Post on 07-Feb-2017
Norepinephrine and epinephrine induced distinct β2 adrenoceptor signaling is
dictated by GRK2 phosphorylation in cardiomyocytes
Yongyu Wang*, Vania De Arcangelis*, Xiaoguang Gao*, Biswarathan Ramani*, Yi-sook Jung*
†, Yang Xiang* #
*Department of Molecular and Integrative Physiology, University of Illinois at Urbana
Champaign, Urbana, IL 61822, †Department of Physiology at School of Medicine and
Department of Molecular Science and Technology, Ajou University, Suwon 442-749, Korea
Corresponding Author:
#Yang Xiang, Department of Molecular and Integrative Physiology, University of Illinois at
Urbana Champaign, 523 Burrill Hall, 407 S. Goodwin Ave, Urbana, IL 61822. Phone 217-265-
9448, Fax: 217-333-1133, Email: kevinyx@uiuc.edu
Running title: GRK2 regulates β2AR signaling in cardiomyocytes
1
http://www.jbc.org/cgi/doi/10.1074/jbc.M705747200The latest version is at JBC Papers in Press. Published on December 3, 2007 as Manuscript M705747200
Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
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Summary
Agonist-dependent activation of G protein-
coupled receptors (GPCR) induces diversified
receptor cellular and signaling properties.
Norepinephrine (NE) and epinephrine (Epi) are
two endogenous ligands that activate
adrenoceptor (AR) signals in a variety of
physiological stress responses in animals. Here
we use cardiomyocyte contraction rate response
to analyze the endogenous β2AR signaling
induced by Epi or NE in cardiac tissue. The Epi-
activated β2AR induced a rapid contraction rate
increase that peaked at 4 minutes after
stimulation. In contrast, the NE-activated β2AR
induced a much slower contraction rate increase
that peaked at 10 minutes after stimulation.
While both drugs activated β2AR coupling to Gs
proteins, only Epi-activated receptors were
capable of coupling to Gi proteins. Subsequent
studies showed that the Epi-activated β2AR
underwent a rapid phosphorylation by G-protein
coupled receptor kinase 2 (GRK2) and
subsequent dephosphorylation on serine residues
355 and 356, which was critical for sufficient
receptor recycling and Gi coupling. In contrast,
the NE-activated β2ARs underwent slow GRK2
phosphorylation, receptor internalization and
recycling, and failed to couple to Gi. Moreover,
inhibiting β2AR phosphorylation by βARKct or
dephosphorylation by okadaic acid prevented
sufficient recycling and Gi coupling. Together,
our data revealed that distinct temporal
phosphorylation of β2AR on serine 355 and 356
by GRK2 plays a critical role for dictating
receptor cellular and signaling properties
induced by Epi or NE in cardiomyocytes.
This study not only helps us understand the
endogenous agonist-dependent β2AR
signaling in animal heart, but also offers an
example of how GPCR signaling may be
finely regulated by GRK in physiological
settings.
Introduction
GPCRs comprise the largest known family
of cell-surface receptors and are
fundamentally involved in mammalian
physiology (1,2). This receptor superfamily
represents the largest single target for
modern drug therapy. A growing body of
evidence indicates that divergent efficacies
of an activated GPCR are both agonist- and
tissue-dependent (3-5), which posts a great
challenge on clinical application when a
specific receptor is targeted with drugs.
Many of the drug-dependent effects have
been attributed to the distinct receptor
conformational changes induced by different
ligands, leading to different subsequent
cellular and signaling properties (5,6). Thus,
there is great interest in elucidating the
mechanisms underlying the drug-dependent
cellular events in physiologically relevant
contexts.
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Interestingly, βARs, a family of prototypical
GPCRs, can be activated by two endogenous ligands
NE and Epi. The receptors play critical roles in the
regulation of cardiovascular (7) and pulmonary
function (8), as well as other physiological
processes. NE is primarily released from
sympathetic nerve terminal on the innervated tissues
whereas Epi is primarily released from adrenal gland
to the circulating plasma. The distinct releasing
routes suggest preferential activation of a βAR
subtype by individual ligand. This notion is
consistent with our recent observations that β1AR
and β2AR have distinct subcellular distribution along
the adrenergic synapses between sympathetic
ganglion neurons and cardiac muscle cells (9).
Meanwhile, it is also speculated that NE and Epi
might activate the same βAR subtype to initiate
distinct signaling for the same physiological
responses such as heart contraction. Evidence
supporting this notion is lacking due to strikingly
similar properties on the βAR activated by these
drugs in vitro. Recently, we have shown that NE and
Epi can induce distinct conformational changes on
β2ARs (10), suggesting that the activated receptors
may recruit different molecules for signal
transduction and function. Therefore, we would like
to study the potential differences of the receptor
signaling activated by NE or Epi for physiological
responses such as cardiomyocyte contraction in the
present study. These studies will not only help us
understand the physiological implications of
receptor signaling activated by these two drugs, but
also offer insights into the utility of a large group of
drugs that either activate or inhibit βARs in a variety
of clinical conditions including heart failure,
hypertension, coronary artery disease, and
asthma. In fact, the specific drug-dependent
signaling properties are proposed to explain the
clinical observations under treatment with
different β-blockers (11). While carvedilol has
been used as an effective long-term therapy for
heart failure, other drugs in the same class have
failed the clinical trials (12).
Studies in cardiac tissue are of prime interest
because adrenergic signaling properties are
not only manipulated with β blockers in
managing heart failure, but are also linked to
the progression of this disease (13). Here,
we analyzed the NE- or Epi-activated β2AR
signaling for physiological contraction
response on primary cardiomyocytes. We
have for the first time uncovered that
agonist-dependent phosphorylation of β2AR
receptor on ser355 and 356 by GRK2 plays
a critical role to differentiate the receptor
signaling activated by Epi or NE to regulate
cardiomyocyte contraction rate response.
Experimental Procedures
Cell Culture and Recombinant Adenoviruses
Spontaneous beating neonatal cardiac
myocytes were prepared from hearts of 1-
day-old WT, β1AR-knockout (β1AR-KO), or
β1β2AR-knockout (β1β2AR-KO) mouse pups
as previously published (14). Neonatal
myocytes were infected with viruses at a
multiplicity of infection (moi) as indicated
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in the text after being cultured for 24 hours.
Recombinant adenoviruses expressing flag-
tagged human or murine β2ARs have been
described previously (15). The receptor
expression levels were equivalent in myocytes
determined by ligand binding assays and
western blot as described before (16).
Adenoviruses expressing βARKct were a gift
from Walter Koch (Thomas Jefferson
University, Philadelphia, PA).
Immunofluorescence Microscopy and
Spectroscopy
Myocyte images were obtained using a Zeiss
Axioplan 2 microscope with Metamorph
software (Universal Imaging). Epitope-tagged
receptors were detected using M1 anti-flag
antibody (Sigma) followed by Alexa-488 or
Alexa-594 conjugated secondary antibodies
(Molecular Probes). Surface receptor levels were
determined with FLISA assay as described
before (10) in the myocytes expressing the
indicated flag-β2ARs. Cells were serum-starved
for 2 hours before stimulation with 10 μM
epinephrine, norepinephrine, or isoproterenol
(Sigma). The recycling of β2AR was done by
washing out agonists after 10 minute of drug
stimulation to allow receptor recovering for an
additional 30 or 60 minutes.
Cardiomyocyte Contraction Rate Assay
Measurement of spontaneous contraction rates
from cardiomyocytes expressing either the
endogenous or the indicated flag-β2ARs was
carried out with or without the use of
pertussis toxin (PTX) as described
previously (14). In some assay, okadaic
acid (OA, 1μM) was applied 30 minutes
before addition of Epi.
Determination of β2AR Phosphorylation or
Dephosphorylation with Phosphoserine-
Specific Antibodies
Antibodies to the C-terminus of the β2AR
and to the phosphorylated serine (355,356)
of β2AR were from Santa Cruz
Biotechnology (Santa Cruz, CA). Neonatal
cardiomyocytes were serum starved for 2
hours prior to addition of epinephrine or
norepinephrine for different time.
Alternatively, myocytes were pretreated
with 1μM OA for 30mins before adding
drugs. The cardiomyocytes were chilled,
washed, and harvested in lysis buffer
(10 mM Tris, pH 7.5, 150 mM NaCl, 2 mM
EDTA, 1% Triton X-100, 20 mM Na4P2O7.
10H2O, 50 mM NaF, 1 mM Na3VO4,
0.1 μM OA and Complete Mini protease
inhibitor (Roche Applied Sciences,
Indianapolis, IN). The lysates were clarified
at 13,200 ×g for 20 mins. The supernatant
were resolved on SDS-PAGE gels, and
blotted with the polyclonal anti-
phosphoserine (355,356) specific β2AR
antibody at 1:500, and revealed with a
IRDye 800CW goat-anti-rabbit secondary
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antibody at 1:5,000 with Odyssey (Li-cor
biosciences, Lincoln, NE). The blots were then
stripped and probed with anti-CT β2AR antibody
at 1:500 to visualize total β2ARs. Control
experiments showed no background signal
remaining after the stripping procedure. The
signals yielded from the anti-pSer (355,356)
β2AR antibodies were first corrected for total
β2AR levels and then either plotted as increase
over basal or percentage of the maximal levels
as indicated in the figure legends.
Statistical Analysis
Curve-fitting and statistical analyses were
performed using Prism (GraphPad Software, Inc.
San Diego, CA).
Results
In neonatal cardiomyocytes, the Epi-activated
β2ARs displayed similar characteristics of
trafficking to those activated by isoproterenol
(Iso, data not shown), showing fast
internalization (Fig1A and (16)) and sufficient
recycling (Fig1B and 1C). In contrast, NE-
activated β2ARs displayed much slower
internalization (Fig1A) and recycling (Fig1B
and 1C), which led to an intracellular
accumulation of the receptor (Fig. 1B, low right
panel). We have previously established that
agonist-dependent β2AR internalization and
recycling is necessary for the receptor to switch
coupling from Gs to Gi proteins to modulate the
myocyte contraction rate (16). We then
examined the potential difference in β2AR
signaling-mediated myocyte contraction rate
response after the endogenous receptors
were stimulated with Epi or NE in β1AR-
KO myocytes. When the β2ARs were
activated by a saturating concentration
(10μM) of Iso, the myocyte contraction rate
response displayed an initial increase
followed by a sustained decrease dropping
the rate below the basal level (Fig. 2A). The
response is due to sequential coupling of the
receptor to Gs and Gi (16). Both Epi and NE
induced a dose-dependent maximum
contraction rate increase, and the maximum
contraction rate increase was attenuated by
membrane permeable peptide PKI, a
selective PKA inhibitor (Fig. 2D and S1).
Moreover, Epi-induced contraction rate
increases (maximized at 4 minutes) were
much faster than NE-induced ones
(maximized at 10 minutes, Fig. 2A and S1).
These data suggest that both NE- and Epi-
activate β2ARs couple to Gs/PKA pathway
to regulate myocyte contraction rate.
At saturating concentrations of 10μM, both
Epi- and NE- induced myocyte contraction
rate responses lacked the secondary decrease
induced by Iso during the late-phase
stimulation (Fig. 2A). The time courses of
contraction rate increases induced by 10μM
NE or Epi were significantly different (Fig.
2A). This was not due to the activation of
α1ARs by NE or Epi since β1β2AR-KO
myocytes treated with these two drugs did
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not display significant change on myocyte
contraction rates (data not shown). The lack of
the secondary decrease of contraction rate in NE
or Epi-treated myocytes implies a minimum role
of the receptor/Gi coupling in the contraction
responses (16). Interestingly, only the Epi- but
not the NE-induced contraction rate response
was further enhanced by inhibiting Gi protein
with PTX, a Gi inhibitor (Fig. 2B and 2C).
Therefore, only Epi-activated β2AR had
sufficient coupling to Gi proteins. This
observation is consistent with the fast
internalization and recycling of β2ARs upon Epi
stimulation, supporting the notion that agonist-
dependent receptor trafficking is necessary for
the efficient receptor coupling to Gi in
cardiomyocytes. This notion is further supported
by an experiment utilizing a mutant β2AR that
can not recycle. Previously, the C-terminal PDZ
motif of β2AR has been shown necessary for
receptor recycling after agonist induced
internalization (16). When the mutant β2AR
lacking this motif was expressed in β1β2AR-KO
myocytes and activated by Epi or NE, the
receptor signaling-mediated myocyte contraction
rate responses were not sensitive to PTX
treatment (Fig. 2E and 2F).
The agonist-dependent GPCR internalization
and recycling is regulated by receptor
phosphorylation by the GRK family and
dephosphorylation by phosphatase 2A (17). We
examined agonist-dependent phosphorylation on
β2AR in cardiomyocytes. Both Epi and NE
induced a dose-dependent phosphorylation
of serine residues 355 and 356 of β2AR in
cardiomyocytes (S2). At 10μM, β2AR
stimulated with Epi displayed a rapid
increase of phosphorylation on these serine
residues that peaked at 5 minutes followed
by a decrease over 60 minutes after drug
administration (Fig. 3A and 3B). In contrast,
β2AR stimulated with 10μM NE displayed a
much slower increase in phosphorylation of
the serine residues that peaked at 15 minutes
and remained at peak level until 60 minutes
after drug treatment (Fig. 3A and 3B). To
rule out the possibility that lower agonist
occupancy by NE accounts for the slower
phosphorylation of the receptor, we titrated
Epi to a concentration equivalent to 10μM
NE in terms of potency to activate Gs
proteins for cAMP accumulation. The
cellular cAMP accumulation induced by Epi
displayed a dose-dependent increase (S3).
At a concentration of 500nM, which was
equivalent to 10μM NE in inducing cAMP,
Epi induced a rapid receptor
phosphorylation at ser355 and 356 (S3). The
time course of the receptor phosphorylation
induced by 500nM of Epi resembled that
induced by 10μM of Epi (Fig.3A and S3). In
addition, inhibiting phosphatase 2A with
okadaic acid (OA) attenuated
dephosphorylation of ser355 and 356 on the
activated β2ARs at 30 minutes of Epi
treatment, but did not alter the
phosphorylation level of these residues on
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the NE activated receptors (S4). To examine
whether the NE-activated β2ARs undergo
dephosphorylation, myocytes were treated with
agonists for 5 minutes before being washed.
Both Epi- and NE-activated receptors displayed
time-dependent dephosphorylation on ser355
and 356 after removal of drugs, which was
partially but significantly blocked by
pretreatment with OA (Fig. 3C and 3D). The
failure of OA treatment to fully restore the
receptor phosphorylation level indicates that
other phosphatases may be involved in the β2AR
dephosphorylation in cardiac myocytes.
Together, Epi and NE induced distinct temporal
phosphorylation of ser355 and 356 on β2AR in
cardiomyocytes. These data are consistent with
the β2AR signaling-mediated myocyte
contraction rate responses under stimulation of
NE and Epi (comparing Fig. 3B and 2A).
We then examined the effect of these agonist-
induced ser355 and 356 phosphorylation on
receptor trafficking and signaling in
cardiomyocytes by inhibiting receptor
dephosphorylation with OA. As expected, OA
treatment reduced the recycling of the Epi-
activated β2ARs after internalization, which
resulted in an intracellular accumulation of the
receptors (Fig. 4A and 4B). In the β1AR-KO
myocytes stimulated by Epi, OA treatment alone
neither significantly changed basal myocyte
contraction rate, nor significantly altered the
Epi-induced contraction rate response (Fig.4C).
However, while additional PTX treatment
enhanced the contraction rate increase
induced by Epi, OA treatment diminished
the effects of PTX on inhibiting Gi signaling
(Fig. 4C and 4E). Without OA treatment,
inhibition of Gi protein with PTX
significantly enhanced both initial maximum
contraction rate increases (Fig. 4D) and the
late-stage contraction rate increases (Fig.
4E) during 30 minutes of stimulation with
Epi. After pretreatment with OA, the PTX-
dependent effect on the initial maximum
contraction rate increases were blunted (Fig.
4D), and the PTX effect on contraction rate
increase during the late stage of stimulation
was completely absent (Fig. 4E), suggesting
a diminished Gi signaling. Meanwhile,
additional OA treatment only slightly
reduced both the initial maximum
contraction rate increase and the late
contraction rate increase induced by NE
(Fig.4D and 4E). These data together with
the data from Figure 3 suggest that OA
treatment blocks the Epi-activated β2AR
dephosphorylation and subsequent recycling
after internalization. The internalized
receptors are accumulated at intracellular
compartments, which results in limited
coupling to Gi protein in cardiomyocytes.
These observations are consistent with the
notion that the agonist-dependent receptor
trafficking is necessary for the efficient
β2AR coupling to Gi in cardiomyocytes.
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To further probe the effects of phosphorylation
of ser355 and 356 on receptor trafficking and
signaling in cardiomyocytes, we used a GRK2
specific inhibitor βARKct to block the agonist-
dependent GRK2 phosphorylation (18).
Overexpressing βARKct blocked both Epi- and
NE-induced GRK2 phosphorylation of β2AR in
cardiomyocyte in a dose-dependent manner (Fig.
5A and 5B). As a control, overexpressing GFP
by adenoviruses did not block Epi- or NE-
induced GRK2 phosphorylation of β2AR in
cardiomyocytes (Fig. 5C and 5D).When
expressed at high level, βARKct, but not the
control GFP, slowed the Epi-induced β2AR
internalization and recycling, which resulted in
an intracellular accumulation of the receptors
(Fig. 5E). Meanwhile, βARKct almost
completely blocked the NE-induced β2AR
internalization (Fig. 5F). When overexpressed in
the β1AR-KO cardiomyocytes, βARKct did not
significantly change the contraction rate
response induced by NE (Fig 5H), suggesting
that the contraction rate response induced by NE
is uncoupled from β2AR internalization after
GRK2-mediated phosphorylation. In contrast,
βARKct selectively enhanced the contraction
rate response induced by Epi (Fig. 5G). βARKct
also blocked the additional effects of PTX on the
Epi-induced myocyte contraction rate increase
(Fig. 5I). These data suggest that inhibiting
agonist-dependent phosphorylation of β2AR by
GRK2 affects receptor trafficking, and blocks
the Epi-activated receptor coupling to Gi in
cardiomyocytes.
Discussion
In this study, we have shown that distinct
β2AR phosphorylation by GRK2 plays a
critical role in differentiating NE- and Epi-
induced receptor signaling to regulate
physiological cardiomyocyte contraction
rate responses. Ligand-dependent
pharmacological and cellular efficacies have
been documented on a growing list of
GPCRs including opioid receptor (19),
angiotensin receptor (20), and adrenoceptor
(21). These efficacies include divergent
signaling pathways activated by the same
GPCR under different drug stimulation (22)
as well as divergent cellular sorting
pathways after agonist induced receptor
internalization (23). In the case of β2AR,
antagonist alprenolol and inverse agonist
ICI118551 fail to activate the β2AR
coupling to Gs protein, but are capable of
activating MAPK signaling cascade in
HEK293 fibroblasts (21). These studies
suggest that diversified signaling pathways
can be potentially activated by the same
GPCR in physiological contexts.
Meanwhile, different clinical outcomes from
long-term therapy of heart failure support
the idea that different β-blockers can induce
distinct cellular effects in patients (24). Epi
and NE are two endogenous ligands that
activate the adrenoceptor family in vivo.
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Recent studies suggest that NE- and Epi-
activated human β2AR can preferentially couple
to distinct G protein signaling pathways when
overexpressed in mouse heart (25). Despite these
in vivo observations, cellular and molecular
mechanisms underlying the physiological
implication of βAR signaling activated by NE
and Epi remain unclear. Here we analyzed the
signaling induced by Epi and NE in cardiac
tissue by examining the effects on
cardiomyocyte contraction rate response. We
have for the first time showed that NE- and Epi-
activated β2ARs induce distinct cellular signals
in regulating physiological cardiomyocyte
contraction responses. At saturating
concentrations, while both drugs induced β2AR
coupling to Gs protein, only Epi-activated
receptors were capable of coupling to Gi
proteins due to its sufficient recycling in
cardiomyocytes. Subsequent studies showed that
a rapid GRK2 phosphorylation and subsequent
dephosphorylation of the Epi-activated β2AR
were critical for sufficient receptor recycling and
Gi coupling. These data suggest that NE and Epi
induce different signaling and functional
properties of β2AR in animal heart.
Myocytes stimulated by Epi at different
concentrations displayed a rapid Gs/PKA
pathway-dependent contraction rate increase
(Fig.2A). After reaching peak level, the
contraction rate underwent an immediate
decrease representing the combination of
receptor desensitization and receptor/Gi
coupling. In contrast, when activated by NE
at different concentrations, the receptor
displayed a much slower Gs/PKA-
dependent contraction rate increase which
peaked around 10 minutes after drug
administration (Fig.2A). This delayed
increase is likely in part due to a slow
desensitization of the NE-activated receptor
by GRK2 phosphorylation (Fig.3A), which
results in a prolonged coupling to Gs
protein. The difference in receptor signaling
and GRK2 phosphorylation was not due to
the difference in these two agonist binding
affinities. In fact, stimulation with 500nM
Epi, a concentration equivalent to 10μM NE
in terms of stimulation of Gs to increase
cAMP, also induced a rapid receptor
phosphorylation (S3), an observation which
is consistent with the receptor
phosphorylation at ser355, 356 reported in
HEK293 cells (26). In our study, the GRK2-
mediated phosphorylation was detected on
human β2AR expressed in mouse myocytes.
Despite different signaling and biochemical
properties reported between human and
mouse β2ARs (27,28), these two receptors
resemble each other. Our recent studies
on β2ARs expressed in mouse
cardiomyocytes show that these two
receptors are remarkably similar in
activating Gs and Gi for myocyte
contraction and undergoing agonist-
dependent internalization and recycling
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(28) . Here, the tight correlation between the
endogenous mouse β2AR-mediated myocyte
contraction change and the exogenously
expressed human β2AR phosphorylation under
agonist stimulation confirms the striking
similarity between these two species. We have
previously reported that Epi and NE can induce
distinct conformational changes on β2AR (10).
Thus the difference in the GRK2
phosphorylation of β2AR may be due to the Epi-
activated receptors having a higher GRK2
binding affinity or serving as better GRK2
substrates than the NE-activated ones.
Alternatively, it may be due to the recruitment of
additional cellular factors to the agonist-
activated β2ARs that regulate the GRK2-
mediated phosphorylation of the receptor.
GRK-mediated phosphorylation of a GPCR has
been implicated in receptor desensitization and
subsequent internalization (29). The
phosphorylated receptors possess increased
binding affinities to a scaffold protein β-arrestin
for internalization. The internalized GPCRs
dissociate from arrestin complexes and undergo
dephosphorylation for recycling (17). Our data
indicates the GRK2-mediated phosphorylation
plays a key role for the agonist-dependent
trafficking (internalization and recycling). The
dephosphorylation of the β2ARs activated by NE
and Epi appeared to be equivalent (Fig.3C and
3D). Thus, in a simple model, when the β2ARs
are activated by Epi, the rapid GRK2
phosphorylation leads to transient Gs
coupling and faster internalization, which is
followed by sufficient dephosphorylation
allowing recycling and Gi coupling in
myocytes. Blocking β2AR
dephosphorylation by okadaic acid inhibits
the receptor coupling to Gi. Disrupting the
GRK2-mediated phosphorylation of β2AR
with βARKct inhibits the receptor
internalization and subsequent recycling,
which enhances the Epi-induced maximum
contraction rate increases, and inhibits the
receptor/Gi coupling for contraction rate
responses. These data are consistent with
recent reports showing that GRK2 and
GRK3 preferentially regulate receptor/G
protein coupling (30,31), but do not rule out
the possible additional role of GRK5 and
GRK6 in β2AR cellular signaling and
trafficking in cardiomyocytes. In fact, the
receptor phosphorylation by other kinases
may contribute to the remaining
internalization of Epi-activated β2ARs after
GRK2 is inhibited by βARKct (Fig.5E).
In contrast, the NE-activated β2ARs undergo
a slow but persistent GRK2
phosphorylation, which may contribute to a
prolonged Gs coupling, slow receptor
trafficking, and minimum Gi coupling. It is
therefore not surprising that disruption of
GRK2 phosphorylation by βARKct has a
minimal effect on the receptor-mediated
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contraction rate response by NE (Fig.5H). This
data also indicates that the NE-mediated acute
myocyte contraction response is not dependent
on the slower receptor phosphorylation by
GRK2 and subsequent trafficking. Since the NE-
activated β2ARs are accumulated inside of cells,
and fail to display sufficient cell surface
recovery and Gi coupling, our data suggest that
the NE-induced β2AR phosphorylation by
GRK2 may play a role in inducing prolonged
β2AR desensitization. These data, however, does
not exclude the possibility that other cellular
kinases are involved in modulating the GRK
phosphorylation of β2AR under NE stimulation,
which may regulate receptor signaling for
contraction rate responses. Based on the
recycling rates, GPCRs can be classified into
two classes: rapid (including β2AR) and slow
recycling receptors (32). The slow recycling
receptors usually form stable complexes with
arrestin in endosomes, which are essential for
initiating additional signaling pathways during a
prolonged period of stimulation (32). Therefore,
it will be interesting to check whether the NE-
activated β2ARs form stable complexes with
arrestin to regulate additional signaling
pathways in cardiomyocytes. Meanwhile, it
remains to be examined whether the
intracellularly accumulated β2ARs, after
stimulation by NE, are targeted to lysosome for
degradation. The slow recycling of the NE-
activated βAR also implies a prolonged
desensitization of receptor at post-synaptic
regions of sympathetic synapses in animal
heart.
Diseases such as heart failure and asthma
are characterized by dysfunction of βAR
signaling, including downregulation and
desensitization of receptors (8,33-35), and
much evidence points to GRK as a culprit
(35-39). Thus, there is great interest in
elucidating the cellular mechanisms by
which GRK-mediated receptor
phosphorylation and function are regulated.
Our studies linked GRK2 phophorylation of
adrenoceptors to agonist-dependent,
physiologically significant receptor
signaling in cardiomyocytes. While our data
indicate that βARKct can block the β2AR/Gi
coupling, it is interesting to point out that
the coupling of β2AR to Gi protein plays a
protective role against insults on cardiac
myocytes (40). Thus, our data appear to be
at odds with the beneficial effects of the
peptide when overexpressed in mouse heart
(36,38). The differences could be due to the
different time course of the results observed
in these two model systems. While the effect
of βARKct in mice is mainly attributed to
the recovery of βAR density and response in
animal heart under chronic conditions
(36,38), our results address the acute effect
of βARKct on βAR signaling in neonatal
cardiomyocytes. Moreover, the observed
difference may imply the different roles of
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GRK2 phosphorylation of βARs in
physiological vs pathological settings. Further
studies along this direction as well as studies
with adult myocytes shall provide more
mechanistic details on the effects of GRK
regulation of βAR signaling in physiological and
pathophysiological conditions.
In summary, we hereby revealed the critical role
of GRK2 phosphorylation underlying the
distinct cellular and signaling properties of
β2AR induced by Epi or NE in cardiomyocytes.
This finding opens the door to further
explore the differential physiological
relevance of these two endogenous ligands
upon binding to adrenoceptors. These data
not only help us understand the
physiological and pathophysiological
significance of βAR activation by Epi and
NE in vivo, but also support the utility of the
combinatory manipulation of βAR and GRK
activities in the treatment of a wide range of
chronic conditions in both cardiovascular
and pulmonary systems.
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Footnote:
This work is supported by NIH R01 HL082846-01. The authors would like to thank members of Xiang
laboratory for critical reading and comments, Kieran Normoyle for manuscript preparation, and Dr.
Walter Koch for the βARKct adenoviruses. We thank Dr. Brian Kobilka for his encouragement during
early stage of this work.
Abbreviations: Norepinephrine, NE; Epinephrine, Epi; G protein-coupled receptor kinase, GRK; Pertussis
toxin, PTX; adrenoceptor, AR; Okadaic acid, OA.
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Figure legend
Figure 1. Epi- and NE-activated β2ARs undergo different trafficking in neonatal cardiomyocytes. (A)
Flag-tagged mouse β2ARs were expressed in the β1β2AR-KO myocytes and visualized by
immunocytochemistry. β2ARs were mainly localized on the cell surface at steady state. Epi induced rapid
receptor internalization whereas NE-activated receptor underwent much slower internalization. Punctate
intracellular staining of flag-β2ARs was observed after 5 mins of Epi stimulation, and after 30 mins of
both Epi and NE stimulation. (B) The β2ARs were stimulated with Epi or NE for 10 mins before recovery
by washing out the drugs. The Epi-activated flag-β2AR efficiently recycled back to the cell surface after
removal of the drug, while the NE-activated β2AR remained inside the cell. (C) The cell-surface receptor
level was measured by FLISA assays after agonist-induced internalization and recycling. The quantitative
data in (C) represent the mean± SE of N different experiments. *, P<0.05 in student’s t-test.
Figure 2. Epi- and NE-activated β2ARs couple to distinct G protein pathways to regulate contraction
rate in neonatal cardiomyocytes. (A)10 μM Iso-, Epi-, or NE-activated endogenous β2AR showed
distinct contraction rate responses in the β1AR-KO myocytes. (B-C) PTX treatment selectively affected
the Epi-induced (B) but not NE-induced (C) contraction rate increase of the β1AR-KO myocytes. (D)
Both Epi and NE possess dose-dependent effects on contraction rate of the β1AR-KO myocytes, and the
maximum contraction rate increase was inhibited by 20μM PKI, a specific PKA inhibitor. (E-F) Epi
(E) and NE (F) induced contraction rate responses mediated by β2AR lacking the C-terminal
PDZ motif (β2ARΔPDZ). Flag-tagged mouse β2ARΔPDZ was expressed in the β1β2AR-KO
cardiomyocytes and stimulated with Epi (E) or NE (F) to induce contraction rate increase.
Additional PTX treatment did not affect the contraction rate responses induced by Epi (E) and
NE (F). The contraction response curves represent the mean± SE of N beating dishes from M different
myocyte preparations. *, P<0.05; time-course curves were significantly different between Epi and NE
(A), and between Epi and Epi + PTX (B) by two-way ANOVA. **, P<0.05 in student’s t-test.
Figure 3. Epi and NE induce β2AR phosphorylation at serine 355 and 356 in cardiomyocytes. (A) Flag-
tagged human β2AR were expressed in the β1β2AR-KO myocytes and stimulated with 10 μM Epi or NE
for indicated time to examine the phosphorylation on serine 355 and 356 of the receptor. (B) Quantitative
analysis of the levels of phospho-β2AR in (A) by normalizing the phospho-β2AR signal against the total
β2AR. (C-D)Flag-β2AR was stimulated with Epi (C) or NE (D) for 5 minutes followed by removal of
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drug. Levels of phospho-β2AR at serine 355 and 356 were examined after a 5-minute stimulation or after
drug removal to examine β2AR dephosphorylation in cardiomyocytes. Okadaic acid (OA) was used to
prevent β2AR dephosphorylation at serine 355 and 356. Quantitative analysis of the levels of the
phospho-β2AR in (C) and (D) are listed below each panel. *, P<0.05; time-course curves were
significantly different by two-way ANOVA. **, P<0.05 in student’s t-test.
Figure 4. Okadaic acid inhibits Epi-mediated β2AR recycling and coupling to Gi in cardiomyocytes. (A)
Flag-tagged mouse β2ARs were expressed in the β1β2AR-KO myocytes and stimulated with Epi before
removal of the drugs for recycling. Epi-activated β2AR underwent internalization and recycling in
cardiomyocytes; pretreatment with OA blocked sufficient receptor recycling. (B) The cell surface receptor
densities were determined with FLISA assay. (C-E) Cardiomyocytes from the β1AR-KO mice were
pretreated with OA and/or PTX before stimulation with Epi or NE. (C) OA did not affect Epi-induced
contraction rate in β1AR-KO neonatal cardiac myocytes, but blocked the additional inhibitory effects of
PTX on Gi signaling. The effects of OA and PTX on initial maximum contraction rate increase (D) and
the late-stage (30 mins after drug stimulation) contraction rate increase (E) were analyzed after Epi or NE
stimulation on β1AR-KO cardiomyocytes. The contraction response curves in (C) represent the mean ±
SE of N beating dishes from M different myocyte preparations. *, P<0.05 in student’s t-test. **, P<0.05;
time-course curves were significantly different between the PTX + Epi and the others (C) by two-way
ANOVA.
Figure 5. βARKct, a GRK2 specific inhibitor, affects β2AR trafficking and the receptor signaling
mediated contraction rate response in neonatal cardiomyocytes. (A) Flag-β2AR and βARKct were co-
expressed in β1β2AR-KO myocytes. Epi- or NE-induced GRK2 phosphorylation at serine 355 and 356
was blocked by βARKct in an expression level-dependent manner. (B) Quantitative analysis of the levels
of phospho-β2AR in (A) by normalizing as percentage of no infected control. (C) Epi- or NE-induced
GRK2 phosphorylation at serine 355 and 356 was selectively blocked by βARKct expression, but not the
GFP control. (D) Quantitative analysis of the levels of phospho-β2AR in (C) by normalizing as percentage
of no infected control. (E) βARKct reduced β2AR internalization and recycling after Epi stimulation, and
caused intracellular accumulation of receptor in β1β2AR-KO myocytes. (F) βARKct almost completely
blocked β2AR internalization after NE stimulation. (G-I) βARKct enhanced the contraction rate increase
mediated by Epi-activated β2AR signaling in β1AR-KO myocytes (G), but blocked the additional effect of
PTX on contraction rate (I). In contrast, βARKct did not alter the contraction rate increase mediated by
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NE-activated β2AR signaling in β1AR-KO myocytes (H). The contraction response curves represent the
mean ± SE of N beating dishes from M different myocyte preparations. *, P < 0.05; time course curves
were significantly different between Epi and βARKct + Epi (G) by two-way ANOVA. **, P < 0.05 in
student’s t-test.
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Epi NE
1 2 3 4 1 2 3 470
80
90
100
110
Epi (N=6)
1. Con2. 10 min3. 10 min + 30 min recycle4. 10 min + 60 min recycle
NE (N=6)
Cel
l sur
face
rec
epto
r(%
)
Co
n10
min
10 m
in +
30 m
in re
cycl
e10
min
+60
min
recy
cle
Fig. 1
B
C
**
0 m
in5
min
30 m
in
Epi (10uM) NE (10uM)A
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-5
0 10 20 30 40-10
0
10
20
30
40Epi (N=18, M=10)Epi + PTX (N=15, M=10)
Time(min)
Con
trac
tion
rate
incr
ease
ove
r ba
sal l
evel
(bea
ts/m
in)
0 10 20 30 40-10
0
10
20
30
40 NE (N=7, M=4)NE + PTX (N=5, M=4)
Time(min)
Con
trac
tion
rate
incr
ease
ove
r ba
sal l
evel
(bea
ts/m
in)
0
10
20
30
40
Max
con
trac
tion
rate
incr
ease
over
bas
al le
vel (
beat
s/m
in)
Fig. 2
-6 -7 -8 -5 -6 -7-5 -5
Epi NEPKI- - - - + - - - +
*
****
A
C
B
D
Epi
NE
(LogM)
0 10 20 30 40-10
0
10
20
30
40Epi (N=18, M=10)NE (N=16, M=10)ISO (N=11, M=10)
Time(min)
Con
trac
tion
rate
incr
ease
ove
r ba
sal l
evel
(bea
ts/m
in)
Epi
10 20 30 40-10
0
10
20
30
40 β2ARΔPDZ + Epi (N=5, M=3)β2ARΔPDZ + Epi + PTX (N=5, M=3)
Time(min)Con
trac
tion
rate
incr
ease
ove
r ba
sal l
evel
(be
ats/
min
)
10 20 30 40-10
0
10
20
30
40β2ARΔPDZ + NE (N=6, M=3)β2ARΔPDZ + NE + PTX (N=4, M=3)
Time(min)Con
trac
tion
rate
incr
ease
ove
r ba
sal l
evel
(be
ats/
min
)
NE
E F
Stim
*
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β2ARp355,356
β2ARp355,356
β2AR
β2AR
NE
0 2 5 15 30 60 min
Epi
0 5 10 15 20 25 300
50
100
150
200
250
EpiNE
Time (min)
pSer
ine
(355
,356
) lev
elov
er b
asal
(arb
itory
uni
t )
β2AR
β2ARp355,356
(%of
max
imum
at 5
min
)
pSer
ine
(355
,356
) lev
el
Fig 3
A
B
C D
β2AR
β2ARp355,356
Epi (min) 5 5 5 5Washout(min) 30 60 60
OA +
(%of
max
imum
at 5
min
)
pSer
ine
(355
,356
) lev
el
NE (min) 5 5 5 5Washout(min) 30 60 60
OA +
*
0
20
40
60
80
100
0
20
40
60
80
100
0 0− −− − − −
− −− − − −
** **
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*
*
*
Epi Epi +OA
Co
n10
min
10m
in +
60 m
in re
cycl
e
Fig.4
EpiA
B
C
D
E
1 2 3 1 2 370
80
90
100
110
Cel
l sur
face
rec
epto
r(%
)
*
Epi (N=6) OA + Epi (N=4)
1. Con2. 10 min3. 10 min + 60 min recycle
0 10 20 30 40-10
0
10
20
30
40Epi + OA (N=16, M=8)Epi + OA + PTX (N=5, M=5)
Epi + PTX (N=10, M=5)Epi (N=14, M=8)
Time(min)
Con
trac
tion
rate
incr
ease
ove
r ba
sal l
evel
(bea
ts/m
in)
Epi
Epi +P
TX
Epi + O
A
Epi +O
A +PTX NE
NE+OA-10
0
10
20
30
40
Late
con
trac
tion
rate
cha
nge
over
bas
al le
vel (
beat
s/m
in)
Epi
Epi +P
TX
Epi + O
A
Epi +O
A +PTX NE
NE+OA OA
-10
0
10
20
30
40
Max
con
trac
tion
rate
cha
nge
over
bas
al le
vel (
beat
s/m
in)
**
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β2AR β2AR + βARKct
Co
nEp
i 10
min
Epi 1
0 m
in +
60m
in re
cycl
eCon 0 100 200 300
βARKct (moi)
Fig. 5
A B
DC
Epi
p355, 356
βARKct
β2AR
Con 0 100 200 300
βARKct (moi)
NE
** **
βARKct (moi)NEEpi
β2AR + GFPE
p355, 356
βARKct
β2AR
NEEpi
**
**
Void
Epi NE
GFP Con βARKct Void GFP Con βARKct
GFP
ConVoid GFP
βARKct
ConVoid GFP
βARKct
0102030405060708090
100110
Con 0 100 200 300 Con 0 100 200 3000
102030405060708090
100110
pSer
ine
(355
,356
) le
vel (
%)
pSer
ine
(355
,356
) le
vel (
%)
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Fig. 5
F
Epi
*
G
NE
β2AR β2AR + βARKctC
on
NE
10 m
inN
E 10
min
+60
min
recy
cle
H
I
10 20 30 40-10
0
10
20
30
40
50GFP + Epi (N=8, M=8)βARKct + Epi (N=11, M=8)
Epi (N=6, M=6)
Time (min)
Con
trac
tion
rate
incr
ease
ove
r ba
sal (
beat
s/m
in)
10 20 30 40-10
0
10
20
30
40
50GFP + NE(N=7, M=5)βARKct + NE (N=7, M=5)
NE (N=6, M=5)
Time (min)
Con
trac
tion
rate
incr
ease
over
bas
al (
beat
s/m
in)
10 20 30 40-10
0
10
20
30
40
50 βARKct + Epi (N=11, M=6)βARKct + Epi + PTX (N=10, M=6)
Time (min)
Con
trac
tion
rate
incr
ease
ove
r bas
al (
beat
s/m
in) Epi
β2AR + GFP
*
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and Yang XiangYongyu Wang, Vania De Arcangelis, Xiaoguang Gao, Biswarathan Ramani, Yi-sook Jung
dictated by GRK2 phosphorylation in cardiomyocytes2 adrenoceptor signaling isβNorepinephrine and epinephrine induced distinct
published online December 3, 2007J. Biol. Chem.
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