This Article Abstract Full Text (PDF) All Versions of this Article: 97/3/244    most recent 01.RES.0000176764.38934.86v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhu, W.-Z. Articles by Xiao, R.-P. Search for Related Content PubMed PubMed Citation Articles by Zhu, W.-Z. Articles by Xiao, R.-P. Related Collections Signal transduction Cardiovascular Pharmacology Receptor pharmacology

(Circulation Research. 2005;97:244.)
© 2005 American Heart Association, Inc.

Cellular Biology

Heterodimerization of ß₁- and ß₂-Adrenergic Receptor Subtypes Optimizes ß-Adrenergic Modulation of Cardiac Contractility

Wei-Zhong Zhu, Khalid Chakir, Shengjun Zhang, Dongmei Yang, Catherine Lavoie, Michel Bouvier, Terence E. Hébert, Edward G. Lakatta, Heping Cheng, Rui-Ping Xiao

From 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 Montréal, Canada; Département de biochimie (C.L., T.E.H., M.B.), Université de Montréal, Canada; and the Institute of Molecular Medicine (R.-P.X.), Peking University, Beijing, China.

Correspondence to Dr Rui-Ping Xiao, Laboratory of Cardiovascular Science, Gerontology Research Center, NIA, NIH, 5600 Nathan Shock Dr, Baltimore, MD 21224. E-mail xiaor{at}grc.nia.nih.gov

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 ß-adrenergic receptor (ßAR) subtypes expressed in the heart, ß₁AR, and ß₂AR, and its physiological relevance. In intact adult-mouse cardiac myocytes lacking native ß₁AR and ß₂AR, coexpression of both ßAR 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 ßAR 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 ßAR subtypes resulted in sensitization of these receptors in response to agonist stimulation. In contrast, the presence of ß₁AR greatly suppressed ligand-independent spontaneous activity of coexisting ß₂ARs. Thus, heterodimerization of ß₁AR and ß₂AR in intact cardiac myocytes creates a novel population of ßARs with distinct functional and pharmacological properties, resulting in enhanced signaling efficiency in response to agonist stimulation while silencing ligand-independent receptor activation, thereby optimizing ß-adrenergic modulation of cardiac contractility.

Key Words: receptor dimerization • ß-adrenergic receptor • G protein-coupled receptors • cardiac contractility • ligand binding

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.^1–7 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.^1–7,9–11 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, ß-adrenergic receptors (ßAR) consist of 3 pharmacologically and genetically distinct subtypes, ß₁AR, ß₂AR, and ß₃AR, which are often coexpressed in many types of cells and tissues. In cardiomyocytes, mainly ß₁AR and ß₂AR subtypes are coexpressed and fulfill distinct functional roles via activation of subtype-specific signaling pathways.¹² Our previous studies have shown that heterodimerization between ß₁AR and ß₂AR subtypes inhibits ß₂AR 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 ßAR subtypes in the heart and its impacts on the functional and signaling properties of these receptors. To create "pure" ß₁AR, ß₂AR, or ß₁ß₂ coexistent systems with a matched total receptor density, we expressed either the mouse ß₁AR or ß₂AR, or both subtypes in cardiomyocytes from the adult ß₁AR and ß₂AR double knockout (ß₁ß₂AR DKO) mice,¹³ in conjunction with adenoviral gene transfer techniques.¹⁴

Our results indicate that ß₁AR and ß₂AR 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 ß₂AR spontaneous activity in the absence of agonist stimulation. Thus, we conclude that ß₁AR and ß₂AR heterodimerization is required for optimal ß-adrenergic regulation of cardiac contractility.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cardiac Myocyte Adenoviral Infection and Cell Contraction Measurement
Single cardiomyocytes were isolated from the hearts of 2- to 3-month-old male ß₁ß₂AR DKO or ß₁AR 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 µg of membrane proteins using saturating amounts of the ßAR specific ligand [¹²⁵I]cyanopindolol (¹²⁵I-CYP). Nonspecific binding was determined in the presence of 20 µmol/L propranolol. B_max 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 ß₁ß₂AR DKO cells infected by either adv-ß₁AR tagged with hemagglutinin (HA), or adv-ß₂AR, 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 ß₁AR and ß₂AR staining, respectively. Immunofluorescence was then detected by a confocal microscope (LSM-510, Zeiss) with an optical section thickness of 1.0 µm.

Coimmunoprecipitation and Western Blotting
Myocytes expressing HA-tagged ß₁AR, Flag-tagged ß₂AR, or both receptors were lysed in RIPA buffer (in mmol/L: 50 Tris pH 7.4, 150 NaCl, 20 ß-glycerophosphate, 20 NaF, 0.2 Na₃VO₄, 5 EDTA, 5 EGTA, 10 benzamidine, 0.5 PMSF, 1 PMSF, 25 µg/mL leupeptin, 1% Triton X-100, and 0.5% sodium deoxycholate) for 30 minutes at 4°C. For immunoprecipitation, 100 to 200 µg of protein was incubated with 1 to 2 µg of anti-Flag (1:100) or anti-HA (1:100) overnight at 4°C to pull-down Flag-tagged ß₂AR or HA-tagged ß₁AR, 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 ß₁AR or ß₂AR with the anti-HA monoclonal antibody or the anti-Flag antibody, respectively. In addition, we have quantified the relative percentage of heterodimer of ß₁/ß₂AR (pull-down with anti-HA antibody or anti-Flag antibody, normalized by anti-body pull-down efficiency) compared with the total ß₁/ß₂AR 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 ß₂AR 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
Colocalization of ßAR Subtypes in Cardiomyocytes
To investigate possible intermolecular interactions between ß₁AR and ß₂AR subtypes, we expressed either or both ßAR subtypes in cultured ventricular myocytes from ß₁ß₂AR DKO mice using adenovirus-mediated gene transfer at moi of 100. After 24 hours infection, the densities of ß₁AR and ß₂AR were comparable to that of cells expressing both ßAR subtypes (Table 1). Using confocal immunocytochemical imaging, we visualized that the specific immunofluorescence of ß₁AR or ß₂AR 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-ß₁AR and ß₂AR revealed an excellent pixel-to-pixel correlation (r²=0.78, Figure 1B), an indication of colocalization of ß₁AR and ß₂AR at optical resolution.

View this table: [in this window] [in a new window]   Table 1. Table 1. Densities and Ratios of the Coexpressed ß1AR:ß2AR in WT, ß1AR KO or Adenovirus-Transfected DKO Mouse Cardiomyocytes

View larger version (42K): [in this window] [in a new window]   Figure 1. Colocalization and coimmunoprecipitation of ßAR subtypes in cardiac myocytes. A, Intracellular distribution of HA-tagged mouse ß1AR or untagged ß2AR in adult DKO mouse cardiac myocytes infected by both adv-HA-ß1AR and adv-ß2-AR (moi 50 for each). B, Pixel-to-pixel correlation of ß1AR and ß2AR immunofluorescence. Color encodes the number of pixels corresponding to each data point. The line refers to a linear regression of the data (r2=0.78). Negative controls (in the absence of primary antibody) showed negligible immunofluorescence (not shown). C, Coimmunoprecipitation of coexpressed ßAR subtypes. Myocytes were infected with adenoviruses encoding HA-ß1AR or Flag-ß2AR individually or in combination for 24 hours. ß2ARs were immunoprecipitated using a rabbit polyclonal anti-Flag antibody and Western blots were performed to confirm pull-down of Flag-tagged ß2AR in the inmmunoprecipitates. The presence of HA-ß1AR in the immunoprecipitate was detected by Western blot using a mouse monoclonal anti-HA antibody. D, Typical Western blot of total cellular extracts using the anti-HA antibody. E, Reverse coimmunoprecipitation of coexpressed ßAR subtypes. The experimental protocols were similar to that used in panel C. ß1AR was immunoprecipitated using the anti-HA antibody, and Western blots were performed to confirm pull-down of HA-tagged ß1AR in the inmmunoprecipitates. The presence of Flag-ß2AR in the immunoprecipitate was assayed by Western blot with the anti-Flag antibody. F, Typical Western blot of total cellular extracts using the anti-Flag antibody. Throughout all of the experiments, immunocomplexes were analyzed by SDS-PAGE (4% to 20% gradient gels). C through F, the Western blots are representative of at least 3 experiments with each on cells from 34 mouse hearts.

Coimmunoprecipitaiton of ß₁AR and ß₂AR
To directly demonstrate physical association of ßAR subtypes, we expressed HA-tagged ß₁AR or Flag-tagged ß₂AR or both in the null background of DKO myocytes and then performed immunoprecipitation and Western blot assays. Total cellular proteins containing either or both ßAR subtypes were first immunoprecipitated with a rabbit polyclonal anti-Flag antibody. The pull-down of Flag-tagged ß₂AR in the immunoprecipitate was confirmed by Western blot using the anti-Flag antibody (Figure 1C, top). Notably, the presence of ß₁AR 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-ß₁AR (Figure 1D). The 50 kDa species likely represents the monomeric form of HA-ß₁AR, 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-ß₁-AR. As a negative control, there was no detectable HA-immunoreactivity in myocytes expressing either ß₂AR alone or ß-gal (Figure 1D). Conversely, we performed the coimmunoprecipitation experiments with the anti-HA antibody to pull-down ß₁ARs and detected coimmunoprecipitated Flag-tagged ß₂ARs 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-ß₂AR but not in those expressing HA-ß₁AR alone or ß-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 ß₁ARs and ß₂AR populations, as indexed by their coimmunoprecipitation. These results indicate that intermolecular interactions occur between ß₁AR and ß₂AR in adult-mouse cardiomyocytes.

Suppression of Spontaneous ß₂AR Activation by ß₁AR Coexpression
To determine the functional consequences of ß₁AR-ß₂AR heterodimerization in adult-mouse ventricular myocytes, we first examined constitutive ßAR activity in the absence of agonist stimulation. The baseline contractility of cells expressing ß₂AR was enhanced by 1.6-fold relative to myocytes expressing ß₁AR or those uninfected cells from WT or DKO mice (Figure 2A). In contrast, expression of ß₁AR at a receptor density that matched the ß₂AR density did not alter basal ligand-independent myocyte contraction amplitude (Figure 2A). ICI 118 551 (ICI, 5x10^–7 mol/L), a ß₂AR inverse agonist, completely reversed the enhanced basal contraction (Figure 2B), without altering the baseline contraction in cells expressing ß₁AR (data not shown). These results are consistent with the previous notion that ß₂AR,^16–20 but not ß₁AR,^16,21 exhibits spontaneous activity. Surprisingly, the Adv-ß₂AR infection-induced augmentation in the baseline contractility was fully prevented when cells were coinfected with Adv-ß₁AR (50 moi for each) (Figure 2A). This was not caused by a reduction in ß₂AR subtype density, because Adv-ß₂AR alone (50 moi) significantly elevated, in an ICI-sensitive manner, the baseline contractility relative to that of uninfected WT or DKO cells or Adv-ß₁AR infected myocytes (Figure 2A). It is noteworthy that there was no significant difference in the baseline contraction amplitude between WT and ß₁AR 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 ß₂ARs in the ß₁AR KO cells, because the spontaneous activity of the ß₂AR is receptor density-dependent.^16–20

View larger version (21K): [in this window] [in a new window]   Figure 2. The presence of ß1AR suppresses spontaneous activity of coexpressed ß2AR. A, The open bars illustrate baseline concentration amplitudes for groups, as indicated, (n=34 to 80 cells from at least 10 mouse hearts for each group; *P<0.01 vs Adv-ß1AR infected, or Adv-ß1AR and Adv-ß2AR coinfected, or uninfected WT or DKO myocytes). The solid bars show effects of ICI 118 551 (0.5 µmol/L) on the baseline contraction amplitude in myocytes infected with Adv-ß2AR at moi of 100 or 50, or in those coinfected by Adv-ß1AR and Adv-ß2AR (moi of 50 for each). B, Typical time course of the ICI 118 551 effect on basal contraction amplitude in DKO myocytes infected by Adv-ß2AR (50 moi, top) or those coinfected by Adv-ß1AR and Adv-ß2AR (50 moi for each, bottom).

Figure 2B shows representative examples of the inhibitory effect of ICI in a cell expressing either ß₂AR or both ßAR subtypes (top and bottom, respectively). Clearly, coexpression of ß₁AR virtually abolished spontaneous ß₂AR activity, as manifested by the inability of the ß₂AR inverse agonist to reduce basal myocyte contraction (Figure 2A and 2B).

Heterodimerization of ßAR Subtypes Enhances Cardiomyocyte Contractile Response to ßAR Agonist Stimulation
Next, we determined the potential impact of coexpression of these receptors on the myocyte contractile response to agonist-induced ßAR stimulation. In cells expressing either ß₁AR or ß₂AR, stimulation of these ßAR 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 ß₁AR- and ß₂AR-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 ß₁AR and ß₂AR 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 ß₁AR or the ß₂AR group). Thus, ßAR subtype heterodimerization sensitizes the contractile response to ligand-induced receptor stimulation in cardiac myocytes.

View larger version (16K): [in this window] [in a new window]   Figure 3. Synergy between ß1AR- and ß2AR-mediated positive inotropic effects in adult mouse cardiomyocytes. A, Shows myocyte contractile response to a nonselective ßAR agonist, isoproterenol (ISO) in ß1ß2AR DKO cardiomyocytes infected with Adv-ß1AR or Adv-ß2-AR or both, as indicated. B and C illustrate the normalized concentration-responses of myocyte contraction to ISO (data are presented as % of control and % of the maximal response, respectively) (n=10 to 14 cells from 810 hearts for each data point).

Cellular cAMP Responses in ß₁AR- or ß₂AR- or Mixed ß₁ß₂AR-Expressing Cardiomyocytes
Because both ß₁AR- and ß₂AR-induced positive inotropic effects are mediated by a cAMP-dependent mechanism,^21–23 we next investigated the cAMP response to ßAR stimulation in DKO cardiomyocytes expressing either or both ßAR subtypes. Compared with that of uninfected WT or DKO cells, the baseline cAMP level was unchanged in myocytes expressing ß₁AR, but augmented by 2.1-fold in cells infected with Adv-ß₂AR (100 moi) (Figure 4A), caused by spontaneous ß₂AR activity.^16–20 Coexpression of the ß₁AR with ß₂AR fully suppressed the ß₂AR-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 ß₂AR was increased versus that in cells expressing ß₁AR (Figure 4B). However, the relative response of cAMP formation (% of basal level) to ISO was greater in ß₁AR-expressing cells compared with that in those expressing ß₂AR (Figure 4C). Remarkably, when these ßAR subtypes were coexpressed in ß₁ß₂AR 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 ß₁AR or ß₂AR alone (Figure 4B and 4C), consistent with the profile of myocyte contractile response to either spontaneous or ligand-induced ßAR subtype activation.

View larger version (16K): [in this window] [in a new window]   Figure 4. Coexpression of ß1AR and ß2AR potentiates the response of myocyte cAMP to ISO stimulation for 10 minutes. A, Displays basal cAMP levels in ß1ß2AR DKO mouse cardiomyocytes expressing either or both ßAR subtypes, as indicated. *P<0.01 vs the value in myocytes expressing ß1AR or both ßAR subtypes or uninfected cells from WT or DKO mice. B and C Illustrate the concentration-response relations of intracellular cAMP formation to ISO as the absolute change or the percent increase, respectively, (n=3 to 4 experiments in each myocytes obtained from 3 mouse hearts). *P<0.01 vs ß1AR or both ßAR subtypes; P<0.01 vs ß1AR or ß2AR subtype alone.

Ligand Binding Profiles of ß₁AR, ß₂AR, and Coexpressed ßAR 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 ßAR subtypes were individually expressed or coexpressed. There were 2 ßAR subpopulations in WT mouse heart with 73.6±2.7% and 26.4±2.9% for ß₁AR for ß₂AR, respectively. Most importantly, radioligand binding assays revealed that the binding affinity of separately expressed ß₁AR or ß₂AR 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 ßAR 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 ßAR subtypes, because it occurred in both WT and coinfected DKO mouse myocytes regardless of their different densities or the ratio of the coexisting ßAR subtypes (Tables 1 and 2 ). That coexpression enhances the affinity of both ß₁AR and ß₂AR for their selective ligand binding further supports the notion that of these receptors form heterodimers in intact adult-mouse cardiomyocytes.

View this table: [in this window] [in a new window]   Table 2. Table 2. Competition of 125I-CYP Binding With ß1AR- or ß2AR-Specific Antagonists, ICI 118 551 and CGP 20712A, Respectively, in Adult Mouse Cardiomyocytes

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
ß₁AR and ß₂AR 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 ß₁AR and ß₂AR in intact adult mouse cardiac myocytes. Heterodimerization of these receptors suppresses constitutive ligand-independent ß₂AR signaling, but facilitates receptor-ligand interactions, thereby increasing the signaling efficiency of both ßAR subtypes and optimizing ßAR-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 ßARs, ß₁AR and ß₂AR are physically associated with each other, as manifested by their coimmunoprecipitation and intracellular colocalization at optical resolution. Second, coexpression of ß₁AR and ß₂AR creates a novel population of ßARs 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 ß₁AR fully quenches the spontaneous activity of coexpressed ß₂ARs, as evidenced by a full reversal of agonist-independent ß₂AR-mediated augmentation in basal myocyte cAMP production and contractility. This finding provides compelling evidence for constitutive intermolecular interactions between these ßAR 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 ßAR 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 ß₁AR and ß₂AR relative to those expressing a single subtype.²⁵ Moreover, coexpression of both ßAR 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 ßAR-Mediated Modulation of Cardiac Contractility by Subtype Heterodimerization
Although a large number of other GPCRs have been previously reported to undergo homodimerization or heterodimerization,^1–10 the present study provides the first demonstration that ß₁AR and ß₂AR, prototypical members of the GPCR superfamily, form heterodimers in the physiological context of intact cardiac myocytes. When ß₁AR and ß₂AR are coexpressed, myocyte contractile or cAMP response to ßAR agonist stimulation is profoundly sensitized compared with the "pure" ß₁AR or ß₂AR system, indicating that ß₁AR and ß₂AR interact in a synergistic fashion. Similar functional synergy between ß₁AR and ß₂AR 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 ß₁AR and ß₂AR 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 ß₁AR, stimulation of the native cardiac ß₂AR with ISO was unable to elicit a positive inotropic effect in vivo.²⁷ This further supports the perception that ß₁AR and ß₂AR exhibit a synergistic interaction in their modulation of cardiac contractility.

It is noteworthy that heterodimerization of ß₁AR and ß₂AR, although enhancing agonist-induced signaling, silences the spontaneous activation of ß₂AR, 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 ß₁AR on ß₂AR spontaneous activation awaits future investigation. Altogether, our present and previous studies^11,24,25 have demonstrated that intermolecular interactions between ß₁AR and ß₂AR 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 ß₁AR with little or no change in ß₂AR density.^28–30 The heart failure-associated decrease in the ratio of ß₁AR to ß₂AR might alter the heterodimerization of the remaining ßARs, thus contributing to the diminution of ßAR contractile support or an upregulation of anti-apoptotic ß₂AR signaling.^31–33 These hypotheses merit further investigation.

Heterodimerization Between ßAR and Members From Other GPCR Families
In addition to the complicated impacts of heterodimerization of the closely related ß₁AR and ß₂AR on their trafficking and signaling properties, recent studies have revealed evidence for heterodimerization of ß₂AR with other members of adrenergic receptor family, including ß₃AR, _2AAR, and _1DAR, in HEK 293 cells.^34–36 Interestingly, whereas either ß₂AR or ß₃AR alone couples to both G_s and G_i proteins, the ß₂AR-ß₃AR heterodimer is unable to activate G_i signaling.³⁴ Equally appealing, the heterodimerization of ß₂AR with either _2AAR or _1DAR leads to cross-internalization of the receptors on agonist stimulation of either ß₂AR 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 ß₂AR also alters receptor trafficking and signal transduction.^37–39 Additionally, it has been demonstrated that intermolecular interactions between ßAR 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 ß₁AR and ß₂AR 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 ß₂AR activity. The heterodimeric ß₁AR-ß₂AR may represent a pharmacologically and functionally distinct population of ßARs. Thus, many well-established paradigms for ßAR 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 ß₁AR or both ß₁AR and ß₂AR. The authors are also grateful to Dr Hal Spurgeon and B. Ziman for their excellent technique support.

	Footnotes

 
This manuscript was sent to H. Michael Piper, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Original received March 14, 2005; revision received June 24, 2005; accepted June 29, 2005.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Jones KA, Borowsky B, Tamm JA, Craig DA, Durkin MM, Dai M, Yao WJ, Johnson M, Gunwaldsen C, Huang LY. GABAB receptors function as a heteromeric assembly of the subunits GABABR1 and GABABR2. Nature. 1998; 396: 674–679.[CrossRef][Medline] [Order article via Infotrieve]
2. Kaupmann K, Malitschek B, Schuler V, Heid J, Froestl W, Beck P, Mosbacher J, Bischoff S, Kulik A, Shigemoto R. GABA_B-receptor subtypes assemble into functional heteromeric complexes. Nature. 1998; 396: 683–687.[CrossRef][Medline] [Order article via Infotrieve]
3. White JH, Wise A, Main MJ, Green A, Fraser NJ, Disney GH, Barnes AA, Emson P, Foord SM, Marshall FH. Heterodimerization is required for the formation of a functional GABA_B receptor. Nature. 1998; 396: 679–682.[CrossRef][Medline] [Order article via Infotrieve]
4. Hebert TE, Moffett S, Morello JP, Loisel TP, Bichet DG, Barret C, Bouvier M. A peptide derived from a ß₂-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation. J Biol Chem. 1996; 271: 16384–16392.[Abstract/Free Full Text]
5. Cvejic S, Devi LA. Dimerization of the delta opioid receptor: implication for a role in receptor internalization. J Biol Chem. 1997; 272: 26959–26964.[Abstract/Free Full Text]
6. Bai M, Trivedi S, Brown EM. Dimerization of the extracellular calcium-sensing receptor (CaR) on the cell surface of CaR-transfected HEK293 cells. J Biol Chem. 1998; 273: 23605–23610.[Abstract/Free Full Text]
7. Rocheville M, Lange DC, Kumar U, Patel SC, Patel RC, Pate YC. Receptors for dopamine and somatostatin: formation of hetero-oligomers with enhanced functional activity. Science. 2000; 288: 154–157.[Abstract/Free Full Text]
8. Agnati LF, Fuxe K, Zoli M, Rondanini C, Ogren SO. New vistas on synaptic plasticity: the receptor mosaic hypothesis of the engram. Med Biol. 1982; 60: 183–190.[Medline] [Order article via Infotrieve]
9. AbdAlla S, Lother H, Quitterer U. AT1-receptor heterodimers show enhanced G-protein activation and altered receptor sequestration. Nature. 2000; 407: 94–98.[CrossRef][Medline] [Order article via Infotrieve]
10. Gines S, Hillion J, Torvinen M, Le Crom S, Casado V, Canela EI, Rondin S, Lew JY, Watson S, Zoli M, Agnati LF, Verniera P, Lluis C, Ferre S, Fuxe K, Franco R. Dopamine D1 and adenosine A1 receptors form functionally interacting heteromeric complexes. Proc Natl Acad Sci U S A. 2000; 9: 8606–8611.
11. Lavoie C, Mercier JF, Salahpour A, Umapathy D, Breit A, Villeneuve LR, Zhu WZ, Xiao RP, Lakatta EG, Bouvier M, Hebert TE. ß₁/ß₂-adrenergic receptor heterodimerization regulates ß₂-adrenergic receptor internalization and ERK signaling efficacy. J Biol Chem. 2002; 277: 35402–35410.[Abstract/Free Full Text]
12. Xiao RP ß-adrenergic signaling in the heart: dual coupling of the ß₂-adrenergic receptor to G_s and G_i proteins. Sci STKE. 2001; 104: RE15.
13. Rohrer DK, Chruscinski A, Schauble EH, Bernstein D, Kobilka BK. Cardiovascular and metabolic alterations in mice lacking both ß₁- and ß₂-adrenergic receptors. J Biol Chem. 1999; 274: 16701–16708.[Abstract/Free Full Text]
14. Zhou YY, Wang SQ, Zhu WZ, Chruscinski A, Kobilka BK, Ziman B, Wang S, Lakatta EG, Cheng H, Xiao RP. Culture and adenoviral infection of adult mouse cardiac myocytes: methods for cellular genetic physiology. Am J Physiol Heart Circ Physiol. 2000; 279: H429–H436.[Abstract/Free Full Text]
15. Xiao RP, Ji X, Lakatta EG. Functional coupling of the ß₂-adrenoceptor to a pertussis toxin-sensitive G protein in cardiac myocytes. Mol Pharmacol. 1995; 47: 322–329.[Abstract]
16. Zhou YY, Yang D, Zhu WZ, Zhang SJ, Wang DJ, Rohrer DK, Devic E, Kobilka BK, Lakatta EG, Cheng H, Xiao RP. Spontaneous activation of ß₂- but not ß₁-adrenoceptors expressed in cardiac myocytes from ß₁ ß₂ double knockout mice. Mol Pharmacol. 2000; 58: 887–894.[Abstract/Free Full Text]
17. Milano CA, Allen LF, Rockman HA, Dolber PC, McMinn TR, Chien KR, Johnson TD, Bond RA, Lefkowitz RJ. Enhanced myocardial function in transgenic mice overexpressing the ß₂-adrenergic receptor. Science. 1994; 264: 582–586.[Abstract/Free Full Text]
18. Bond RA, Leff P, Johnson TD, Milano CA, Rockman HA, McMinn TR, Apparsundaram S, Hyek MF, Kenakin TP, Allen LF, Lefkowitz RJ. Physiological effects of inverse agonists in transgenic mice with myocardial overexpression of the ß₂-adrenoceptor. Nature. 1995; 374: 272–276.[CrossRef][Medline] [Order article via Infotrieve]
19. Xiao RP, Avdonin P, Zhou YY, Cheng H, Akhter SA, Eschenhagen T, Lefkowitz RJ, Koch WJ, Lakatta EG. Coupling of ß₂-adrenoceptor to G_i proteins and its physiological relevance in murine cardiac myocytes. Circ Res. 1999; 84: 43–52.[Abstract/Free Full Text]
20. Zhou YY, Cheng H, Bogdanov KY, Hohl C, Altschuld R, Lakatta EG, Xiao RP. Localized cAMP-dependent signaling mediates ß₂-adrenergic modulation of cardiac excitation-contraction coupling. Am J Physiol Heart Circ Physiol. 1997; 273: H1611–H1618.[Abstract/Free Full Text]
21. Chakir K, Xiang Y, Yang D, Zhang SJ, Cheng H, Kobilka BK, Xiao RP. The third intracellular loop and the carboxyl terminus of ß₂-adrenergic receptor confer spontaneous activity of the receptor. Mol Pharmacol. 2003; 64: 1048–1058.[Abstract/Free Full Text]
22. Kuschel M, Zhou YY, Spurgeon HA, Bartel S, Karczewski P, Zhang SJ, Krause EG, Lakatta EG, Xiao RP. ß₂-adrenergic cAMP signaling is uncoupled from phosphorylation of cytoplasmic proteins in canine heart. Circulation. 1999; 99: 2458–2465.[Abstract/Free Full Text]
23. Kuschel M, Zhou YY, Cheng H, Zhang SJ, Chen Y, Lakatta EG, Xiao RP. G_i protein-mediated functional compartmentalization of cardiac ß₂-adrenergic signaling. J Biol Chem. 1999; 274: 22048–22052.[Abstract/Free Full Text]
24. Mercier JF, Salahpour A, Angers S, Breit A, Bouvier M. Quantitative assessment of ß₁- and ß₂-adrenergic receptor homo- and heterodimerization by bioluminescence resonance energy transfer. J Biol Chem. 2002; 277: 44925–44931.[Abstract/Free Full Text]
25. Lavoie C, Hebert TE. Pharmacological characterization of putative ß₁-ß₂-adrenergic receptor heterodimers. Can J Physiol Pharmacol. 2003; 81: 186–195.[CrossRef][Medline] [Order article via Infotrieve]
26. Zhong H, Guerrero SW, Esbenshade TA, Minneman KP. Inducible expression of ß₁- and ß₂-adrenergic receptors in rat C6 glioma cells: functional interactions between closely related subtypes. Mol Pharmacol. 1996; 50: 175–184.[Abstract]
27. Rohrer DK, Desai KH, Jasper JR, Stevens ME, Regula DP Jr, Barsh GS, Bernstein D, Kobilka BK. Targeted disruption of the mouse ß₁-adrenergic receptor gene: developmental and cardiovascular effects. Proc Natl Acad Sci U S A. 1996; 93: 7375–7380.[Abstract/Free Full Text]
28. Bristow MR, Ginsburg R, Umans V, Fowler M, Minobe W, Rasmussen R, Zera P, Menlove R, Shah P, Jamieson S. ß₁- and ß₂-adrenergic-receptor subpopulations in nonfailing and failing human ventricular myocardium: coupling of both receptor subtypes to muscle contraction and selective ß₁-receptor down-regulation in heart failure. Circ Res. 1986; 59: 297–309.[Abstract/Free Full Text]
29. Kiuchi K, Shannon RP, Komamura K, Cohen DJ, Bianchi C, Homcy CJ, Vatner SF, Vatner DE. Myocardial ß-adrenergic receptor function during the development of pacing-induced heart failure. J Clin Invest. 1993; 91: 907–914.[Medline] [Order article via Infotrieve]
30. Xiao RP, Zhang SJ, Chakir K, Avdonin P, Zhu W, Bond RA, Balke CW, Lakatta EG, Cheng H. Enhanced G_i signaling selectively negates ß₂-adrenergic receptor (AR)–but not ß₂-AR-mediated positive inotropic effect in myocytes from failing rat hearts. Circulation. 2003; 108: 1633–1639.[Abstract/Free Full Text]
31. Chesley A, Lundberg MS, Asai T, Xiao RP, Ohtani S, Lakatta EG, Crow MT. The ß₂-adrenergic receptor delivers an antiapoptotic signal to cardiac myocytes through G_i-dependent coupling to phosphatidylinositol 3'-kinase. Circ Res. 2000; 87: 1172–1179.[Abstract/Free Full Text]
32. Zhu WZ, Zheng M, Koch WJ, Lefkowitz RJ, Kobilka BK, Xiao RP. Dual modulation of cell survival and cell death by ß₂-adrenergic signaling in adult mouse cardiac myocytes. Proc Natl Acad Sci U S A. 2001; 98: 1607–1612.[Abstract/Free Full Text]
33. Communal C, Singh K, Sawyer DB, Colucci WS. Opposing effects of ß₁- and ß₂-adrenergic receptors on cardiac myocyte apoptosis: role of a pertussis toxin-sensitive G protein. Circulation. 1999; 100: 2210–2212.[Abstract/Free Full Text]
34. Breit A, Lagace M, Bouvier M. Hetero-oligomerization between ß₂- and ß₃-adrenergic receptors generates a ß-adrenergic signaling unit with distinct functional properties. J Biol Chem. 2004; 279: 28756–28765.[Abstract/Free Full Text]
35. Xu J, He J, Castleberry AM, Balasubramanian S, Lau AG, Hall RA. Heterodimerization of _2A- and ß₁-adrenergic receptors. J Biol Chem. 2003; 278: 10770–10777.[Abstract/Free Full Text]
36. Uberti MA, Hague C, Oller H, Minneman KP, Hall RA Heterodimerization with ß₂-adrenergic receptors promotes surface expression and functional activity of _1D-adrenergic receptors. J Pharmacol Exp Ther. 2004; [Epub ahead of print].
37. Jordan BA, Trapaidze N, Gomes I, Nivarthi R, Devi LA. Oligomerization of opioid receptors with ß₂-adrenergic receptors: A role in trafficking and mitogen-activated protein kinase activation. Proc Natl Acad Sci U S A. 2001; 98: 343–348.[Abstract/Free Full Text]
38. McVey M, Ramsay D, Kellett E, Rees S, Wilson S, Pope AJ, Milligan G. Monitoring receptor oligomerization using time-resolved fluorescence resonance energy transfer and bioluminescence resonance energy transfer. The human -opioid receptor displays constitutive oligomerization at the cell surface, which is not regulated by receptor occupancy. J Biol Chem. 2001; 276: 14092–14099.[Abstract/Free Full Text]
39. Ramsay D, Kellett E, McVey M, Rees S, Milligan G. Homo- and hetero-oligomeric interactions between G-protein-coupled receptors in living cells monitored by two variants of bioluminescence resonance energy transfer (BRET): hetero-oligomers between receptor subtypes form more efficiently than between less closely related sequences. Biochem J. 2002; 365: 429–440.[CrossRef][Medline] [Order article via Infotrieve]
40. Barki-Harrington L, Luttrell LM, Rockman HA. Dual inhibition of ß-adrenergic and angiotensin II receptors by a single antagonist: a functional role for receptor-receptor interaction in vivo. Circulation. 2003; 108: 1611–1618.[Abstract/Free Full Text]

This article has been cited by other articles:

				N. J. Evans and J. W. Walker Endothelin Receptor Dimers Evaluated by FRET, Ligand Binding, and Calcium Mobilization Biophys. J., July 1, 2008; 95(1): 483 - 492. [Abstract] [Full Text] [PDF]

				I. Ahmet, M. Krawczyk, W. Zhu, A. Y.-H. Woo, C. Morrell, S. Poosala, R.-p. Xiao, E. G. Lakatta, and M. I. Talan Cardioprotective and Survival Benefits of Long-Term Combined Therapy with {beta}2 Adrenoreceptor (AR) Agonist and {beta}1 AR Blocker in Dilated Cardiomyopathy Postmyocardial Infarction J. Pharmacol. Exp. Ther., May 1, 2008; 325(2): 491 - 499. [Abstract] [Full Text] [PDF]

				B. J. Holycross, M. Kukielka, Y. Nishijima, R. A. Altschuld, C. A. Carnes, and G. E. Billman Exercise training normalizes beta-adrenoceptor expression in dogs susceptible to ventricular fibrillation Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H2702 - H2709. [Abstract] [Full Text] [PDF]

				T. E. Hebert Anti-{beta}1AR antibodies in dilated cardiomyopathy: Are these a new class of receptor agonists? Cardiovasc Res, October 1, 2007; 76(1): 5 - 7. [Full Text] [PDF]

				E. N. Nikolov and T. T. Ivanova-Nikolova Dynamic Integration of {alpha}-Adrenergic and Cholinergic Signals in the Atria: ROLE OF G PROTEIN-REGULATED INWARDLY RECTIFYING K+ CHANNELS J. Biol. Chem., September 28, 2007; 282(39): 28669 - 28682. [Abstract] [Full Text] [PDF]

				S. Brickson, D. P. Fitzsimons, L. Pereira, T. Hacker, H. Valdivia, and R. L. Moss In vivo left ventricular functional capacity is compromised in cMyBP-C null mice Am J Physiol Heart Circ Physiol, April 1, 2007; 292(4): H1747 - H1754. [Abstract] [Full Text] [PDF]

				J.-P. Pin, R. Neubig, M. Bouvier, L. Devi, M. Filizola, J. A. Javitch, M. J. Lohse, G. Milligan, K. Palczewski, M. Parmentier, et al. International Union of Basic and Clinical Pharmacology. LXVII. Recommendations for the Recognition and Nomenclature of G Protein-Coupled Receptor Heteromultimers Pharmacol. Rev., March 1, 2007; 59(1): 5 - 13. [Abstract] [Full Text] [PDF]

				S. J. Wilson, J. K. Dowling, L. Zhao, E. Carnish, and E. M. Smyth Regulation of Thromboxane Receptor Trafficking Through the Prostacyclin Receptor in Vascular Smooth Muscle Cells: Role of Receptor Heterodimerization Arterioscler. Thromb. Vasc. Biol., February 1, 2007; 27(2): 290 - 296. [Abstract] [Full Text] [PDF]

				Highlights from the Literature Physiology, December 1, 2005; 20(6): 369 - 373. [Full Text] [PDF]

				W. Zhu, X. Zeng, M. Zheng, and R.-P. Xiao The Enigma of {beta}2-Adrenergic Receptor Gi Signaling in the Heart: The Good, the Bad, and the Ugly Circ. Res., September 16, 2005; 97(6): 507 - 509. [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 97/3/244    most recent 01.RES.0000176764.38934.86v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend