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INTRODUCTION |
The physiological actions of epinephrine and norepinephrine are
mediated via the activation of the following three distinct classes of
G protein-coupled receptors
(GPCR)1:
1-,
2-, and
-adrenergic receptors. Each class of
adrenergic receptor (AR) is comprised of three closely related subtypes
as follows:
1A-,
1B-, and
1DAR, which couple primarily to Gq to stimulate phospholipase activity;
2A-,
2B-, and
2CAR, which couple primarily to
Gi to inhibit adenylyl cyclase activity; and
1-,
2-, and
3AR, which
couple primarily to Gs to stimulate adenylyl cyclase
activity (1). The adrenergic receptor subtypes are differentially
distributed across various tissues, and tissue responses to epinephrine
and norepinephrine are believed to be dependent upon the relative
ratios of the various adrenergic receptors they express.
Because
- and
2-adrenergic receptors couple to G
proteins with opposing actions on adenylyl cyclase activity, the two
receptors might be expected to purely antagonize each other's
signaling when they are co-stimulated in the same cell. However, it has been shown that
2AR co-stimulation can in some cases
paradoxically sensitize
-adrenergic signaling in brain tissue
(2-4). Moreover, the pharmacological properties of
ARs in brain
tissue are known to be regulated by
2ARs (5, 6), and
reciprocally the pharmacological properties of
2ARs in
brain tissue are known to regulated by
ARs (7, 8). These examples of
cross-talk and mutual regulation between
- and
2-adrenergic receptors have been well known for more
than 20 years, but the underlying molecular mechanisms remain unclear.
GPCRs have traditionally been thought to exist as monomers, but recent
studies (9) have revealed that they can exist in the plasma membrane as
both homodimers and heterodimers. At present, a key question in this
field is: how widespread is the phenomenon of receptor
heterodimerization? The most clear-cut case of the importance of GPCR
heterodimerization comes from the GABAB receptor, a
pharmacologically defined entity that is now known to be comprised of
two distinct GPCRs, GABABR1 and GABABR2 (10).
Because GABABR1 and GABABR2 are not functional
when expressed by themselves, they represent a clear example of the
physiological importance of receptor heterodimerization. Although other
heptahelical receptors may not absolutely require heterodimerization to
be functional in the same way that the GABAB receptor does,
heterodimerization of other receptors may underlie some phenomena that
are major question marks in our present understanding of
neurotransmitter and hormone receptors, such as unexplained forms of
cross-talk between different receptor subtypes.
We wondered if the previously reported cross-talk between
ARs and
2ARs in brain tissue might be due in part to a physical association between these two receptor types. Many early studies (11-13) of GPCR dimerization focused on the
2AR. We
have found recently (14) that the
1AR also exhibits
robust homodimerization in cells. Furthermore, it has been shown
recently (15) that
1AR and
2AR can
heterodimerize in a functionally important manner.
1AR
is the most abundantly expressed
AR in brain (16, 17), a tissue
where
2ARs are found at particularly high levels (18). The most widely expressed
2AR subtype,
2AAR, is known to be localized both pre- and
post-synaptically in a number of brain regions (18), where its pattern
of expression overlaps significantly with that of the
1AR (17). Based on the previously reported functional
interactions between
2ARs and
ARs, as well as the overlapping distribution patterns of
2AAR and
1AR, we examined the possibility that
1AR
might be able to heterodimerize with
2AAR. Our findings
reveal that
1AR and
2AAR robustly
associate in cells and that
2AAR can regulate
1AR internalization and ligand binding.
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MATERIALS AND METHODS |
Plasmids--
FLAG-
1AR was kindly provided by
Robert J. Lefkowitz (Duke University). HA-
2AAR was
kindly provided by Lee Limbird (Vanderbilt University Medical Center).
HA-
1AR was kindly provided by Hitoshi Kurose (University
of Tokyo). The N15A mutant
1AR was prepared via PCR
amplification from the native human
1AR cDNA using a mutant sequence oligonucleotide (CTG GGC GCC TCC GAG CCC GGT
GCC CTG TCG TCG GCC GCA CCG CTC). The N10A/N14A mutant
2AAR was also prepared via PCR amplification from the
wild-type construct in a two-step process, first using a mutant
sequence oligonucleotide (CC CTG CAG CCG GAA GCG GGC GCC
GCG AGC TGG AAT GGG ACA GAG G) to make the N10A mutation, and second
using a second oligonucleotide (GCG GGC GCC GCG AGC TGG
GCT GGG ACA GAG GCG CCG GGG GGC) to make the N14A mutation
using the N10A mutant construct as a template. The point mutations were
confirmed by ABI sequencing.
Cell Culture and Transfection--
All tissue culture media and
related reagents were purchased from Invitrogen. HEK-293 cells were
maintained in complete medium (Dulbecco's modified Eagle's medium
plus 10% fetal bovine serum and 1% penicillin/streptomycin) in a
37 °C, 5% CO2 incubator. For heterologous expression of
receptors, 2 µg of DNA was mixed with LipofectAMINE (15 µl) and
Plus reagent (20 µl) (from Invitrogen) and added to 5 ml of
serum-free medium in 10-cm tissue cultures plates containing cells at
~50-80% confluency. Following a 4-h incubation, the medium was
removed, and 10 ml of fresh complete medium was added. After another
12-16 h, the medium was changed again, and the cells were harvested
24 h later.
Western Blotting--
Samples (5 µg per lane) were run on
4-20% SDS-PAGE gels (Invitrogen) for 1 h at 150 V and then
transferred to nitrocellulose. The blots were blocked in "blot
buffer" (2% non-fat dry milk, 0.1% Tween 20, 50 mM
NaCl, 10 mM Hepes, pH 7.4) for at least 30 min and then
incubated with primary antibody in blot buffer for 1 h at room
temperature. The primary antibodies utilized were either a 12CA5
monoclonal anti-HA antibody (Roche Molecular Biochemicals) or an M2
monoclonal anti-FLAG antibody (Sigma). The blots were then washed three
times with 10 ml of blot buffer and incubated for 1 h at room
temperature with a horseradish peroxidase-conjugated secondary antibody
(Amersham Biosciences) in blot buffer. Finally, the blots were washed
three more times with 10 ml of blot buffer and visualized via
enzyme-linked chemiluminescence using the ECL kit from Amersham Biosciences.
Immunoprecipitation--
Cells were harvested and lysed in 500 µl of ice-cold lysis buffer (10 mM Hepes, 50 mM NaCl, 0.5% Triton X-100, 5 mM EDTA, and the
protease inhibitor mixture from Roche Molecular Biochemicals). The
lysate was solubilized via end-over-end rotation at 4 °C for 30 min
and clarified via centrifugation at 14,000 rpm for 15 min. A small
fraction of the supernatant was taken at this point and incubated with
SDS-PAGE sample buffer in order to examine expression of proteins in
the whole cell extract. The remaining supernatant was incubated with 30 µl of beads covalently linked to anti-FLAG antibodies (Sigma) for
2 h with end-over-end rotation at 4 °C. After five washes with
1.0 ml of lysis buffer, the immunoprecipitated proteins were eluted
from the beads with 1× SDS-PAGE sample buffer, resolved by SDS-PAGE,
and subjected to Western blot analyses.
Enzymatic Deglycosylation--
For enzymatic deglycosylation of
receptors, immunoprecipitates were separated from beads by boiling for
10 min in a denaturing buffer (0.5% SDS containing 1%
-mercaptoethanol). After cooling, Nonidet P-40 was added to the
supernatants to a final concentration of 1%, and
Na2HPO4/NaH2PO4 buffer
(pH 7.5) was added to lysates to a final concentration of 50 mM. N-glycosidase F (1,500 units; New England
Biolabs) was added to a 30-µl reaction volume, and the sample was
incubated for 1 h at 37 °C.
Cyclic AMP Assay--
Intracellular cAMP was measured by using a
non-acetylation cAMP enzyme immunoassay kit (Amersham Biosciences).
Briefly, cultured cells were transfected with either
FLAG-
1AR alone or
FLAG-
1AR/HA-
2AAR in combination. After
24 h, cells were split into 6-well culture dishes with fresh
medium. After another 48 h, cells were treated with varying
concentrations of isoproterenol for 10 min and harvested with cell
harvest buffer (50 mM Tris, pH 7.4, 250 µM Ro
20-1724 (Tocris, Ellisville, NJ), 5 mM MgCl2, 1 mM ATP, and 1 µM GTP). Cell lysates were
sonicated, transferred to a 96-well assay plate coated with anti-rabbit
IgG, and incubated with an anti-cAMP antibody at 4 °C for 2 h
along with a series of cAMP standards. A cAMP-peroxidase conjugate was
then added to the microtiter plate and incubated at 4 °C for 1 h. The plate was then washed four times with 400 µl of wash buffer,
and the wells were incubated with 150 µl of enzyme substrate at room
temperature for 1 h. When the samples were within the linear range
of the standards, the reaction was stopped by adding 100 µl of 1.0 M sulfuric acid. Absorbance was determined in a
plate reader at 450 nm, and cAMP levels were determined using standard curves.
Surface Expression Assay--
Transfected cells were split into
35-mm dishes, grown for 48 h, and then incubated in the absence
and presence of agonist for 10 min. The cells were then rinsed in PBS
and fixed with 4% paraformaldehyde in PBS for 30 min and then rinsed
three times in PBS and blocked with blocking buffer (2% non-fat dry
milk in PBS, pH 7.4) for 30 min. The fixed cells were then incubated
with primary antibody in blocking buffer for 1 h at room
temperature. The dishes were subsequently washed three times with 2 ml
of block buffer and incubated for 1 h at room temperature with
horseradish peroxidase-conjugated secondary antibody (Amersham
Biosciences) in blocking buffer. Finally, the dishes were washed three
times with 2 ml of blocking buffer and one time with 2 ml of PBS and then incubated with 2 ml of ECL reagent (Pierce) for exactly 15 s.
The luminescence, which corresponds to the amount of receptor on the
cell surface, was determined by placing the plate inside a TD 20/20
luminometer (Turner Designs).
Ligand Binding Assays--
For preparation of membranes to be
used in ligand binding assays, transfected cells grown on 100-mm dishes
were rinsed twice with 10 ml of PBS and then scraped into 1 ml of
ice-cold binding buffer (10 mM Hepes, 1 mM
MgCl2, 1 mM ascorbic acid, pH 7.4). Cells were
then washed three times with 1 ml of binding buffer, sonicated for
10 s, and resuspended in fresh binding buffer for use in
radioligand binding assays. Membranes were incubated with increasing
concentrations of [3H]DHA or [3H]RX821002
in binding buffer for saturation binding studies, or with 1 nM [3H]DHA or [3H]RX821002 in
binding buffer in the absence or presence of various unlabeled ligands
to generate inhibition curves. The samples were incubated for 15 min at
37 °C. Nonspecific binding was defined as [3H]DHA or
[3H]RX821002 binding in the presence of either 1 mM isoproterenol or 1 mM clonidine,
respectively, and represented less than 10% of total binding in all
experiments. Incubations were terminated via filtration through GF/C
filter paper using a Brandel cell harvester. Filters were rapidly
washed three times with ice-cold wash buffer (10 mM Hepes),
and radioactive ligand retained by the filters was quantified via
liquid scintillation counting. The fitting of curves for one site
versus two sites was performed using Prism software
(GraphPad, San Diego, CA). Goodness of fit was quantified using
F tests, comparing sum-of-squares values for the one-site
versus two-site fits.
Immunofluorescence Microscopy--
HEK-293 cells were
transiently transfected with pcDNA3/FLAG-
1AR and
pcDNA3/HA-
2AAR. Forty eight hours after
transfection, cells were washed three times with Dulbecco's PBS and
then incubated for 10 min at 37 °C in the absence or presence of 10 µM isoproterenol or 10 µM UK 14,304 (Sigma). Following this incubation, cells were fixed in 4%
paraformaldehyde in PBS for 30 min at room temperature. To visualize
the subcellular localization of
1AR and
2AAR, cells were blocked and permeabilized with a buffer
containing 2% bovine serum albumin and 0.04% saponin in PBS
("saponin buffer") for 30 min at room temperature. The cells were
then incubated with anti-
1AR polyclonal antibody (Santa
Cruz Biotechnology) at 1:500 dilution and anti-HA monoclonal antibody
(12CA5; Roche Molecular Biochemicals) at 1:1000 dilution for 1 h
at room temperature. After three washes (1 min) with saponin buffer,
the cells were incubated with a rhodamine red-conjugated anti-rabbit
IgG at 1:200 dilution and FITC-conjugated anti-mouse IgG at 1:200
dilution (Jackson ImmunoResearch Laboratories) for 1 h at room
temperature. After three washes (1 min) with saponin buffer and one
wash with PBS, coverslips were mounted, and rhodamine red-labeled
1AR and FITC-labeled
2AAR were visualized
with a Zeiss LSM-410 laser confocal microscope. Multiple control
experiments, utilizing either transfected cells in the absence of
primary antibody or untransfected cells in the presence of primary
antibody, revealed a very low level of background staining, indicating
that the primary antibody-dependent immunostaining observed
in the transfected cells was specific.
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RESULTS |
Co-immunoprecipitation of
2A- and
-Adrenergic
Receptors--
To assess the potential physical association of
2A- and
-adrenergic receptors, HA-
2AAR
was expressed in HEK-293 cells either alone or in combination with
FLAG-
1AR or FLAG-
2AR. As shown in Fig.
1, Western blotting for
HA-
2AAR in cell lysates revealed multiple bands, with
major species at ~65 and 120 kDa. The higher order bands presumably
represent receptor complexes resistant to separation by SDS-PAGE, as is
commonly observed for many GPCRs (9). When the FLAG-tagged
ARs were
immunoprecipitated with an anti-FLAG antibody, the co-transfected
HA-
2AAR was robustly co-immunoprecipitated. All of the
bands of HA-
2AAR immunoreactivity were evident in
FLAG-
AR immunoprecipitates. Somewhat more co-immunoprecipitation was
observed with
1AR than with
2AR, and thus
further experiments in this area focused on the
1AR/
2AAR interaction. No changes in the
extent of co-immunoprecipitation were observed when cells were
stimulated before harvesting with various adrenergic receptor agonists
(data not shown). In related control experiments,
HA-
2AAR and FLAG-
1AR were transfected
separately into different plates of cells, which were harvested,
prepared as detergent-solubilized lysates, and then mixed together.
Immunoprecipitation of FLAG-
1AR in these experiments did
not yield any detectable co-immunoprecipitation of
HA-
2AAR (data not shown), revealing that the two
receptors need to be expressed in the same cell in order to physically
associate.

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Fig. 1.
Co-immunoprecipitation of
2A- and
1AR and
2AR. HEK-293 cells were
transfected with empty vector (lane 1),
HA- 2AAR alone (lane 2),
HA- 2AAR/FLAG- 1AR (lane 3), or
HA- 2AAR/FLAG- 2AR (lane 4). The
expression of HA- 2AAR in detergent-solubilized lysates
prepared from the transfected cells is shown in the 1st 4 lanes of the Western blot (IB) shown in this figure.
Several nonspecific bands were evident in untransfected cell lysates
(lane 1), whereas specific immunoreactivity for
HA- 2AAR (lanes 2-4) was observed as major
bands at ~65 and 120 kDa (arrows). The lysates were
incubated with anti-FLAG affinity agarose to immunoprecipitate the
FLAG-tagged -adrenergic receptors, and the resultant
immunoprecipitates (IP) were examined via Western blot for
anti-HA immunoreactivity. As shown in the last 2 lanes of
this figure, specific co-immunoprecipitation of HA- 2AAR
was observed with both FLAG- 1AR and
FLAG- 2AR. The positions of molecular mass standards are
indicated on the left side of the figure. This experiment
was repeated five times with nearly identical results.
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Co-internalization of
2A- and
1-Adrenergic Receptors--
As a second method of
assessing the physical association between
2AAR and
1AR, we expressed the two receptors in cells and studied
their co-internalization. Agonist stimulation of many GPCRs induces
significant internalization from the cell surface, and this process is
known to be important in the desensitization and resensitization of
GPCR responses (19). HA-
2AAR and FLAG-
1AR were expressed either separately or together in HEK-293 cells and then
stimulated with one of three agonist conditions: the
AR agonist
isoproterenol alone ("Iso"), the
2AR agonist UK
14,034 alone ("UK"), or Iso + UK together. When endocytosis of
2AAR was examined via a quantitative luminometer-based
assay (Fig. 2A), no
significant internalization was observed in response to Iso under any
condition, whereas internalization in response to UK was ~15%
whether Iso was co-applied or not. When endocytosis of
1AR was examined (Fig. 2B), ~25-30%
receptor internalization was observed in response to Iso. The extent of
internalization was not significantly different for
1AR
expressed alone as compared with
1AR expressed in the
presence of
2AAR. In response to UK, no significant
internalization was observed for
1AR expressed alone,
which is the expected result because UK does not activate
1AR. Strikingly, however,
1AR coexpressed
with
2AAR exhibited ~15% internalization in response
to UK stimulation. These data indicate that stimulation of
2AAR can cause co-internalization of
1AR.

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Fig. 2.
Co-internalization of
1AR with
2AAR. HA- 2AAR and
FLAG- 1AR were expressed either separately (solid
bars) or together (striped bars) in HEK-293 cells. The
internalization of 2AAR (A) and
1AR (B) was examined using a
luminometer-based assay following 10-min stimulations with the
-adrenergic agonist isoproterenol (Iso; 10 µm), the
2-adrenergic agonist UK 14,304 (UK; 10 µM), or a combination of the two agonists together. As
shown in A, 2AAR exhibited ~15%
internalization in response to UK stimulation but no significant
internalization in response to isoproterenol under any condition.
Conversely, as shown in B, 1AR exhibited
~25-30% internalization in response to isoproterenol but also
exhibited ~15% internalization in response to stimulation with UK.
This effect was only observed, however, when 2AAR was
coexpressed (** indicates significantly different from
1AR alone, p < 0.01). These data
suggest that 1AR can co-internalize with
agonist-activated 2AAR. The bars and
error bars represent the means ± S.E. for 4-5
independent experiments for each condition, with each experiment being
performed in triplicate.
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The internalization of
2AAR and
1AR was
also studied via immunofluorescence confocal microscopy. In cells
co-transfected with HA-
2AAR and FLAG-
1AR,
immunostaining for both receptors was concentrated into a smooth rim
along the edge of the cells, which presumably corresponds to receptor
localization in the plasma membrane of the cells (Fig.
3, A-C). Stimulation with Iso
resulted in the development of significant intracellular immunostaining for FLAG-
1AR (Fig. 3D) but had no apparent
effect on the pattern of immunostaining for HA-
2AAR
(Fig. 3E). In contrast, stimulation with UK resulted in
mobilization of both HA-
2AAR and FLAG-
1AR inside the cell (Fig. 3, G and H), where the two
receptors exhibited significant co-localization (Fig. 3I).
These data are consistent with the findings obtained using the
luminometer-based assay (Fig. 2) and offer additional evidence that
2AAR and
1AR co-internalize from the cell
surface following stimulation with
2-adrenergic agonists.

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Fig. 3.
Immunofluorescence confocal microscopy
reveals agonist-promoted co-internalization of
2A- and
1-adrenergic receptors.
HA- 2AAR (red) and FLAG- 1AR
(green) were co-transfected into HEK-293 cells and
visualized using secondary antibodies coupled to rhodamine and FITC,
respectively. In the absence of agonist stimulation, immunostaining for
both receptors was found predominantly in the plasma membrane
(A-C). Stimulation with isoproterenol (Iso) for
10 min induced significant mobilization of 1AR inside
the cell (D) but had no significant effect on the
subcellular distribution of 2AAR (E and
F). Stimulation with UK 14,034 (UK), in
contrast, resulted in significant internalization of both
2AAR (H) and 1AR
(G) and marked co-localization of the two receptors in
intracellular regions (I, with co-localization indicated in
yellow). The specificity of staining was determined in
control (Con) experiments using both untransfected and
transfected cells incubated in the absence and presence of the relevant
primary antibodies. These data are representative of 3-5 experiments
for each condition.
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Ligand Binding and Signaling of Coexpressed
2A- and
1-Adrenergic Receptors--
Because coexpressed
2AAR was able to regulate
1AR
internalization, we next examined if coexpression with
2AAR was able to regulate
1AR
pharmacological properties. The binding of the
AR-selective
antagonist [3H]DHA to lysed membranes derived from cells
transfected with either
1AR alone or
1AR/
2AAR was examined. Saturation binding
studies revealed that [3H]DHA bound with comparable
affinity to
1AR expressed in the absence and presence of
2AAR coexpression (KD = 2.8 ± 0.5 nM, Bmax = 39.3 ± 7.7 pmol/mg for
1AR alone; KD = 2.3 ± 0.4 nM, Bmax = 33.0 ± 6.5 pmol/mg for
1AR/
2AAR). However, studies
examining the displacement of [3H]DHA binding by a
variety of
AR-selective ligands revealed that many of these
compounds exhibited altered affinity for
1AR coexpressed with
2AAR relative to
1AR expressed
alone. Inhibition curves for displacement of [3H]DHA
binding to membranes expressing
1AR alone were fit
extremely well by assuming one binding site (Fig.
4; Table I).
In contrast, curves for displacement of
[3H]DHA binding to membranes expressing
1AR/
2AAR were in most cases fit
significantly better by two-site analyses rather than one-site
analyses. The appearance of a significant low affinity component for
the displacement of [3H]DHA by metoprolol, labetalol,
bisoprolol, dobutamine, and isoproterenol suggests that these ligands
bind with substantially lower affinity to
1AR/
2AAR heterodimers than to
1AR alone. On the other hand, norepinephrine, an
endogenous agonist for both
- and
-adrenergic receptors,
exhibited slightly enhanced affinity for binding to the
1AR in the presence of
2AAR coexpression
as compared with
1AR expressed alone, whereas
epinephrine, which is also an endogenous agonist for both receptors,
exhibited no significant change in its apparent affinity for
1AR alone versus
1AR/
2AAR. In control experiments,
membranes derived from cells expressing
1AR alone and
2AAR alone were mixed together, as in the control
co-immunoprecipitation experiments described above. In these mixing
experiments, no changes in the ligand binding properties of
1AR were observed for any of the ligands examined (data
not shown), suggesting that
1AR and
2AAR
must be expressed in the same cell for the modulation of
1AR pharmacological properties to occur. Furthermore,
the effects of
2AAR coexpression on
1AR
ligand binding properties were not blocked by treatment of the cells
with pertussis toxin prior to harvesting (data not shown), suggesting
that these effects are not due to activation of
Gi/Go-dependent intracellular
signaling pathways by the coexpressed
2AAR. In related
experiments, the binding of various
2AR-selective
ligands to membranes expressing
2AAR alone
versus
1AR/
2AAR was examined.
No differences in the binding properties of the
2AR-selective agonist UK 14,034, the
2AR-selective partial agonist clonidine, or the
2AR-selective partial agonist guanfacine were observed
(Table II), indicating that although
1AR possesses altered pharmacological properties when
expressed in the presence of
2AAR, it does not seem to
reciprocally be the case that
2AAR possesses altered
pharmacological properties when expressed in the presence of
1AR.

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Fig. 4.
Coexpression with
2AAR alters
1AR pharmacological properties.
HEK-293 cells were transfected with either FLAG- 1AR
alone (filled squares, solid line) or
HA- 2AAR/FLAG- 1AR (open triangles,
dashed line). Membranes were prepared, and the binding of the
-adrenergic antagonist [3H]DHA was studied in the
presence of increasing concentrations of the -adrenergic antagonist
bisoprolol. The apparent binding affinity of bisoprolol decreased in
the presence of 2AAR coexpression (please see Table I
for a summary of other ligands examined in these experiments). Notably,
the curve for bisoprolol inhibition of [3H]DHA binding
was well fit by assuming a single binding site in the case of
1AR alone but was a significantly better fit by a
two-site analysis in the case of the
1AR/ 2AAR co-transfected samples. The
points and error bars shown are the means ± S.E. for 4 independent determinations each.
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Table I
Ligand binding properties of 1AR expressed in the absence
and presence of 2AAR
The binding of [3H]DHA to lysed membranes was studied in the
presence of increasing concentrations of various adrenergic receptor
ligands. The estimated Ki values (in nM)
are shown for each ligand. One-site and two-site fits of each data set
were performed as described under "Materials and Methods." The
inhibition curves for 1AR/ 2AAR were significantly
better fit by two-site fits rather than one-site fits for all ligands
except for propranolol, norepinephrine, and epinephrine, whereas the
inhibition curves for 1AR alone were not significantly
better fit in any case by two-site fits relative to one-site fits.
Hence, two Ki values (KH for the
high affinity component and KL for the low affinity
component) are provided for binding to 1AR/ 2AAR
for most of the ligands, whereas only a single Ki
value is provided for binding to 1AR expressed alone. Note
that for all of the two-site fits, the KH value for
binding to 1AR/ 2AAR is similar to the single
Ki value for binding to 2AAR alone,
suggesting that the majority of binding sites in the membranes
expressing 1AR/ 2AAR possess binding properties
similar to the binding sites in membranes expressing 1AR
alone. However, membranes expressing 1AR/ 2AAR
also exhibit, in most cases, a small low affinity component
(KL), which was estimated between 10 and 25% of
total binding sites in all cases, as shown in the right-hand column.
The data for these inhibition curves were derived from 3 to 5 independent determinations for each ligand.
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Table II
Ligand binding properties of 2AAR expressed in the absence
and presence of 1AR
The binding of [3H]RX821002 to lysed membranes was studied in
saturation binding studies, and no significant differences were found
for 2AAR/ 1AR relative to 2AAR alone
(KD = 6.0 ± 2.1 nM,
Bmax = 7.7 ± 1.8 pmol/mg for 2AAR
alone; KD = 4.6 ± 1.5 nM,
Bmax = 6.8 ± 1.6 pmol/mg for
2AAR/ 1AR). The binding of [3H]RX821002
to lysed membranes was also studied in the presence of increasing
concentrations of several other -adrenergic receptor ligands. The
estimated Ki values (in nM) are shown
for each ligand. The levels of significance of differences in ligand
binding to 2AAR/ 1AR relative to 2AAR
alone were assessed via t tests, and no significant
differences were found between any of the matched sets. Moreover,
one-site versus two-site fits were performed as described
under "Materials and Methods" and in no cases were two-site fits
significantly better than one-site fits. Thus, 2AAR ligand
binding properties showed no obvious differences when 2AAR
was examined in the absence and presence of 1AR
coexpression. The data for these inhibition curves were derived from
three independent determinations for each ligand.
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We next examined the ability of
2AAR to modulate
1AR signaling. We utilized a transfection-based approach
to study
1AR stimulation of cAMP production in the
absence and presence of
2AAR coexpression in HEK-293
cells. These studies revealed that isoproterenol was
significantly less potent at stimulating cAMP production when
1AR was expressed in the presence of
2AAR
than when
1AR was expressed alone (Fig.
5). The maximal extent of cAMP
production, however, was comparable in both cases, and the expression
level of
1AR was unaltered by coexpression of
2AAR. Moreover, the effect of
2AAR
coexpression on isoproterenol-induced cAMP production was not
attributable to constitutive coupling of
2AAR to
Gi, because the effect was not blocked by pertussis toxin
treatment (data not shown). These data reveal that isoproterenol has a
higher potency at
1AR expressed alone versus
1AR coexpressed with
2AAR. These findings
are consistent with the ligand binding data presented in Table I, which
indicate that coexpression with
2AAR results in reduced
1AR affinity for isoproterenol and other
AR-selective
ligands.

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Fig. 5.
Coexpression of
1AR with
2AAR alters the potency of
isoproterenol-induced stimulation of adenylyl cyclase. HEK-293
cells were transfected with either 1AR alone
(filled squares, solid line) or
1AR/ 2AAR (open triangles, dotted
line). Expression levels of the 1AR were identical
for the two transfection conditions, as assessed by Western blot. The
cells were stimulated with increasing concentrations of
isoproterenol, and agonist-induced rises in cellular cyclic AMP were
quantified. The maximal extent of cyclic AMP produced in the
1AR/ 2AAR cells was 106 ± 8% of
that produced in the cells transfected with only 1AR.
The EC50 for isoproterenol stimulation of
1AR alone was 0.16 ± 0.02 nM, as
compared with 0.68 ± 0.17 for
1AR/ 2AAR (significantly different
from 1AR alone, p < 0.01). The
points and error bars represent the means and
S.E. values for four independent determinations.
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Regulation of
2A-AR/
1AR
Heterodimerization by Receptor Glycosylation--
Glycosylation of G
protein-coupled receptors can have variable effects on receptor
trafficking and signaling (20). The
1AR contains one
consensus site for N-linked glycosylation on its extracellular amino terminus (Asn-15). We mutated this site to alanine,
creating a mutant receptor (N15A) that exhibited a significant decrease
in apparent size on SDS-PAGE (Fig.
6A). Enzymatic deglycosylation with N-glycosidase F also decreased the apparent size of the
wild-type
1AR on SDS-PAGE but had no effect on the
apparent size of the N15A mutant receptor suggesting that Asn-15 is the
sole site of
1AR N-linked glycosylation (21).
We examined the capacity of the N15A mutant receptor for
heterodimerization with the
2AAR. HA-
2AAR
was coexpressed with either FLAG-
1AR wild-type or
FLAG-
1AR N15A, which exhibited equivalent levels of
total cellular expression as shown in Fig. 6A. The
FLAG-tagged
1ARs were immunoprecipitated with an
anti-FLAG antibody, and the amount of co-immunoprecipitated HA-
2AAR was examined via Western blot (Fig.
6B) and quantified (as shown in Fig. 6C).
Strikingly,
2AAR was co-immunoprecipitated much more
efficiently with the N15A mutant
1AR than with the wild-type
1AR. These data reveal that blockade of
1AR glycosylation enhances
1AR
heterodimerization with
2AAR.

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Fig. 6.
Heterodimerization of
2AAR and
1AR is enhanced when receptor
glycosylation is blocked. HEK-293 cells were transfected with
HA- 2AAR and FLAG- 1AR wild-type
(lane 1), HA- 2AAR wild-type and the
FLAG- 1AR N15A mutant (lane 2), or empty
vectors (lane 3). The total expression levels of all the
transfected proteins are shown in A. For the
anti-FLAG- 1AR samples (right), note the
decrease in the apparent size of the band for the N15A mutant,
corresponding to the decreased glycosylation of this mutant receptor.
For both wild-type and N15A mutant 1AR, several higher
order immunoreactive bands were evident in transfected cell lysates,
but only the lowest molecular weight band (~54 kDa) is shown here to
demonstrate the comparable levels of expression of the wild-type and
mutant receptors. The transfected cells were harvested, solubilized,
and incubated with anti-FLAG affinity resin to immunoprecipitate
FLAG- 1AR. The resultant immunoprecipitates
(IP) were run on 4-20% SDS-PAGE gels and probed on Western
blots (IB) to detect both anti-HA (B, left blot)
and anti-FLAG (B, right blot) immunoreactivity.
The FLAG-tagged wild-type and N15A mutant 1-adrenergic
receptors were immunoprecipitated equally (B, right blot).
The amount of HA- 2AAR that was co-immunoprecipitated
(B, left blot) differed markedly, however, with
the N15A mutant pulling down an average of nearly 3-fold more
HA- 2AAR than the wild-type 1AR. These
data reveal that 1AR/ 2AAR
heterodimerization is enhanced for the N15A mutant 1AR
relative to wild-type 1AR. Quantification of all of
these data is shown in C. Similar experiments were performed
in the experiments illustrated in D-F, except
that the three lanes that are shown correspond to
HA- 2AAR and FLAG- 1AR wild-type
(lane 1), HA- 2AAR N10A/N14A mutant and
FLAG- 1AR wild-type (lane 2), and empty
vectors (lane 3). Blockade of 2AAR
glycosylation, like blockade of 1AR glycosylation,
resulted in enhanced heterodimerization between the two receptors. The
bars and error bars shown in C and
F represent the means ± S.E. for 4 independent
determinations for each condition. WT, wild type.
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Because
2AAR/
1AR heterodimerization was
more efficient for the N15A mutant, we examined whether or not
2AAR might exert a more robust regulation of
N15A-
1AR relative to wild-type
1AR. However, we found that agonist-promoted
1AR
internalization following a 10-min stimulation with UK 14,304 was not
significantly different for wild-type
1AR
versus the N15A mutant (wild type = 16.8 ± 3.5%;
N15A = 18.0 ± 4.4% in matched plates examined side-by-side; n = 3). Similarly, the changes in ligand binding
properties induced by coexpression with
2AAR were
comparable for the wild-type
1AR and the N15A mutant
(data not shown). Thus, although blockade of
1AR
glycosylation results in a clear enhancement of
2AAR/
1AR heterodimerization, it may not
lead to an enhancement in heterodimerization of functional receptors on
the cell surface. This observation may be related to the fact that the
N15A mutant
1AR is deficient in its ability to traffic
to the cell surface relative to the wild-type receptor (21).
The
2AAR is also known to be glycosylated on its amino
terminus, on residues Asn-10 and Asn-14 (22). We therefore prepared a
mutant version of the
2AAR (N10A/N14A) that cannot be
glycosylated. Transfection of this mutant construct into HEK-293 cells
resulted in the expression of receptors with significantly decreased
apparent size on SDS-PAGE gels relative to wild-type
2AAR (Fig. 6D), as reported previously (22).
The heterodimerization of the HA-tagged N10A/N14A mutant with wild-type
FLAG-
1AR was assessed in side-by-side experiments in
comparison to the heterodimerization of wild-type
2AAR
with wild-type FLAG-
1AR (Fig. 6E). The
N10A/N14A mutant
2AAR exhibited an ~3-fold enhancement
over wild-type
2AAR in heterodimerization with
1AR (Fig. 6F). Taken together with the experiments described above examining the N15A
1AR,
these findings indicate that blockade of glycosylation of both
2AAR and
1AR results in enhanced
2AAR/
1AR heterodimerization.
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DISCUSSION |
Our findings reveal a functionally important heterodimerization
between
2A- and
1-adrenergic receptors.
The evidence for the physical association of these two receptor
subtypes is derived from both co-immunoprecipitation and
co-internalization assays. The co-internalization experiments not only
represent evidence for the physical association of the two receptors,
they also represent a specific mechanism by which
2AAR
stimulation may influence
1AR function. Although
2ARs and
ARs couple primarily to G proteins with
opposing cellular effects on cAMP production, it is known that agonist
activation of
2ARs can in some cases paradoxically sensitize
AR signaling in brain tissue (2-4). Because
internalization of GPCRs is known to play a key role in promoting GPCR
resensitization (19), our observation that
2AAR
stimulation can promote
1AR internalization provides a
specific molecular mechanism that could potentially account for the
previously reported ability of
2AR stimulation to
sensitize
AR-mediated responses in native tissues.
Coexpression of
2AAR and
1AR in our
studies not only allowed for
2AAR regulation of
1AR internalization, it also resulted in altered
1AR pharmacological properties. We found that the curves
for displacement of [3H]DHA binding to membranes
expressing
1AR alone were fit well by one-site analyses
for all ligands examined. Whereas it is true that analyses of agonist
binding to
-adrenergic receptors often require resolution into two
sites, which correspond to G protein-coupled (high affinity)
versus uncoupled (low affinity) states (23), our analyses of
agonist binding to
1AR alone were fit well by assuming a
single site. This is probably due to the fact that the transfected
1AR was expressed in our cells at much higher levels
than the endogenous G proteins, meaning that the G protein-coupled (high affinity) component of agonist binding to the receptors in our
assays represented only a tiny and unresolvable component of the
inhibition curves. In any case, coexpression of
1AR with
2AAR resulted in the appearance of a significant low
affinity component of binding for many of the
AR-selective ligands.
Our interpretation of these data is that a proportion of the
1AR in the cells assembled with
2AAR to
form heterodimers that exhibited unaltered affinity for some ligands
(such as DHA and propranolol), substantially reduced affinity for other
ligands (such as metoprolol, labetalol, bisoprolol, isoproterenol and
dobutamine), and slightly increased affinity for yet other ligands
(such as the endogenous agonist norepinephrine). Many of the ligands
examined share significant structural similarity, and it is therefore
uncertain why the binding properties of the various ligands should be
differentially altered by
1AR coexpression with
2AAR. Moreover, the affinity constant values derived for
the low affinity component of binding in these studies must be
considered as rough estimates, because it is difficult to derive
accurate affinity constant estimates from curves where the size of the
low affinity component (10-25%) represents such a small minority of
the total population of binding sites.
A question of interest is why coexpression of
1AR with
2AAR should result in ligand binding curves that are
best fit by two sites, rather than simply resulting in a single
population of novel binding sites as might be expected if every
1AR were to heterodimerize with an
2AAR
to form receptors with novel pharmacological properties. The most
likely explanation for the observed mixed population of binding sites
is that, regardless of how efficiently
2AAR
heterodimerizes with
1AR, it is unlikely that
all
1AR in a given cell will form heteromeric
complexes with coexpressed
2AAR. A large proportion of
cellular
1-adrenergic receptors are likely to exist
either as monomers or homodimers, with only a fraction of the total
1AR population assembling with other receptors such as
2AAR. Thus, studies examining the binding of ligands to
co-transfected
2AAR/
1AR are almost
certainly studying mixed populations of receptors, complicating
attempts to estimate the true changes in
1AR
pharmacological properties induced by heterodimerization with
2AAR. This is a general problem shared by all studies
examining pharmacological changes induced by heterodimerization of GPCRs.
Over the past several years, heterodimerization of a number of
different types of GPCRs has been reported. Examples where heterodimerization is required for the formation of functional receptors include the GABA receptors GABABR1 and
GABABR2 (10) and the taste receptor combinations T1R1/T1R3
and T1R2/T1R3, which have been reported to form receptors for
umami and sweet stimuli, respectively (24-26). Examples where
heterodimerization allows for cross-regulation between receptors but is
not required for receptor function include the following:
1- and
2-adrenergic receptors (15);
and
opioid receptors (27);
and µ opioid receptors (28, 29);
opioid and
2-adrenergic receptors (30, 31);
muscarinic acetylcholine M2 and M3 receptors (32); angiotensin AT1 and
bradykinin B2 receptors (33); dopamine D1 and adenosine A1 receptors
(34); dopamine D2 and somatostatin SSTR5 receptors (35); dopamine
D2 and adenosine A2 receptors (36); mGluR1 glutamate and A1 adenosine
receptors (37); SSTR1 and SSTR5 somatostatin receptors (38); SSTR2A and
SSTR3 somatostatin receptors (39); and µ opioid and SSTR2A
somatostatin receptors (40). Many of these receptor/receptor
interactions have been found to result in altered pharmacological
properties for one or both receptors (27-29, 32, 35, 38, 39), similar
to what we have found for the
2AAR/
1AR
interaction. Additionally, several of the previously reported (30, 36,
40) receptor-receptor interactions have been found to facilitate
receptor co-internalization, similar to our observation that
stimulation of
2AAR can lead to co-internalization of
1AR.
In both the luminometer-based surface expression assays and the
immunofluorescence microscopy experiments performed on
2AAR/
1AR co-transfected cells, we
observed that
2AAR stimulation resulted in
internalization of both
2AAR and
1AR,
whereas stimulation of
1AR resulted in internalization
of only
1AR. The reason for this difference is not
clear. It may be case that the
2AAR/
1AR heterodimer has internalization properties that are distinct from either of the two individual receptors. Alternatively,
2AAR/
1AR heterodimerization may be
impaired by
-adrenergic agonist stimulation, allowing
1AR to temporarily internalize in the absence of
2AAR co-internalization. Our co-immunoprecipitation
studies, however, did not reveal any consistent effects of agonist
stimulation on the amount of HA-
2AAR
co-immunoprecipitated with FLAG-
1AR. It is uncertain,
however, whether or not this technique is sensitive enough to detect
changes in
2AAR/
1AR
co-immunoprecipitation in the range of 10-20%, as might be expected
if receptor internalization were correlated with a temporary release
from heterodimerization. One thing that is interesting to note is the
similarity between our findings for the effects of
2AAR/
1AR heterodimerization on receptor
ligand binding properties versus internalization; we found
that assembly with
2AAR influenced
1AR
pharmacological properties, whereas conversely assembly with
2AAR did not result in any evident change in the ligand
binding properties of
2AAR. Similarly, we found that
2AAR stimulation led to
1AR
internalization, but conversely
1AR stimulation did not
lead to any evident internalization of
2AAR. Thus, for
both ligand binding and internalization,
2AAR was able
to influence
1AR, but
1AR was not able to
influence
2AAR.
If
1-adrenergic receptors can form
1AR/
1AR homodimers (14),
1AR/
2AR heterodimers (15), and
1AR/
2AAR heterodimers, as the present
data reveal, it is a point of significant interest to understand the
factors that regulate the proportion of cellular homodimers
versus heterodimers. Studies on the dimerization of other
GPCRs have provided evidence that agonist stimulation can regulate
dimerization (9). However, as mentioned above, our co-immunoprecipitation experiments did not reveal any consistent effects of agonist stimulation on
2AAR/
1AR heterodimerization. Association
with cytoplasmic scaffold proteins is another factor that might
potentially regulate heterodimer formation. The
1-adrenergic receptor is known to associate with
PSD-95/Discs-large/ZO-1 homology domain-containing scaffold proteins
such as PSD-95 (14, 41, 42) and MAGI-2 (14). However, we have not
observed any significant effects of PSD-95 or MAGI-2 coexpression on
the extent of either
1AR/
1AR
homodimerization (14) or
2AAR/
1AR
heterodimerization (data not shown). The
2A-adrenergic
receptor is known to associate with cytoplasmic proteins such as 14-3-3 (43) and spinophilin (44), but we have not examined the effects of
these interactions on
2AAR/
1AR heterodimerization.
One additional way that receptor heterodimerization might be regulated
is via post-translational receptor modifications. A post-translational
modification common to many GPCRs is receptor glycosylation (20). We
have found that the
1AR is N-glycosylated on
a single residue, Asn-15, and that
2AAR/
1AR heterodimerization is markedly
enhanced via mutation of Asn-15 to an amino acid that cannot
be glycosylated. Moreover, we have also found that
2AAR/
1AR heterodimerization is enhanced
by blocking glycosylation of the
2AAR. These data could
indicate that lack of glycosylation alters the conformations of the
2AAR and
1AR such that the efficiency of
their heterodimerization is increased. Alternatively, it is possible
that the enhanced heterodimerization of the N10A/N14A
2AAR and N15A
1AR is the result of a more
global alteration in the trafficking and processing of the mutant
receptors. Interestingly, we have found previously (21) that
1AR homodimerization is impaired for the
1AR-N15A mutant relative to the wild-type receptor. Thus, our data indicate that blockade of glycosylation has differential effects on
1AR homo- versus
heterodimerization. In any case, it is known that the glycosylation
state of transmembrane receptors can vary significantly in different
tissue types (45, 46). The glycosylation state of the
1AR in particular is known to be regulated via
polymorphic variation (47). Thus, the extent of
2AAR/
1AR heterodimerization may be
regulated differentially between tissues and between individuals via
differences in
1AR glycosylation state.
In summary, we have found a physical association between
2A- and
1-adrenergic receptors. This
heterodimerization alters
1AR pharmacological properties
and facilitates cross-internalization of
1AR following
2AAR agonist stimulation. Both
2AAR and
1AR are abundantly expressed in the brain, and
heterodimerization of these two receptors might therefore underlie
previously reported functional cross-talk between endogenous
2- and
-adrenergic receptors in brain tissue (2-8).
Therapeutic drugs acting on
2ARs (such as clonidine) and
-adrenergic receptors (such as propranolol and metoprolol) are
commonly utilized in the treatment of hypertension and are known to
exhibit significant clinical interactions (48, 49). The
heterodimerization of
2AAR and
1AR
described here may help to provide new insights into both physiological
cross-talk between
2- and
-adrenergic receptors and
clinical interactions between therapeutic drugs acting on these
receptor subtypes.