Agonist Dose-dependent Phosphorylation by Protein Kinase A and G Protein-coupled Receptor Kinase Regulates β₂ Adrenoceptor Coupling to G_i Proteins in Cardiomyocytes

Abstract

Adrenoceptors receptors (ARs) play a pivotal role in regulating cardiovascular response to catecholamines during stress. β₂ARs, prototypical G protein-coupled receptors (GPCRs), expressed in animal hearts, display dual coupling to both G_s and G_i proteins to control the adenylyl cyclase-cAMP dependent protein kinase A (PKA) pathway to regulate contraction responses. Here, we showed that the β₂AR coupling to G_i proteins was agonist dose-dependent and occurred only at high concentrations in mouse cardiac myocytes. Both the β₂AR-induced PKA activity, measured by fluorescence resonance energy transfer (FRET) imaging, and the increase in myocyte contraction rate displayed sensitivity to the G_i inhibitor pertussis toxin (PTX). Further studies revealed that activated β₂ARs underwent PKA phosphorylation at a broad range of agonist concentrations. Disruption of the PKA phosphorylation sites on the β₂AR blocked receptor/G_i coupling. However, a sufficient β₂AR/G_i coupling was also dependent on the G protein-coupled receptor kinase (GRK)-mediated phosphorylation of the receptors, which only occurred at high concentrations of agonist (≥100 nm). Disruption of the GRK phosphorylation sites on the β₂AR blocked receptor internalization and coupling to G_i proteins, probably by preventing the receptor's transportation to access G_i proteins. Furthermore, neither PKA nor GRK site mutated receptors displayed sensitivity to the G_i-specific inhibitor, G_iCT. Together, our studies revealed distinct roles of PKA and GRK phosphorylation of the β₂AR for agonist dose-dependent coupling to G_i proteins in cardiac myocytes, which may protect cells from overstimulation under high concentrations of catecholamines.

Introduction

Adrenoceptors (ARs)³ play a pivotal role in regulating cardiovascular response to catecholamines during stress. Both β₁- and β₂ARs are prototypical GPCRs expressed in animal hearts, which mediate the increases in cardiac contraction upon agonist stimulation through the G_s-adenylyl cyclase-cAMP-dependent PKA pathway (1). β₂ARs are also known to uniquely couple to inhibitory G_i proteins, which inhibits adenylyl cyclases to reduce cardiac contraction and initiates anti-apoptotic and cell growth signaling (2, 3). Although β₂AR coupling to G_i has been studied in reconstituted systems and in fibroblasts (4, 5), little is known about the regulation process and the circumstances under which this coupling occurs in cardiac cells, along with its physiological consequences.

Various studies indicate that phosphorylation of β₂AR plays a critical role in regulating differential G protein coupling. The β₂AR phosphorylation by PKA mediates the switch of coupling from G_s to G_i (6, 7), presumably operating in a feedback after receptor activation. Our previous studies have revealed that β₂AR/G_i coupling is also dependent on receptor internalization and recycling (8, 9). Meanwhile, Wang et al. have shown that direct inhibition of GRK2 prevents β₂AR/G_i coupling in mouse cardiac myocytes (8), supporting a role of the β₂AR phosphorylation by GRK in receptor/G_i coupling.

Studies have also revealed that β₂ARs are differentially phosphorylated by PKA and GRK in fibroblasts in an agonist dose-dependent manner (10–13). Although the PKA-mediated phosphorylation is sensitive to stimulation with subnanomolar concentrations, the GRK-mediated phosphorylation happens only when β₂ARs are stimulated with saturated concentrations (10–13). This differential regulation of receptor phosphorylation by different kinases and their effects on subsequent receptor trafficking indicate that the β₂AR/G_i coupling may be finely regulated in an agonist concentration-dependent manner.

Here, using a fluorescence resonance energy transfer (FRET) assay with a biosensor for PKA activity and a physiological cardiac contraction rate assay, we show for the first time that β₂AR/G_i coupling occurs only at a saturated concentration of isoproterenol (Iso) in cardiac cells. Stimulation induces the PKA-mediated phosphorylation of the β₂AR over a wide range of agonist concentrations, whereas the GRK-mediated phosphorylation occurs only at sufficiently high concentrations (≥100 nm) of Iso and is necessary for receptor internalization. We further demonstrate that mutations of the specific PKA or GRK phosphorylation sites on the β₂AR abolish receptor coupling to G_i protein. Our data suggest that the PKA-mediated phosphorylation is necessary for β₂AR/G_i coupling, but a sufficient receptor/G_i coupling is also dependent on the GRK-mediated phosphorylation and subsequent trafficking of the receptors.

MATERIALS AND METHODS

Site-directed Mutagenesis and Recombinant Adenoviruses

pcDNA3.1-FLAG-tagged murine β₂AR was used as a template for mutagenesis. A protein kinase A mutant (PKAmut) β₂AR lacking protein kinase A (PKA) phosphorylation sites (serines 261, 262, 345, and 346) was generated by replacing the four serines with alanines. A GRK mutant (GRKmut) β₂AR lacking the GRK phosphorylation sites (serines 355, 356, and 358) was also created by replacing serines with alanine. These phosphorylation sites are determined by Tran et al. (13). Both β₂AR mutants were sequenced and transformed into adenovector for producing adenoviruses, as described previously (14). The titers of the viruses were assessed by comparison of receptor expression levels with both Western blot and ligand binding, as described previously (15). The GFP-G_iCT virus used in contraction assays was a kind gift from Dr. Walter Koch (Thomas Jefferson University, Philadelphia, PA).

Cardiac Myocyte Contraction Assay

Measurement of spontaneous myocyte contraction was performed as described previously (16). Neonatal cardiac myocytes were isolated from mice lacking either β₁AR (β₁AR-knock-out; β₁AR- KO) or both β₁ and β₂AR genes (β₁β₂AR-KO). β₁β₂AR-KO myocytes were infected with murine β₂AR, PKAmut β₂AR, or GRKmut β₂AR at a multiplicity of infection of 100 and stimulated with 10 μm Iso. Cardiac myocytes were treated with G_i inhibitor pertussis toxin (PTX) at 0.3 μg/ml, 3 h before stimulation. GFP-G_iCT virus was co-infected at a multiplicity of infection of 100 or, as indicated, along with the wild type or mutant β₂AR viruses. Contraction rate assays were performed the next day as described previously (16).

Receptor Phosphorylation

Neonatal cardiac myocytes were infected with FLAG-tagged wild type or mutant β₂ARs at a multiplicity of infection of 100. After 48 h of expression, cells were serum-starved for 2 h and stimulated with different doses of Iso for 5 min. For the time course experiments, cells were stimulated with 10 μm Iso for different times as indicated. Cells were placed into lysis buffer (10 mm Tris, pH 8.0, 1% Nonidet P-40, 150 mm NaCl, 2 mm EDTA, and protease and phosphatase inhibitor mixture (Thermo Scientific)) for 30 min at 4 °C. Samples were centrifuged, and the supernatant was resolved by SDS-PAGE for Western blotting. The β₂AR phosphorylation by PKA at Ser(P)^261/262 was detected with a monoclonal antibody from Dr. Richard Clark (University of Texas Health Science Center, Houston, TX). The β₂AR phosphorylation by GRK at Ser(P)^355/356 was detected with a polyclonal antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). IRDye 680CW goat anti-mouse and IRDye 800CW goat anti-rabbit secondary antibodies were used to detect Ser(P)^261/262 and Ser(P)^355/356 phosphorylation, respectively. Western blots were visualized with an Odyssey scanner (Li-cor Biosciences, Lincoln, NE) and quantified before normalization against the base-line levels.

Receptor Internalization and Recycling

After expressing the FLAG-β₂AR, the β₁β₂AR-KO myocytes were serum-starved for 2 h and stimulated with 10 nm or 10 μm Iso for different times. For receptor recycling, cells were stimulated with Iso for 10 min, rinsed, and refed with serum-free medium for different times. Cells were then fixed, permeabilized, and stained with anti-FLAG antibody, which was revealed with an Alexa-488 conjugated goat anti-mouse antibody (Invitrogen). Fluorescence images were taken with a CCD camera on a Zeiss microscope with Metamorph software. HEK293 cells were transfected with plasmids expressing the FLAG-β₂AR, PKAmut β₂AR, and GRKmut β₂AR. Receptor internalization and recycling was quantified in HEK293 cells by using a fluorescence-linked immunosorbent assay (FLISA) to determine cell surface receptor levels as previously described (17). In brief, the FLAG-tagged receptors were labeled with anti-FLAG M1 antibody, which was revealed by the Alexa-488 goat-anti-mouse secondary antibody and detected by Spectramax M2 fluorometer.

PKA Activity Measurement through FRET Assay

β₁AR-KO or β₁β₂AR-KO cardiac myocytes were infected with virus expressing A-kinase activity reporter (AKAR2.2) as previously described (18). For β₁β₂AR-KO myocytes, cells were also infected with receptor virus for overnight expression. The procedure for PKA FRET activity was carried out as previously described (18).

Statistical Analysis

Curve fitting and statistical analyses were performed using Prism software (GraphPad Software, Inc. San Diego, CA).

RESULTS

Stimulation of the β₂AR Induces an Agonist Concentration-dependent Receptor/G_i Coupling in Cardiac Myocytes

By activation of the endogenous β₂AR with Iso in β₁AR- KO cardiac myocytes, we analyzed the receptor-induced PKA activity through a FRET assay using the fluorescent biosensor AKAR2.2. Activation of the β₂AR with 10 nm Iso induced a transient peak in PKA activity, which returned to base-line levels within 5 min (Fig. 1A). Pretreatment with PTX, which specifically inhibits G_i proteins, did not significantly change the β₂AR-induced FRET response. However, a saturated concentration of 10 μm Iso resulted in a more sustained PKA FRET response, which was further enhanced with PTX pretreatment (Fig. 1B). This finding indicates that 10 μm Iso, but not 10 nm Iso, selectively induces β₂AR/G_i coupling for PKA activity in cardiac myocytes. We then used a cardiac contraction rate assay to examine the physiological consequences of this dose-dependent receptor/G_i protein coupling. We found that at 10 nm Iso, the β₂AR-induced increase in contraction rate was not PTX-sensitive (Fig. 1C), whereas PTX significantly enhanced the increase in contraction rate induced by 10 μm Iso (Fig. 1D). These data again suggest that the β₂AR couples to G_i proteins only after stimulation with high concentrations of agonist. Consistent with this notion, the PKA-mediated phosphorylation of phospholamban, a PKA substrate that is critical for the βAR-induced cardiac contractile response, was enhanced by PTX only after stimulation with 10 μm Iso (Fig. 1E). Previous reports have suggested that the PKA-mediated phosphorylation of the β₂AR enhances the receptor coupling to G_i protein (6, 7), and the GRK-dependent phosphorylation of the β₂AR occurs only after stimulation with high concentrations of agonist (13), making the phosphorylation of β₂AR a potential regulator of this dose-dependent G_i coupling in cardiac myocytes.

FIGURE 1.

Stimulation of the β₂AR induces agonist dose-dependent coupling to G_i in cardiac myocytes. A and B, β₁AR-KO cardiac myocytes expressing the PKA biosensor AKAR2.2 were stimulated with 10 nm (A) or 10 μm (B) Iso. Myocytes were treated with G_i protein inhibitor PTX for 2 h before stimulation to examine the effect on PKA FRET activity induced by agonist. C and D, β₁AR-KO cardiac myocytes were stimulated with 10 nm (C) or 10 μm (D) Iso with and without PTX pretreatment, and the increases in contraction rate over base-line levels were measured as a function of time. E, β₁AR-KO myocytes were stimulated with 10 nm or 10 μm Iso with and without PTX treatment. The phosphorylation of phospholamban was detected and normalized against total phospholamban (n = 4). **, p < 0.01 when compared with the group without PTX treatment by two-way ANOVA. ***, p < 0.001 when compared with the group without PTX treatment by Student's t test.

Agonist Stimulation Induces a Dose-dependent Distinct PKA and GRK Phosphorylation for β₂AR Internalization and Signaling in Cardiac Myocytes

Analysis of the phosphorylation of β₂AR in cardiac myocytes showed that the PKA-dependent phosphorylation at serines 261/262 occurred at subnanomolar concentrations of Iso and displayed a dose-dependent increase (Fig. 2, A and B). In contrast, the β₂AR phosphorylation by GRK at serines 355/356 occurred only at 100 nm Iso or at higher doses (Fig. 2, A and C). After stimulation with saturated 10 μm Iso, the kinetics of the GRK- and PKA-mediated phosphorylation of the β₂AR differed (Fig. 2D). The PKA-mediated phosphorylation showed a rapid increase, followed by a slow decrease, whereas the GRK-mediated phosphorylation was slower but more sustained over time (Fig. 2, E and F). It has been well documented that the GRK-mediated phosphorylation is involved in the endocytosis of β₂AR in fibroblasts (19, 20), prompting us to explore the role of β₂AR phosphorylation in receptor internalization in cardiac myocytes. The activated β₂ARs showed little punctuate staining upon stimulation with 10 nm Iso in cardiac myocytes (Fig. 3A). However, stimulation with 10 μm Iso induced a time-dependent clustering of β₂ARs in the cells, similar to those reported previously (8), which is a characteristic of receptor internalization (Fig. 3A). Moreover, the removal of Iso after 10 min of stimulation allowed the internalized receptor to return to the cell surface (Fig. 3A). These observations were further confirmed by quantification with a FLISA assay (Fig. 3B).

FIGURE 2.

Stimulation of the β₂AR induces agonist dose-dependent and dynamic receptor phosphorylation by PKA and GRK. A, the β₁β₂AR-KO cardiac myocytes expressing FLAG-β₂ARs were stimulated with different concentrations of Iso and blotted for phosphorylation by PKA (Ser(P)^261/262) or GRK (Ser(P)^355/356). The Western blots in A were quantified and plotted as -fold increases over base-line levels for the PKA-dependent phosphorylation (B) and GRK-dependent phosphorylation (C). D, myocytes expressing FLAG-β₂ARs were stimulated with 10 μm Iso for the indicated time to examine the PKA- and GRK-mediated phosphorylation. E and F, the Western blots in D were quantified and plotted as -fold increases over base-line levels. *, p < 0.05; **, p < 0.01 when compared with the base-line levels by one-way ANOVA. a.u., arbitrary units.

FIGURE 3.

Stimulation of the β₂AR induces agonist dose-dependent internalization in cardiac myocytes. A, the β₁β₂AR-KO cardiac myocytes expressing FLAG-β₂AR were stimulated with 10 nm or 10 μm Iso for 10 or 30 min or 10-min Iso stimulation followed by drug removal for 30 or 60 min. The FLAG-β₂ARs were visualized through immunofluorescence imaging. These data are representative of at least three experiments. B, the cell surface FLAG-β₂ARs were quantified by FLISA in HEK293 cells after stimulation up to 10 or 30 min or after 10 min Iso stimulation followed by drug removal for 30 or 60 min. Receptor levels were normalized against base-line levels (n = 5). **, p < 0.01 by Student's t test. Con, control.

We then directly tested the role of phosphorylation in receptor internalization and signaling by using mutant β₂ARs with the PKA or GRK phosphorylation sites replaced by alanines. The expressions of both PKAmut and GRKmut were quantified and equalized through Western blot and ligand binding (supplemental Fig. S1). Western blots of the mutant β₂ARs also confirmed abolishment of the specific receptor phosphorylation events upon stimulation with 10 μm Iso (supplemental Fig. S2). Immunostaining of the wild type and PKAmut β₂ARs showed time-dependent internalization of the receptors after stimulation with 10 μm Iso (Fig. 4A). However, the GRKmut β₂ARs failed to sufficiently internalize under Iso stimulation. Washout of Iso after 10 min of stimulation showed that the internalized receptors underwent recycling to the cell surface (Fig. 4B). Quantification through the FLISA assay confirmed that the wild type and PKAmut β₂ARs but not the GRKmut β₂ARs were significantly internalized upon 10 min of stimulation with 10 μm Iso (Fig. 4C), supporting the necessary role of GRK phosphorylation in the internalization of the β₂AR in cardiac myocytes. Together, the characterization of the mutant β₂ARs revealed distinct actions of PKA and GRK on the receptor phosphorylation and trafficking in cardiac myocytes upon agonist stimulation, deeming the phosphorylation-deficient mutants suitable for closer examination on the effects on the receptor signaling in these cells.

FIGURE 4.

Stimulation of wild type and mutant β₂ARs induces different receptor internalization and recycling. A, the FLAG-tagged β₂AR, PKAmut β₂AR, and GRKmut β₂AR expressed in cardiac myocytes were stimulated with 10 μm Iso for different times before they were visualized by immunofluorescence imaging. B, the wild type and mutant β₂ARs were visualized for recycling after stimulation for 10 min, followed by drug removal for the indicated times. These data are representative of at least three experiments. C, the cell surface wild type and mutant β₂ARs were quantified through FLISA after 10 min of Iso stimulation and recycling after drug removal for 30 and 60 min in HEK293 cells (n = 3). Receptor levels were normalized against base-line levels. **, p < 0.01 by Student's t test. Con, control.

By introducing either wild type or mutant β₂AR into β₁β₂AR-KO cardiac myocytes, we were able to examine the physiological effects of the phosphorylation of the β₂AR upon receptor activation. Stimulation of the PKAmut β₂AR induced a stronger contraction rate increase over that of the wild type (Fig. 5A). Stimulation of the GRKmut β₂AR showed a contraction rate increase similar to that of the wild type, but this increase was much more sustained (Fig. 5A). Although the base-line contraction rates between the wild type and mutant β₂ARs were not significantly different (Fig. 5B), the maximal contraction rate increase induced by the PKAmut β₂AR was significantly higher than that of wild type (Fig. 5C). These data strongly suggest that the phosphorylation of the β₂AR has significant consequences in regulating myocyte contraction rate responses.

FIGURE 5.

Activation of wild type and mutant β₂ARs induces distinct responses in contraction rates in cardiac myocytes. A, the β₁β₂AR-KO cardiac myocytes expressing β₂AR, PKAmut β₂AR, or GRKmut β₂AR were stimulated with 10 μm Iso for contraction rate measurement. The base-line levels of contraction rates (B) and the maximal increases in contraction rate after stimulation (C) were plotted. ***, p < 0.001 when compared with the wild type β₂AR by two-way ANOVA. *, p < 0.05 by Student's t test.

PKA and GRK Phosphorylation Differentially Regulate β₂AR/G_i Coupling in Cardiac Myocytes

By analyzing PKA activity in cardiac myocytes induced by these mutant β₂ARs, we were able to observe the kinetics of upstream signaling, which leads to contraction induced by the receptors. Stimulation of the wild type, PKAmut β₂AR, or GRKmut β₂AR with 10 μm Iso induced a stronger FRET increase in PKA activity, although the increase induced by the GRKmut β₂AR was less than those of the wild type and PKAmut receptors (Fig. 6A). Inhibition of G_i proteins with PTX pretreatment significantly enhanced the PKA FRET increase induced by activation of the wild type β₂AR (Fig. 6B). However, the FRET response induced by the PKAmut β₂AR was insensitive to PTX (Fig. 6C), indicating that this receptor was unable to couple to G_i protein. Interestingly, the FRET response induced by the GRKmut β₂AR was also PTX-insensitive (Fig. 6D), indicating that the GRK-dependent phosphorylation was necessary for β₂AR coupling to G_i protein.

FIGURE 6.

Wild type and mutant β₂ARs differentially couple to G_i proteins to regulate agonist-induced PKA FRET responses. A, the β₁β₂AR-KO cardiac myocytes expressing the PKA FRET biosensor AKAR2.2 together with the β₂AR, PKAmut β₂AR, or GRKmut β₂AR were stimulated with 10 μm Iso as indicated. The PKA FRET ratio was measured. B–D, myocytes were also treated with PTX to access the receptor/G_i coupling in regulating PKA FRET response after Iso stimulation. **, p < 0.01; ***, p < 0.001 when compared with the wild type receptor by two-way ANOVA. YFP, yellow fluorescent protein; CFP, cyan fluorescent protein.

Extending the PKA FRET results into the contraction rate assay reinforced the physiological effects of this phosphorylation-dependent G protein-coupling phenomenon. Although pretreatment with PTX significantly enhanced the wild type β₂AR-induced increase in contraction rate upon stimulation with 10 μm Iso (Fig. 7, A and E), it did not significantly affect the PKAmut β₂AR-induced increase in contraction rate (Fig. 7, B and E). Moreover, PTX did not enhance the GRKmut β₂AR-induced increase in contraction rate; instead, it lowered the contraction rate increase (Fig. 7, C and E), which could be in part due to the enhanced base-line contraction rate by PTX treatment (Fig. 7D). These data suggest that both PKA and GRK phosphorylation are required for β₂AR coupling to G_i protein, which thereafter affects myocyte contraction rate response.

FIGURE 7.

Wild type and mutant β₂ARs differentially couple to G_i proteins to regulate agonist-induced contraction rate increases. A–C, the β₁β₂AR-KO cardiac myocytes expressing the β₂AR, PKAmut β₂AR, or GRKmut β₂AR were stimulated with 10 μm Iso in the presence or absence of PTX. The response in myocyte contraction rate was measured. D and E, the base-line levels of contraction rates and the maximal increases in contraction rates after stimulation in the presence or absence of PTX were plotted. ***, p < 0.001 when compared with the group without PTX treatment by two-way ANOVA. *, p < 0.05 when compared with the group without PTX treatment in Student's t test.

We further employed a specific inhibitor of the G_i protein consisting of the C-terminal portion of Gα_i2 (G_iCT) (21) to probe receptor/G_i coupling by either wild type or mutant β₂AR. We first determined that infection with G_iCT virus at a multiplicity of infection of 100 optimally inhibited receptor/G_i coupling and enhanced the β₂AR-induced increase in contraction rate (Fig. 8A). We then introduced G_iCT along with wild type or mutant β₂AR into β₁β₂AR-KO cardiac myocytes. Overexpressing G_iCT significantly enhanced the wild type β₂AR-induced increase in contraction rate (Fig. 8, B and F), which was similar to that obtained with PTX treatment. However, G_iCT did not affect the PKAmut β₂AR-induced increase in contraction rate (Fig. 8, C and F). Similar to that of PTX treatment (Fig. 7C), inhibition of G_i protein with G_iCT also reduced the GRKmut β₂AR-induced increase in contraction rate (Fig. 8, D and F). Overexpression of G_iCT did not affect the base-line contraction rates (Fig. 8E). Together, we showed that inhibition of G_i with G_iCT significantly enhanced the maximal contraction rate induced by the wild type but not by the PKAmut and GRKmut β₂ARs.

FIGURE 8.

G_iCT inhibits β₂AR/G_i coupling for myocyte contraction rate response upon agonist stimulation. A, the maximal responses in contraction rate induced by the β₂AR were enhanced by expression of G_iCT along with the wild type β₂AR in β₁β₂AR-KO cardiac myocytes. B–D, the β₁β₂AR-KO cardiac myocytes expressing the β₂AR, PKAmut β₂AR, or GRKmut β₂AR were stimulated with 10 μm Iso in the presence or absence of G_iCT. The response in myocyte contraction rate was measured. E and F, the base-line levels of contraction rates and the maximal increases in contraction rates after stimulation in the presence or absence of G_iCT were plotted. **, p < 0.01; ***, p < 0.001 when compared with the wild type receptor by two-way ANOVA. *, p < 0.05 when compared with the group without G_iCT in Student's t test.

DISCUSSION

By measuring PKA activity and cardiac contraction rate increase, we show for the first time that β₂AR activation of G_i proteins occurs selectively at high concentrations but not at low concentrations of agonist. Moreover, we show that the receptor coupling to G_i proteins is differentially regulated by the PKA- and GRK-mediated phosphorylation of the activated β₂ARs. At both low and high concentrations of agonist, the activated β₂ARs undergo the PKA-mediated phosphorylation. In comparison, only high concentrations of agonist induce the GRK-mediated phosphorylation of the β₂ARs for subsequent internalization, which is also necessary for sufficient receptor coupling to G_i proteins (8, 9). Our studies link together various components of the β₂AR, including receptor phosphorylation, receptor trafficking, and differential receptor/G protein coupling in cardiac cells, which may allow the activation of the β₂AR signaling pathway to function as either a stimulatory or protective mechanism for cardiac cells under different levels of stress.

It has been well documented that the PKA-mediated phosphorylation of β₂AR enhances the receptor coupling affinity to G_i protein in a reconstituted system, and mutation of the PKA phosphorylation sites blocks receptor coupling to G_i in HEK293 fibroblasts (6, 7). In this study, we have further resolved the necessity of the PKA-dependent phosphorylation of β₂AR in coupling to G_i proteins in cardiac myocytes. Activation of the murine β₂AR by both low and high concentration of isoproterenol leads to phosphorylation of the receptor by PKA. At high concentrations of isoproterenol, the phosphorylation levels of the β₂AR by PKA are further enhanced in comparison with those at low concentrations. This is probably due to dissociation of phosphodiesterase 4D isoforms from the activated receptors (data not shown), which leads to higher local PKA activity for receptor phosphorylation. However, under stimulation with low concentrations of isoproterenol, the activated β₂ARs do not couple to G_i, even if the PKA-mediated phosphorylation occurs (Figs. 1 and 2). However, disruption of the PKA phosphorylation sites on the β₂AR with mutagenesis blocks the receptor coupling to G_i (Figs. 6–8). Together, these data indicate that PKA phosphorylation is necessary but not sufficient for the activated β₂AR coupling to G_i in cardiac myocytes.

In contrast, the β₂ARs undergo GRK-dependent phosphorylation only under stimulation with high concentrations of Iso (Fig. 2). Disruption of the GRK sites prolongs the Iso-induced myocyte contraction rate response (Fig. 5A), which is consistent with the notion that the phosphorylation of β₂AR by GRK is required for rapid receptor desensitization (22). We do not observe obvious change in the overall PKA activity induced by the GRKmut (Fig. 6A), probably because the change is small and occurs in the local environment of the receptor and thus is not sensed by PKA probes presented throughout the cytosol. However, similar to those of the PKAmut, the PKA FRET and contraction rate responses induced by the GRKmut β₂AR do not show sensitivity to the inhibition of G_i protein (Figs. 6–8), indicating that the GRK-mediated phosphorylation is required for sufficient coupling to G_i. One possibility is that the GRK-phosphorylated receptors undergo internalization to promote the access of the receptor to G_i protein. Another possibility is that the GRK-mediated phosphorylation of the β₂AR directly enhances the binding affinity of the receptor to G_i protein. However, evidence so far argues that the GRK phosphorylation-dependent receptor trafficking is more likely the key for sufficient receptor/G_i coupling. To support this notion, either blocking receptor internalization or disrupting receptor recycling through point mutation on the PDZ motif of the β₂AR blocks the receptor/G_i coupling, which yields enhanced myocyte contraction responses (9, 15). Moreover, Wang et al. (8) have shown that direct inhibition of GRK2 activity blocks the receptor coupling to G_i proteins in myocytes. Furthermore, the GRK phosphorylation-dependent recruitment of arrestin-PDE4D complexes to the activated β₂AR has been implicated in receptor/G_i coupling (23). These studies, together with our data, suggest that the GRK-mediated phosphorylation of β₂AR and subsequent receptor trafficking are critical for sufficient access of the receptor to G_i proteins. Consistent with this notion, a G_i-specific inhibitor, G_iCT, selectively blocks the receptor coupling to G_i without affecting the receptor coupling to G_s. These data suggest that the G_iCT peptide did not compete against the unphosphorylated receptors in binding to G_s protein but blocks the phosphorylated receptor in binding to G_i protein, which reinforces the idea that β₂AR/G_i coupling is dependent on the agonist-induced phosphorylation of the receptor. Together, our data suggest that although the PKA-mediated phosphorylation is required for high affinity binding to G_i protein, the GRK-mediated phosphorylation is necessary for receptor trafficking that appears to control the accessibility of β₂AR to G_i protein in cardiac myocytes.

Cardiac contraction in response to adrenergic stimulation is a tightly controlled process to ensure steady increases in cardiac output without overstimulation. Our observation of an agonist concentration-dependent switch of the activated β₂AR coupling from G_s and G_i may be critical for such fine regulation. At low concentrations of catecholamines, the activated receptors will couple only to G_s for a stimulatory effect on cAMP/PKA activities for increasing cardiac contraction. However, in the presence of high concentrations of catecholamines, the activated β₂ARs will switch from G_s to G_i to reduce cAMP/PKA activities in cardiac tissues while preventing the overstimulation of cardiac contraction. The mechanism for how high concentrations of agonists can induce receptor phosphorylation by different kinases for subsequent switch of G protein coupling is currently not known. Structural studies reveal that higher concentrations of agonists induce conformational changes distinct from those induced by low concentrations (17), which may enhance the receptor binding affinity to GRK. Alternatively, low concentrations may selectively activate the G_s protein-precoupled pool of receptors with high affinity binding, whereas higher concentrations of agonists activate both precoupled and non-coupled pools of receptors. The latter pool of receptors may be involved in coupling to G_i proteins. The mechanism thus remains to be addressed. This protective mechanism by receptor/G_i coupling may be essential for preventing a high frequency of contractions, such as fibrillation during stress. In addition, β₂AR/G_i coupling can play a protective role against the apoptotic effects induced by adrenergic/G_s stimulation (24–27).

More than a simple extension of the biochemical characterization of the β₂AR, this study provides insightful information on the physiological effects of receptor phosphorylation by PKA and GRK. More importantly, it opens up questions about the nature of phosphorylation and its role in receptor desensitization, a concept that has been established in the last couple of decades. Undoubtedly, phosphorylation seems to play a role in diminishing the effects of receptor activation in cardiac myocytes, since abolishing those phosphorylation sites enhanced the contraction rate over the wild type upon stimulation (Fig. 4). However, we find that receptor phosphorylation appears to be critical in regulating contraction through coupling to different G proteins to protect cardiac cells from overwhelming agonist stimulation. Our study provides a clear step toward understanding the nature of receptor regulation by PKA- and GRK-mediated phosphorylation in a physiological context.