From the Molecular Pharmacology Group, Division of
Biochemistry and Molecular Biology, Institute of Biomedical and Life
Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland,
¶ SmithKline Beecham Pharmaceuticals, Harlow, Essex CM19 5AD,
England, and the § Biological Chemistry Unit, Glaxo Wellcome
Research and Development, Gunnels Wood Road, Stevenage,
Herts SG1 2NY, England, United Kingdom
Received for publication, September 29, 2000, and in revised form, January 16, 2001
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ABSTRACT |
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Oligomerization of the human
Recent studies have started to provide a significant body of
evidence to support a concept of constitutive homo-oligomerization of a
range of G protein-coupled receptors
(GPCRs)1 (1, 2). GPCRs for
which such evidence exists include the The earliest studies on GPCR oligomerization relied on the capacity to
co-immunoprecipitate co-expressed but differentially epitope-tagged
forms of a GPCR (see Ref. 15 for review). Because of the hydrophobic
nature of the seven trans-plasma membrane helices of GPCR family
members, care must be taken, however, to exclude nonspecific
interactions between GPCR pairs resulting from detergent dissolution of
cellular membranes. More recent studies have employed various forms of
either fluorescence resonance energy transfer (FRET) (13, 14) or
bioluminescence resonance energy transfer (BRET) (4) to explore GPCR
homo- and hetero-oligomerization in living cells.
Herein we use each of co-immunoprecipitation, BRET, and time-resolved
FRET to examine constitutive homo-oligomerization of the human
As the time-resolved FRET assays were designed to detect only cell
surface oligomers, these studies also demonstrate for the first time
the presence of All materials for tissue culture were supplied by Life
Technologies Inc (Paisley, United Kingdom). [3H]DADLE
(55.3 Ci/mmol) was purchased from PerkinElmer Life Sciences. [3H]Dihydroalprenolol (64 Ci/mmol),
[3H]diprenorphine (66 Ci/mmol),
[3H]adenine, and [3H]cAMP were from
Amersham Pharmacia Biotech. Coelenterazine was from Prolume
(Pittsburgh, PA). Eu3+ and APC-labeled antibodies for
time-resolved fluorescence were from Wallac or Packard. Antisera for
immunoprecipitation and immunoblotting studies were from Santa Cruz
Biotechnology Inc. (Wembley, UK) or Sigma. All other chemicals were
from Sigma or Fisons plc (Loughborough, UK) and were of the highest
purity available. Oligonucleotides were synthesized by Cruachem Limited
(Glasgow, UK).
Construction of Receptor Plasmids--
The human Cell Culture and Transfection--
HEK293 cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% newborn
calf serum and 2 mM L-glutamine. Transient
transfections were performed on cells that were at 70-80% confluence
with LipofectAMINE reagent (Life Technologies, Inc.) according to the
manufacturer's instructions. Cells were harvested 48 h after transfection.
Co-immunoprecipitation--
48 h after transfection, cell
lysates were prepared. The cells were washed three times with 6 ml of
PBS, followed by resuspension in 800 µl of RIPA buffer (RIPA: 50 mM HEPES, 150 mM NaCl, 0.5% sodium
deoxycholate, 0.1% SDS, 1% Triton X-100, 10 mM NaF, 5 mM EDTA, 0.1 mM NaPO4, 5% ethylene
glycol). The cells were lysed for 1 h at 4 °C on a rotating
wheel before removal of the cell debris by a 10-min spin on a benchtop
centrifuge at 13,000 rpm. 500 µg of cell lysate protein was mixed
with protein G-Sepharose (Sigma) for 1 h before removal of the
protein G-Sepharose and its replacement by protein G-Sepharose with the
appropriate antibody for the immunoprecipitation (1.6 µg of
anti-c-Myc antibody (A14, Santa Cruz), 6.8 µg of anti-FLAGTM (M5
monoclonal, Sigma), or 1.6 µg of anti- Immunoblotting--
Samples were resolved by SDS-PAGE on 7%
Tris acetate gels (Novex, Frankfurt, Germany) before being transferred
onto nitrocellulose for Western blot analysis. For Western blotting M5
anti-FLAGTM antibody was diluted 1:2000, the A14 anti-c-Myc antibody
was diluted 1:1000, and the anti- BRET--
Cells were harvested 48 h after transfection.
Media were removed from cell culture dishes, and cells were washed
twice with PBS before cells were detached using TEM buffer (75 mM Tris, 1 mM EDTA, 12 mM
MgCl2, pH 7.4). All samples were subjected to
fluorescence-activated cell sorting analysis to confirm the presence of
eYFP in cells transfected with acceptor-tagged receptors. Approximately
4 × 106 cells in 1.5 ml of TEM buffer were then added
to a glass cuvette; an equal volume of TEM containing 10 µM coelenterazine was then added and the contents of the
cuvette mixed. The emission spectrum (400-600 nM) was
immediately acquired using a Spex fluorolog spectrofluorimeter with the
excitation lamp turned off. For comparisons between experiments, emission spectra were normalized with the peak emission from
Renilla luciferase in the region of 480 nm being defined as
an intensity of 1.00. In some cases a BRET signal was calculated by
measuring the area under the curve between 500 and 550 nm. Background
was taken as the area of this region of the spectrum when examining emission from the isolated Renilla luciferase.
Time-resolved FRET--
Cells were harvested 48 h after
transfection. A 2-h incubation was performed at room temperature with
500,000 cells in a total volume of 100 µl containing 15 nM Eu3+ -labeled anti-c-Myc antibody (Wallac)
and 45 nM APC-labeled M5 anti-FLAGTM antibody (in house)
and 50% newborn calf serum/PBS. After incubation the cells were washed
twice with PBS and resuspended in 30 µl of PBS before placing into
wells of a 384-well microtiter plate for FRET analysis using a
Victor2 (Wallac) configured for time-resolved fluorescence.
The Eu3+-labeled anti-c-Myc antibody was excited at 320 nm
and emissions monitored at 615 nm (Eu3+ emission) and 665 nm (energy transfer). A 200-µs reading was taken after a 50-µs
delay to allow for decay of short-lived endogenous fluorescence signals.
[3H]Ligand Binding Studies (BRET)--
As the BRET
experiments cannot distinguish between receptors present at the cell
surface and in intracellular membranes, membrane preparations were used
to obtain total cell expression levels.
All binding experiments were terminated by filtration through Whatman
GF/C filters followed by three washes with ice-cold TEM.
For each assay equivalent unlabeled cells were counted and membranes
prepared to calculate receptor levels per cell (see Table I).
[3H]Ligand Binding Studies (Time-resolved
FRET)--
As the time-resolved FRET experiments monitor only
receptors delivered to the cell surface, intact cells were used for
these studies in conditions akin to those used for the FRET experiments (see above). [3H]DADLE (5 nM), in the absence
or presence of naloxone (300 µM), was used to define
total and nonspecific binding.
Intact Cell Adenylyl Cyclase Activity Measurements--
Were
performed essentially as described in Refs. 19 and 20. Cells were split
into wells of a 24-well plate, and the cells were allowed to reattach.
Cells were then incubated in medium containing
[3H]adenine (0.5 µCi/well) for 16-24 h. The generation
of [3H]cAMP in response to treatment of the cells with
various ligands and other reagents was then assessed.
The human -opioid receptor and its regulation by ligand occupancy were
explored following expression in HEK293 cells using each of
co-immunoprecipitation of differentially epitope-tagged forms of
the receptor, bioluminescence resonance energy transfer and
time-resolved fluorescence resonance energy transfer. All of the
approaches identified constitutively formed receptor oligomers, and the
time-resolved fluorescence studies confirmed the presence of such
homo-oligomers at the cell surface. Neither the agonist ligand
[D-Ala2,D-Leu5]enkephalin
nor the inverse agonist ligand ICI174864 were able to modulate the
oligomerization status of this receptor. Interactions between
co-expressed
-opioid receptors and
2-adrenoreceptors were observed in
co-immunoprecipitation studies. Such hetero-oligomers could also be
detected using bioluminescence resonance energy transfer although the
signal obtained was substantially smaller than for homo-oligomers of
either receptor type. Signal corresponding to the
-opioid
receptor-
2-adrenoreceptor hetero-oligomer was increased
in the presence of agonist for either receptor. However, substantial
levels of this hetero-oligomer were not detected at the cell surface
using time-resolved fluorescence resonance energy transfer. These
studies demonstrate that, following transient transfection of HEK293
cells, constitutively formed oligomers of the human
-opioid receptor
can be detected by a variety of approaches. However, these are not
regulated by ligand occupancy. They also indicate that time-resolved
fluorescence resonance energy transfer represents a means to detect
such oligomers at the cell surface in populations of intact cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenoreceptor
(3, 4), the D2 dopamine receptor (5), the M3
muscarinic acetylcholine receptor (6), the V2 vasopressin receptor (7), the
-opioid (8, 9) and
-opioid receptors (9), the
histamine H2 receptor (10), and the CCR5 receptor (11).
Furthermore, recent evidence has also indicated a requirement for the
constitutive hetero-oligomerization of distinct GPCRs, such as between
the GABABR1 and GABABR2 receptors, to generate a functional receptor expressed at the cell surface (12). Two important issues, however, remain contentious. The first
of these is whether ligand occupancy alters the extent of GPCR
oligomerization, and the second is the likely extent of GPCR
hetero-oligomerization. In a range of reports, GPCR
homo-oligomerization has been reported to be increased (3, 4, 11),
decreased (8), or unaffected (6, 7) by the addition of receptor
ligands. Similarly, in GPCR heterodimerization studies, interactions
have been indicated to be unaffected (12), regulated (13), or almost
entirely dependent upon (14) the addition of receptor agonists and
hetero-oligomers have recently been reported to form between quite
distinct (14), as well as between closely related (12, 13), GPCR sequences.
-opioid receptor and its possible regulation by agonist and inverse
agonist ligands. We demonstrate that such constitutive oligomerization
can be observed for this GPCR using each of the three approaches but
that ligands do not regulate these interactions appreciably. We also
demonstrate that it is possible to detect a hetero-oligomeric
interaction between the human
-opioid receptor and the human
2-adrenoreceptor in co-immunoprecipitation studies. However, the energy transfer approaches indicated such interactions to
be less prevalent than homo-oligomerization between either of the two
receptor types.
-opioid receptor homo-oligomers at the plasma membrane.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-opioid
receptor in pcDNA4 was used as the starting point for the
construction of the opioid receptor plasmids used in this study.
Primers were made to introduce an ApaI site, followed by the
FLAGTM epitope tag at the 5' end
(5'-AAAAAAGGGCCCGCCACCATGGACTACAAGGACGACGATGATAAGGAACCGGCCCCCTCCGCC-3') and to remove the stop codon and add an XbaI site at the 3'
end (5'-TGCTCTAGAGGCGGCAGCGCC-3') of the receptor. The resulting
fragment was cloned into pcDNA3.1(
) (Stratagene). PCR of both
Renilla reniformis luciferase and enhanced yellow
fluorescent protein (eYFP) was performed to construct the fusion
plasmids using primers, which introduced an XbaI and an
XhoI site at the 5' and 3' ends, respectively. The primers
were as follows: Renilla luciferase (forward,
5'-GCGTCTAGAACTTCGAAAGTTTATG-3'; reverse,
5'-TCGCTCGAGTTATTGTTCATTTT-3'), eYFP (forward,
5'-TGATCTAGAATGGTGAGCAAGGGCGA-3'; reverse,
5'-AGACTCGAGTTACTTGTACAGCTCGTC-3'). The resulting products were
cloned into the plasmid containing the FLAG epitope-tagged
-opioid
receptor to give the plasmids p
ORluc and p
ORYFP. All plasmids
were sequenced to ensure fidelity of the PCR amplification and the
maintenance of the correct open reading frame. Equivalent constructs
containing an N-terminal c-Myc epitope tag sequence were generated in a
similar fashion. FLAGTM- and fluorescent protein-tagged forms of the
human
2-adrenoreceptor were produced as described
previously (16, 17). A positive control vector for BRET was constructed
by linking together Renilla luciferase and eYFP as
previously described by Xu et al. (18).
2-adrenoreceptor
(polyclonal, Santa Cruz). Samples were incubated overnight at 4 °C
on a rotating wheel. The protein G-Sepharose was then washed with RIPA
buffer before resuspending the pellet in reducing SDS-PAGE sample buffer.
2-adrenoreceptor
antibody diluted 1:2000. Immunoreactivity was detected using
horseradish peroxidase-linked anti-mouse (1:5000 dilution) for
anti-FLAGTM or anti-rabbit (1:10,000 dilution) for
anti-
2-adrenoreceptor or anti-c-Myc (Amersham Pharmacia Biotech) followed by ECL detection (Pierce).
-Opioid Receptor Binding Assays--
For binding to the
-opioid receptor, a single concentration (4 nM) of
[3H] diprenorphine in the absence and presence of 300 µM naloxone was used to define total and nonspecific
binding. Assays were performed at 25 °C for 1 h in a buffer
comprising 50 mM Tris, 5 mM EDTA, 15 mM CaCl2, 5 mM MgCl2, 5 mM KCl, 120 mM NaCl, pH 7.4.
2-Adrenoreceptor Binding Assays--
For binding
to the
2-adrenoreceptor, a single concentration (2 nM) of [3H]dihydroalprenolol in the absence
and presence of 10 µM propranolol was used to define
total and nonspecific binding. Assays were performed at 30 °C for
1 h in a TEM buffer comprising 75 mM Tris, 5 mM EDTA, 12.5 mM MgCl2, pH 7.4.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-opioid receptor was modified at the N
terminus to include either a c-Myc or a FLAGTM epitope tag. Following
transient expression of either form of the receptor in HEK293 cells,
these could be immunoprecipitated with appropriate anti-c-Myc or
anti-FLAGTM antibodies (Fig.
1a). No immunoprecipitation
was observed, however, when the antibody/epitope-tagged GPCR
combinations were reversed, confirming the specificity of
immunoprecipitation (Fig. 1a and data not shown).
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Fig. 1.
Co-immunoprecipitation of differentially
epitope-tagged forms of the human -opioid
receptor: evidence for constitutive homo-oligomerization. a,
the FLAGTM
-opioid receptor was expressed transiently in HEK293
cells in the presence (lanes 1 and 4)
or absence (lanes 2 and 3) of the
c-Myc
-opioid receptor. Cell lysates were immunoprecipitated with
anti-FLAGTM (lanes 1 and 2) or
anti-c-Myc (lanes 3 and 4) antibodies,
the samples resolved by SDS-PAGE and then immunoblotted with
anti-FLAGTM (lanes 1 and 2) or
anti-c-Myc (lanes 3 and 4) antibodies.
b, membranes (lanes 1 and
2) or cell lysates (lanes 3-5) were
prepared from HEK293 cells transiently expressing the c-Myc
-opioid
receptor (lanes 1, 3, and
5) or the FLAGTM
-opioid receptor (lanes
2-4). The cell lysates were immunoprecipitated with
anti-FLAGTM antibodies and these samples and the membrane fractions
resolved by SDS-PAGE and immunoblotted using the anti-c-Myc antibody as
primary reagent.
Immunoblotting of SDS-PAGE resolved membrane fractions expressing the
c-Myc-tagged -opioid receptor with the antic-Myc antibody resulted in detection of a 60-kDa polypeptide (Fig. 1b).
Such a polypeptide was not detected by the anti-c-Myc antibody in
membranes expressing the FLAGTM-tagged form of the receptor (Fig.
1b). Co-expression of the c-Myc and the FLAGTM
epitope-tagged forms of the
-opioid receptor followed by
immunoprecipitation with the anti-FLAGTM antiserum and immunoblotting
with the anti-c-Myc antibody also resulted in detection of the 60-kDa
c-Myc-tagged
-opioid receptor (Fig. 1b). Equivalent
results were obtained when the protocol was reversed and
immunoprecipitation of cells co-expressing the two epitope-tagged forms
of the
-opioid receptor was performed with the anti-c-Myc antibody
followed by immunoblotting with the anti-FLAGTM antibody (data not
shown). However, expression of either the c-Myc or FLAGTM-tagged
-opioid receptor alone failed to result in detection of the 60-kDa polypeptide using either of these two protocols (Fig. 1b and
data not shown). Separate expression of the c-Myc- and the FLAGTM
epitope-tagged forms of the
-opioid receptor followed by physical
mixing of cell lysates prior to immunoprecipitation with either
antibody also failed to result in co-immunoprecipitation of the two
forms of the receptor (data not shown). Such results confirm previous data on the ability to detect homo-oligomers of co-expressed but differentially tagged forms of the
-opioid receptor (8).
When the c-Myc-tagged -opioid receptor was co-expressed along with
the human
2-adrenoreceptor, co-immunoprecipitation
experiments akin to those described above, but now using combinations
of the anti-c-Myc antibody and an anti-
2-adrenoreceptor
antibody, were able to provide evidence for the presence of
hetero-interactions between these two GPCRs (Fig.
2). Immunoprecipitation of the
2-adrenoreceptor resulted in the presence of the
c-Myc-tagged
-opioid receptor in the precipitated sample, which
could be detected by immunoblotting following resolution of the sample
by SDS-PAGE. A second polypeptide with mobility consistent with a dimer
containing the c-Myc-tagged
-opioid receptor was also detected (Fig.
2). Neither of these bands was detected when the human
2-adrenoreceptor was expressed in the absence of the
c-Myc-tagged
-opioid receptor and then immunoprecipitated (Fig. 2).
Equivalent results were obtained when the c-Myc-tagged
-opioid
receptor was co-expressed with a form of the
2-adrenoreceptor that had been C-terminally tagged with
eYFP or with a form of the
2-adrenoreceptor tagged at
the N terminus with the FLAGTM epitope and at the C terminus with green fluorescent protein (GFP) (Fig. 2).
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Immunoprecipitation of either of these modified forms of the
2-adrenoreceptor resulted in co-precipitation of the
c-Myc-tagged
-opioid receptor and detection of both monomeric and
potential dimeric species. These rather unexpected observations led us
to consider whether such co-immunoprecipitation approaches following transient transfection of cells might produce artifactual results following solubilization of GPCRs from the membrane environment.
Recently, 2-adrenoreceptor homo-oligomerization has been
observed in intact cells by monitoring the interactions between forms
of this GPCR C-terminally epitope-tagged with either Renilla luciferase or eYFP (4). We thus constructed C-terminally tagged Renilla luciferase and eYFP forms of the
-opioid receptor
and the
2-adrenoreceptor as well as a Renilla
luciferase and eYFP fusion protein akin to that described by Xu
et al. (18) to act as a positive control for BRET. Transient
expression of either isolated Renilla luciferase (data not
shown) or
2-adrenoreceptor-Renilla luciferase
in HEK293 cells, followed by the addition of the cell permeant
luciferase substrate coelenterazine, resulted in emission of light with
a single peak centred at 480 nm (Fig.
3a). Expression of the
Renilla luciferase-eYFP fusion construct and addition of coelenterazine resulted in both a peak at 480 nm and the appearance of
a second peak centred at 527 nm (Fig. 3a). This second peak represents energy transfer from Renilla luciferase
to eYFP and its subsequent emission with lower energy. Co-expression
of
2-adrenoreceptor-Renilla luciferase and
2-adrenoreceptor-eYFP followed by addition of coelenterazine again produced the dual peak consistent with energy transfer between the BRET partners, which is reliant on their close
physical proximity (Fig. 3a). However, co-expression of the
isolated Renilla luciferase along with
2-adrenoreceptor-eYFP did not result in energy transfer
upon addition of coelenterazine (data not shown), indicating that there
were not direct interactions between these two constructs. Furthermore,
separate expression of
2-adrenoreceptor-Renilla luciferase and
2-adrenoreceptor-eYFP followed by mixing of the cells
prior to addition of coelenterazine also failed to produce an energy
transfer signal (Fig. 3b), demonstrating a requirement for
physical proximity for energy transfer. Equivalent studies with the
co-expression of
-opioid receptor-Renilla luciferase and
-opioid receptor-eYFP again produced a pattern of light emission following addition of coelenterazine consistent with energy transfer and thus the proximity of the BRET partners and their associated GPCRs
(Fig. 4a). As before, no
energy transfer signals were observed when the Renilla
luciferase and eYFP-tagged forms of this GPCR were transiently
expressed in different populations of HEK293 cells, which were then
mixed prior to addition of coelenterazine (data not shown).
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Fluorescence-activated cell sorting analysis of cells to detect
-opioid receptor-eYFP expression demonstrated an average of 35% of
the cell population was successfully transfected.
3H-labeled ligand binding studies using the
-opioid
receptor antagonist [3H]diprenorphine as radioligand
indicated that an average of 200,000 ± 10,000 copies of the
receptor binding sites were present in each successfully transfected
cell following co-expression of the
-opioid
receptor-Renilla luciferase and
-opioid receptor-eYFP constructs (Table I). Addition of either
the agonist DADLE or the inverse agonist ICI174864 (20, 21) (both up to
10 µM) failed to produce a statistically significant
alteration in the
-opioid receptor energy transfer signal (Fig. 4,
a and b), indicating that these ligands were not
altering the extent of
-opioid receptor oligomerization or the
relative proximity of the two forms of this GPCR.
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Co-expression of 2-adrenoreceptor-Renilla
luciferase and the
-opioid receptor-eYFP construct resulted in a
small energy transfer signal upon addition of coelenterazine (Fig. 4,
b and c). Combinations of
[3H]diprenorphine binding and direct monitoring of the
fluorescence of eYFP clearly indicated that the
-opioid
receptor-eYFP construct had been expressed successfully in these
experiments and at similar levels as in the receptor
homo-oligomerization studies (Table I and data not shown). Equivalent
ligand binding studies used the
2-adrenoreceptor
antagonist [3H]dihydroalprenolol to identify
2-adrenoreceptor-Renilla luciferase. In cells
that produced a positive BRET signal, an estimated 76,000 ± 14,000 copies of this
2-adrenoreceptor construct were
expressed (Table I). Such results indicate that these two GPCRs can
hetero-oligomerize in intact cells but that the extent of these
interactions is substantially lower than for homo-oligomerization of
either the
-opioid receptor or the
2-adrenoreceptor.
Addition of either the opioid receptor agonist DADLE or the
2-adrenoreceptor agonist isoprenaline (both at 10 µM) resulted in statistically significant
(p < 0.05) increases in the energy transfer signal
consistent with agonist-induced formation of a
2-adrenoreceptor-
-opioid receptor hetero-oligomer (Fig. 4b), but these signals remained small compared with
those indicative of
-opioid receptor homo-oligomer formation (Fig. 4, b and c).
Neither the co-immunoprecipitation nor BRET studies can provide direct
information on the cellular location of the detected receptor
oligomers. To address whether -opioid homo-oligomers were present at
the cell surface and if at least this fraction of the GPCR
homo-oligomer population would be regulated by agonist or inverse
agonist treatment, we expressed combinations of N-terminally c-Myc- and
FlagTM-tagged forms of the
-opioid receptor in HEK293 cells. We then
added combinations of a europium3+-labeled anti-c-Myc
antibody as energy donor and an allophycocyanin (APC)-labeled
anti-FlagTM antibody as acceptor to intact cells and used this pairing
in time-resolved FRET studies monitored by light emission at 665 nm
from APC. No increase in signal above that observed in untransfected
HEK293 cells was detected upon individual expression of either the
c-Myc or FlagTM forms of the
-opioid receptor. However,
co-expression of these two forms of the
-opioid receptor resulted in
strong time-resolved FRET (Fig. 5a), which provided a
substantially greater signal to noise ratio than obtained in the BRET
studies. Such a signal was only observed with addition of both the
europium3+-labeled and APC-labeled antibodies. The absence
of any of the four elements resulted in no energy transfer signal being
detected (data not shown). Again, no time-resolved FRET signal was
obtained if cells separately expressing either the c-Myc or FlagTM
forms of the
-opioid receptor were simply mixed prior to addition of the antibodies (Fig. 5a).
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Despite the substantially greater capacity to observe -opioid
receptor homo-oligomers in intact cells using time-resolved FRET
compared with BRET, co-expression of the c-Myc-tagged
-opioid receptor and the FLAGTM-
2-adrenoreceptor-GFP did not
result in the production of a time-resolved FRET signal upon addition
of the appropriate antibodies (Fig. 5a). These observations
indicate that, at least at the cell surface, levels of a potential
-opioid receptor-
2-adrenoreceptor hetero-oligomer
were not detectable. Despite the clear evidence for the presence of
cell surface constitutive
-opioid receptor homo-oligomers, addition
of neither DADLE nor ICI174864 (both at 100 nM) altered the
time-resolved FRET signal (Fig. 5b). It was clearly possible
that ligands would not be able to bind to the GPCRs in the presence of
the antibodies required for time-resolved FRET. However, specific
binding to the
-opioid receptor of [3H]DADLE was
unaffected by the presence of the antibodies (Fig. 5c).
Furthermore, the presence of the epitope tag antibodies did not reduce
the capacity of DADLE to mediate inhibition of cAMP production
monitored in intact cell adenylyl cyclase activity assays (Table
II).
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DISCUSSION |
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It is now clear that many GPCRs have the capacity to oligomerize
(1-2, 15). However, key issues that remain unresolved or contentious
are how widespread this phenomenon is, whether such oligomers persist
at the plasma membrane or reflect only a chaperonin-like strategy to
deliver the GPCRs to the cell surface, and whether oligomerization is
regulated by the binding of agonist ligands. Early studies utilized
co-immunoprecipitation strategies to infer oligomerization of GPCRs and
reports such as with the 2-adrenoreceptor (3) indicated
the extent of oligomerization to be increased in the presence of
agonist ligands. As this has also been observed for a number of
chemokine receptors (11, 22, 23), this increased the possibility of it
being a widespread regulatory feature. However, related studies with
the
-opioid receptor indicated that agonist ligands reduced the
extent of oligomerization of this GPCR (8, 9), whereas, for the
-opioid receptor (9) and the M3 muscarinic receptor (6),
no effects of ligands were observed. Many of these above studies were
unable to address the cellular location of the GPCR oligomers. However, the demonstration that appropriate membrane delivery and production of
a functional GABAB receptor requires the co-expression of
two distinct GPCR gene products suggested a chaperonin role for GPCR dimerization (Refs. 12, 24, and 25; see Ref. 26 for review). Recently,
a considerable range of such GPCR hetero-oligomers have been detected
(2) and, at least in certain cases such as with a
/
-opioid
receptor hetero-oligomer, these have been reported to have
pharmacological characteristics distinct from (presumably) homo-oligomers of these receptors (9). Moreover, in a number of reports
on hetero-oligomer formation, their effective detection has required
the presence of agonist ligands (13, 14).
Considerable efforts have recently been given to the development of
assays able to monitor the presence and regulation of GPCR oligomers in
intact cells. At least partially this reflects concerns of the
possibility of potential artifacts being produced in studies that rely
entirely on the co-immunoprecipitation of highly hydrophobic proteins.
Energy transfer approaches have been the systems of choice. Recently,
FRET between forms of the -factor receptor of the yeast
Saccharomyces cerevisiae, C-terminally tagged with energy
transfer competent cyan and yellow fluorescent proteins, has been used
to demonstrate constitutive oligomerization of this receptor and that
this is unaffected by the presence of ligand (27). A variation of this
procedure, termed BRET, has been used to re-examine ligand regulation
of oligomerization of the
2-adrenoreceptor. Here Anger
et al. (4) observed both basal oligomerization of this
receptor and an increase in this signal upon addition of the agonist
isoproterenol. Although these results were consistent with
agonist-induced oligomerization, the authors were careful to note that
it could also represent only a re-orientation of the GPCR constructs. A
third variation, FRET with photobleaching, has been applied to study
homo- and hetero-oligomerization of somatostatin receptor subtypes (13)
and between the somatostatin SSTR5 receptor and the D2
dopamine receptor (14). Some, but not all, somatostatin receptor
subtypes could form hetero-oligomers on addition of agonist (13) and
the interaction between the SSTR5 receptor and the D2
dopamine receptor could be achieved on addition of agonists for either
receptor (14).
Herein, we have used combinations of GPCR co-immunoprecipitation
studies, BRET, and a further variant of FRET, which takes advantage of
the long-lived fluorescence characteristics of certain lanthanide
chelates to allow time-resolved fluorescence to be employed to
re-explore constitutive oligomerization of the -opioid receptor and
the possible effects of agonist and inverse agonist ligands. This
approach has recently been applied to the analysis of the polypeptide
makeup of the GABAA Cl
ion channel (28). All
three approaches provided evidence of constitutive oligomerization of
the
-opioid receptor. However, unlike the studies of Devi and
colleagues (8, 9), we were unable to observe consistent regulation of
-opioid receptor homo-oligomers by either a synthetic opioid peptide
agonist or the classical inverse agonist at this receptor. It thus
appears that, at least for this receptor in intact cells,
oligomerization status is not related to the R and
R* equilibria, which determine receptor activation state
(29). Devi and co-workers (8) have also proposed that
-opioid
receptor monomerization might be required for agonist-induced endocytosis. However, at least in the case of the S. cerevisiae
-factor receptor, recent elegant studies have
indicated that it is internalized as a dimer (30). Both
co-immunoprecipitation and the BRET studies are unable to provide
information on the cellular location of the GPCR oligomers that produce
the signals, but the Eu3+- and APC-labeled antibody pairs
used for the time-resolved FRET studies only have access to GPCRs
successfully delivered to the plasma membrane and these studies
confirmed the presence of preformed homo-oligomers at the cell surface
and their lack of regulation by ligands (Fig. 5).
We also attempted to explore the specificity of GPCR
oligomerization using each of the three approaches. In
co-immunoprecipitation studies, apparent interactions could be observed
between the co-expressed -opioid receptor and the
2-adrenoreceptor (Fig. 2). However, in such
co-immunoprecipitation studies, samples can be exposed to film using
enhanced chemiluminescence for the period required to obtain a signal.
By contrast, we were unable to observe any significant interactions
between these two receptors in the time-resolved FRET-based assays
(Fig. 5). We were able, however, to observe a BRET signal consistent
with interactions between co-expressed
-opioid receptors and
2-adrenoreceptors (Fig. 4, b and
c). This signal was small when compared with those obtained
when monitoring homo-oligomerization of either of these two receptors.
However, in contrast to the situation with
-opioid receptor
homo-oligomers, signal consistent with hetero-oligomerzation between
the
-opioid receptor and the
2-adrenoreceptor was
increased in the presence of agonists at either of the receptors.
However, even this signal was still small compared with that
observed for either
-opioid receptor or
2-adrenoreceptor homo-oligomers. This measured increase in either
-opioid receptor-
2-adrenoreceptor
interaction or orientation relative to each other in response to
agonist ligands is intriguing. It is possible that transient expression
of the same or closely related receptors, e.g. opioid
receptor subtypes, may result in constitutive interaction based
primarily on effects of mass action and a significant level of mutual
affinity, whereas less closely related receptors may require ligand
binding to promote interactions. However, it must be stressed that the
signal to noise ratio in the BRET assay is poor (as also noted by
Angers et al. (Ref. 4)), at least in part because
Renilla luciferase and eYFP are not optimal BRET partners
and this limits the current sensitivity of the approach. Further
studies that take advantage of novel and rapidly improving energy
transfer techniques will be required to validate and unravel the basis
for these observations. A general issue in all studies of this nature,
particularly when performed using transient transfection, is whether
artifacts may be produced due to high level expression of the potential
interacting partners. In the current energy transfer studies, we have
maintained levels of expression of the receptor constructs as low as
possible (Table I) with the proviso that signal had to be sufficient to
monitor the interactions.
Evidence from construction of the functional GABAB receptor dimer indicates that the GABABR1 displays little capacity to move to the cell surface without co-expression of the GABABR2 (12). In this example dimer formation takes place in the endoplasmic reticulum with the GABABR2 acting both to mask an ER retention sequence in the GABABR1 (31) and to allow plasma membrane delivery of the functional receptor, even though the GABABR2 appears not be be directly involved in recognition of the agonist GABA. These requirements for interactions between the GABABR1 and GABABR2 at the ER to allow delivery of the functional receptor suggest that the GABABR2 functions as a chaperonin for the GABABR1. This may be a specialized and extremely well studied example of a common process in which GPCRs dimerize in the ER to act as mutual chaperonins (32). Evidence in favor of such a model is provided by the example in which co-expression of the D3 dopamine receptor with a naturally occurring splice variant named D3nf, which lacks transmembrane regions VI and VII and functions akin to a dominant negative mutant, blocks delivery of the wild type receptor to the plasma membrane (33).
In many regards time-resolved FRET provided the most useful approach
employed herein and certainly provided excellent signal to noise
ratios. As noted above, a key feature of this approach is that it is
only able to monitor the proximity of GPCRs which have matured and had
reached the cell surface. In transient expression studies, this is
often not achieved by a significant fraction of the expressed protein.
The maturation process of the -opioid receptor is appreciated to
cause problems in effective cell surface delivery for this GPCR (34)
and such intracellularly retained receptors cannot be resolved from
those at the plasma membrane using BRET or the co-immunoprecipitation
approaches. Second, the time-resolved nature of the fluorescence assays
allows fluorescence derived from excitation of other cell components to
decay prior to monitoring the signal. This substantially increases the
signal to noise ratio obtained in the assay. However, despite these
advantages, we were unable to detect
-opioid
receptor-
2-adrenoreceptor interactions in this mode or
to monitor regulation of
-opioid receptor homo-oligomerization by ligands.
One potential caveat of the time-resolved FRET approach is that the
antibodies used to identify the epitope-tagged receptors are bivalent
and thus might be anticipated to cluster receptors. Indeed, a
monoclonal anti-2-adrenoreceptor antibody with
agonist-like properties has been described in which Fab fragments
behave as antagonists (35). This is at least consistent with the idea that clustering of receptors might be required for signal transduction. However, although this is an interesting issue, it is unlikely to be of
importance to the results of this study. The bivalency of the anti-FLAG
antibody can only cause potential dimerization of the FLAG-tagged
version of a receptor and the anti-c-Myc antibody likewise only
potential dimerization of a c-Myc-tagged version of the receptor.
However, to obtain a time-resolved FRET signal requires interaction
between a FLAG-tagged opioid receptor and a c-Myc-tagged one to
produce a pairing that can generate an energy transfer signal. If
antibody-induced clustering were sufficient to provide sufficient
proximity of the differentially tagged receptors, we would have
anticipated that signal would also be produced for the
-opioid
receptor and
2-adrenoreceptor pair used in the
time-resolved FRET format. This was not observed (Fig.
5a).
These studies demonstrate delivery of preformed -opioid receptor
oligomers to the surface of HEK293 cells following transient transfection and indicate that time-resolved FRET currently represents the most sensitive means to detect such oligomers at the cell surface
in populations of intact cells.
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FOOTNOTES |
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* 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 all correspondence should be addressed: Davidson
Bldg., University of Glasgow, University Ave., Glasgow G12 8QQ,
Scotland, United Kingdom. Tel.: 44-141-330-5557; Fax:
44-141-330-4620; E-mail: g.milligan@bio.gla.ac.uk.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M008902200
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ABBREVIATIONS |
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The abbreviations used are: GPCR, G protein-coupled receptor; APC, allophycocyanin; FRET, fluorescence resonance energy transfer; BRET, bioluminescence resonance energy transfer; PAGE, polyacrylamide gel electrophoresis; RIPA, radioimmune precipitation assay; DADLE, [D-Ala2, D-Leu5]enkephalin; eYFP, enhanced yellow fluorescent protein; GFP, green fluorescent protein; ER, endoplasmic reticulum; PBS, phosphate-buffered saline.
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