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Originally published In Press as doi:10.1074/jbc.M705747200 on December 3, 2007

J. Biol. Chem., Vol. 283, Issue 4, 1799-1807, January 25, 2008
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Norepinephrine- and Epinephrine-induced Distinct β2-Adrenoceptor Signaling Is Dictated by GRK2 Phosphorylation in Cardiomyocytes*Formula

Yongyu Wang{ddagger}, Vania De Arcangelis{ddagger}, Xiaoguang Gao{ddagger}, Biswarathan Ramani{ddagger}, Yi-sook Jung{ddagger}1, and Yang Xiang{ddagger}2

From the {ddagger}Department of Molecular and Integrative Physiology, University of Illinois at Urbana Champaign, Urbana, Illinois 61822

Received for publication, July 12, 2007 , and in revised form, November 30, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Agonist-dependent activation of G protein-coupled receptors 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 min after stimulation. In contrast, the NE-activated β2AR induced a much slower contraction rate increase that peaked at 10 min after stimulation. Whereas 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 βARK C terminus 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 events 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 G protein-coupled receptor signaling may be finely regulated by GRK in physiological settings.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
GPCRs3 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 presents 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 events and signaling properties (5, 6). Thus, there is great interest in elucidating the mechanisms underlying the drug-dependent cellular events in physiologically relevant contexts.

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 an 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 because of 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, in this work we study the potential differences of the receptor signaling activated by NE or Epi for physiological responses such as cardiomyocyte contraction. 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, specific drug-dependent signaling properties are proposed to explain the clinical observations under treatment with different β-blockers (11). Whereas 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).


Figure 1
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  		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 min of Epi stimulation and after 30 min of both Epi and NE stimulation. B, β2ARs were stimulated with Epi or NE for 10 min before recovery by washing out the drugs. The Epi-activated FLAG-β2AR efficiently recycled back to the cell surface after removal of the drug, although the NE-activated β2AR remained inside the cell. C, the cell-surface receptor level was measured by FLISAs after agonist-induced internalization and recycling. The quantitative data in C represent the mean ± S.E. of N different experiments. Con, control. *, p < 0.05 in Student's t test.

 
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 Ser-355 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
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Recombinant Adenoviruses—Spontaneous beating neonatal cardiac myocytes were prepared from hearts of 1-day-old wild type, β1AR-knock-out (β1AR-KO), or β1β2AR-knock-out (β1β2AR-KO) mouse pups as published previously (14). Neonatal myocytes were infected with viruses at a multiplicity of infection as indicated in the text after being cultured for 24 h. 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).

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 as described before (10) in the myocytes expressing the indicated FLAG-β2ARs. Cells were serum-starved for 2 h before stimulation with 10 µM epinephrine, norepinephrine, or isoproterenol (Sigma). The recycling of β2AR was done by washing out agonists after 10 min of drug stimulation to allow receptor recovery for an additional 30 or 60 min.

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, 1 µM) was applied 30 min 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 h prior to addition of epinephrine or norepinephrine for different times. Alternatively, myocytes were pretreated with 1 µM OA for 30 min 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 Science)). The lysates were clarified at 13,200 x g for 20 min. The supernatants were resolved on SDS-polyacrylamide 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 antibody at 1:5000 with Odyssey (Li-cor Biosciences, Lincoln, NE). The blots were then stripped and probed with anti-C-terminal 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-Ser(P) (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.


Figure 2
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  		FIGURE 2.
Epi- and NE-activatedβ2ARs couple to distinct G protein pathways to regulate contraction rate in neonatal cardiomyocytes. A, 10 µM isoproterenol (Iso)-, Epi-, or NE-activated endogenous β2AR showed distinct contraction rate responses in the β1AR-KO myocytes. B and 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 20 µM PKI, a specific PKA inhibitor. E and F, Epi (E) and NE (F) induced contraction rate responses mediated by β2AR lacking the C-terminal PDZ motif (β2AR{Delta}PDZ). FLAG-tagged mouse β2AR{Delta}PDZ was expressed in the β1β2AR-KO cardiomyocytes and stimulated (Stim) 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 ± S.E. 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.

 
Statistical Analysis—Curve-fitting and statistical analyses were performed using Prism (GraphPad Software, Inc., San Diego).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
In neonatal cardiomyocytes, the Epi-activated β2ARs displayed similar characteristics of trafficking to those activated by isoproterenol (Iso) (data not shown), showing fast internalization (Fig. 1A) (16) and sufficient recycling (Fig. 1, B and C). In contrast, NE-activated β2ARs displayed much slower internalization (Fig. 1A) and recycling (Fig. 1, B and C), which led to an intracellular accumulation of the receptor (Fig. 1B, lower 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 (10 µM) 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 because of 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 supplemental Fig. S1). Moreover, Epi-induced contraction rate increases (maximized at 4 min) were much faster than NE-induced ones (maximized at 10 min, Fig. 2A and supplemental Fig. S1). These data suggest that both NE- and Epi-activated β2ARs couple to the Gs/PKA pathway to regulate myocyte contraction rate.

At saturating concentrations of 10 µM, 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 10 µM NE or Epi were significantly different (Fig. 2A). This was not because of the activation of {alpha}1ARs by NE or Epi because β1β2AR-KO myocytes treated with these two drugs did 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 Epibut not the NE-induced contraction rate response was further enhanced by inhibiting the Gi protein with PTX, a Gi inhibitor (Fig. 2, B and C). 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 cannot 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. 2, E and F).


Figure 3
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  		FIGURE 3.
Epi and NE induce β2AR phosphorylation at serine 355 and 356 in cardiomyocytes. A, FLAG-tagged human β2ARs were expressed in the β1β2AR-KO myocytes and stimulated with 10 µM Epi or NE for the indicated times 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 and D, FLAG-β2AR was stimulated with Epi (C) or NE (D) for 5 min followed by removal of drug. Levels of phosphor-β2AR at serine 355 and 356 were examined after a 5-min 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 phosphor-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.

 
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 10 µM, β2AR stimulated with Epi displayed a rapid increase of phosphorylation on these serine residues that peaked at 5 min followed by a decrease over 60 min after drug administration (Fig. 3, A and B). In contrast, β2AR stimulated with 10 µM NE displayed a much slower increase in phosphorylation of the serine residues that peaked at 15 min and remained at peak level until 60 min after drug treatment (Fig. 3, A and B). 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 10 µM NE in terms of potency to activate Gs proteins for cAMP accumulation. The cellular cAMP accumulation induced by Epi displayed a dose-dependent increase (supplemental Fig. S3). At a concentration of 500 nM, which was equivalent to 10 µM NE in inducing cAMP, Epi induced a rapid receptor phosphorylation at Ser-355 and Ser-356 (supplemental Fig. S3). The time course of the receptor phosphorylation induced by 500 nM of Epi resembled that induced by 10 µM of Epi (Fig. 3A and supplemental Fig. S3). In addition, inhibiting phosphatase 2A with okadaic acid (OA) attenuated dephosphorylation of Ser-355 and -356 on the activated β2ARs at 30 min of Epi treatment but did not alter the phosphorylation level of these residues on the NE-activated receptors (supplemental Fig. S4). To examine whether the NE-activated β2ARs undergo dephosphorylation, myocytes were treated with agonists for 5 min before being washed. Both Epi- and NE-activated receptors displayed time-dependent dephosphorylation on Ser-355 and -356 after removal of drugs, which was partially but significantly blocked by pretreatment with OA (Fig. 3, C and D). 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 Ser-355 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 (compare Fig. 3B and 2A).

We then examined the effect of these agonist-induced Ser-355 and -356 phosphorylations 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. 4, A and B). 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, whereas additional PTX treatment enhanced the contraction rate increase induced by Epi, OA treatment diminished the effects of PTX on inhibiting Gi signaling (Fig. 4, C and E). 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 min 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. 4, D and E). These data together with the data from Fig. 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.


Figure 4
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  		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, cell-surface receptor densities were determined with FLISA. Con, control. 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 min 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 ± S.E. 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.

 
To further probe the effects of phosphorylation of Ser-355 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 cardiomyocytes in a dose-dependent manner (Fig. 5, A and B). As a control, overexpressing GFP by adenoviruses did not block Epi- or NE-induced GRK2 phosphorylation of β2AR in cardiomyocytes (Fig. 5, C and D). When expressed at high levels, 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
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
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 adrenoreceptor (21). These efficacies include divergent signaling pathways activated by the same GPCR under different drug stimulations (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 failed to activate the β2AR coupling to Gs protein but are capable of activating the mitogen-activated protein kinase (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 adrenoreceptor family in vivo. 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 shown for the first time that NE- and Epi-activated β2ARs induce distinct cellular signals in regulating physiological cardiomyocyte contraction responses. At saturating concentrations, whereas both drugs induced β2AR coupling to Gs protein, only Epi-activated receptors were capable of coupling to Gi proteins because of 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.


Figure 5
Figure 5
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  		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 (Con). 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 NE-activated β2AR signaling in β1AR-KO myocytes (H). The contraction response curves represent the mean ± S.E. 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.

 
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 that peaked around 10 min after drug administration (Fig. 2A). This delayed increase is likely due in part because of 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 because of the difference in these two agonist binding affinities. In fact, stimulation with 500 nM Epi, a concentration equivalent to 10 µM NE in terms of stimulation of Gs to increase cAMP, also induced a rapid receptor phosphorylation (supplemental Fig. S3), an observation that is consistent with the receptor phosphorylation at Ser-355 and Ser-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 (15, 27), 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 (15). 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 indicate 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. 3, C and D). 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 contraction rate response by NE (Fig. 5H). These data also indicate that the NE-mediated acute myocyte contraction response is not dependent on the slower receptor phosphorylation by GRK2 and subsequent trafficking. Because 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, do 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 down-regulation 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 phosphorylation of adrenoceptors to agonist-dependent, physiologically significant receptor signaling in cardiomyocytes. Although 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 (28). 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. Although 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 GRK2 phosphorylation of βARs in physiological versus pathological settings. Further studies along this direction as well as studies with adult myocytes will 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 events 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.


   FOOTNOTES
 
* This work is supported by National Institutes of Health R01 HL082846-01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4. Back

1 Present address: Dept. of Physiology, School of Medicine, and Department of Molecular Science and Technology, Ajou University, Suwon 442-749, Korea. Back

2 To whom correspondence should be addressed: Dept. of Molecular and Integrative Physiology, University of Illinois at Urbana Champaign, 523 Burrill Hall, 407 S. Goodwin Ave, Urbana, IL 61822. Tel.: 217-265-9448; Fax: 217-333-1133; E-mail: kevinyx{at}uiuc.edu.

3 The abbreviations used are: GPCR, G protein-coupled receptor; NE, norepinephrine; Epi, epinephrine; GRK, G protein-coupled receptor kinase; PTX, pertussis toxin; AR, adrenoceptor; OA, okadaic acid; PKA, cAMP-dependent protein kinase; Iso, isoproterenol; GFP, green fluorescent protein; βARKct, βARK C terminus; FLISA, fluorescence-linked immunosorbent assay; ANOVA, analysis of variance. Back


   ACKNOWLEDGMENTS
 
We thank members of the 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 encouragement during the early stage of this work.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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