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J. Biol. Chem., Vol. 279, Issue 27, 28756-28765, July 2, 2004

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Hetero-oligomerization between ₂- and ₃-Adrenergic Receptors Generates a -Adrenergic Signaling Unit with Distinct Functional Properties^*

Andreas Breit, Monique Lagacé, and Michel Bouvier, Holds a Canada Research Chair in Signal Transduction and Molecular Pharmacology

From the Département de Biochimie et Groupe de Recherche sur le Système Nerveux Autonome, Université de Montréal, Montréal, Québec H3C 3J7, Canada

Received for publication, December 5, 2003 , and in revised form, April 6, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The ability of the closely related ₂- and ₃-adrenergic receptors (AR) to form hetero-oligomers was assessed by bioluminescence resonance energy transfer. Quantitative bioluminescence resonance energy transfer titration curves revealed that the ₂AR has identical propensity to hetero-oligomerize with the ₃AR than to form homo-oligomers. To determine the influence of heterooligomerization, a HEK293 cell line stably expressing an excess of ₃AR over ₂AR was generated so that all ₂AR are engaged in hetero-oligomerization with ₃AR, providing a tool to study the effect of hetero-oligomerization on ₂AR function in the absence of any ₂AR homooligomer. The hetero-oligomerization had no effect on the ligand binding properties of various ₂AR ligands and did not affect the potency of isoproterenol to stimulate adenylyl cyclase. Despite the unaltered ligand binding properties of the _2/3AR hetero-oligomer, the stable association of the ₂AR with the ₃AR completely blocked agonist-stimulated internalization of the ₂AR. Given that the ₃AR is resistant to agonist-promoted endocytosis, the results indicate that the ₃AR acted as a dominant negative of the ₂AR endocytosis process. Consistent with this notion, the _2/3AR hetero-oligomer displayed a lower propensity to recruit -arrestin-2 than the ₂AR. The hetero-oligomerization also led to a change in G protein coupling selectivity. Indeed, in contrast to ₂AR and ₃AR, which regulate adenylyl cyclase and extracellular signal-regulated kinase activity through a coupling to G_s and G_i/o, no G_i/o coupling was observed for the _2/3AR hetero-oligomer. Together, these results demonstrate that hetero-oligomerization between ₂AR and ₃AR forms a -adrenergic signaling unit that possesses unique functional properties.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell surface receptors, which mediate their biological effects via coupling to G proteins, control major functions of eucaryotic organisms like neurotransmission, immune response, cell growth, and metabolism (1). Over the past few years, several lines of evidence have supported the notion that G protein-coupled receptors (GPCR)¹ exist and act as homo- or heterooligomeric signaling units (2–5). Hetero-oligomerization among GPCR has been shown to modulate ligand binding, G protein-coupling, endocytosis, and desensitization of the receptors (6–19).

Because of their clear and distinct properties, the ₂ adrenergic receptor (₂AR) and ₃AR offer an ideal receptor pair to investigate the pharmacological and functional consequences of hetero-oligomerization. Indeed, despite their high degree of sequence homology (49%), each receptor displays characteristic ligand binding properties that can be easily distinguished (20). Also, although the ₂AR undergoes rapid and efficient agonist-promoted internalization (21–23), the ₃AR is resistant to these regulatory processes (24–26). The fact that these two closely related receptor subtypes are naturally co-expressed in adipocytes (27–29) suggests the possibility of formation of hetero-oligomers in native tissues and makes the characterization of this hetero-oligomer of potential physiological interest.

One difficulty when investigating hetero-oligomerization of GPCR is the heterogeneity of ligand-binding sites and signaling units that can occur if two receptors are co-expressed in the same cell. To analyze the properties of a hetero-oligomer in the presence of two homo-oligomers, one must ensure that the hetero-oligomer represents the major subpopulation among the expressed receptors. Also, a good correlation between the extent of hetero-oligomerization and the functional changes attributed to the formation of hetero-oligomers should be established. Unfortunately, quantitative assessment of hetero-oligomers is not a trivial issue. The amount of hetero-oligomers formed will be a function of the affinity of the individual receptors for one another and of their relative expression levels. Because of the difficulty in getting good estimation for these parameters, studies investigating GPCR hetero-oligomerization remained largely qualitative.

In the present study, we established experimental conditions that ensured that the entire population of ₂AR heterologously expressed in HEK293 cells is engaged in hetero-oligomerization with the ₃AR, thus allowing the functional characterization of the ₂AR within the _2/3AR hetero-oligomer in the absence of any ₂AR homo-oligomer.

Our study reveals that in contrast to the robust agonist-promoted endocytosis characteristic of the ₂AR, the _2/3AR hetero-oligomer was not internalized upon agonist stimulation, suggesting that the ₃AR-negative endocytotic phenotype prevailed in the hetero-oligomer. This dominant-negative effect of the ₃AR occurred without any change in the ligand binding properties of the receptors and most likely results from a diminished -arrestin-2 recruitment to the hetero-oligomer. When considering the coupling properties of the hetero-oligomer, we found that unlike ₂AR and ₃AR, which can couple to G_i/o (30, 31), the _2/3AR hetero-oligomer cannot engage this signaling pathway. Taken together, our results indicate that the _2/3AR hetero-oligomer is a AR-like signaling unit distinct from ₂AR or ₃AR expressed alone.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, L-glutamine, and geneticin (G418) were obtained from Wisent Laboratories. Alprenolol, isoproterenol (ISO), fenoterol (FEN), ICI118551, CGP12177A, 3-isobutyl-1-methylxanthine (IBMX), and methotrexate were purchased from Sigma. FuGENE 6 was from Roche Applied Science. Anti-rabbit-HRP or anti-mouse-HRP antibodies were from Amersham Biosciences, and anti-phospho-ERK1/2 (E-4, sc-7383) or anti-ERK1/2 (K-23, sc-94) was from Santa Cruz Biotechnology. ECL, DeepBlueC, ¹²⁵I-cyanopindolol, and [³H]adenine were from PerkinElmer Life Sciences, whereas coelenterazine H was from Molecular Probes.

Eucaryotic Expression Vectors—Constructions of the pGFP-₂AR-GFP10 (where GFP10 is a variant form of the green fluorescent protein (GFP) containing the following mutations: P64L, S147P, and S202P), pcDNA3.1-₂AR-Rluc, and pcDNA3.1--arrestin-2-YFP vectors were reported previously (32, 33). The pcDNA3-₃AR-Rluc construct was kindly provided by PerkinElmer Life Sciences. For pGFP-₃AR-GFP10, the ₃AR coding sequence without its stop codon was amplified by PCR using primers harboring unique HindIII or AgeI restriction sites. This PCR fragment was subcloned into the pGFP10-C1 vector (obtained from PerkinElmer Life Sciences) in a way that fused the 3'-end of the ₃AR-cDNA onto the 5'-end of the GFP10-cDNA.

Cell Culture and Transfection—HEK293 or COS-1 cells were cultured in DMEM supplemented with 10% FBS, 100 units/ml penicillin/streptomycin, and 2 nM L-glutamine. For transient expression of recombinant proteins, cells were seeded at a density of 2 x 10⁶ cells per 100-mm dish, cultured for 24 h, and then transfected by the calcium phosphate precipitation method for HEK293 cells (33) or by using the FuGENE 6 reagent according to the manufacturer's protocol for COS-1 cells. 48 h after transfection, cells were washed twice with phosphate-buffered saline (PBS), detached with 5 mM EDTA in PBS, and used immediately. HEK293 cell clones stably expressing ₂AR-Rluc or ₃AR-Rluc were obtained from transfected cells selected with 400 µg/ml G418. The expression of the receptor-Rluc fusion protein was controlled by measuring luciferase activity. To obtain HEK293 cells stably co-expressing ₂AR-Rluc and ₃AR-GFP, a cell clone already expressing ₂AR-Rluc was co-transfected with the ₃AR-GFP construct along with the pED_mtxr vector harboring a point-mutated dihydrofolate reductase gene that transfers resistance to methotrexate (34). After selection with 400 nM methotrexate, clones co-expressing ₂AR-Rluc and ₃AR-GFP were isolated, and co-expression of both proteins was monitored by luminescence, fluorescence, and BRET measurement (33).

BRET Measurement—To monitor receptor-receptor interactions in living cells, BRET² assays were performed using a TopCount NXT^TM (PerkinElmer Life Sciences) as described before (33). Briefly, after catalytic degradation of the substrate DeepBlueC by the energy donor Renilla luciferase (Rluc), light is emitted with a peak at 400 nm. The energy acceptor green fluorescent protein (GFP) is excited by nonradiative energy transfer if GFP is located within a distance of less than 100 Å from the energy donor. As a result, fluorescence is re-emitted by GFP with a peak at 510 nm. The ratio of the light intensity emitted at 500–530 nm over 370–450 nm is defined as the BRET² signal. For the detection of the -arrestin-2 recruitment by ₂AR, the BRET¹ technology (Fusion^TM -FM, PerkinElmer Life Sciences) was used as described before (32). Briefly, after the substrate coelenterazine H was added, light intensity was sequentially integrated in the 510–590 and 440–500 nm window. The BRET¹ signal was defined as the ratio of the light intensity measured at 510–590 nm over 440–500 nm. The expression level of the energy donor (Rluc) or acceptor (GFP or YFP) was controlled by measuring luminescence and fluorescence for each BRET experiment as described before (33).

Membrane Preparation—HEK293 cells were homogenized in ice-cold buffer (5 mM Tris/HCl, pH 7.4, 2 mM EDTA, 5 mg/ml leupeptin, 10 mg/ml benzamidine, and 5 mg/ml soybean trypsin inhibitor) using a Polytron (Ultra-Turrax T24, IKA) for 5–10 s at maximum speed. Lysates were centrifuged at 500 x g for 10 min at 4 °C. The resulting supernatant was centrifuged at 45,000 x g for 20 min (4 °C), and pellets were washed twice using the same buffer. Protein amount was determined (Bio-Rad Protein Assay), and membranes were stored at -80 °C.

Radioligand Binding Assay—For saturation binding, 2 µg of total membrane preparation were incubated with increasing concentrations (0.002–10 nM) of the -AR antagonist ¹²⁵I-labeled cyanopindolol (¹²⁵I-CYP) in 5 mM Tris/HCl, pH 7.4, and 0.1% bovine serum albumin. Specific binding of ¹²⁵I-CYP was determined as the amount of ¹²⁵I-CYP binding inhibited by 10 µM alprenolol. Samples were incubated for 1 h at 37 °C; the reaction was stopped by rapid filtration over Whatman GF/C glass-fiber filters, and the retained radioactivity was measured (1271 RIAgamma Counter, LKB Wallac). Competition binding assays were performed under the same conditions using 25 pM of ¹²⁵I-CYP to occupy only ₂AR (this concentration binds 1% or less of the ₃AR sites) or 250 pM of ¹²⁵I-CYP to ensure the occupancy of ₂AR and ₃AR. Specific binding of ¹²⁵I-CYP was determined in the presence of increasing concentrations of the competing ligands.

Internalization Assay—To induce receptor endocytosis, cells were stimulated for 30 min with 1 µM ISO or 50 nM FEN at 37 °C. After washing the cells twice with ice-cold PBS, receptor sequestration was measured by detecting total binding of 25 pM ¹²⁵I-CYP (CYP_tot) at 13 °C for 3 h. Specific binding of ¹²⁵I-CYP on the plasma membrane was determined as the binding inhibited by 100 nM of the hydrophilic ligand CGP12177A (CYP_CGP), although total specific binding was defined as the binding inhibited by 10 µM of the hydrophobic ligand alprenolol (CYP_Alp). The internalization rate of the receptor in percentage was calculated based on the following equation: ((CYP_tot - CYP_CGP)/(CYP_tot - CYP_Alp) x 100)_basal - ((CYP_tot - CYP_CGP)/(CYP_tot - CYP_Alp) x 100)_ISO/FEN. Endocytosis was also assessed after cell fractionation as described previously (25). Briefly, after stimulation of whole cells for 30 min at 37 °C with 1 µM ISO, cells were detached and membranes prepared as described above. Membranes were then placed at the top of a sucrose cushion (35%) and centrifuged at 150,000 x g for 90 min. The light endosomal fraction containing internalized receptor (Rluc_light) was found at the 0–35% interface, whereas the heavy plasma membrane fraction (Rluc_heavy) sedimented at the bottom of the tube. Each membrane fraction was collected, centrifuged at 200,000 x g for 60 min, and resuspended in 5 mM Tris/HCl, pH 7.4, and the amount of receptor-Rluc was determined by measuring Rluc activity in each fraction. Endocytosis was calculated in percentage using the following equation: (Rluc_heavy/(Rluc_heavy + Rluc_light) x 100)_basal - (Rluc_heavy/(Rluc_heavy + Rluc_light) x 100)_ISO.

cAMP Production—To determine cAMP accumulation in living cells, 200,000 cells were seeded in 12-well microplates (coated with 0.1% poly-D-lysine) 24 h prior to the experiment and labeled for 4–6 h in DMEM without FBS containing 2 µCi/ml of [³H]adenine. Cells were stimulated for 30–45 min at 37 °C in DMEM containing 2.5 µM IBMX and different drugs at the indicated concentrations. The reaction was terminated by removing the DMEM/IBMX/ligand solution and the addition of ice-cold 5% trichloroacetic acid. [³H]ATP and [³H]cAMP were separated by sequential chromatography (Dowex resin/aluminum oxide), and the accumulation of cAMP was expressed as the ratio of [³H]cAMP/([³H]cAMP + [³H]ATP).

Adenylyl Cyclase Activity—Adenylyl cyclase activity of membranes freshly prepared (see above) from HEK293 cells was measured using the cAMP AlphaScreen^TM assay from PerkinElmer Life Sciences (for details see www.perkinelmer.com/lifesciences) with slight modifications. Briefly, membranes (2 µg of proteins) were incubated for 30 min at room temperature in the reaction buffer (5 mM Tris/HCl, pH 7.4, 2 mM EDTA, 53 µM GTP, 112 µM ATP, 2.7 mM phosphoenolpyruvate, 50 units/ml myokinase, and 10 units/ml pyruvate kinase) containing the tested activators (ISO, FEN, forskolin) and the anti-cAMP-conjugated acceptor bead. The reaction was stopped by adding a buffer containing 0.3% Triton X-100, biotinylated cAMP, and the streptavidin-coated donor bead. After 1 h of incubation at room temperature, the interaction between donor and acceptor beads was detected by measuring the emission of the acceptor bead (520–620 nm) after excitation of the donor bead (670–690 nm) by using the Fusion^TM -FM from PerkinElmer Life Sciences.

Extracellular Signal-regulated Kinase (ERK) Activity—HEK293 cells were seeded on 6-well microplates at a density of 200,000 cells per well. After 24 h, cells were serum-starved for 24 h. ERK1/2 phosphorylation was induced by the addition of -AR agonists or FBS for 5 min at 37 °C. The reaction was stopped by washing the cells twice with ice-cold PBS and adding 200 µl of Laemmli buffer (35). Samples were then analyzed by SDS-PAGE and Western blotting by using anti-phospho-ERK1/2 antibodies from mouse (1:5000) and anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies from sheep (1:10,000). The immunoactivity was then revealed by chemiluminescence using ECL. After stripping the membranes with 25 mM glycine, pH 2.2, 1% SDS for 1 h, the amount of total ERK1/2 proteins was detected using anti-ERK1/2 antibodies from rabbit (1:10,000) and anti-rabbit HRP-conjugated secondary antibodies from donkey (1:10,000). ERK1/2 phosphorylation was expressed as the ratio of the signal provided by the phospho-ERK1/2 antibody over the signal obtained with the total ERK1/2 antibody.

Data Analysis—Data obtained in binding experiments were analyzed using Prism3.0. Isotherms for saturation or competition binding assays were plotted for one or two binding sites, and the best fit was then used to calculate K_D, K_i, or EC₅₀ values. Data obtained in ERK1/2 activity experiments were digitized on a flatbed scanner and analyzed using the Quantity One (Bio-Rad) software. Statistical significance of the differences was assessed by the two-tailed Student's t test.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Detection of _2/3AR Hetero-oligomers by BRET—To determine whether ₂AR and ₃AR can form hetero-oligomers, we took advantage of the BRET² technology and fused both receptors on their C terminus to Rluc or GFP10. After transient co-expression of ₂AR-Rluc and ₂AR-GFP in HEK293 cells, an energy transfer resulting in a BRET signal of 0.14 ± 0.02 was detected in the absence of ligand stimulation (Fig. 1A), consistent with the formation of constitutive ₂AR homo-oligomers described previously (32, 33, 36, 37). Co-expression of ₃AR-Rluc and ₃AR-GFP also led to constitutive energy transfer (BRET signal, 0.16 ± 0.02), providing the first evidence that ₃AR can form homo-oligomers in living cells (Fig. 1A). To assess whether ₂AR and ₃AR could also form hetero-oligomeric complexes, ₂AR-Rluc/₃AR-GFP and ₃AR-Rluc/₂AR-GFP pairs were co-expressed, and the resulting energy transfer was measured. Both combinations resulted in significant BRET signals (₂AR-Rluc/₃AR-GFP, 0.15 ± 0.02; ₃AR-Rluc/₂AR-GFP, 0.17 ± 0.03) indicating that constitutive _2/3AR hetero-oligomers can assemble (Fig. 1A). No significant energy transfer was detected between ₂AR-Rluc and GABA_BR₂-GFP (BRET signal, 0.02 ± 0.005) when these receptors were expressed at levels comparable with those of ₂AR-Rluc and ₃AR-GFP, thus indicating that the energy transfer observed for the ₂AR-Rluc/₃AR-GFP pair did not result from an overexpression of energy donor and acceptor.

View larger version (25K): [in this window] [in a new window]   FIG. 1. Detection of 2/3AR hetero-oligomers by BRET2. A, the energy donor 2AR-Rluc or 3AR-Rluc and the energy acceptor 2AR-GFP, 3AR-GFP, or GABABR2-GFP were transiently co-expressed in HEK293 cells and BRET2 signals measured following the addition of the luciferase substrate DeepBlueC as described under "Experimental Procedures." Numbers above the bars indicate the mean of the expression ratios of receptor-GFP over receptor-Rluc for each pair. Results are expressed as the mean ± S.E. of five different experiments. B, 2AR-Rluc and 3AR-GFP were transiently co-expressed in HEK293 cells and the BRET2 signals measured. The transfections were carried out to obtain increasing numbers of total receptor numbers while keeping the 3AR/2AR ratio relatively constant. The receptor expression levels appear below each bar and were determined by monitoring the luminescence and fluorescence signals. These values were then transformed into picomoles of receptor/mg of proteins using a standard curve correlating the fluorescence and luminescence signals to the number of 125I-CYP-binding sites determined in a radioligand binding assay (33). The dotted line indicates the maximal BRET signal that can be achieved. Data from six different experiments were compiled. C, increasing amounts of 2AR-GFP, 3AR-GFP, or GABABR2-GFP were transiently expressed in a cell line stably expressing 2AR-Rluc (410 fmol/mg) and BRET2 signals measured 48 h after transfection. Expression levels of 3AR-GFP varied between 100 and 2000 fmol/mg. BRET2 signals are plotted as a function of the expression ratio of receptor-GFP over 2AR-Rluc obtained by measuring the total luminescence and fluorescence in each condition. The expression level of the 2AR-Rluc was not significantly affected by the transient expression of the receptor-GFP fusion proteins. The curve was drawn from data obtained in three independent experiments. The dashed line indicates the 3AR-GFP over 2AR-Rluc expression ratio observed in HEK293-2/3AR cells stably co-expressing both receptor subtypes, and the bar illustrates their BRET2 signal obtained from five different experiments.

To assess the propensity of ₂AR to either homo- or heterooligomerize with ₃AR, BRET titration curves were carried out in HEK293 cells stably expressing 410 fmol/mg of ₂AR-Rluc by increasing the concentrations of ₂AR-GFP or ₃AR-GFP transiently expressed. As reported previously (33), increasing the GFP fusion proteins expressed led to a hyperbolic increase in the BRET signal that reached a maximum BRET signal once all ₂AR-Rluc molecules that can engage in oligomerization are in a complex with either ₂AR- or ₃AR-GFP. The apparent affinity of the ₂AR-GFP and ₃AR-GFP for the ₂AR-Rluc can then be determined by estimating the GFP concentration required to attain 50% of the maximal BRET signal (BRET₅₀). Titration curves for the ₂AR homo-oligomer and the _2/3AR hetero-oligomer gave similar BRET₅₀ values of 2.30 ± 0.54 and 2.45 ± 0.67, respectively (Fig. 1C), indicating that ₂AR had similar propensity to form homo-oligomers and hetero-oligomers with ₃AR. In contrast, co-expression of GABA_BR₂-GFP in ₂AR-Rluc-expressing cells led to a marginal BRET signal that could not be fitted to a hyperbolic function, confirming the selectivity of interaction among the AR subtypes (Fig. 1C).

To generate a cell system that would allow us to reliably assess the properties of the _2/3AR hetero-oligomer, we selected a HEK293 cell clone (HEK293-_2/3AR cells) that stably expressed a high ratio of ₃AR-GFP/₂AR-Rluc. The high ratio observed for the HEK293-_2/3AR cells (3.6 ± 0.2) corresponds to a GFP/Rluc ratio that would be expected to cause near-saturation of the ₂AR-Rluc by ₃AR-GFP (Fig. 1C). This prediction is further supported by the high level of constitutive BRET signal observed in these cells. Indeed, the BRET signal of 0.26 ± 0.02 observed was not significantly different from the maximum BRET signal (0.28 ± 0.03) obtained in the titration experiments (Fig. 1C). These data indicate that most if not all of the ₂AR-Rlucs, which can engage in oligomerization, are interacting with ₃AR-GFP molecules, excluding the existence of a significant proportion of ₂AR homo-oligomers in these cells. Since previous studies have suggested that if GPCR monomers exist, they represent a very small fraction of the total receptor population (33, 39), our data indicate that the HEK293-_2/3AR cells could provide a powerful tool to study _2/3AR hetero-oligomers in the absence of appreciable ₂AR homo-oligomers or monomers. However, the saturating BRET data presented here do not allow us to formally exclude the possibility that a fraction of the ₂AR may be incapable of forming oligomers and thus that a small population of ₂AR monomer could still exist in these cells. However, as will be seen from the extent of the effect of ₃AR co-expression on the agonist-promoted endocytosis of the ₂AR, this is unlikely.

Agonist-promoted Internalization of _2/3AR Hetero-oligomers—One of the major functional differences between ₂AR and ₃AR relates to their subcellular distribution following agonist stimulation. Indeed, as indicated in the Introduction, agonist treatment leads to a rapid endocytosis of the ₂AR while the ₃AR remains at the cell surface, even following sustained stimulation (24–26). We thus investigated whether the _2/3AR hetero-oligomer could undergo agonist-promoted endocytosis. In a first series of experiments, internalization was determined by assessing the translocation of receptor-Rluc fusion proteins into endosomes following cell fractionation. As expected, agonist stimulation of HEK293 cells expressing the ₂AR-Rluc alone led to 52 ± 5% receptor internalization, whereas no ₃AR-Rluc internalization (4 ± 3%) was observed in cells expressing this receptor subtype (Fig. 2A). In HEK293-_2/3AR cells, the agonist stimulation failed to promote any detectable internalization (3 ± 2%) of the ₂AR-Rluc, indicating that co-expression of the two receptors inhibited ₂AR endocytosis. This dominant-negative effect of ₃AR on ₂AR endocytosis was further confirmed by radioligand binding studies assessing the loss of cell surface-binding sites for the hydrophilic ligand CGP12177(40, 41). In these experiments, the CGP12177binding to ₂AR was determined by competition against a concentration of the lipophilic ligand ¹²⁵I-CYP (25 pM) that binds almost exclusively to ₂AR (this concentration occupies only 1% of the ₃AR sites) in HEK293-_2/3AR cells. As shown in Fig. 2B, although the nonselective -AR agonist ISO promoted a loss of 49 ± 8% of the CGP12177binding sites in HEK293-₂AR cells, no significant loss of cell surface ₂AR (4 ± 2%) was observed in HEK293-_2/3AR cells. This inhibitory effect of ₃AR on ₂AR sequestration was not due to heterologous cross-talk resulting from ₃AR activation because the ₂AR-selective agonist FEN used at a concentration (50 nM) that does not activate ₃AR (see Fig. 8) also failed to induce sequestration of ₂AR in HEK293-_2/3AR cells (Fig. 2B). Considering that ₃AR is able to block ₂AR sequestration without being directly activated, we conclude that ₃AR inhibits ₂AR sequestration as a result of their hetero-oligomerization. The total inhibition of ₂AR sequestration observed in HEK293-_2/3AR cells is a strong indication that, as suggested by the BRET data, all ₂AR are engaged in _2/3AR hetero-oligomers and that no functional ₂AR monomers or homo-oligomers are detectable in these cells. The lack of ₂AR endocytosis in HEK293-_2/3AR cells does not represent an idiosyncrasy of the cell clone studied because identical data were obtained with an independent cell clone expressing a similarly high ₃AR-GFP/₂AR-Rluc ratio (data not shown).

View larger version (23K): [in this window] [in a new window]   FIG. 2. Agonist-promoted 2AR endocytosis. A, HEK293 cells stably expressing 2AR-Rluc, 2AR-Rluc/3AR-GFP, or 3AR-Rluc were incubated with 1 µM ISO for 30 min at 37 °C. Receptor internalization was then assessed following cell fractionation by measuring the proportion of the luciferase activity associated with the endosomal fraction as described under "Experimental Procedures." B, agonist-promoted receptor internalization was also determined by radioligand binding studies. Cell surface receptors were quantified by assessing the proportion of 125I-CYP-binding sites accessible to the hydrophilic ligand CGP12117 The extent of internalization is expressed as a percentage of the cell surface receptor lost following treatments with 1 µM ISO or 50 nM FEN for 30 min at 37 °C. The binding experiments were carried out with 25 pM of 125I-CYP, a concentration that selectively labels 2AR without binding to 3AR. Results are expressed as the mean ± S.E. of 3–5 independent experiments carried out in triplicate. Asterisk indicates a significant (p < 0.05) difference of the 2AR endocytosis observed in 2/3AR versus 2AR expressing cells.

View larger version (15K): [in this window] [in a new window]   FIG. 8. PTX sensitivity of receptor-stimulated cAMP accumulation. HEK293-2AR, HEK293-2/3AR, and HEK293-3AR cells pre-treated or not with PTX were stimulated for 30 min at 37 °C with 50 nM or 1 µM FEN. The agonist-stimulated cAMP production was assessed by measuring the accumulation of [3H]cAMP in cells prelabeled with [3H]adenine and expressed as the ratio of [3H]cAMP/([3H]cAMP + [3H]ATP). Data represent the mean ± S.E. of five independent experiments carried out in triplicate. Asterisk indicates a significant (p < 0.05) difference between PTX-treated and -untreated cells.

View this table: [in this window] [in a new window]   TABLE I Ligand binding properties of 2/3AR hetero-oligomers Saturation binding or competition binding studies using 125I-CYP were performed with membranes derived from HEK293 cells stably expressing 2/3AR, 2AR, or 3AR. 125I-CYP concentrations for saturation binding experiments varied from 0.002 to 10 nM. 25 pM (isoproterenol/norepinephrine) or 250 pM (fenoterol/ICI118551) of 125I-CYP were used for competition binding experiments. Binding curves were analyzed by using the program Prism 3.0 and the curves best-fitted to either one or two binding sites. KD and Ki values are expressed in nM, and the Bmax are given in pmol/mg. When two sites were identified in saturation binding experiments, they were defined as binding site 1 and 2. In competition binding experiments, high and low affinity binding states for agonists were defined as Ki(H) and Ki(L), respectively. Results represent the mean ± S.D. of 2–3 independent experiments performed in triplicates. ND, not determined.

View larger version (27K): [in this window] [in a new window]   FIG. 3. Receptor-stimulated cAMP accumulation. A, HEK293-2AR, HEK293-2/3AR, and HEK293-3AR cells were stimulated for 45 min at 37 °C with increasing concentrations of ISO in the absence (filled symbols) or presence (open symbols) of the 2AR selective inverse agonist ICI118551 (10 nM). The cAMP production was assessed by measuring the accumulation of [3H]cAMP in cells prelabeled with [3H]adenine and expressed as % of the maximal accumulation observed in each cell line. B, spontaneous cAMP accumulation in HEK293-2AR and HEK293-2/3AR cells was measured in the presence of increasing concentrations of the inverse agonist ICI118551. Results are expressed as the mean ± S.E. of three independent experiments carried out in triplicate.

View larger version (25K): [in this window] [in a new window]   FIG. 4. -Arrestin-2 recruitment by the 2/3AR hetero-oligomer in HEK293 cells. HEK293 cells transiently expressing 2AR-Rluc and -arrestin-2-YFP in the presence or absence of co-expressed 3AR were stimulated or not with 1 µM (A) or with increasing concentrations (10-3 to 10-10 M) of ISO (B) for 30 min at room temperature, and the interaction between 2AR-Rluc and -arrestin-2-YFP was monitored by BRET1 following the addition of coelenterazine H. The -arrestin-2-YFP/2AR-Rluc expression ratios are indicated below the bars (A). Results are expressed as the mean ± S.E. of three independent experiments performed in duplicate. Asterisk indicates a significant (p < 0.05) difference between ISO-treated and -untreated cells.

The correlation between the reduced ₂AR endocytosis and the reduced ability of the _2/3AR hetero-oligomer to recruit -arrestin was further investigated in an additional cell system. For this purpose, COS-1 cells expressing ₂AR-Rluc and -arrestin-2-YFP in the presence or absence of excess ₃AR were used to measure the agonist-promoted ₂AR endocytosis and -arrestin recruitment, in parallel, in the same cells. In these experiments, COS-1 cells expressed 1.4 ± 0.4 pmol/mg of ₂AR-Rluc and 6.2 ± 1.6 pmol/mg ₃AR, resulting in a ₃AR/₂AR ratio of 4.4 ± 0.8. As shown in Fig. 5A and in agreement with the results obtained in the HEK293-_2/3AR cells, the robust ISO-promoted internalization of ₂AR observed in cells expressing the ₂AR was inhibited by 95 ± 7.8% in cell co-expressing an excess of ₃AR. This confirms that the dominant-negative effect of the ₃AR on ₂AR endocytosis is not limited to HEK293 cells and is most likely a general phenomenon. The endocytosis inhibition was paralleled by a 90 ± 4.8% decrease in agonist-promoted -arrestin recruitment in cells co-expressing the ₃AR (Fig. 5B). Taken together, these data support the hypothesis that the dominant-negative effect of the ₃AR on ₂AR endocytosis could result from a reduced ability of the hetero-oligomer to recruit -arrestin efficiently.

View larger version (26K): [in this window] [in a new window]   FIG. 5. ISO-promoted -arrestin-2 recruitment and 2/3AR hetero-oligomer endocytosis in COS-1 cells. COS-1 cells transiently expressing 2AR-Rluc and -arrestin-2-YFP in the presence or absence of co-expressed 3AR were stimulated or not for 30 min with ISO (1 µM). A, ISO-promoted endocytosis of the 2AR-Rluc was measured by assessing the proportion of 125I-CYP-binding sites accessible to the hydrophilic ligand CGP12117and expressed as % internalization, as described under "Experimental Procedures." Results are expressed as the mean ± S.E. of three independent experiments performed in triplicate. Asterisk indicates a significant (p < 0.05) difference. B, ISO-induced -arrestin-2-YFP recruitment by 2AR-Rluc was monitored by BRET1 following the addition of coelenterazine H to cells derived form the transfection experiments described in A. The -arrestin-2-YFP/2AR-Rluc expression ratios are indicated below the bars. Results are expressed as the mean ± S.E. of three independent experiments performed in triplicate. Asterisk indicates a significant (p < 0.05) difference between ISO-treated and -untreated cells. The transfection experiments were carried out to favor an excess of 3AR. The expression of the receptors was determined by radioligand binding assays and found to be 1.4 ± 0.4 pmol/mg for 2AR-Rluc and 6.2 ± 1.6 pmol/mg for 3AR.

View larger version (17K): [in this window] [in a new window]   FIG. 6. ISO-promoted desensitization of the 2/3AR hetero-oligomer. HEK293-2AR (triangle) or HEK293-2/3AR cells (circle) were pretreated (open symbols) or not (closed symbols) with 10 µM ISO for 1 h at 37 °C. Receptor desensitization was then assessed by measuring the ISO-promoted AC activity in membranes derived from naive or ISO-pre-stimulated cells, as described under "Experimental Procedures." Data represent the mean ± S.E. of three independent experiments carried out in duplicate.

View larger version (27K): [in this window] [in a new window]   FIG. 7. Receptor-stimulated ERK1/2 activation. HEK293-2AR, HEK293-2/3AR, and HEK293-3AR cells pretreated or not with pertussis toxin (PTX) were stimulated for 5 min at 37 °C with 1 µM ISO, 50 nM FEN, or FBS. ERK1/2 activity was then assessed following cell lysis using phospho-specific ERK1/2 antibodies. Densitometric analysis of the phospho-ERK1/2 bands were carried out and normalized to the intensity of the bands obtained for the total ERK1/2 population following reprobing with anti-ERK1/2 antibodies. The graphical representation of the ERK1/2 activity is expressed as fold over basal. Asterisk indicates a significant (p < 0.05) difference between PTX-treated and -untreated cells.

Effect of PTX on FEN-mediated cAMP Accumulation in Cells Expressing ₂AR, ₃AR, or _2/3AR—To determine whether the lack of G_i/o dependence for the _2/3AR-stimulated ERK1/2 activation could also be observed in the AC signaling, we tested the effect of PTX on agonist-induced cAMP accumulation. As shown in Fig. 8, PTX treatment increased the net agonist-stimulated cAMP production generated by a nonselective concentration of FEN (1 µM) in ₂AR-, ₃AR-, and _2/3AR-expressing cells, indicating that it alleviated an inhibitory influence of G_i/o on the receptor-mediated responses. However, cAMP production induced by a concentration of FEN (50 nM), which activates only the ₂AR, was PTX-sensitive in HEK293-₂AR but not in HEK293-_2/3AR cells, whereas ₃AR-expressing cells did not respond at all (Fig. 8). Thus, in contrast to ₂AR or ₃AR, _2/3AR-stimulated AC activity is not limited by a concomitant activation of G_i/o proteins, confirming that the hetero-oligomer has a distinct G protein coupling pattern.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Resonance energy transfer approaches similar to the BRET assay used in the present study are becoming tools of choice to monitor protein-protein interactions in living cells (32, 54). Because BRET between Rluc and GFP can occur only if the two molecules are within 100 Å from one another, the occurrence of BRET between Rluc- and GFP-bearing proteins indicates a molecular proximity that is consistent with oligomerization. Although dimerization is the simplest form of oligomerization, the detection of BRET does not provide any direct information about the stoichiometric arrangement of the protomers and the proportion of molecules involved in the oligomeric assembly. Thus, the term oligomer rather than dimer, which is often used to offer a simple model, is used throughout this article. Despite these limitations, proper quantitative analysis can provide useful information about the oligomerization process. For instance, BRET₅₀ values obtained from titration assays can provide information about the relative affinity of the partners involved (33). Also, by assuming that all the considered molecules are free to oligomerize, the maximal BRET values can be used to estimate the proportion of Rluc-bearing molecules that are engaged in oligomerization with the GFP-bearing molecules. Here, we took advantage of some of these features of BRET to quantitatively assess the formation of _2/3AR hetero-oligomers.

The similar BRET₅₀ observed in the present study for the ₂AR homo-oligomer and the _2/3AR hetero-oligomer indicate that the ₂AR has similar propensity to homo- and to hetero-oligomerize with the ₃AR. This is not unique to this receptor pair because similar high affinity hetero-oligomerization has been reported for several closely related GPCR subtypes (33, 55–57). The similar propensity of the ₂AR to form homo and _2/3AR hetero-oligomers suggests that rather small changes in the relative expression levels of the two subtypes could significantly affect the homo/hetero-oligomer ratio. We took advantage of this characteristic to generate a cell line where the overexpression of the ₃AR would favor the formation of _2/3AR hetero-oligomers so that the properties of the hetero-oligomer could be studied in the absence of significant amounts of ₂AR homo-oligomers or monomers. One line of evidence suggesting that the HEK293-_2/3AR cells fulfill this criterion was provided by the quantitative analysis of the BRET data. Indeed, the BRET signals observed between ₂AR-Rluc and ₃AR-GFP in HEK293-_2/3AR cells correspond to the theoretical value expected for the hetero-oligomerization of all ₂AR-Rluc with ₃AR-GFP. Although one cannot exclude the possibility that a proportion of ₂AR could be incapable of forming homo- or hetero-oligomers (leading to the persistence of monomer despite a saturation of the BRET signal), the observation that the excess ₃AR expressed in HEK293-_2/3AR cells completely inhibited the agonist-promoted endocytosis and G_i/o coupling of the ₂AR strongly indicates that all functional ₂AR were engaged in hetero-oligomerization. Thus, HEK293-_2/3AR cells provided a tool to characterize selectively the property of _2/3AR hetero-oligomers without the confounding effects of the ₂AR homo-oligomers, thus solving a problem often encountered in studies assessing the functional outcome of GPCR hetero-oligomerization.

The analysis of HEK293-_2/3AR cells revealed that the _2/3AR hetero-oligomeric complex maintains some of the properties of the individual protomers but acquired distinct ones. The identical properties includes unchanged ligand binding profiles and a preserved positive coupling to both AC and ERK signaling pathways. The hetero-oligomer differs from the ₃AR by its ability to undergo agonist-promoted desensitization and from the ₂AR by its lack of efficient -arrestin recruitment and absence of endocytosis in response to activation. Finally, the _2/3AR hetero-oligomer differs from both ₂AR and ₃AR by its inability to functionally interact with G_i/o.

Although several studies (7, 11, 12, 18, 58) reported that hetero-oligomerization changes the ligand binding properties of one or both protomers, no such effect was observed for the _2/3AR hetero-oligomer. Indeed, neither the binding affinity, potency, nor efficacy of any of the ₂AR ligands tested was found to be affected by _2/3AR hetero-oligomerization. This lack of changes in the pharmacological properties suggests that each protomer maintains its cognate ligand binding pocket. Given the role of the transmembrane domains in defining binding affinity and selectivity among -AR subtypes, the preservation of a ₂AR-like binding profile argues against a hetero-oligomerization model involving swapping of transmembrane segments between ₂AR and ₃AR and would be more compatible with a contact type hetero-oligomer, where each protomer maintains its original transmembrane domain organization (59, 60).

Despite the apparent lack of effect on ligand binding, hetero-oligomerization had a significant impact on receptor endocytosis. The association of the endocytosis-prone ₂AR with the endocytosis-resistant ₃AR led to a complex that could not be internalized upon agonist stimulation, indicating that the ₃AR acted as a dominant-negative of the ₂AR endocytosis. Changes in the internalization profile of a given receptor upon co-expression of a distinct receptor have often been reported and attributed to hetero-oligomerization (10, 12, 13, 17–19, 61). In the case of the ₂AR, co-expression with ₁AR (16), -opioid receptors (15), or V₂-vasopressin receptors (62) has been shown to inhibit its internalization. In the latter case, the inhibition required the concomitant activation of the ₂AR and V₂-vasopressin receptors and was attributed to the sequestration of -arrestin by the V₂-vasopressin receptors resulting in a decreased concentration of -arrestins available for the ₂AR (62). Such a mechanism cannot be invoked to explain the inhibitory effect of the ₃AR on the ₂AR internalization because: 1) the ₃AR blocked ₂AR internalization promoted by a concentration of FEN that does not activate the ₃AR, and 2) even when activated, ₃AR is not able to recruit -arrestin (48). The observation that endocytosis was blocked in the absence of ₃AR activation also rules out a possible involvement of downstream signaling cross-talk in the dominant-negative effect of the ₃AR. Thus, as in the case of the ₂AR/-opioid receptor co-expression, the inhibition of the ₂AR internalization by the ₃AR most likely results from their hetero-oligomerization.

Two main hypotheses could be proposed to explain how hetero-oligomerization with an endocytosis-resistant receptor can block ₂AR internalization: 1) the hetero-oligomerization leads to a complex that cannot efficiently interact with -arrestin, or 2) the interaction of the complex with -arrestin occurs but does not result in the targeting of the hetero-oligomer to the clathrin-coated pits and endosomes. The results obtained in the BRET-based -arrestin recruitment studies clearly favor the first hypothesis. Indeed, the very low propensity of the _2/3AR to recruit -arrestin indicates that the ₃AR efficiently blocks the interaction between -arrestin and ₂AR via hetero-oligomerization, hence inhibiting -arrestin-mediated endocytosis of the ₂AR. The difference in the potency of ISO to promote -arrestin recruitment in face of an unaltered receptor binding and AC stimulation potency suggests that the hetero-oligomerization selectively affected the ISO-promoted interaction with -arrestin but not with G_s. Because G protein-coupled receptor kinase-mediated phosphorylation of the ₂AR has been shown to increase its affinity for -arrestin, it could be hypothesized that hetero-oligomerization prevents efficient G protein-coupled receptor kinase-promoted phosphorylation.

Despite the reduced recruitment of -arrestin by the _2/3AR hetero-oligomer, this oligomeric complex was found to undergo agonist-promoted desensitization. This is somewhat surprising when considering the important role played by -arrestin in this regulatory process. However, several -arrestin-independent processes such as cAMP-dependent protein kinase-mediated receptor phosphorylation (63, 64) and endocytosis-independent receptor degradation (65) have been suggested previously. Additional studies will be needed to determine the precise mechanism underlying the hetero-oligomer desensitization.

It has been suggested previously that receptor endocytosis and/or -arrestin recruitment contributes to the activation of ERK1/2 by ₂AR (49–52). Consistent with this notion, the inhibition of ₂AR endocytosis upon hetero-oligomerization with either ₁AR or -opioid receptors was accompanied by an abolition of the ₂AR-stimulated ERK1/2 activation (15, 16). In contrast with these studies, the _2/3AR, which failed to undergo endocytosis, still evoked ERK1/2 activation in response to the ₂AR-selective agonist FEN, indicating that endocytosis is not required for the activation of ERK1/2 by the _2/3AR complex. The low apparent affinity of the _2/3AR for -arrestin also suggests that the ERK1/2 activation promoted by the hetero-oligomer occurs in a -arrestin independent manner. These characteristics are reminiscent of the ₃AR-stimulated ERK1/2 that also takes place in the absence of -arrestin recruitment and receptor endocytosis (31, 66). These results raise the intriguing possibility that agonist binding to the ₂AR could transactivate the ₃AR within the _2/3AR hetero-oligomer. Such transactivation within receptor oligomers has been proposed recently for GPCR of the family A (67, 68) and family C (69). However, as reported previously (31, 66, 70), the ₃AR-mediated ERK1/2 activation was found to be PTX-sensitive, whereas the _2/3AR-promoted activation was not. Although this does not exclude the possibility of transactivation within the _2/3AR, it clearly indicates that the _2/3AR hetero-oligomer induces ERK1/2 activity by a mechanism that differs from the ₃AR.

The lack of PTX sensitivity of the _2/3AR-stimulated ERK1/2 also clearly distinguishes the response of the hetero-oligomer from that observed for the ₂AR expressed alone. Indeed, in the present study as well as in numerous previous studies (71–78), ₂AR was found to couple to both G_s and G_i/o. This dual coupling of the ₂AR, observed in both ERK1/2 and AC pathways, was entirely lost for the _2/3AR hetero-oligomer. Similar loss of G_i/o coupling upon hetero-oligomerization has been suggested previously for the CCR2/CCR5 (79) and /µ-opioid receptor (10) pairs. In these two studies, coupling of the hetero-oligomer to a G protein that is not classically engaged by each of the protomers, G_q/11 in the case of the CCR2/CCR5 and not identified for the /µ-opioid receptors, was invoked to explain the maintained signaling. In the present study, no coupling to an additional G protein was observed. Rather, all the signaling properties of the _2/3AR could be explained by its maintained coupling to G_s.

The data presented here demonstrate that upon heterologous co-expression, human ₂AR and ₃AR can associate to form a hetero-oligomeric complex that maintains some of the properties of each of the protomers but acquires distinct ones. The distinct signaling characteristics could have important functional consequences on the catecholamine signaling efficacy in cells endogenously co-expressing these two receptors. Most interesting, mammalian adipocytes co-express ₂AR and ₃AR with a high ₃AR/₂AR expression ratio that resembles the one observed in HEK293-_2/3AR cells established in the present study (27–29, 80). Moreover, the hetero-oligomerization between human ₂AR and ₃AR could be observed at expression levels that are similar to those reported for human adipocytes (38). If, as in the case of HEK293-_2/3AR cells, hetero-oligomerization with the ₃AR blocks the G_i/o coupling of the ₂AR in adipocytes, it would undoubtedly lead to a more efficient activation of AC and ensuing lipolysis (27, 28, 38). The hetero-oligomerization would thus contribute to increase the AC responsiveness of this tissue to the endogenous catecholamines. The inhibition of both -arrestin recruitment and ₂AR endocytosis, resulting from the hetero-oligomerization with the ₃AR, would also be predicted to increase the adipocyte responsiveness to catecholamine stimulation. Although the experiments were not performed in the adipocytes, the present work carried out in heterologous expression systems allowed us to make predictions about the potential functional consequences of hetero-oligomerization in cells endogenously co-expressing ₂AR and ₃AR. The testing of these predictions in adipocytes derived from knock-out mice lacking either one of the two receptors should now help to determine the physiological importance of hetero-oligomerization for these receptor subtypes.

	FOOTNOTES

 
^* This work was supported in part by grants from the Canadian Institute for Health Research and from the Heart and Stroke Foundation of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a post-doctoral fellowship from AstraZeneca (Montréal).

To whom correspondence should be addressed. Tel.: 514-343-6372; Fax: 514-343-2210; E-mail: michel.bouvier{at}umontreal.ca.

¹ The abbreviations used are: GPCR, G protein-coupled receptors; AR, adrenergic receptor(s); AC, adenylyl cyclase; CYP, cyanopindolol; BRET, bioluminescence resonance energy transfer; DMEM, Dulbecco's modified Eagle's medium; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; FEN, fenoterol; GFP, green fluorescent protein; HRP, horseradish peroxidase; IBMX, 3-isobutyl-1-methylxanthine; ISO, isoproterenol; PBS, phosphate-buffered saline; PTX, pertussis toxin; Rluc, Renilla luciferase; YFP, yellow fluorescent protein.

	ACKNOWLEDGMENTS

 
We are grateful to Dr. Ali Salahpour for the critical reading of the manuscript.

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