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INTRODUCTION |
The standard model describing signaling in G protein-coupled
receptors (GPCRs),1 where the
receptor functions strictly as a monomer, is no longer tenable. In the
past few years, a number of studies have demonstrated that
oligomerization of GPCRs may play important roles in receptor trafficking and signaling (for reviews, see Refs. 1 and 2). In addition
to forming homodimers, several receptors have been shown to
heterodimerize with other receptor subtypes. In some cases, such as the
metabotropic GABAB (3-7) and the gustatory receptors (8,
9), heterodimerization between closely related subtypes was found to be
essential for the formation of functional receptors. Although only a
few examples for such obligatory heterodimerization are available to
date, an increasing number of reports suggest the occurrence of
heterodimerization between more or less closely related family members
(10-19). In some of these cases, heterodimerization has been proposed
to lead to receptors with pharmacological and/or functional properties
that are different from those of the individual receptors.
The potential regulatory influences that these oligomeric assemblies
may have on the function of receptors co-existing in the same cell led
us to investigate whether receptors that are ubiquitously expressed in
many tissues and cell types could function as heterodimers. Two such
widely distributed receptors are the 
1AR and
2AR, which are co-expressed in a large number of tissues and cell types (20-24). Interestingly, the two receptor subtypes were
shown to form homodimers when expressed individually in heterologous expression systems (25-27). Although the two receptors display more
than 50% sequence homology (28) and share transmembrane domain motifs
proposed as a dimerization interface (25), no study has directly
investigated their potential for heterodimerization.
Although the two receptors are known to couple to Gs
important differences in their functional properties have been
reported. For instance, the 2AR has been shown to be
more efficiently coupled than some variants of the 1AR
to adenylyl cyclase (29-31). Also, 2AR activation leads
to a more efficient stimulation of various MAPK signaling pathways (32,
33). In further contrast to the 2AR, which undergoes
rapid internalization following agonist stimulation, the
1AR was found to remain largely localized at the cell
surface for extended periods following agonist stimulation (34-36).
Since these differences represent intrinsic properties that were
determined for the individual receptors, they offer useful readouts to assess the functional consequences
of heterodimerization.
In the present study, the occurrence of heterodimerization between the
1AR and 2AR was assessed by
co-immunoprecipitation and bioluminescence resonance energy transfer
(BRET) in living human cells co-expressing the two receptors. The
potential functional consequences of the heterodimerization were also
assessed by determining the influence of receptor co-expression on the
ability of the receptors to stimulate adenylyl cyclase and MAPK
pathways as well as to undergo agonist-promoted internalization. We
found that co-expression of the two receptor subtypes in HEK 293 cells
lead to their heterodimerization and inhibited both agonist-promoted 2AR internalization and ERK1/2 MAPK stimulation. This
suggests that heterodimerization may represent a regulatory cross-talk process arising through the creation of a receptor form that has distinct functional properties.
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EXPERIMENTAL PROCEDURES |
Materials
Unless otherwise stated, all chemicals were of reagent grade or
higher and were obtained from Sigma.
Eukaryotic Expression Vectors
1AR-GFP10--
The 1AR coding
sequence without its stop codon was amplified from the pBC12BI-human
1AR plasmid (28) using sense and antisense primers
harboring unique SacI and AgeI sites. The
fragment was then subcloned in frame into the
SacI/AgeI site of the turquoise variant
GFP-Sapphire vector (pGFP-N1-Sapphire; Packard Instrument Co.)
to give the plasmid pGFP-N1-1AR-Sapphire. Finally, the
GFP-Sapphire was replaced by a green GFP variant (GFP10) containing the
following mutations: P64L, S147P, and S202P. For this purpose,
an AgeI/BsrGI fragment of the GFP10
was subcloned into the AgeI/BsrGI site of the pGFP-N1-1AR-Sapphire to finally yield
pGFP-N1-1AR-GFP10.
2AR-GFP10--
The GFP10
AgeI/BsrGI fragment was subcloned into the
AgeI/BsrGI site of
pGFP-N1-His2AR-YFP (26). For the sake of
simplicity, the constructs are referred to as 1AR-GFP and
2AR-GFP, respectively.
HA-1AR--
The
HindIII/XhoI fragment of the plasmid
pCMV-HA-1AR, which was a generous gift of Dr. Brian
Kobilka (Stanford University) (37), was subcloned into the
HindIII/XhoI site of pcDNA3 (RSV) plasmid to
yield pcDNA3 (RSV)-HA-1AR. This 1AR
construct contains an arginine at position 389, the most common
polymorphism at this position (38).
HA-2AR--
The human His2AR
coding sequence was amplified using a sense primer containing a
BamHI restriction site followed by the HA tag sequence and
an antisense primer. The amplified HA-2AR sequence was
digested with BamHI/EcoNI and then subcloned into
BamHI/EcoNI-digested pcDNA3
(RSV)-His-2AR vector, resulting in pcDNA3
(RSV)-HisHA-2AR plasmid.
1AR-Rluc--
The
pcDNA3.1-1AR:6:hRluc was a generous gift from
BioSignal Packard Bioscience. This fusion protein contains a
linker of 6 amino acids linking the carboxyl tail of the human
1AR to humanized Rluc.
2AR-Rluc--
The humanized Rluc
coding sequence (Packard Instrument Co.) was amplified using
sense and antisense primers and then subcloned into the PCR Blunt II
Topo vector (Invitrogen). The hRluc fragment was excised by
digestion with KpnI/XbaI and subcloned into
the KpnI/XbaI-digested pcDNA3.1
Zeo vector to generate the pcDNA3.1 Zeo/hRluc plasmid.
The human His2AR coding sequence was amplified without
its stop codon using sense and antisense primers. The PCR product was
subcloned into PCR Blunt II Topo Vector and then excised by double
digestion with HindIII/KpnI and ligated into the
HindIII/KpnI-digested expression vector
pcDNA3.1Zeo/hRluc. The resulting construct encodes a
6-amino acid linker between the carboxyl tail of the 2AR
and the humanized Rluc sequence. In some experiments, a
c-myc-tagged version (N-terminal) of the 2AR-Rluc construct was used.
HA-GABABR2--
The
pcDNA3.1-HA-GABABR2 was a generous gift from GlaxoSmithKline.
Cell Culture and Transfection
HEK 293 cells were maintained in Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum (Wisent or BioMedia), 100 units/ml penicillin/streptomycin, 2 mM
L-glutamine (from Wisent or Invitrogen). For
transfection experiments, cells were seeded at a density of 2 × 106 cells/100-mm dish and cultured for 24 h. Transient
transfections were then performed using either the calcium phosphate
precipitation protocol (39) or LipofectAMINE (Invitrogen) according to
the manufacturer's recommendations. The cells were then cultured in the same medium for 48-72 h. In some experiments, transient
transfections were carried out in HEK 293 cells stably expressing the
human 2AR (a generous gift from Dr. Phil Wedergaertner,
Thomas Jefferson University).
Immunoprecipitation and Western Blot Analysis
For immunoprecipitation experiments, cells co-expressing
HA-tagged receptors and the 2AR-GFP were harvested
72 h post-transfection, washed three times in PBS, and incubated
with blocking buffer (PBS containing 0.2% bovine serum albumin) for
1 h on ice. Subsequently, the cells were incubated with the mouse
monoclonal anti-HA (12CA5) antibody (1:250 dilution) in blocking buffer
on ice for an additional 1 h. After two washes in blocking buffer
and two washes in PBS, cells were lysed, and proteins were
solubilized in radioimmune precipitation buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet
P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM
iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, 0.1 mg/ml benzamidine, 2.5 µg/ml leupeptin for 30 min on ice and
centrifuged at 12,000 × g for 15 min at 4 °C to
remove cellular debris. For immunoprecipitation of the total receptor
pool, lysates were incubated overnight at 4 °C with additional 12CA5
antibodies (1:200 dilution) before the addition of protein G-Sepharose
for 3 h. For immunoprecipitation of cell surface receptors, only
protein G-Sepharose was added. Protein
G-Sepharose-antibody-antigen complexes were then collected by
centrifugation at 12,000 × g. The immunoprecipitates
were washed four times with cold radioimmune precipitation buffer and
resuspended in sample buffer containing 60 mM Tris-HCl, pH
6.8, 2% SDS, 4 M urea, and 100 mM
dithiothreitol and heated at 50 °C. Protein concentration to be used
in the immunoprecipitation was assessed using the DC Protein assay kit
(Bio-Rad) with bovine serum albumin as a standard. Protein samples were
resolved by 10% SDS-polyacrylamide gel electrophoresis, transferred to
nitrocellulose (Protran), and subjected to immunoblotting using rabbit
polyclonal anti-GFP antibody (CLONTECH, 1:100
dilution). The Renaissance chemiluminescence kit (PerkinElmer Life
Sciences) was used for Western blot development.
BRET Assay
Forty-eight hours post-transfection, cells were washed twice in
PBS, detached with PBS/EDTA, and resuspended in PBS plus 0.1% glucose.
Cells were then distributed in 96-well microplates (white Optiplate
from BioSignal Packard Bioscience) at a density of ~100,000 cells/well. Deep Blue C coelenterazine (BioSignal Packard
Bioscience) was added at a final concentration of 5 µM,
and readings were collected using a modified Top-count apparatus
(BRETCount) that allows the sequential integration of the signals
detected in the 370-450- and 500-530-nm windows using filters with
the appropriate band pass (Chroma). The BRET signal is determined by
calculating the ratio of the light emitted by the receptor-GFP
(500-530 nm) over the light emitted by the receptor-Rluc
(370-450 nm). The values were corrected by subtracting the background
signal detected when the Receptor-Rluc constructs were
expressed alone. On a routine basis, the protein concentration of the
samples was determined to control for the number of cells using a
Bradford assay (Bio-Rad) with bovine serum albumin as a standard. To
determine the maximal BRET level detectable between each partner,
preliminary experiments were carried out by co-transfecting increasing
amounts of the receptor-GFP plasmids with a constant quantity of
receptor-Rluc construct.
Membrane Preparation
Membranes were prepared from cells 48 h after transfection
as described previously (25, 40). Briefly, cells were washed twice with
ice-cold PBS. They were then disrupted by homogenization with a
Polytron homogenizer in 10 ml of ice-cold buffer containing 5 mM Tris-HCl, pH 7.4, 2 mM EDTA, 5 µg/ml
leupeptin, 10 µg/ml benzamidine, and 5 µg/ml soybean trypsin
inhibitor. Lysates were centrifuged at 500 × g for 5 min at 4 °C to remove nuclei and unbroken cells. The supernatant was
then centrifuged at 45,000 × g for 20 min, and the
pellet was washed twice in the same buffer. Membrane preparations were
used immediately for adenylyl cyclase and binding assays.
Receptor Quantification
Membranes were prepared and washed as described above. Total
AR number was calculated from binding experiments using a saturating concentration (300 pM) of [125I]cyanopindolol
(CYP; PerkinElmer Life Sciences) as the radioligand. Briefly, membrane
preparations (10 µg of protein) were incubated with
[125I]CYP for 90 min at room temperature in a total
volume of 0.5 ml in the presence or absence of 10 µM
alprenolol or propranolol (Sigma) to define specific binding. In some
experiments, the proportion of 1AR and
2AR expressed were determined using a concentration (0.1 µM) of the selective 2- or
1-specific antagonists ICI 118,551 (Tocris) or betaxolol
(Tocris) that fully occupies one of the subtypes but blocks less than
10% of the other.
Surface receptor expression was determined using whole cell binding
assays with the hydrophilic ligand [3H]CGP 12177 (CGP; PerkinElmer Life Sciences) as described (41). Cells were
transiently transfected with either 2AR-GFP10 or
2AR-GFP10 and HA-1AR. Briefly,
2AR-specific internalization was determined using 5 × 10
8 M betaxolol to block CGP binding to
the 1AR, and nonspecific surface binding was determined
using 10 µM propranolol and subtracted from values
determined in the presence of betaxolol. These experiments (except for
washing steps) were conducted at 37 °C. Cells were treated with 10 µM isoproterenol or vehicle (104
M ascorbic acid) for 2 h and then washed with cold PBS
to remove ligand. For cell surface binding, cells were incubated with
CGP for 30 min, and for total binding, cells were incubated with CYP for 60 min. Cells were then filtered with a Brandel cell harvester as
for the membrane receptor assay described above.
Measurement of Adenylyl Cyclase Activity
Adenylyl cyclase activity was assayed in the same membrane
preparation according to the method of Salomon et al. (42)
using 50 µg of protein in a total volume of 50 µl. Enzyme
activities were determined following a 15-min incubation in the
presence of 1 nM to 100 µM isoproterenol, 100 µM forskolin, 10 mM NaF, or the vehicle at
37 °C. Data were calculated as pmol of cAMP produced/min/mg of
protein, normalized with respect to forskolin-stimulated adenylyl
cyclase activity and analyzed by least squares regression using
GraphPad PRISM.
Detection of Phosphorylated MAPK Isoforms (p38 and
p42/p44 ERK)
Cells were serum-starved for 16 h and treated for the
indicated times with the nonselective -adrenergic agonist
isoproterenol or phorbol 12-myristate 13-acetate at 37 °C. Cells
were then lysed in a buffer containing 25 mM HEPES (pH
7.5), 150 mM NaCl, 0.25% sodium deoxycholate, 10%
glycerol (v/v), 25 mM NaF, 10 mM
MgCl2, 1 mM EGTA, 1 mM
Na3VO4, 10 µg/ml leupeptin, 10 mM
benzamidine, 0.5 µM microcystin, 1% Triton X-100 (v/v),
0.1 mM phenylmethylsulfonyl fluoride, and 5 mM
dithiothreitol. The samples were then prepared for denaturing SDS-PAGE,
and the activity of the p38 and p42/p44 (ERK1/2) MAPK was determined by
immunoblotting with anti-phospho-p38 or anti-phospho-ERK (both at
1:1000 dilution; Cell Signaling). The blots were then stripped and
reblotted with the anti-ERK1/2 or anti-p38 (1:2000; StressGen) to
control for the total amount of kinases loaded. Data from separate
experiments were digitized on a flatbed scanner and analyzed using
Quantity One (Bio-Rad) software.
Confocal Microscopy
Cells were cultured on coverslips overnight in Dulbecco's
modified Eagle's medium and stimulated for the indicated times with 1 µM isoproterenol at 37 °C. They were then
permeabilized and fixed with 3% paraformaldehyde (v/v) and 0.2%
Triton X-100 (v/v) for 20 min at room temperature. After three washes
with PBS, the cells were treated with a blocking solution containing
2% BSA and 5% preimmune normal donkey serum (Jackson Laboratories)
for 1 h. Rabbit polyclonal anti-2AR (1:100
dilution; Santa Cruz Biotechnology) or anti-1AR (a
generous gift from Dr. Brian Kobilka, Stanford University; 1:100
dilution), diluted in a solution of 2.5% normal donkey serum and 1%
BSA, were subsequently incubated with fixed, permeabilized cells for
16 h at 4 °C. Following three washes with PBS, CY5-labeled
secondary donkey anti-rabbit antiserum (1:500 dilution; Jackson
Laboratories) was incubated with the samples for 60 min in the dark
followed again by three washes with PBS. For colocalization
experiments, cells were transfected with 2AR-GFP and
1AR. GFP fluorescence was stimulated with a 488-nm argon laser, and these signals were overlaid with CY5 fluorescence
(stimulated with a 633-nm helium-neon laser) after labeling with
anti-1AR. Confocal microscopy was performed using a
Zeiss LSM-510 system with a highly corrected objective (Zeiss
Plan-Apochromat ×63, numerical aperture 1.4 under oil). Control
experiments were performed in the absence of primary antibodies and
revealed a low level of background staining, indicating the specificity
of the primary antibodies used.
Enzyme-linked Immunosorbent Assay to Detect Receptors Expressed
at the Cell Surface
Cells were transfected with 2AR-Rluc
bearing a c-myc tag on the extracellular N terminus alone or
with 1AR-GFP10 and transferred to six-well
polylysine-coated plates 12 h after transfection. 24 h
post-transfection, cells were treated with 10 µM
isoproterenol or vehicle for 60 min at 37 °C. Cells were kept on ice
for all subsequent steps. After three washes with cold PBS, cells were blocked with PBS with 1% BSA (w/v) for 30 min. Blocked cells were then
incubated with anti-myc (9E10) antibodies (1:500 dilution) for 60 min and subsequently washed again three times with PBS plus 1%
BSA. Cells were then fixed with 3% paraformaldehyde (v/v) for 15 min
and washed three times with PBS. Cells were blocked again for 15 min
with PBS plus 1% BSA, incubated with anti-mouse/horseradish peroxidase
conjugate (Amersham Biosciences; 1:1000) for 30 min, and finally washed
with PBS plus 1% BSA three times. The substrate o-phenylenediamine dihydrochloride (Sigma) was added
according to the manufacturer's instructions for 4-7 min. The
reaction was stopped with 200 µl of 3 N HCl, and
extinction was measured at 492 nm. Data were plotted as percentage of
signal (isoproterenol) relative to time-matched control (vehicle).
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RESULTS |
Characterization of the
1AR/2AR Heterodimer by
Co-immunoprecipitation--
In a first attempt to determine whether
the 2AR could form a heterodimer with the
1AR, a co-immunoprecipitation experiment between
1AR and 2AR bearing different epitope
tags (HA and GFP, respectively) was designed. The ability of the
2AR to form homodimers or to heterodimerize with the
1AR was assessed in HEK 293 cells co-expressing the
2AR-GFP with either HA-2AR or
HA-1AR (Fig. 1,
lanes 3-6). After cell surface (S) or
total cell extract (T) immunoprecipitation of the HA-tagged
receptors using a mouse monoclonal anti-HA antibody, the presence of
2AR-GFP in the immunoprecipitate was probed using a
rabbit anti-GFP antibody. Two major species corresponding to monomeric
and oligomeric forms of the 2AR were detected in both
HA-2AR/2AR-GFP- and
HA-1AR/2AR-GFP-expressing cells. In
contrast, no specific GFP immunoreactivity was detected at the cell
surface or in the total extract of cells co-expressing the unrelated
HA-tagged GABAB-R2 receptor and the 2AR-GFP
(Fig. 1, lanes 7 and 8), confirming
the selectivity of the immunoprecipitation approach. Immunoreactivity
for the HA-GABAB-R2 was, however, detected in the anti-HA
immunoprecipitate (data not shown), confirming that this receptor was
efficiently expressed and immunoprecipitated. Taken together, these
results confirm, as previously reported (25-27), that the
2AR and 1AR can form homodimers and
indicate that stable and selective intermolecular interactions can also occur between the two receptors as well. The higher molecular weight
species detected in Western analyses correspond to SDS-resistant complexes that may either represent dimers or possibly higher order
oligomers such as trimers that were not fully denatured by SDS (25).
The low resolution in this region of the gels and the possibility of
aberrant mobility make it difficult to unambiguously distinguish
between these possibilities. For the sake of simplicity, we will
therefore refer to stable receptor-receptor complexes as dimers.
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Fig. 1.
Immunodetection of co-immunoprecipitated
1AR/2AR.
72 h after transfection, HEK 293 cells were harvested, and
immunoprecipitations were carried out on cell surface (S) or
total extracts (T) in nontransfected cells (lanes
1 and 2) and in cells transiently co-transfected
with HA-2AR and 2AR-GFP (lanes
3 and 4), HA-1AR and
2AR-GFP (lanes 5 and
6), and HA-GABAB-R2 and 2AR-GFP
(lanes 7 and 8). HA-tagged receptors
were immunoprecipitated with a mouse monoclonal anti-HA (12CA5)
antibody. Immunoreactive bands were revealed using a rabbit polyclonal
anti-GFP antibody. Immunocomplexes were analyzed by SDS-PAGE (10%).
Western blots are representative of at least three independent
experiments. Arrow, high molecular weight oligomeric
species; open circle, fully glycosylated forms of the
2AR-GFP. IP, immunoprecipitation;
IB, immunoblot.
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1- and 2-Adrenergic Receptors Form
Homo- and Heterodimers in Living Cells--
Although
co-immunoprecipitation is a generally accepted method to document
protein-protein interactions, the interpretation of these experiments
for hydrophobic membrane proteins such as receptors is complicated by
detergent solubilization that could promote artifactual aggregation. To
assess whether 1AR/2AR heterodimers could
be detected in living cells, BRET was used. This technique is a
proximity assay based on the nonradiative transfer of energy between a
bioluminescent donor (Rluc) and a fluorescent acceptor (GFP)
that allows real time monitoring of protein-protein interaction in
living cells (26, 43). BRET has been used to complement biochemical
approaches to studying receptor/receptor interactions for
2AR (26), 
-opioid (17), and thyrotropin-releasing
hormone receptor (44) homodimerization. In these experiments, we have used a slight modification of the previously published assay. The new
BRET2 technology (BioSignal Packard Bioscience)
takes advantage of the spectral properties of a distinct luciferase
substrate known as Deep Blue coelenterazine (Deep Blue C), which
permits a better separation between Rluc and GFP emission spectra (see
"Experimental Procedures").
To assess 2AR and 1AR homo- and
heterodimerization, fusion constructs linking the receptor
carboxyl-terminal tails to either Rluc or GFP were
co-transfected in HEK 293 cells, and the transfer of energy between the
two partners was assessed following the addition of Deep Blue C. Upon
oxidation of the luciferase substrate, the enzyme emits light with a
peak at 400 nm that can excite GFP, which, in turn, re-emits
fluorescence with a peak at 510 nm but only if the two partners are
within the permissive distance (<100 Å). The BRET signal is
determined by calculating the ratio of the light emitted by the
receptor-GFP (500-530 nm) over the light emitted by the
receptor-Rluc (370-450 nm).
Fig. 2 presents the BRET level measured
for the different partners considered. A strong BRET signal was
detected for both 1AR and 2AR homodimers,
and a smaller but significant BRET was also observed in cells
expressing 1AR-Rluc/2AR-GFP or
2AR-Rluc/1AR-GFP, indicating
that heterodimers between the receptor subtypes also form in living
cells. No significant BRET was detected when either 1AR-Rluc or
2AR-Rluc was co-expressed with soluble GFP
expressed at a similar fluorescence level as 1AR- or
2AR-GFP, confirming the selectivity of the detected
signals. The small difference between the BRET signals observed for the
homo- and heterodimers does not necessarily reflect a smaller number of
heterodimers, since the level of BRET does not depend strictly on the
number of dimers formed but also on the relative orientation and
distance between the energy donor and acceptor within the dimers. It
follows that differences in the structural organization of the dimers could account for the different BRET levels. No significant changes were detected in the measured BRET levels when the cells were stimulated with 10 µM isoproterenol (data not shown).
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Fig. 2.
Homo- and heterodimers exist in living
cells. HEK 293 cells were transiently co-transfected with
1AR-Rluc or
2AR-Rluc in combination with
1AR-GFP, 2AR-GFP, or the soluble GFP
(sGFP). For each pair considered, the quantities of DNA used
for 1AR-Rluc and
2AR-Rluc were selected to yield equivalent
luminescent signals (370-450 nm), whereas those for
1AR-GFP and 2AR-GFP were selected to
obtain the maximum BRET levels. The BRET level for the soluble GFP was
measured at a fluorescence level equivalent to those of
1AR-GFP and 2AR-GFP. Cells were harvested
48 h post-transfection, counted, and transferred to 96-well plates
(100,000 cells/well). The energy transfer reaction was initiated by
adding 5 µM Deep Blue C coelenterazine to each well, and
BRET was assessed in a BRETCount microplate reader with the filter
settings described under "Experimental Procedures." The results
represent mean ± S.E. of 3-6 independent experiments performed
in triplicate.
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Functional Characterization of
1AR/2AR Interactions--
We
next determined the functional consequences of
1AR/2AR heterodimerization by measuring
receptor-mediated stimulation of several effector pathways. For this
purpose, cells were transfected with the 1AR,
2AR, or both receptors together. Transfection conditions
were such that an equivalent total number of receptors were expressed
in each case (1AR, 2.0 ± 0.7; 2AR,
1.8 ± 0.5; 1AR + 2AR, 1.5 ± 0.6 pmol/mg). For cells expressing the two receptor subtypes,
competition radioligand binding with the selective antagonists
betaxolol and ICI-118,551 revealed equivalent proportions of
1AR and 2AR. In a first series of
experiments, the ability of the nonselective -adrenergic agonist,
isoproterenol, to stimulate the adenylyl cyclase was tested. In the
absence of transfected receptor, isoproterenol promoted a modest
concentration-dependent increase in cAMP production that
most likely reflects the presence of endogenously expressed
2AR (~0.01 pmol/mg) in HEK 293 cells (Fig.
3). Transfection of the
1AR, 2AR, or both receptors together led
to similar increases in the agonist-stimulated adenylyl cyclase activity. EC50 and maximal stimulated values were 60 ± 24 nM and 20.6 ± 3.3 pmol/min·mg for
1AR (n = 4), 48 ± 33 nM and 21.6 ± 1.4 pmol/min·mg for
2AR (n = 4), and 128 ± 54 nM and 25.2 ± 4.7 pmol/min·mg for
1AR + 2AR (n = 7),
respectively. In mock-transfected cells, the EC50 and
maximal stimulated values were 171 ± 179 nM and
8.1 ± 5.5 pmol/min·mg (n = 3). No differences
in the ability of NaF or forskolin to stimulate adenylyl cyclase were
detected whether the receptors were expressed alone or together (data
not shown). Since no significant difference in either the efficacy or the potency of isoproterenol was detected, we conclude
that 1) the two receptor subtypes had similar abilities to couple with the adenylyl cyclase pathway and 2) that the formation of
1AR/2AR heterodimers had no effect on
this signaling pathway (Fig. 3).
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Fig. 3.
Stimulation of adenylyl cyclase by
1AR,
2AR, and
1AR/2AR.
HEK 293 cells expressing 1AR, 2AR, or the
two receptors together were assayed for adenylyl cyclase activity.
Expression of both receptors was confirmed by ligand binding and were
as follows ± S.D.: 1AR, 2 ± 0.7 pmol/mg
(n = 6); 2AR, 1.8 ± 0.5 pmol/mg
(n = 7); and 1AR/2AR,
1.5 ± 0.6 pmol/mg (n = 7). The ratio of
1AR to 2AR in HEK 293 cells expressing
both receptors was ~1:1, as determined using 2- or
1-selective ligand ICI 118,551 or betaxolol
(1AR, 51.2 ± 3.2%; 2AR, 48.7 ± 3.2). The level of endogenous 2AR in HEK 293 cells
was 0.01 ± 0.01 pmol/mg (n = 3). Membranes
were prepared from HEK 293 cells as described under "Experimental
Procedures." Control cells expressing GFP vector (Mock)
alone displayed a markedly lower stimulation (presumably due to the low
levels of endogenous 2AR in these cells) of adenylyl
cyclase under the same conditions used for receptor-transfected cells
(data not shown). Data were normalized to forskolin stimulation and
represent mean ± S.E. for 1AR (n = 4), 2AR (n = 4),
1AR/2AR (n = 7), and mock
(n = 3).
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The efficacy of each receptor subtype expressed individually or
together to stimulate the MAPKs p38 and ERK1/2 was then assessed. p38
was modestly activated by the 1AR, and this was not
altered by co-expression with the 2AR (Fig.
4E). In HEK 293 cells, we could not detect a consistent stimulation of p38 by the
2AR alone. In contrast, a dramatic difference was
observed between 1AR and 2AR in their
ability to promote phosphorylation of ERK1/2. Indeed, as seen by other
groups, agonist stimulation of the 2AR results in a
consistent activation of ERK1/2, which peaked at 5 min and returned to
almost basal levels by 30 min (Figs. 4A and
5A). However, no increase in
the phosphorylation of ERK1/2 could be detected upon isoproterenol
treatment in cells expressing the 1AR (Fig. 4B). This difference did not result from aberrant ERK1/2
activities in the 1AR-expressing cells, since the
phorbol ester-stimulated ERK1/2 phosphorylation was normal in these
cells. Interestingly, in a similar fashion to what was observed for the
1AR-expressing cells, no -adrenergic-stimulated
ERK1/2 activity could be detected in cells co-expressing both
1AR and 2AR (Fig. 4C). For
both the 1AR alone and 1AR and
2AR together, no stimulation was seen after 30 min of
agonist stimulation (Fig. 5B), demonstrating that the loss
of ERK1/2 stimulation shown at 5 min was not simply due to changes in
the time course of receptor activation of the ERK pathway (Fig. 5). In
fact, we could not detect changes in ERK1/2 activation even after
2 h of agonist stimulation when the two receptors were expressed
together (data not shown). Given that the two receptors were expressed
at equivalent levels (~52% 1AR), these results
suggest that heterodimerization inhibited the
2AR-promoted ERK1/2 activation. Data from several
experiments are summarized in Fig. 4D. To confirm that
expression of the 1AR by itself did not reduce levels of
2AR in the transient co-transfections, we also
transfected 1AR into a stable cell line expressing the 2AR. As determined by confocal microscopy, the
transfection efficiency for the 1AR into the
2AR stable cell line was always between 60 and 70% in
these experiments. As shown in Fig. 5, a similar loss of
isoproterenol-stimulated ERK1/2 activity was also seen upon
co-expression of the two receptor subtypes. Again, no stimulation was
seen when the 1AR/2AR were co-expressed
at any of the times measured. These data are summarized in Fig.
5C.
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Fig. 4.
Stimulation of ERK1/2 phosphorylation by
1AR,
2AR, and
1AR/2AR.
ERK1/2 activation is shown in the upper part of
each panel (as measured by anti-phospho-ERK1/2 antibodies)
in response to stimulation by 10 µM isoproterenol for 5 min of serum-starved HEK 293 cells transiently transfected with
2AR (A), 1AR (B),
or the two receptors together (C). As a loading control, the
total ERK1/2 pool in the lysate was measured using anti-ERK1/2
antibodies, and the anti-phospho-ERK data are normalized with respect
to the total ERK pool. Expression of both receptors was confirmed by
ligand binding, Western blotting, and immunocytochemistry. 200 µg of
cell lysate was loaded into each lane. SDS-PAGE was followed by
immunoblot, developed with enhanced chemiluminescence. Representative
experiments are shown. D, p42/44 ERK data from separate
experiments were digitized on a flatbed scanner and analyzed using
Quantity One (Bio-Rad) software and are presented here as mean ± S.E. for 1AR (n = 4), 2AR
(n = 3), and 1AR/2AR
(n = 4). *, significant differences (p < 0.05 using Student's t test) between control and
isoproterenol-stimulated samples. E, representative example
(n = 3) of p38 MAPK activation (as measured using
anti-phospho-p38 antibodies) by 1AR (28 ± 10%
increase over basal), 2AR (20 ± 0.07%
decrease from basal), or the two receptors together (35 ± 20% increase over basal).
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Fig. 5.
Stimulation of ERK1/2 phosphorylation by
stably transfected 2AR in absence
and presence of transiently co-transfected
1AR. ERK1/2 activation is shown in
the upper part of each panel (as
measured by anti-phospho-ERK1/2 antibodies) in response to stimulation
by 10 µM isoproterenol for various times of serum-starved
HEK 293 cells stably transfected with 2AR in the absence
(A) and presence (B) of transiently
co-transfected 1AR. Expression of both receptors was
confirmed by ligand binding, Western blotting, and immunocytochemistry.
Estimates using confocal microscopy of transfection efficiency for
transiently expressed 1AR were between 60 and 70% using
our procedures. 200 µg of cell lysate was loaded into each lane.
SDS-PAGE was followed by immunoblot, developed with enhanced
chemiluminescence. Representative experiments are shown and were
repeated at least three times. C, p42/44 ERK data from
separate experiments were digitized on a flatbed scanner and analyzed
using Quantity One (Bio-Rad) software and are presented here as
mean ± S.E. for 2AR (n = 5) and
1AR/2AR (n = 5). *,
significant differences (p < 0.07 using Student's
t test) between control and isoproterenol-stimulated
samples.
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Given the distinct internalization profiles previously reported for the
two receptors, the consequences of heterodimerization on the
internalization of each receptor were then assessed. Confocal immunocytochemistry analysis revealed that, under control conditions, both receptors are found primarily at the plasma membrane (Fig. 6, a and b).
Stimulation of the 2AR expressing cells with
isoproterenol led to a rapid internalization indicated by the
disappearance of receptor from the plasma membrane that could be
detected as early as 5 min poststimulation (Fig. 6A,
top panel). For 1AR-expressing cells, the same treatment had no detectable effect on the expression of
the receptor at the cell surface (Fig. 6B, top
panel). No significant internalization of the
1AR was detected even following a 2-h treatment with the
agonist, confirming earlier studies that indicated that the
1AR is resistant to agonist-promoted internalization (34-36). In cells co-expressing the two receptor subtypes,
isoproterenol stimulation failed to promote significant internalization
of either the 1AR or the 2AR (Fig. 6,
A and B, bottom panels),
indicating that expression of the internalization-resistant
1AR inhibited the internalization of the
2AR. We also performed colocalization experiments in
cells transiently transfected with both 1AR and 2AR-GFP. Under control conditions, both receptors are
expressed at the cell surface, where they colocalize (Fig.
7A). As described above for
the untagged 2AR, isoproterenol-promoted internalization was readily observed in cells only or predominantly expressing 2AR-GFP (Fig. 7, B and C). In
contrast, no such internalization of 2AR-GFP was
observed in those cells co-expressing significant amounts of the
1AR (Fig. 7, B and C). The modest
2AR-GFP internalization, seen in some of cells
co-expressing both receptors, most likely reflects internalization of
2AR homodimers.
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Fig. 6.
Distribution and trafficking of
1AR or
2AR when expressed alone or as part of
a putative heterodimer. Confocal images were taken from fixed
and permeabilized HEK 293 cells expressing 1AR,
2AR, or the two receptors together after 0, 5, and 120 min of stimulation with 1 µM isoproterenol. A,
distribution of 2AR when the receptor is expressed alone
(top panel) or with the 1AR
(bottom panel). B, distribution of
1AR when the receptor is expressed alone (top
panel) or with the 2AR (bottom
panel). These images are representative of three separate
experiments. In addition to confocal microscopy, we also confirmed the
presence of both receptors by Western blotting (data not shown) to
assure that both receptors are represented in the double
transfections.
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Fig. 7.
Colocalization of transiently
co-transfected 1AR and
2AR-GFP in HEK 293 cells in response to
agonist stimulation. Confocal images were taken from fixed and
permeabilized cells expressing 2AR-GFP and
1AR before (A) and after (B and
C) stimulation for various times with 10 µM
isoproterenol. Images of GFP fluorescence and staining with
CY5-conjugated secondary antibody following labeling with
anti-1AR. Shown in order from top to
bottom are anti-1AR signals, GFP signals, and
the overlay. A representative image is shown for unstimulated cells
(A), which demonstrates cell surface colocalization of both
receptors, and cells 5 min (B) and 120 min (C)
after stimulation with 10 µM isoproterenol. Note that, in
cells expressing both receptors, receptors remain colocalized at the
cell surface (red arrows), whereas cells
expressing only or predominantly 2AR show rapid receptor
internalization (yellow arrows).
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To quantify the proportion of 2AR internalized in the
presence and absence of the 1AR, we took two separate
approaches using transient transfections of the 2AR in
the absence or presence of co-transfected 1AR. First,
using the hydrophilic ligand CGP, we determined that 54.5 ± 4.5%
of 2AR (as measured in the presence of 5 × 108 M betaxolol to isolate the
2AR signal) remained at the surface after 2 h of
agonist stimulation (Fig. 8A).
In the presence of the 1AR, the amount of
2AR that remained at the cell surface after 2 h of
agonist stimulation was 108 ± 25% confirming our observations
made using confocal microscopy. No changes were seen in the total
numbers of receptors under these conditions as determined with CYP
(data not shown). To further verify these results, we also measured
cell surface expression of extracellularly tagged (N terminus)
2AR using an enzyme-linked immunosorbent assay. Again in
the presence of the 1AR, the amount of
2AR internalized is markedly reduced. When the
2AR is expressed alone, 52 ± 10% of
c-myc-tagged receptors remain at the surface after 60 min of 10 µM isoproterenol in comparison with 84 ± 6.6%
when the 1AR is present (Fig. 8B). Taken
together, these results demonstrate that 2AR trafficking
is altered in the presence of the 1AR.
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Fig. 8.
Quantification of
2AR internalization in the absence and
presence of co-transfected
1AR. A, loss of cell
surface (CGP 12177) binding to 2AR after 120 min of
stimulation at 37 °C with 10 µM isoproterenol was
determined in the presence of 5 × 108 M
betaxolol to isolate the 2AR signal. Shown are mean ± S.E. for percentage of cell surface 2AR remaining
after agonist stimulation in the presence and absence of co-expressed
1AR (n = 3 for both conditions). Data
were compared with cell surface binding in unstimulated cells, which
was normalized to a value of 100%. Samples were controlled for changes
in total receptor binding as determined with CYP. B,
enzyme-linked immunosorbent assay to measure cell surface
2AR. Shown are mean ± S.E. for percentage of
N-terminally tagged myc-2AR-Rluc
receptors remaining at the cell surface after 60 min of agonist
stimulation at 37 °C in the presence and absence of co-expressed
1AR-GFP10 (n = 3 for both conditions).
Data are shown relative to cells stimulated with vehicle. Total AR
expression levels in all of these experiments were between 1 and 3 pmol/mg of membrane protein. At these levels, the 2AR
internalizes efficiently when expressed alone, suggesting that our
results with co-expression of the 1AR are not simply due
to saturation of the internalization mechanism. *, significant
differences (p < 0.05 using Student's t
test) between 2AR expressed alone and
1AR/2AR co-expressed together.
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DISCUSSION |
We have demonstrated that, in addition to forming homodimers,
1AR and 2AR can also form heterodimers
when co-expressed in HEK 293 cells. These interactions were shown both
by co-immunoprecipitation and BRET assays in living cells. Cell surface
co-immunoprecipitation experiments revealed that the heterodimer was
expressed at the plasma membrane. Further, an analysis of the
processing of receptors, as assessed by their sensitivity to
endoglycosidase H or peptide N-glycosidase F (data
not shown) indicated that fully mature receptors were forming dimers.
We have demonstrated that co-expression of the two receptors leads to
altered functional properties in the putative heterodimer, suggesting
that the 1AR and 2AR interact as a
heterodimer to yield a novel AR subtype with unique functional
properties. This is manifested by the complete loss of ERK1/2 MAPK
stimulation by isoproterenol when the two receptors were co-expressed.
Given that the 1AR, when expressed alone, was also
incapable of stimulating ERK1/2, our results suggest that the
heterodimers display a coupling pattern characteristic of the
1AR. Previous studies have also demonstrated that the
1AR is less effective than 2AR in
stimulating ERK1/2 MAPK (32, 33). To our knowledge, however, this is
the first report indicating that cells co-expressing the two receptors may have blunted ERK1/2 responses to -adrenergic stimulation.
Both the 1AR and 2AR are expressed in the
cardiovascular system, and more particularly, both receptors are
co-expressed in cardiomyocytes. One potential consequence of this
cross-talk regulation of the ERK1/2 pathway through receptor
heterodimerization may manifest itself in the modulation of signaling
by -adrenergic stimulation. For example, recent reports have
demonstrated that phospholipase A2 translocation to the
plasma membrane, stimulated by the 2AR, is an important
mediator of 2AR-mediated positive inotropy in
embryonic chick cardiomyocytes (45, 46). In these cells,
2AR stimulation leads to arachidonic acid release via phospholipase A2, resulting in an increased release of
calcium from sarcoplasmic reticulum stores. Further, these authors have also demonstrated that this 2AR response is
ERK1/2-dependent. These effects of 2AR
stimulation on intracellular free calcium and positive inotropy in
embryonic chick heart myocytes might be masked by activation of
1AR, and this possibility was considered by the authors
of the studies cited above (46). It is likely that this type of
regulation may also occur in mammalian cardiomyocytes. It has been
demonstrated that AR stimulation of ERK1/2 may play a role in the
development of cardiac hypertrophy (47). Up-regulation of a
2AR-specific signal may also be important when one
considers the proapoptotic effects of 1AR stimulation
and the antiapoptotic effects of 2AR stimulation on the
myocardium (32, 48). This may be particularly important during the
progression to heart failure when the 1AR is selectively
down-regulated, potentially unmasking and increasing the impact of
signaling pathways specific to the 2AR.
Several lines of evidence derived from transgenic and knockout mice
also support the functional importance of
1AR/2AR heterodimerization. For example,
high levels of 2AR overexpression led to an impairment of cardiac 1AR function, which is relieved using
2AR inverse agonists such as ICI 118,551 (49). Also,
responses to 2-specific agonist stimulation were lost in
mice with homozygous deletion of the 1AR, although
cardiac 2AR number and distribution remained similar to
wild type animals (37).2
Taken together, these data are consistent with the notion that each
receptor in some way depends on the other for function.
The fact that 1AR and 2AR had similar
abilities to modulate adenylyl cyclase precludes determination of
whether or not heterodimerization affects this pathway. Previous
studies had suggested that the 1AR had a reduced
efficacy to stimulate adenylyl cyclase when compared with the
2AR (29-31). It was later demonstrated that this
decreased efficacy was characteristic of a particular
1AR variant (glycine at position 389), whereas the most
common polymorphism (Arg389) has similar efficacy as
the 2AR (38). Since we were using the Arg389
1AR variant, it is not surprising that
1AR and 2AR had similar abilities to
stimulate adenylyl cyclase. In the case of p38 MAPK, we again
demonstrate that the 1AR phenotype dominates over the 2AR when the two receptors are co-expressed in HEK 293 cells, although -adrenergic stimulation of p38 MAPK is much more
modest than for ERK1/2 MAPK. We could not detect stimulation of p38
MAPK via the 2AR alone. This may not be the case for all
cell types, since stimulation of both 1AR and
2AR were shown to be coupled to the activation of p38
MAPK in adult murine cardiomyocytes (50, 51).
We have demonstrated that the loss of p42/44 ERK MAPK stimulation in
the heterodimer is accompanied by a reduced ability for the receptor to
be internalized. A number of previous studies have demonstrated that
internalization of the 2AR is required for these distal
signaling events to occur (16, 52-54). Further experiments will be
needed to determine whether the presence of the 1AR in
the putative heterodimer is directly responsible for the alteration in
receptor trafficking and ERK activation. Another recent study has shown
that a 2AR/
-opioid receptor heterodimer was also
defective for both p42/44 ERK MAPK activation and agonist-mediated receptor internalization (16). These authors also showed that a
2AR/-opioid receptor heterodimer that could be
internalized could still stimulate the ERK pathway. Taken together,
these results suggest that novel heterodimeric receptors can have
altered trafficking itineraries, which have functional consequences.
Several studies have demonstrated that 2AR-stimulated
ERK1/2 activity requires the recruitment of -arrestin, which acts as
a scaffolding protein to assemble the ERK1/2 signaling cascades (see
Ref. 55 for a review). The fact that the 1AR has been shown to recruit -arrestin much less efficiently than the
2AR (35, 36) may provide a mechanistic explanation for
the inhibitory effect of receptor heterodimerization on ERK1/2
activation. The heterodimer may therefore also have a reduced ability
to promote the assembly of the ERK1/2 signaling complex.
Although co-expression of the 1AR completely inhibited
2AR-stimulated ERK1/2 MAPK activity in HEK 293 cells,
2AR stimulation does result in MAPK activation in
cardiac tissue and in isolated cardiomyocytes. Given that these cells
express both 1AR and 2AR, there must be a
mechanism of sequestering the 2AR away from the 1AR. One can suppose that there ar
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ABSTRACT
INTRODUCTION
