DAMGO-treatment bypasses the nPLD2-mediated block of
receptor-induced activation of PLD2 and led to a robust µ receptor
internalization in HAMOR1-nPLD2-expressing cells. In addition, it
should be noted that morphine, which failed to induce µ-opioid
receptor internalization, did not activate PLD2. However, heterologous
PLD2 activation by PMA permitted morphine to induce robust µ receptor
internalization suggesting that both the agonist-induced conformational
change and stimulation of PLD2 activity were required for receptor
endocytosis. Desensitization of the µ-opioid receptor after
agonist-treatment is induced by a rapid receptor phosphorylation and
-arrestin binding resulting in an uncoupling of the receptor from G
proteins (15, 35-37). Since morphine failed to induce µ-opioid
receptor internalization, it was speculated that morphine restrains the
MOR1 receptor in a conformation that is recalcitrant to GRK-mediated
phosphorylation and subsequent
-arrestin binding (15, 38). This is
supported by our previous finding that morphine failed to induce MOR1
phosphorylation, whereas splice variants (MOR1D and MOR1E) of the mouse
µ-opioid receptor, which are internalized after morphine treatment,
revealed a marked morphine-induced receptor phosphorylation (15).
Moreover,
-arrestin was shown to interact with
2-adaptin (AP-2)
leading to the initiation of clathrin-mediated receptor endocytosis
(39). Furthermore, it was demonstrated that the recruitment of AP-2 is
facilitated by the acidic phospholipid-enriched membrane resulting from
PA production by ARF-activated PLD2 (40, 41). This indicates that
receptor endocytosis can be modulated not only by receptor phosphorylation and
-arrestin binding but also by the PLD2-mediated PA production which, in turn, facilitates recruitment of AP-2 and
clathrin to the plasma membrane. Consistent with this hypothesis we
observed that inhibition of PLD2 activity led to a marked decrease in
receptor endocytosis, whereas activation of PLD2 by PKC resulted in an
enhanced agonist-induced endocytosis of the µ-opioid receptor even
after morphine treatment. Thus, it can be suggested that activation of
PLD2 may be a key step during the induction of agonist-mediated endocytosis of the µ-opioid receptor.
 |
ACKNOWLEDGEMENTS |
We thank Evelyn Kahl, Sandra Grosseheilmann,
Michaela Böx, and Dana Mayer for excellent technical assistance.
 |
FOOTNOTES |
*
This work was supported by Deutsche Forschungsgemeinschaft
Grants HO 1027/10-1 (to V. H.), SCHU 924/4-3 (to S. S.), and SFB 426/A2 (V. H.).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
and Toxicology, Otto-von-Guericke University, 39120 Magdeburg, Leipziger Str. 44, Germany. Tel.: 49-391-671-5372; Fax:
49-391-671-5869; E-mail: Thomas.Koch@Medizin.Uni-Magdeburg.de.
Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M206709200
 |
ABBREVIATIONS |
The abbreviations used are:
PLD, phospholipase
D;
ARF, ADP-ribosylation factor;
DAMGO, [D-Ala2, Me Phe4,
Glyol5]enkephalin;
HA, hemagglutinin;
BFA, brefeldin A;
HAMOR1, HA epitope-tagged rat µ-opioid receptor isoform 1;
HEK293, human embryonic kidney 293;
MOR1, rat µ-opioid receptor isoform 1;
PA, phosphatidic acid;
PC, phosphatidylcholine;
PKC, protein kinase C;
PMA, phorbol-12-myristate-13-acetate;
ANOVA, analysis of variance;
ELISA, enzyme-linked immunosorbent assay;
PX, phosphoinositide-binding
Phox homologous domain.
 |
REFERENCES |
1.
|
Morris, A. J.,
Frohman, M. A.,
and Engebrecht, J.
(1997)
Anal. Biochem.
252,
1-9[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Liscovitch, M.,
and Cantley, L. C.
(1994)
Cell
77,
329-334[Medline]
[Order article via Infotrieve]
|
3.
|
Liscovitch, M.,
Czarny, M.,
Fiucci, G.,
and Tang, X.
(2000)
Biochem. J.
345,
401-415[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Kiss, Z.
(1996)
Chem. Phys. Lipids
80,
81-102[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Natarajan, V.,
Scribner, W. M.,
and Vepa, S.
(1996)
Chem. Phys. Lipids
80,
103-116[CrossRef][Medline]
[Order article via Infotrieve]
|
6.
|
Exton, J. H.
(1998)
Biochim. Biophys. Acta
1436,
105-115[Medline]
[Order article via Infotrieve]
|
7.
|
Hammond, S. M.,
Altshuller, Y. M.,
Sung, T. C.,
Rudge, S. A.,
Rose, K.,
Engebrecht, J.,
Morris, A. J.,
and Frohman, M. A.
(1995)
J. Biol. Chem.
270,
29640-29643[Abstract/Free Full Text]
|
8.
|
Park, S. K.,
Provost, J. J.,
Bae, C. D.,
Ho, W. T.,
and Exton, J. H.
(1997)
J. Biol. Chem.
272,
29263-29271[Abstract/Free Full Text]
|
9.
|
Colley, W. C.,
Sung, T. C.,
Roll, R.,
Jenco, J.,
Hammond, S. M.,
Altshuller, Y.,
Bar-Sagi, D.,
Morris, A. J.,
and Frohman, M. A.
(1997)
Curr. Biol.
7,
191-201[Medline]
[Order article via Infotrieve]
|
10.
|
Kodaki, T.,
and Yamashita, S.
(1997)
J. Biol. Chem.
272,
11408-11413[Abstract/Free Full Text]
|
11.
|
Sung, T. C.,
Zhang, Y.,
Morris, A. J.,
and Frohman, M. A.
(1999)
J. Biol. Chem.
274,
3659-3666[Abstract/Free Full Text]
|
12.
|
Slaaby, R.,
Jensen, T.,
Hansen, H. S.,
Frohman, M. A.,
and Seedorf, K.
(1998)
J. Biol. Chem.
273,
33722-33727[Abstract/Free Full Text]
|
13.
|
Shen, Y.,
Xu, L.,
and Foster, D. A.
(2001)
Mol. Cell. Biol.
21,
595-602[Abstract/Free Full Text]
|
14.
|
Chen, C. A.,
and Okayama, H.
(1988)
BioTechniques
6,
632-638[Medline]
[Order article via Infotrieve]
|
15.
|
Koch, T.,
Schulz, S.,
Pfeiffer, M.,
Klutzny, M.,
Schröder, H.,
Kahl, E.,
and Höllt, V.
(2001)
J. Biol. Chem.
276,
31408-31414[Abstract/Free Full Text]
|
16.
|
Pfeiffer, M.,
Koch, T.,
Schröder, H.,
Klutzny, M.,
Kirscht, S.,
Kreienkamp, H. J.,
Höllt, V.,
and Schulz, S.
(2001)
J. Biol. Chem.
276,
14027-14036[Abstract/Free Full Text]
|
17.
|
Pfeiffer, M.,
Koch, T.,
Schröder, H.,
Laugsch, M.,
Höllt, V.,
and Schulz, S.
(2002)
J. Biol. Chem.
277,
19762-19772[Abstract/Free Full Text]
|
18.
|
Kotter, K.,
and Klein, J.
(1999)
J. Neurochem.
73,
2517-2523[CrossRef][Medline]
[Order article via Infotrieve]
|
19.
|
Skippen, A.,
Jones, D. H.,
Morgan, C. P.,
Li, M.,
and Cockcroft, S.
(2002)
J. Biol. Chem.
277,
5823-5831[Abstract/Free Full Text]
|
20.
|
McCulloch, D. A.,
Lutz, E. M.,
Johnson, M. S.,
Robertson, D. N.,
MacKenzie, C. J.,
Holland, P. J.,
and Mitchell, R.
(2001)
Mol. Pharmacol.
59,
1523-1532[Abstract/Free Full Text]
|
21.
|
Shinomura, T.,
del Rio, E.,
Breen, K. C.,
Downes, C. P.,
and McLaughlin, M.
(2000)
Br. J. Pharmacol.
131,
1011-1018[Abstract/Free Full Text]
|
22.
|
Kanumilli, S.,
Toms, N. J.,
Venkateswarlu, K.,
Mellor, H.,
and Roberts, P. J.
(2002)
Neuropharmacology
42,
1-8[CrossRef][Medline]
[Order article via Infotrieve]
|
23.
|
Sandmann, J.,
Peralta, E. G.,
and Wurtman, R. J.
(1991)
J. Biol. Chem.
266,
6031-6034[Abstract/Free Full Text]
|
24.
|
Ambar, I.,
and Sokolovsky, M.
(1993)
Eur. J. Pharmacol.
245,
31-41[CrossRef][Medline]
[Order article via Infotrieve]
|
25.
|
MacNulty, E. E.,
McClue, S. J.,
Carr, I. C.,
Jess, T.,
Wakelam, M. J.,
and Milligan, G.
(1992)
J. Biol. Chem.
267,
2149-2156[Abstract/Free Full Text]
|
26.
|
Senogles, S. E.
(2000)
Mol. Pharmacol.
58,
455-462[Abstract/Free Full Text]
|
27.
|
Xu, Y.,
Seet, L. F.,
Hanson, B.,
and Hong, W.
(2001)
Biochem. J.
360,
513-530[CrossRef][Medline]
[Order article via Infotrieve]
|
28.
|
Phillips, S. A.,
Barr, V. A.,
Haft, D. H.,
Taylor, S. I.,
and Haft, C. R.
(2001)
J. Biol. Chem.
276,
5074-5084[Abstract/Free Full Text]
|
29.
|
Haft, C. R.,
de la Luz Sierra, M.,
Barr, V. A.,
Haft, D. H.,
and Taylor, S. I.
(1998)
Mol. Cell. Biol.
18,
7278-7287[Abstract/Free Full Text]
|
30.
|
Mitchell, R.,
McCulloch, D.,
Lutz, E.,
Johnson, M.,
MacKenzie, C.,
Fennell, M.,
Fink, G.,
Zhou, W.,
and Sealfon, S. C.
(1998)
Nature
392,
411-414[CrossRef][Medline]
[Order article via Infotrieve]
|
31.
|
Moritz, A.,
De Graan, P. N.,
Gispen, W. H.,
and Wirtz, K. W.
(1992)
J. Biol. Chem.
267,
7207-7210[Abstract/Free Full Text]
|
32.
|
Honda, A.,
Nogami, M.,
Yokozeki, T.,
Yamazaki, M.,
Nakamura, H.,
Watanabe, H.,
Kawamoto, K.,
Nakayama, K.,
Morris, A. J.,
Frohman, M. A.,
and Kanaho, Y.
(1999)
Cell
99,
521-532[Medline]
[Order article via Infotrieve]
|
33.
|
Ktistakis, N. T.,
Brown, H. A.,
Sternweis, P. C.,
and Roth, M. G.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
4952-4956[Abstract]
|
34.
|
Ktistakis, N. T.,
Brown, H. A.,
Waters, M. G.,
Sternweis, P. C.,
and Roth, M. G.
(1996)
J. Cell Biol.
134,
295-306[Abstract]
|
35.
|
Zhang, J.,
Ferguson, S. S.,
Barak, L. S.,
Bodduluri, S. R.,
Laporte, S. A.,
Law, P. Y.,
and Caron, M. G.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
7157-7162[Abstract/Free Full Text]
|
36.
|
Kovoor, A.,
Nappey, V.,
Kieffer, B. L.,
and Chavkin, C.
(1997)
J. Biol. Chem.
272,
27605-27611[Abstract/Free Full Text]
|
37.
|
Bohn, L. M.,
Gainetdinov, R. R.,
Lin, F. T.,
Lefkowitz, R. J.,
and Caron, M. G.
(2000)
Nature
408,
720-723[CrossRef][Medline]
[Order article via Infotrieve]
|
38.
|
Whistler, J. L.,
Chuang, H. H.,
Chu, P.,
Jan, L. Y.,
and von Zastrow, M.
(1999)
Neuron
23,
737-746[CrossRef][Medline]
[Order article via Infotrieve]
|
39.
|
Laporte, S. A.,
Miller, W. E.,
Kim, K. M.,
and Caron, M. G.
(2002)
J. Biol. Chem.
277,
9247-9254[Abstract/Free Full Text]
|
40.
|
Liscovitch, M.,
and Cantley, L. C.
(1995)
Cell
81,
659-662[Medline]
[Order article via Infotrieve]
|
41.
|
De Camilli, P.,
Emr, S. D.,
McPherson, P. S.,
and Novick, P.
(1996)
Science
271,
1533-1539[Abstract]
|
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