Norepinephrine- and Epinephrine-induced Distinct β2-Adrenoceptor Signaling Is Dictated by GRK2 Phosphorylation in Cardiomyocytes*

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.

GPCRs 3 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)(4)(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, ␤ARs, 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 ␤AR subtype by an individual ligand. This notion is consistent with our recent observations that ␤ 1 AR and ␤ 2 AR 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 ␤AR 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 ␤AR activated by these drugs in vitro. Recently, we have shown that NE and Epi can induce distinct conformational changes on ␤ 2 ARs (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 ␤ARs 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 ␤-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).
Studies in cardiac tissue are of prime interest because adrenergic signaling properties are not only manipulated with ␤-blockers in managing heart failure but are also linked to the progression of this disease (13). Here we analyzed the NE-or Epi-activated ␤ 2 AR signaling for physiological contraction response on primary cardiomyocytes. We have for the first time uncovered that agonist-dependent phosphorylation of ␤ 2 AR 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
Cell Culture and Recombinant Adenoviruses-Spontaneous beating neonatal cardiac myocytes were prepared from hearts of 1-day-old wild type, ␤ 1 AR-knock-out (␤ 1 AR-KO), or ␤ 1 ␤ 2 AR-knock-out (␤ 1 ␤ 2 AR-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 FLAGtagged human or murine ␤ 2 ARs 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 ␤ARKct 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). Epitopetagged 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-␤ 2 ARs. Cells were serum-starved for 2 h before stimulation with 10 M epinephrine, norepinephrine, or isoproterenol (Sigma). The recycling of ␤ 2 AR 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-␤ 2 ARs was carried out with or without the use of pertussis toxin (PTX) as described previously (14). In some assay, okadaic acid (OA, 1 M) was applied 30 min before addition of Epi.
Determination of ␤ 2 AR Phosphorylation or Dephosphorylation with Phosphoserine-specific Antibodies-Antibodies to the C terminus of the ␤ 2 AR and to the phosphorylated serine (355, 356) of ␤ 2 AR 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 M 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 Na 4 P 2 O 7 . 10H 2 O, 50 mM NaF, 1 mM Na 3 VO 4 , 0.1 M OA, and Complete Mini protease inhibitor (Roche Applied Science)). The lysates were clarified at 13,200 ϫ g for 20 min. The supernatants were resolved on SDS-polyacrylamide gels and blotted with the polyclonal anti-phosphoserine (355, 356)-specific A, FLAG-tagged mouse ␤ 2 ARs were expressed in the ␤ 1 ␤ 2 AR-KO myocytes and visualized by immunocytochemistry. ␤ 2 ARs 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-␤ 2 ARs was observed after 5 min of Epi stimulation and after 30 min of both Epi and NE stimulation. B, ␤ 2 ARs were stimulated with Epi or NE for 10 min before recovery by washing out the drugs. The Epi-activated FLAG-␤ 2 AR efficiently recycled back to the cell surface after removal of the drug, although the NE-activated ␤ 2 AR 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 Ϯ S.E. of N different experiments. Con, control. *, p Ͻ 0.05 in Student's t test. ␤ 2 AR 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 ␤ 2 AR antibody at 1:500 to visualize total ␤ 2 ARs. Control experiments showed no background signal remaining after the stripping procedure. The signals yielded from the anti-Ser(P) (355, 356) ␤ 2 AR antibodies were first corrected for total ␤ 2 AR 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).

RESULTS
In neonatal cardiomyocytes, the Epi-activated ␤ 2 ARs displayed similar characteristics of trafficking to those activated by isoproterenol (Iso) (data not shown), showing fast internaliza-tion ( Fig. 1A) (16) and sufficient recycling (Fig. 1, B and C). In contrast, NE-activated ␤ 2 ARs 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 agonistdependent ␤ 2 AR internalization and recycling is necessary for the receptor to switch coupling from G s to G i proteins to modulate the myocyte contraction rate (16). We then examined the potential difference in ␤ 2 AR signaling-mediated myocyte contraction rate response after the endogenous receptors were stimulated with Epi or NE in ␤ 1 AR-KO myocytes. When the ␤ 2 ARs were activated by a saturating concentration (10 M) 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 G s and G i (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 ␤ 2 ARs couple to the G s /PKA pathway to regulate myocyte contraction rate.
At saturating concentrations of 10 M, 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 M NE or Epi were significantly different ( Fig.  2A). This was not because of the activation of ␣ 1 ARs by NE or Epi because ␤ 1 ␤ 2 AR-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/G i coupling in the contraction responses (16). Interestingly, only the Epi-but not the NE-induced contraction rate response was further enhanced by inhibiting the G i protein with PTX, a G i inhibitor (Fig. 2, B and C). Therefore, only Epi-activated ␤ 2 AR had sufficient coupling to G i proteins. This observation is consistent with the fast internalization and recycling of ␤ 2 ARs upon Epi stimulation, supporting the notion that agonist-dependent receptor trafficking is necessary for the efficient receptor coupling to G i in cardiomyocytes. This notion is further supported by an experiment utilizing a mutant ␤ 2 AR that cannot recycle. Previously, the C-terminal PDZ motif of ␤ 2 AR has been shown necessary for receptor recycling after agonist-induced internalization (16). When the mutant ␤ 2 AR lacking this motif was expressed in ␤ 1 ␤ 2 AR-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).
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 ␤ 2 AR in cardiomyocytes. Both Epi and NE induced a dose-dependent phosphorylation of serine residues 355 and 356 of ␤ 2 AR in cardiomyocytes (S2). At 10 M, ␤ 2 AR 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, ␤ 2 AR stimulated with 10 M 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 M NE in terms of potency to activate G s 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 M 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 M 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 ␤ 2 ARs 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 ␤ 2 ARs undergo dephosphorylation, myocytes were treated with agonists for 5 min before being washed. Both Epi-and NE-activated receptors displayed timedependent 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 ␤ 2 AR dephosphorylation in cardiac myocytes. Together, Epi and NE induced distinct temporal phosphorylation of Ser-355 and -356 on ␤ 2 AR in cardiomyocytes. These data are consistent with the ␤ 2 AR 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 ␤ 2 ARs after internalization, which resulted in an intracellular accumulation of the receptors (Fig. 4, A and B). In the ␤ 1 AR-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 G i signaling (Fig. 4, C and E). Without OA treatment, inhibition of G i protein with PTX significantly enhanced both initial maximum contraction rate increases (Fig. 4D) and the late stage contraction rate increases (Fig. 4E)   of stimulation with Epi. After pretreatment with OA, the PTXdependent 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 G i 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 ␤ 2 AR dephosphorylation and subsequent recycling after internalization. The internalized receptors are accumulated at intracellular compartments, which results in limited coupling to G i protein in cardiomyocytes. These observations are consistent with the notion that the agonist-dependent receptor trafficking is necessary for the efficient ␤ 2 AR coupling to G i in cardiomyocytes.
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 ␤ARKct to block the agonistdependent GRK2 phosphorylation (18). Overexpressing ␤ARKct blocked both Epi-and NE-induced GRK2 phosphorylation of ␤ 2 AR 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 ␤ 2 AR in cardiomyocytes (Fig. 5, C and D). When expressed at high levels, ␤ARKct, but not the control GFP, slowed the Epi-induced ␤ 2 AR internalization and recycling, which resulted in an intracellular accumulation of the receptors (Fig. 5E). Meanwhile, ␤ARKct almost completely blocked the NE-induced ␤ 2 AR internalization (Fig. 5F). When overexpressed in the ␤ 1 AR-KO cardiomyocytes, ␤ARKct 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 ␤ 2 AR internalization after GRK2-mediated phosphorylation. In contrast, ␤ARKct selectively enhanced the contraction rate response induced by Epi (Fig. 5G). ␤ARKct also blocked the additional effects of PTX on the Epi-induced myocyte contraction rate increase (Fig. 5I). These data suggest that inhibiting agonistdependent phosphorylation of ␤ 2 AR by GRK2 affects receptor trafficking and blocks the Epi-activated receptor coupling to G i in cardiomyocytes.

DISCUSSION
In this study, we have shown that distinct ␤ 2 AR 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 ␤ 2 AR, antagonist

. Okadaic acid inhibits Epi-mediated ␤ 2 AR recycling and coupling to G i in cardiomyocytes.
A, FLAG-tagged mouse ␤ 2 ARs were expressed in the ␤ 1 ␤ 2 AR-KO myocytes and stimulated with Epi before removal of the drugs for recycling. Epi-activated ␤ 2 AR 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 ␤ 1 AR-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 ␤ 1 AR-KO neonatal cardiac myocytes but blocked the additional inhibitory effects of PTX on G i 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 ␤ 1 AR-KO cardiomyocytes. The contraction response curves in C represent the mean Ϯ S.E. of N beating dishes from M different myocyte preparations. *, p Ͻ 0.05 in Student's t test. **, p Ͻ 0.05; time course curves were significantly different between the PTX ϩ Epi and the others (C) by two-way ANOVA.
alprenolol and inverse agonist ICI118551 failed to activate the ␤ 2 AR coupling to G s 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 ␤-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 ␤ 2 AR 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 ␤AR 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 NEand Epi-activated ␤ 2 ARs induce distinct cellular signals in regulating physiological cardiomyocyte contraction responses. At saturating concentrations, whereas both drugs induced ␤ 2 AR coupling to G s protein, only Epi-activated receptors were capable of coupling to G i proteins because of its sufficient recycling in cardiomyocytes. Subsequent studies showed that a rapid GRK2 phosphorylation and subsequent dephosphorylation of the Epi-activated ␤ 2 AR were critical for sufficient receptor recycling and G i coupling. These data suggest that NE and Epi induce different signaling and functional properties of ␤ 2 AR in animal heart. Myocytes stimulated by Epi at different concentrations displayed a rapid G s /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/G i coupling. In contrast, when activated by NE at different concentrations, the receptor displayed a much slower G s /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 G s protein. The difference in receptor signaling and . ␤ARKct, a GRK2-specific inhibitor, affects ␤ 2 AR trafficking and the receptor signaling mediated contraction rate response in neonatal cardiomyocytes. A, FLAG-␤ 2 AR and ␤ARKct were co-expressed in ␤ 1 ␤ 2 AR-KO myocytes. Epi-or NE-induced GRK2 phosphorylation at serine 355 and 356 was blocked by ␤ARKct in an expression level-dependent manner. B, quantitative analysis of the levels of phospho-␤ 2 AR 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 ␤ARKct expression but not the GFP control. D, quantitative analysis of the levels of phospho-␤ 2 AR in C by normalizing as percentage of no infected control. E, ␤ARKct reduced ␤ 2 AR internalization and recycling after Epi stimulation and caused intracellular accumulation of receptor in ␤ 1 ␤ 2 AR-KO myocytes. F, ␤ARKct almost completely blocked ␤ 2 AR internalization after NE stimulation. G-I, ␤ARKct enhanced the contraction rate increase mediated by Epi-activated ␤ 2 AR signaling in ␤ 1 AR-KO myocytes (G), but blocked the additional effect of PTX on contraction rate (I). In contrast, ␤ARKct did not alter the contraction rate increase mediated by NE-activated ␤ 2 AR signaling in ␤ 1 AR-KO myocytes (H). The contraction response curves represent the mean Ϯ S.E. of N beating dishes from M different myocyte preparations. *, p Ͻ 0.05; time course curves were significantly different between Epi and ␤ARKct ϩ Epi (G) by two-way ANOVA. **, p Ͻ 0.05 in Student's t test.
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 M NE in terms of stimulation of G s 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 GRK2mediated phosphorylation was detected on human ␤ 2 AR expressed in mouse myocytes. Despite different signaling and biochemical properties reported between human and mouse ␤ 2 ARs (15,27), these two receptors resemble each other. Our recent studies on ␤ 2 ARs expressed in mouse cardiomyocytes show that these two receptors are remarkably similar in acti-vating G s and G i for myocyte contraction and undergoing agonistdependent internalization and recycling (15). Here the tight correlation between the endogenous mouse ␤ 2 AR-mediated myocyte contraction change and the exogenously expressed human ␤ 2 AR 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 ␤ 2 AR (10). Thus the difference in the GRK2 phosphorylation of ␤ 2 AR 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 ␤ 2 ARs 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 ␤-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 ␤ 2 ARs activated by NE and Epi appeared to be equivalent (Fig. 3, C and D). Thus, in a simple model, when the ␤ 2 ARs are activated by Epi, the rapid GRK2 phosphorylation leads to transient G s coupling and faster internalization, which is followed by sufficient dephosphorylation allowing recycling and G i coupling in myocytes. Blocking ␤ 2 AR dephosphorylation by okadaic acid inhibits the receptor coupling to G i . Disrupting the GRK2-mediated phosphorylation of ␤ 2 AR with ␤ARKct inhibits the receptor internalization and subsequent recycling, which enhances the Epi-induced maximum contraction rate increases, and inhibits the receptor/G i 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 ␤ 2 AR cellular signaling and trafficking in cardiomyocytes. In fact, the receptor phosphorylation by other kinases may contribute to the remaining internalization of Epi-activated ␤ 2 ARs after GRK2 is inhibited by ␤ARKct (Fig. 5E).
In contrast, the NE-activated ␤ 2 ARs undergo a slow but persistent GRK2 phosphorylation, which may contribute to a prolonged G s coupling, slow receptor trafficking, and minimum G i coupling. It is therefore not surprising that disruption of GRK2 phosphorylation by ␤ARKct 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 ␤ 2 ARs are accumulated inside of cells, and fail to display sufficient cell surface recovery and G i coupling, our data suggest that the NE-induced ␤ 2 AR phosphorylation by GRK2 may play a role in inducing prolonged ␤ 2 AR desensitization. These data, however, do not exclude the possibility that other cellular kinases are involved in modulating the GRK phosphorylation of ␤ 2 AR 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 ␤ 2 AR) 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 ␤ 2 ARs form stable complexes with arrestin to regulate additional signaling pathways in cardiomyocytes. Meanwhile, it remains to be examined whether the intracellularly accumulated ␤ 2 ARs, after stimulation by NE, are targeted to lysosome for degradation. The slow recycling of the NE-activated ␤AR also implies a prolonged desensitization of receptor at postsynaptic regions of sympathetic synapses in animal heart.
Diseases such as heart failure and asthma are characterized by dysfunction of ␤AR signaling, including down-regulation and desensitization of receptors (8,(33)(34)(35), and much evidence points to GRK as a culprit (35)(36)(37)(38)(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 ␤ARKct can block the ␤ 2 AR/G i coupling, it is interesting to point out that the coupling of ␤ 2 AR to G i 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 ␤ARKct in mice is mainly attributed to the recovery of ␤AR density and response in animal heart under chronic conditions (36,38), our results address the acute effect of ␤ARKct on ␤AR signaling in neonatal cardiomyocytes. Moreover, the observed difference may imply the different roles of GRK2 phosphorylation of ␤ARs 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 ␤AR 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 ␤ 2 AR 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 ␤AR activation by Epi and NE in vivo but also support the utility of the combinatory manipulation of ␤AR and GRK activities in the treatment of a wide range of chronic conditions in both cardiovascular and pulmonary systems.