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the Laboratory of Cardiovascular Science (W.-Z.Z., K.C., S.Z., D.Y., E.G.L., H.C., R.-P.X.), Gerontology Research Center, National Institute on Aging, Baltimore, Md
Centre de recherche (C.L.), Institut de Cardiologie de Montreeal, Canada
Deepartement de biochimie (C.L., T.E.H., M.B.), Universitee de Montreeal, Canada
the Institute of Molecular Medicine (R.-P.X.), Peking University, Beijing, China.
Abstract
Intermolecular interactions between members of both similar and divergent G protein-coupled receptor subfamilies have been shown in various experimental systems. Here, we demonstrate heterodimerization of predominant -adrenergic receptor (AR) subtypes expressed in the heart, 1AR, and 2AR, and its physiological relevance. In intact adult-mouse cardiac myocytes lacking native 1AR and 2AR, coexpression of both AR subtypes led to receptor heterodimerization, as evidenced by their coimmunoprecipitation, colocalization at optical resolution, and markedly increased binding affinity for subtype-selective ligands. As a result, the dose-response curve of myocyte contraction to AR agonist stimulation with isoproterenol (ISO) was shifted leftward by 1.5 orders of magnitude, and the response of cellular cAMP formation to ISO was enhanced concomitantly, indicating that intermolecular interactions of AR subtypes resulted in sensitization of these receptors in response to agonist stimulation. In contrast, the presence of 1AR greatly suppressed ligand-independent spontaneous activity of coexisting 2ARs. Thus, heterodimerization of 1AR and 2AR in intact cardiac myocytes creates a novel population of ARs with distinct functional and pharmacological properties, resulting in enhanced signaling efficiency in response to agonist stimulation while silencing ligand-independent receptor activation, thereby optimizing -adrenergic modulation of cardiac contractility.
Key Words: receptor dimerization -adrenergic receptor G protein-coupled receptors cardiac contractility ligand binding
Introduction
G protein-coupled receptors (GPCRs) represent the largest family of transmembrane molecules involved in cell signal transduction. GPCRs have traditionally been thought to function as monomers, but increasing evidence suggests that GPCRs may exist as both homodimers or heterodimers.1eC7 The idea that GPCRs might undergo dimerization was first proposed in 1982.8 The physical interaction of GPCRs within or among different families leads to a multitude of changes in ligand binding and signaling properties of these receptors.1eC7,9eC11 However, a close inspection of previous studies reveals that most have been conducted in naive cell lines or in vitro experimental settings. An important question is, however, whether GPCR dimerization occurs in a physiological context such as the intact cardiomyocyte and, if so, whether this has physiological or pathophysiological relevance.
As prototypical members of the GPCR superfamily, -adrenergic receptors (AR) consist of 3 pharmacologically and genetically distinct subtypes, 1AR, 2AR, and 3AR, which are often coexpressed in many types of cells and tissues. In cardiomyocytes, mainly 1AR and 2AR subtypes are coexpressed and fulfill distinct functional roles via activation of subtype-specific signaling pathways.12 Our previous studies have shown that heterodimerization between 1AR and 2AR subtypes inhibits 2AR internalization and its ability to activate ERK1/2 MAPK signaling in HEK293 cells.11 The present study is aimed to characterize potential heterodimerization of these AR subtypes in the heart and its impacts on the functional and signaling properties of these receptors. To create "pure" 1AR, 2AR, or 12 coexistent systems with a matched total receptor density, we expressed either the mouse 1AR or 2AR, or both subtypes in cardiomyocytes from the adult 1AR and 2AR double knockout (12AR DKO) mice,13 in conjunction with adenoviral gene transfer techniques.14
Our results indicate that 1AR and 2AR are able to form heterodimers in intact cardiomyocytes, and that the heterodimeric receptors exhibit altered ligand binding profiles, enhanced signaling efficiency in regulating myocyte cAMP production and contractility, and suppressed 2AR spontaneous activity in the absence of agonist stimulation. Thus, we conclude that 1AR and 2AR heterodimerization is required for optimal -adrenergic regulation of cardiac contractility.
Materials and Methods
Cardiac Myocyte Adenoviral Infection and Cell Contraction Measurement
Single cardiomyocytes were isolated from the hearts of 2- to 3-month-old male 12AR DKO or 1AR KO or wild-type (WT) mice with an enzymatic technique, then cultured and infected with adenoviral vectors for 24 hours, as described previously.14 Cultured cells were then perfused with a HEPES-buffered solution (in mmol/L: NaCl 137, KCl 5.4, MgCl2 1.2, NaH2PO4 1, CaCl2 1, glucose 20, and HEPES 20, pH 7.4), and electrically stimulated at 0.5 Hz at 23°C. Cell contraction was measured by the percent shortening of cell length in response to electrical stimulation.15
cAMP Measurement
Intracellular cAMP levels were assayed by radioimmunoassay, as previously described.16 Briefly, cultured mouse cardiomyocytes were incubated with isoproterenol (ISO) for 10 minutes, and cellular cAMP formation was determined using a radioimmunoassay kit from Amersham with a duplicate in each experiment.
Radioligand-Binding Assay
As described previously,16 binding assays were performed on 25 e of membrane proteins using saturating amounts of the AR specific ligand [125I]cyanopindolol (125I-CYP). Nonspecific binding was determined in the presence of 20 eol/L propranolol. Bmax for ICYP were determined by Scatchard analysis of saturation binding isotherms. Data of competition experiments were analyzed using 1- or 2-site competition binding curves with GraphPad PRISM.14,16
Immunocytochemical Staining and Confocal Imaging
Immunocytochemical staining and confocal imaging were performed in 12AR DKO cells infected by either adv-1AR tagged with hemagglutinin (HA), or adv-2AR, or a combination of both, at multiplicity of infection (moi) 100 for 24 hours, as described previously.16 Horse anti-mouse IgG secondary antibodies and goat anti-rabbit IgG secondary antibodies were used for 1AR and 2AR staining, respectively. Immunofluorescence was then detected by a confocal microscope (LSM-510, Zeiss) with an optical section thickness of 1.0 e.
Coimmunoprecipitation and Western Blotting
Myocytes expressing HA-tagged 1AR, Flag-tagged 2AR, or both receptors were lysed in RIPA buffer (in mmol/L: 50 Tris pH 7.4, 150 NaCl, 20 -glycerophosphate, 20 NaF, 0.2 Na3VO4, 5 EDTA, 5 EGTA, 10 benzamidine, 0.5 PMSF, 1 PMSF, 25 e/mL leupeptin, 1% Triton X-100, and 0.5% sodium deoxycholate) for 30 minutes at 4°C. For immunoprecipitation, 100 to 200 e of protein was incubated with 1 to 2 e of anti-Flag (1:100) or anti-HA (1:100) overnight at 4°C to pull-down Flag-tagged 2AR or HA-tagged 1AR, respectively. Immunocomplexes were isolated by incubation with 10% vol/vol protein G-Sepharose for 2 to 3 hours. The immunoprecipitate was then treated with 100 mmol/L DTT in the sample buffer and subjected to SDS/PAGE and Western blotting to detect the presence of 1AR or 2AR with the anti-HA monoclonal antibody or the anti-Flag antibody, respectively. In addition, we have quantified the relative percentage of heterodimer of 1/2AR (pull-down with anti-HA antibody or anti-Flag antibody, normalized by anti-body pull-down efficiency) compared with the total 1/2AR pool (cell lysate).
Materials
Unless otherwise indicated, all chemicals were purchased from Sigma. [125I]Cyanopindolol was purchased from NEN Life Science Products, Inc. (Boston, Mass). Anti-HA monoclonal antibody, anti-Flag, and 2AR polyclonal antibody were purchased from Berkeley Antibody Co. (Berkeley, Calif) and Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif), respectively. The secondary antibodies were purchased from Vector Laboratories (Burlingame, Calif).
Statistical Analysis
Data were expressed as mean±SE. Statistical comparisons used 1-way ANOVA followed by the Bonferroni procedure for multiple-group comparisons. A P<0.05 was considered statistically significant.
Results
Colocalization of AR Subtypes in Cardiomyocytes
To investigate possible intermolecular interactions between 1AR and 2AR subtypes, we expressed either or both AR subtypes in cultured ventricular myocytes from 12AR DKO mice using adenovirus-mediated gene transfer at moi of 100. After 24 hours infection, the densities of 1AR and 2AR were comparable to that of cells expressing both AR subtypes (Table 1). Using confocal immunocytochemical imaging, we visualized that the specific immunofluorescence of 1AR or 2AR was largely concentrated on cell surface membranes, including transverse tubules, with enriched staining of the perinuclear area (Figure 1A). An overlay of the images of HA-1AR and 2AR revealed an excellent pixel-to-pixel correlation (r2=0.78, Figure 1B), an indication of colocalization of 1AR and 2AR at optical resolution.
Coimmunoprecipitaiton of 1AR and 2AR
To directly demonstrate physical association of AR subtypes, we expressed HA-tagged 1AR or Flag-tagged 2AR or both in the null background of DKO myocytes and then performed immunoprecipitation and Western blot assays. Total cellular proteins containing either or both AR subtypes were first immunoprecipitated with a rabbit polyclonal anti-Flag antibody. The pull-down of Flag-tagged 2AR in the immunoprecipitate was confirmed by Western blot using the anti-Flag antibody (Figure 1C, top). Notably, the presence of 1AR in the immunoprecipitate was detected by Western blot using a mouse monoclonal anti-HA antibody (Figure 1C, bottom). Three major species (Mr: 52 kDa, 70 kDa, and 150 kDa) were visualized with the Western blot. Three similar immunoreactive species (Mr: 50, 70, 150 kDa) were illustrated by Western blot using anti-HA in the total extracts from cells expressing HA-1AR (Figure 1D). The 50 kDa species likely represents the monomeric form of HA-1AR, and the 150 kDa form likely represents SDS-resistant homodimeric receptors (or oligomers). The 70-kDa species might represent the monomeric core glycosylated form of HA-1-AR. As a negative control, there was no detectable HA-immunoreactivity in myocytes expressing either 2AR alone or -gal (Figure 1D). Conversely, we performed the coimmunoprecipitation experiments with the anti-HA antibody to pull-down 1ARs and detected coimmunoprecipitated Flag-tagged 2ARs by Western blot using rabbit polyclonal anti-Flag (Figure 1E). The specificity of anti-Flag was confirmed by the immunoreactive signals in the total extracts from cells expressing Flag-2AR but not in those expressing HA-1AR alone or -gal (Figure 1F). In an attempt to quantify the relative proportions of receptor heterodimers, we determined the pull-down efficiency of each antibody (by comparing pull-down with whole cell lysate) and measured the relative amount of the other tagged receptor coimmunoprecipitated. Heterodimers represented 18.9±4.6% (n=3) and 21.0±5.6% (n=3) of the total 1ARs and 2AR populations, as indexed by their coimmunoprecipitation. These results indicate that intermolecular interactions occur between 1AR and 2AR in adult-mouse cardiomyocytes.
Suppression of Spontaneous 2AR Activation by 1AR Coexpression
To determine the functional consequences of 1AR-2AR heterodimerization in adult-mouse ventricular myocytes, we first examined constitutive AR activity in the absence of agonist stimulation. The baseline contractility of cells expressing 2AR was enhanced by 1.6-fold relative to myocytes expressing 1AR or those uninfected cells from WT or DKO mice (Figure 2A). In contrast, expression of 1AR at a receptor density that matched the 2AR density did not alter basal ligand-independent myocyte contraction amplitude (Figure 2A). ICI 118 551 (ICI, 5x10eC7 mol/L), a 2AR inverse agonist, completely reversed the enhanced basal contraction (Figure 2B), without altering the baseline contraction in cells expressing 1AR (data not shown). These results are consistent with the previous notion that 2AR,16eC20 but not 1AR,16,21 exhibits spontaneous activity. Surprisingly, the Adv-2AR infection-induced augmentation in the baseline contractility was fully prevented when cells were coinfected with Adv-1AR (50 moi for each) (Figure 2A). This was not caused by a reduction in 2AR subtype density, because Adv-2AR alone (50 moi) significantly elevated, in an ICI-sensitive manner, the baseline contractility relative to that of uninfected WT or DKO cells or Adv-1AR infected myocytes (Figure 2A). It is noteworthy that there was no significant difference in the baseline contraction amplitude between WT and 1AR KO groups (3.6±0.7 and 3.7±0.5% of resting cell length, n=35 and 38 cells, respectively) (Figure 2A). This might be explained by the relatively modest density of the native 2ARs in the 1AR KO cells, because the spontaneous activity of the 2AR is receptor density-dependent.16eC20
Figure 2B shows representative examples of the inhibitory effect of ICI in a cell expressing either 2AR or both AR subtypes (top and bottom, respectively). Clearly, coexpression of 1AR virtually abolished spontaneous 2AR activity, as manifested by the inability of the 2AR inverse agonist to reduce basal myocyte contraction (Figure 2A and 2B).
Heterodimerization of AR Subtypes Enhances Cardiomyocyte Contractile Response to AR Agonist Stimulation
Next, we determined the potential impact of coexpression of these receptors on the myocyte contractile response to agonist-induced AR stimulation. In cells expressing either 1AR or 2AR, stimulation of these AR subtypes with the same agonist, isoproterenol (ISO), produced comparable maximal contractile responses despite their distinct basal contraction amplitudes (Figure 3A). When the concentration-response curves were normalized by their corresponding basal level or maximal response (Figure 3B and Figure 3C, respectively), it is clear that the concentration-response curves of 1AR- and 2AR-mediated increases in myocyte contractility virtually overlapped with each other with pD2 (-log EC50) of 9.02±0.03 and 8.23±0.29 (n=6 to 8 for both groups), respectively (Figure 3B and 3C). However, in myocytes coexpressing 1AR and 2AR at a similar total receptor density, the concentration-response curve of ISO-induced relative increase in myocyte contractility was shifted leftward by 1.5 orders of magnitude (pD2 10.41±0.49; P<0.01 versus the 1AR or the 2AR group). Thus, AR subtype heterodimerization sensitizes the contractile response to ligand-induced receptor stimulation in cardiac myocytes.
Cellular cAMP Responses in 1AR- or 2AR- or Mixed 12AR-Expressing Cardiomyocytes
Because both 1AR- and 2AR-induced positive inotropic effects are mediated by a cAMP-dependent mechanism,21eC23 we next investigated the cAMP response to AR stimulation in DKO cardiomyocytes expressing either or both AR subtypes. Compared with that of uninfected WT or DKO cells, the baseline cAMP level was unchanged in myocytes expressing 1AR, but augmented by 2.1-fold in cells infected with Adv-2AR (100 moi) (Figure 4A), caused by spontaneous 2AR activity.16eC20 Coexpression of the 1AR with 2AR fully suppressed the 2AR-induced increase in basal cAMP production (Figure 4A), as was the case for the baseline contractility (Figure 2). The absolute increase in cAMP formation in response to ISO in cells expressing 2AR was increased versus that in cells expressing 1AR (Figure 4B). However, the relative response of cAMP formation (% of basal level) to ISO was greater in 1AR-expressing cells compared with that in those expressing 2AR (Figure 4C). Remarkably, when these AR subtypes were coexpressed in 12AR DKO cardiomyocytes at matched levels of total receptor expression, the ISO-induced absolute or relative increase in cAMP formation was almost 2-fold greater than that in cells expressing either 1AR or 2AR alone (Figure 4B and 4C), consistent with the profile of myocyte contractile response to either spontaneous or ligand-induced AR subtype activation.
Ligand Binding Profiles of 1AR, 2AR, and Coexpressed AR Subtypes
In addition to the aforementioned immunocytochemical, physiological, and biochemical data, differences in ligand-receptor interactions would provide strong pharmacological evidence for receptor dimerization. In this regard, we examined ligand-binding profiles in WT or in DKO mouse cardiomyocytes when AR subtypes were individually expressed or coexpressed. There were 2 AR subpopulations in WT mouse heart with 73.6±2.7% and 26.4±2.9% for 1AR for 2AR, respectively. Most importantly, radioligand binding assays revealed that the binding affinity of separately expressed 1AR or 2AR in DKO cells for their selective ligands, CGP 20712A or ICI 118 551, was reduced by 30-fold and 10-fold, respectively, compared with that in WT cells or DKO myocytes expressing both AR subtypes (Table 2). The coexpression-induced increase in the binding affinity for subtype-selective ligands was not influenced by the absolute densities or the ratio of these AR subtypes, because it occurred in both WT and coinfected DKO mouse myocytes regardless of their different densities or the ratio of the coexisting AR subtypes (Tables 1 and 2 ). That coexpression enhances the affinity of both 1AR and 2AR for their selective ligand binding further supports the notion that of these receptors form heterodimers in intact adult-mouse cardiomyocytes.
Discussion
1AR and 2AR Heterodimerization Underlying Altered Signaling Properties of These Receptors
In the present study, using immunocytochemical, biochemical, physiological, and pharmacological approaches, we have provided the first documentation of intermolecular interactions between 1AR and 2AR in intact adult mouse cardiac myocytes. Heterodimerization of these receptors suppresses constitutive ligand-independent 2AR signaling, but facilitates receptor-ligand interactions, thereby increasing the signaling efficiency of both AR subtypes and optimizing AR-mediated modulation of cardiac contractility. This conclusion is based on several independent lines of evidence. First, when coexpressed in adult-mouse cardiac myocytes lacking native ARs, 1AR and 2AR are physically associated with each other, as manifested by their coimmunoprecipitation and intracellular colocalization at optical resolution. Second, coexpression of 1AR and 2AR creates a novel population of ARs endowed with increased binding affinity for subtype-specific ligands regardless of the total receptor density or the ratio of these receptor subtypes (Tables 1 and 2 ). Perhaps most importantly, the presence of 1AR fully quenches the spontaneous activity of coexpressed 2ARs, as evidenced by a full reversal of agonist-independent 2AR-mediated augmentation in basal myocyte cAMP production and contractility. This finding provides compelling evidence for constitutive intermolecular interactions between these AR subtypes, because downstream regulatory events are unlikely to be involved in the absence of agonist stimulation. The enhanced ligand binding might contribute, at least in part, to the sensitization of myocyte contractile and cAMP responses to AR agonist stimulation (Figures 3 and 4 HREF="#FIG4">).
Interestingly, there are important differences between the present study in native cardiac myocytes and our previous studies performed in HEK 293 cells.11,24,25 Specifically, high-affinity binding to subtype-selective ligands was reduced, rather than increased, in HEK 293 cells coexpressing both 1AR and 2AR relative to those expressing a single subtype.25 Moreover, coexpression of both AR subtypes is not associated with an increased cAMP formation in response to agonist stimulation in HEK 293 cells.11 These obvious discrepancies between intact cardiomyocytes and naive cells underscore that cell-specific factors certainly influence the properties of receptor signaling, and highlights the importance and necessity to validate these issues in a native cellular context.
Optimizing AR-Mediated Modulation of Cardiac Contractility by Subtype Heterodimerization
Although a large number of other GPCRs have been previously reported to undergo homodimerization or heterodimerization,1eC10 the present study provides the first demonstration that 1AR and 2AR, prototypical members of the GPCR superfamily, form heterodimers in the physiological context of intact cardiac myocytes. When 1AR and 2AR are coexpressed, myocyte contractile or cAMP response to AR agonist stimulation is profoundly sensitized compared with the "pure" 1AR or 2AR system, indicating that 1AR and 2AR interact in a synergistic fashion. Similar functional synergy between 1AR and 2AR in regulating cAMP production has been previously reported in cultured rat C6 glioma cells.26 These present findings might imply that in cardiac myocytes, the native 1AR and 2AR may require mutual support from each other to maintain optimal sympathetic control over heart rate and myocardial contractile performance, allowing the heart to increase its output several times within seconds in response to a "fight-or-flight" situation. In this regard, previous studies have demonstrated that in mice lacking native 1AR, stimulation of the native cardiac 2AR with ISO was unable to elicit a positive inotropic effect in vivo.27 This further supports the perception that 1AR and 2AR exhibit a synergistic interaction in their modulation of cardiac contractility.
It is noteworthy that heterodimerization of 1AR and 2AR, although enhancing agonist-induced signaling, silences the spontaneous activation of 2AR, suggesting that heterodimerization might mutually stabilize both receptor subtypes in their respective inactive conformations in the absence of agonist. As a result, it might reduce the signaling background, but optimize the responsiveness of dimeric receptors to agonist stimulation, thus further synchronizing the sympathetic control over cardiac performance in response to exercise or stress. The exact mechanism underlying the inhibitory effect of 1AR on 2AR spontaneous activation awaits future investigation. Altogether, our present and previous studies11,24,25 have demonstrated that intermolecular interactions between 1AR and 2AR create a new population of receptors in terms of their pharmacology, trafficking, signaling, and functionality.
It has been shown that during the progression to heart failure caused by a variety of etiologies, there is a selective downregulation of 1AR with little or no change in 2AR density.28eC30 The heart failure-associated decrease in the ratio of 1AR to 2AR might alter the heterodimerization of the remaining ARs, thus contributing to the diminution of AR contractile support or an upregulation of anti-apoptotic 2AR signaling.31eC33 These hypotheses merit further investigation.
Heterodimerization Between AR and Members From Other GPCR Families
In addition to the complicated impacts of heterodimerization of the closely related 1AR and 2AR on their trafficking and signaling properties, recent studies have revealed evidence for heterodimerization of 2AR with other members of adrenergic receptor family, including 3AR, 2AAR, and 1DAR, in HEK 293 cells.34eC36 Interestingly, whereas either 2AR or 3AR alone couples to both Gs and Gi proteins, the 2AR-3AR heterodimer is unable to activate Gi signaling.34 Equally appealing, the heterodimerization of 2AR with either 2AAR or 1DAR leads to cross-internalization of the receptors on agonist stimulation of either 2AR or the AR subtypes,35,36 and enables 1DAR to regulate intracellular Ca2+ mobilization in response to agonist stimulation.36 Moreover, oligomerization of opioid peptide receptors with 2AR also alters receptor trafficking and signal transduction.37eC39 Additionally, it has been demonstrated that intermolecular interactions between AR and angiotensin II type 1 receptor (AT1R) occurring in the heart leads to a cross-inhibition of their downstream signaling and trafficking by either type of receptor antagonist.40 Thus, oligomerization of GPCRs from the same or different families not only increases the complexity of GPCR signaling and their functional diversity, but also raises important therapeutic considerations.
In summary, the present results indicate that the 1AR and 2AR are able to form heterodimers in adult-mouse cardiomyocytes, and that the heterodimeric receptors exhibit altered pharmacological and signaling properties, resulting in more potent cAMP and contractile responses to agonist stimulation, while silencing ligand-independent spontaneous 2AR activity. The heterodimeric 1AR-2AR may represent a pharmacologically and functionally distinct population of ARs. Thus, many well-established paradigms for AR signaling and function may need to be revisited in the context of the coexistence of multiple receptor subtypes and their homo- or heterodimerization.
Acknowledgments
This work is supported by the National Institutes of Health intramural research grant (W.Z.Z., K.C., S.J.Z., D.Y., H.C., E.G.L., and R.P.X.), and in part by Chinese National Natural Science Foundation (30100215), Peking University 985 Project, Chinese National Key Project 973 (G2000056906), and Chinese Young Investigator Award (30225036). T.E.H. is a MacDonald Scholar of the Heart and Stroke Foundation of Canada (HSFC). The authors would like to thank Dr Brian K. Kolbilka at Stanford University School of Medicine for kindly providing mice lacking native 1AR or both 1AR and 2AR. The authors are also grateful to Dr Hal Spurgeon and B. Ziman for their excellent technique support.
This manuscript was sent to H. Michael Piper, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
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