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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