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Originally published In Press as doi:10.1074/jbc.M313310200 on April 27, 2004

J. Biol. Chem., Vol. 279, Issue 27, 28756-28765, July 2, 2004
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Hetero-oligomerization between {beta}2- and {beta}3-Adrenergic Receptors Generates a {beta}-Adrenergic Signaling Unit with Distinct Functional Properties*

Andreas Breit{ddagger}, 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 {beta}2- and {beta}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 {beta}2AR has identical propensity to hetero-oligomerize with the {beta}3AR than to form homo-oligomers. To determine the influence of heterooligomerization, a HEK293 cell line stably expressing an excess of {beta}3AR over {beta}2AR was generated so that all {beta}2AR are engaged in hetero-oligomerization with {beta}3AR, providing a tool to study the effect of hetero-oligomerization on {beta}2AR function in the absence of any {beta}2AR homooligomer. The hetero-oligomerization had no effect on the ligand binding properties of various {beta}2AR ligands and did not affect the potency of isoproterenol to stimulate adenylyl cyclase. Despite the unaltered ligand binding properties of the {beta}2/3AR hetero-oligomer, the stable association of the {beta}2AR with the {beta}3AR completely blocked agonist-stimulated internalization of the {beta}2AR. Given that the {beta}3AR is resistant to agonist-promoted endocytosis, the results indicate that the {beta}3AR acted as a dominant negative of the {beta}2AR endocytosis process. Consistent with this notion, the {beta}2/3AR hetero-oligomer displayed a lower propensity to recruit {beta}-arrestin-2 than the {beta}2AR. The hetero-oligomerization also led to a change in G protein coupling selectivity. Indeed, in contrast to {beta}2AR and {beta}3AR, which regulate adenylyl cyclase and extracellular signal-regulated kinase activity through a coupling to Gs and Gi/o, no Gi/o coupling was observed for the {beta}2/3AR hetero-oligomer. Together, these results demonstrate that hetero-oligomerization between {beta}2AR and {beta}3AR forms a {beta}-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 (25). Hetero-oligomerization among GPCR has been shown to modulate ligand binding, G protein-coupling, endocytosis, and desensitization of the receptors (619).

Because of their clear and distinct properties, the {beta}2 adrenergic receptor ({beta}2AR) and {beta}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 {beta}2AR undergoes rapid and efficient agonist-promoted internalization (2123), the {beta}3AR is resistant to these regulatory processes (2426). The fact that these two closely related receptor subtypes are naturally co-expressed in adipocytes (2729) 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 {beta}2AR heterologously expressed in HEK293 cells is engaged in hetero-oligomerization with the {beta}3AR, thus allowing the functional characterization of the {beta}2AR within the {beta}2/3AR hetero-oligomer in the absence of any {beta}2AR homo-oligomer.

Our study reveals that in contrast to the robust agonist-promoted endocytosis characteristic of the {beta}2AR, the {beta}2/3AR hetero-oligomer was not internalized upon agonist stimulation, suggesting that the {beta}3AR-negative endocytotic phenotype prevailed in the hetero-oligomer. This dominant-negative effect of the {beta}3AR occurred without any change in the ligand binding properties of the receptors and most likely results from a diminished {beta}-arrestin-2 recruitment to the hetero-oligomer. When considering the coupling properties of the hetero-oligomer, we found that unlike {beta}2AR and {beta}3AR, which can couple to Gi/o (30, 31), the {beta}2/3AR hetero-oligomer cannot engage this signaling pathway. Taken together, our results indicate that the {beta}2/3AR hetero-oligomer is a {beta}AR-like signaling unit distinct from {beta}2AR or {beta}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-{beta}2AR-GFP10 (where GFP10 is a variant form of the green fluorescent protein (GFP) containing the following mutations: P64L, S147P, and S202P), pcDNA3.1-{beta}2AR-Rluc, and pcDNA3.1-{beta}-arrestin-2-YFP vectors were reported previously (32, 33). The pcDNA3-{beta}3AR-Rluc construct was kindly provided by PerkinElmer Life Sciences. For pGFP-{beta}3AR-GFP10, the {beta}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 {beta}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 {beta}2AR-Rluc or {beta}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 {beta}2AR-Rluc and {beta}3AR-GFP, a cell clone already expressing {beta}2AR-Rluc was co-transfected with the {beta}3AR-GFP construct along with the pEDmtxr vector harboring a point-mutated dihydrofolate reductase gene that transfers resistance to methotrexate (34). After selection with 400 nM methotrexate, clones co-expressing {beta}2AR-Rluc and {beta}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 NXTTM (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 {beta}-arrestin-2 recruitment by {beta}2AR, the BRET1 technology (FusionTM {alpha}-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 {beta}-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 {beta}2AR (this concentration binds 1% or less of the {beta}3AR sites) or 250 pM of 125I-CYP to ensure the occupancy of {beta}2AR and {beta}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 (CYPtot) 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 (CYPCGP), although total specific binding was defined as the binding inhibited by 10 µM of the hydrophobic ligand alprenolol (CYPAlp). The internalization rate of the receptor in percentage was calculated based on the following equation: ((CYPtot - CYPCGP)/(CYPtot - CYPAlp) x 100)basal - ((CYPtot - CYPCGP)/(CYPtot - CYPAlp) 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 (Rluclight) was found at the 0–35% interface, whereas the heavy plasma membrane fraction (Rlucheavy) 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: (Rlucheavy/(Rlucheavy + Rluclight) x 100)basal - (Rlucheavy/(Rlucheavy + Rluclight) 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 AlphaScreenTM 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 FusionTM {alpha}-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 {beta}-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 KD, Ki, 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 {beta}2/3AR Hetero-oligomers by BRET—To determine whether {beta}2AR and {beta}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 {beta}2AR-Rluc and {beta}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 {beta}2AR homo-oligomers described previously (32, 33, 36, 37). Co-expression of {beta}3AR-Rluc and {beta}3AR-GFP also led to constitutive energy transfer (BRET signal, 0.16 ± 0.02), providing the first evidence that {beta}3AR can form homo-oligomers in living cells (Fig. 1A). To assess whether {beta}2AR and {beta}3AR could also form hetero-oligomeric complexes, {beta}2AR-Rluc/{beta}3AR-GFP and {beta}3AR-Rluc/{beta}2AR-GFP pairs were co-expressed, and the resulting energy transfer was measured. Both combinations resulted in significant BRET signals ({beta}2AR-Rluc/{beta}3AR-GFP, 0.15 ± 0.02; {beta}3AR-Rluc/{beta}2AR-GFP, 0.17 ± 0.03) indicating that constitutive {beta}2/3AR hetero-oligomers can assemble (Fig. 1A). No significant energy transfer was detected between {beta}2AR-Rluc and GABABR2-GFP (BRET signal, 0.02 ± 0.005) when these receptors were expressed at levels comparable with those of {beta}2AR-Rluc and {beta}3AR-GFP, thus indicating that the energy transfer observed for the {beta}2AR-Rluc/{beta}3AR-GFP pair did not result from an overexpression of energy donor and acceptor.



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FIG. 1.
Detection of {beta}2/3AR hetero-oligomers by BRET2. A, the energy donor {beta}2AR-Rluc or {beta}3AR-Rluc and the energy acceptor {beta}2AR-GFP, {beta}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, {beta}2AR-Rluc and {beta}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 {beta}3AR/{beta}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 {beta}2AR-GFP, {beta}3AR-GFP, or GABABR2-GFP were transiently expressed in a cell line stably expressing {beta}2AR-Rluc (410 fmol/mg) and BRET2 signals measured 48 h after transfection. Expression levels of {beta}3AR-GFP varied between 100 and 2000 fmol/mg. BRET2 signals are plotted as a function of the expression ratio of receptor-GFP over {beta}2AR-Rluc obtained by measuring the total luminescence and fluorescence in each condition. The expression level of the {beta}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 {beta}3AR-GFP over {beta}2AR-Rluc expression ratio observed in HEK293-{beta}2/3AR cells stably co-expressing both receptor subtypes, and the bar illustrates their BRET2 signal obtained from five different experiments.

 
To exclude further the possibility that the BRET signals obtained could result from nonspecific interaction or aggregation due to overexpression in a heterologous expression system, BRET experiments were carried out in HEK293 cells expressing a wide spectrum of receptor numbers (580–6570 fmol/mg) in an ~1:1 {beta}3AR/{beta}2AR ratio. If the BRET signal observed is the result of spurious interactions between the two receptors, it would be expected to increase as a function of the total receptors expressed even at constant energy acceptor/donor ({beta}3AR-GFP/{beta}2AR-Rluc) ratios. As shown in Fig. 1B, the BRET signals detected were constant over the entire range of receptor expression, indicating that overexpression did not drive the heterooligomerization. Most interesting, the lowest receptor density tested (580 fmol/mg) is similar to values reported previously (596 ± 263 fmol/mg) for fully differentiated human brown adipocytes (38), indicating that oligomerization could occur in native tissue.

To assess the propensity of {beta}2AR to either homo- or heterooligomerize with {beta}3AR, BRET titration curves were carried out in HEK293 cells stably expressing 410 fmol/mg of {beta}2AR-Rluc by increasing the concentrations of {beta}2AR-GFP or {beta}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 {beta}2AR-Rluc molecules that can engage in oligomerization are in a complex with either {beta}2AR- or {beta}3AR-GFP. The apparent affinity of the {beta}2AR-GFP and {beta}3AR-GFP for the {beta}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 {beta}2AR homo-oligomer and the {beta}2/3AR hetero-oligomer gave similar BRET50 values of 2.30 ± 0.54 and 2.45 ± 0.67, respectively (Fig. 1C), indicating that {beta}2AR had similar propensity to form homo-oligomers and hetero-oligomers with {beta}3AR. In contrast, co-expression of GABABR2-GFP in {beta}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 {beta}AR subtypes (Fig. 1C).

To generate a cell system that would allow us to reliably assess the properties of the {beta}2/3AR hetero-oligomer, we selected a HEK293 cell clone (HEK293-{beta}2/3AR cells) that stably expressed a high ratio of {beta}3AR-GFP/{beta}2AR-Rluc. The high ratio observed for the HEK293-{beta}2/3AR cells (3.6 ± 0.2) corresponds to a GFP/Rluc ratio that would be expected to cause near-saturation of the {beta}2AR-Rluc by {beta}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 {beta}2AR-Rlucs, which can engage in oligomerization, are interacting with {beta}3AR-GFP molecules, excluding the existence of a significant proportion of {beta}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-{beta}2/3AR cells could provide a powerful tool to study {beta}2/3AR hetero-oligomers in the absence of appreciable {beta}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 {beta}2AR may be incapable of forming oligomers and thus that a small population of {beta}2AR monomer could still exist in these cells. However, as will be seen from the extent of the effect of {beta}3AR co-expression on the agonist-promoted endocytosis of the {beta}2AR, this is unlikely.

Agonist-promoted Internalization of {beta}2/3AR Hetero-oligomers—One of the major functional differences between {beta}2AR and {beta}3AR relates to their subcellular distribution following agonist stimulation. Indeed, as indicated in the Introduction, agonist treatment leads to a rapid endocytosis of the {beta}2AR while the {beta}3AR remains at the cell surface, even following sustained stimulation (2426). We thus investigated whether the {beta}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 {beta}2AR-Rluc alone led to 52 ± 5% receptor internalization, whereas no {beta}3AR-Rluc internalization (4 ± 3%) was observed in cells expressing this receptor subtype (Fig. 2A). In HEK293-{beta}2/3AR cells, the agonist stimulation failed to promote any detectable internalization (3 ± 2%) of the {beta}2AR-Rluc, indicating that co-expression of the two receptors inhibited {beta}2AR endocytosis. This dominant-negative effect of {beta}3AR on {beta}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 {beta}2AR was determined by competition against a concentration of the lipophilic ligand 125I-CYP (25 pM) that binds almost exclusively to {beta}2AR (this concentration occupies only 1% of the {beta}3AR sites) in HEK293-{beta}2/3AR cells. As shown in Fig. 2B, although the nonselective {beta}-AR agonist ISO promoted a loss of 49 ± 8% of the CGP12177binding sites in HEK293-{beta}2AR cells, no significant loss of cell surface {beta}2AR (4 ± 2%) was observed in HEK293-{beta}2/3AR cells. This inhibitory effect of {beta}3AR on {beta}2AR sequestration was not due to heterologous cross-talk resulting from {beta}3AR activation because the {beta}2AR-selective agonist FEN used at a concentration (50 nM) that does not activate {beta}3AR (see Fig. 8) also failed to induce sequestration of {beta}2AR in HEK293-{beta}2/3AR cells (Fig. 2B). Considering that {beta}3AR is able to block {beta}2AR sequestration without being directly activated, we conclude that {beta}3AR inhibits {beta}2AR sequestration as a result of their hetero-oligomerization. The total inhibition of {beta}2AR sequestration observed in HEK293-{beta}2/3AR cells is a strong indication that, as suggested by the BRET data, all {beta}2AR are engaged in {beta}2/3AR hetero-oligomers and that no functional {beta}2AR monomers or homo-oligomers are detectable in these cells. The lack of {beta}2AR endocytosis in HEK293-{beta}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 {beta}3AR-GFP/{beta}2AR-Rluc ratio (data not shown).



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FIG. 2.
Agonist-promoted {beta}2AR endocytosis. A, HEK293 cells stably expressing {beta}2AR-Rluc, {beta}2AR-Rluc/{beta}3AR-GFP, or {beta}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 {beta}2AR without binding to {beta}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 {beta}2AR endocytosis observed in {beta}2/3AR versus {beta}2AR expressing cells.

 



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FIG. 8.
PTX sensitivity of receptor-stimulated cAMP accumulation. HEK293-{beta}2AR, HEK293-{beta}2/3AR, and HEK293-{beta}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.

 
Pharmacological Characterization of {beta}2/3AR Hetero-oligomers—Since previous studies (11, 12, 18) reported that hetero-oligomerization can inhibit ligand binding, alterations in the binding properties of the {beta}2AR could be responsible for the lack of agonist-promoted endocytosis. Thus, to determine whether the ligand binding properties of the {beta}2AR are affected by its hetero-oligomerization with {beta}3AR, both saturation and competition radioligand binding studies were performed. 125I-CYP saturation isotherms carried out with membranes derived from HEK293-{beta}2/3AR cells were found to be biphasic, revealing high (KD1) and low (KD2) affinity components that agreed well with the affinities of CYP determined in cells individually expressing the {beta}2AR and {beta}3AR, respectively (Table I). Based on the fact that all {beta}2AR are engaged in oligomeric assembly with the {beta}3AR in HEK293-{beta}2/3AR cells, the data indicate that the {beta}2AR component of the {beta}2/3AR hetero-oligomer maintains {beta}2AR-like affinity for CYP. The {beta}2AR component of the hetero-oligomer also maintained {beta}2AR-like affinities for the agonists ISO and norepinephrine. Indeed, in competition binding experiments performed with a concentration of 125I-CYP (25 pM) that can only bind the {beta}2AR component, ISO and norepinephrine showed Ki values that are not different from those observed in cells expressing the {beta}2AR alone (Table I). Most interesting, both Ki(H) and Ki(L), which are characteristic of agonist affinities for the G protein-coupled and -uncoupled forms of the receptor (42), were similar, indicating that the {beta}2AR component of the {beta}2/3AR hetero-oligomer can engage G proteins. Next, to confirm that the selectivity of ligands toward {beta}2AR was also preserved in the hetero-oligomer, competition binding experiments with the {beta}2AR selective agonist, FEN, and inverse agonist, ICI118551, were carried out using a 125I-CYP concentration (250 pM) that can detectably bind to both {beta}2AR and {beta}3AR. As shown in Table I, each compound recognized two binding sites on membranes derived from HEK293-{beta}2/3AR cells that corresponded well with their affinities for the individually expressed {beta}2AR and {beta}3AR. Although we cannot exclude that the {beta}2/3AR hetero-oligomer could have altered binding properties for other ligands, our results indicate that hetero-oligomerization with the {beta}3AR does not radically affect the overall binding properties of the {beta}2AR and clearly demonstrate that the lack of agonist-promoted {beta}2/3AR endocytosis does not result from altered binding to ISO or FEN.


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TABLE I
Ligand binding properties of {beta}2/3AR hetero-oligomers Saturation binding or competition binding studies using 125I-CYP were performed with membranes derived from HEK293 cells stably expressing {beta}2/3AR, {beta}2AR, or {beta}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.

 
cAMP Accumulation Induced by {beta}2/3AR Hetero-oligomers—Hetero-oligomerization between AT2 and AT1 angiotensin receptors has been proposed to inhibit AT1 receptor activation (14). Thus to determine whether the lack of internalization could result from a similar inhibition of the {beta}2AR activity by {beta}3AR, the functional characteristic of the {beta}2/3AR hetero-oligomer was assessed. When expressed individually, {beta}2AR and {beta}3AR are well characterized positive regulators of adenylyl cyclase (AC) through their coupling to Gs (43). To test whether the {beta}2/3AR hetero-oligomeric complex is still able to stimulate AC activity, we examined ISO-induced cAMP production in HEK293-{beta}2/3AR cells. As shown in Fig. 3A, the concentration-dependent increase in ISO-stimulated AC activity was very similar for {beta}2/3AR-, {beta}3AR-, and {beta}2AR-expressing cells (EC50, 9.8 ± 2.6, 28.7 ± 5.8, and 13.9 ± 3.9 nM, respectively). Because the AC activity observed in {beta}2/3AR cells could be entirely due to {beta}3AR, the functional integrity of {beta}2AR within the hetero-oligomer was assessed by using the {beta}2AR-selective antagonist ICI118551. The EC50 for ISO in HEK293-{beta}2AR cells increased about 65-fold in the presence of ICI118551 (894 ± 64 nM), whereas the response was not affected in HEK293-{beta}3AR cells (Fig. 3A). For HEK293-{beta}2/3AR cells, the presence of the {beta}2AR-selective antagonist led to a biphasic dose-response curve. The first portion of the curve was insensitive to ICI118551, with an EC50 of 6.5 ± 2.2 nM, and thus represented the {beta}3AR response. The second portion was significantly right-shifted by ICI118551, with an EC50 of 1254 ± 276 nM, and thus reflected a {beta}2AR-mediated cAMP production. Considering the undetectable level of {beta}2AR homo-oligomers in HEK293-{beta}2/3AR cells, the ICI118551-sensitive response most likely resulted from the {beta}2AR component of the {beta}2/3AR hetero-oligomer. Taken with the detection of a high affinity ISO-binding site (Table I), these results indicate that {beta}2/3AR can functionally interact with Gs and thus the lack of endocytosis does not reflect an inactive receptor. As in the case of the {beta}2AR expressed alone (44), the spontaneous (agonist-independent) activity of the {beta}2/3AR hetero-oligomer could be efficiently inhibited by the inverse agonist ICI118551 (Fig. 3B), confirming the unaltered functional properties of the {beta}2AR within the hetero-oligomer.



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FIG. 3.
Receptor-stimulated cAMP accumulation. A, HEK293-{beta}2AR, HEK293-{beta}2/3AR, and HEK293-{beta}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 {beta}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-{beta}2AR and HEK293-{beta}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.

 
{beta}-Arrestin Recruitment by {beta}2/3AR Hetero-oligomers—Because agonist-induced {beta}-arrestin recruitment is a crucial step in the {beta}2AR internalization process (45), the lack of agonist-promoted internalization of {beta}2/3AR raises the question of whether {beta}2/3AR hetero-oligomers are able to recruit {beta}-arrestin. To address this question, we monitored the recruitment of {beta}-arrestin-2-YFP by {beta}2AR-Rluc in the absence or presence of co-expressed {beta}3AR using the BRET1 technology (32, 46, 47) after transient expression in HEK293 cells. As shown in Fig. 4A, stimulation with 1 µM ISO promoted a significant BRET signal between {beta}2AR-Rluc and {beta}-arrestin-2-YFP, reflecting the translocation of {beta}-arrestin-2 to the receptor. In contrast, no BRET signal could be observed between {beta}3AR-Rluc and {beta}-arrestin-2-YFP under the same conditions (data not shown), an observation consistent with the lack of agonist-promoted internalization of this receptor. Co-expression of the {beta}3AR with {beta}2AR-Rluc and {beta}-arrestin-2-YFP significantly reduced the ISO-promoted BRET signal (Fig. 4A). Given that the {beta}-arrestin-2-YFP/{beta}2AR-Rluc expression ratio was unaffected by the {beta}3AR co-expression, the decreased BRET signal most likely reflects a decreased ability of the hetero-oligomer to recruit {beta}-arrestin-2. However, because the extent of energy transfer does not only reflect the number of BRET pairs generated but also the distance and orientation between the energy donor and acceptor within the pairs, one cannot exclude the possibility that the reduced BRET signal could reflect a different conformational arrangement of the {beta}2AR-Rluc/{beta}-arrestin-2-YFP complex within the hetero-oligomer.



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FIG. 4.
{beta}-Arrestin-2 recruitment by the {beta}2/3AR hetero-oligomer in HEK293 cells. HEK293 cells transiently expressing {beta}2AR-Rluc and {beta}-arrestin-2-YFP in the presence or absence of co-expressed {beta}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 {beta}2AR-Rluc and {beta}-arrestin-2-YFP was monitored by BRET1 following the addition of coelenterazine H. The {beta}-arrestin-2-YFP/{beta}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.

 
To determine whether the decreased BRET signal results from a reduced ability of the hetero-oligomer to recruit {beta}-arrestin-2 or from an altered conformation of the complex, the potency of ISO to recruit {beta}-arrestin-2-YFP to {beta}2AR-Rluc was assessed in the presence or absence of co-expressed {beta}3AR. Indeed, given that the hetero-oligomerization with {beta}3AR did not affect the affinity of the {beta}2AR for ISO (Table I), the potency of ISO to promote the BRET signal will reflect the relative affinity of {beta}-arrestin-2 for the receptor. In the absence of {beta}3AR, the potency of ISO to promote {beta}-arrestin-2 recruitment was 17.6 ± 4.1 nM (Fig. 4B), a value consistent with the potency of ISO to stimulate AC (13.9 ± 3.9 nM; Fig. 3A) and with its high affinity binding site (15.6 ± 3.6 nM; Table I). In cells co-expressing {beta}3AR, the ISO-promoted BRET signal between {beta}2AR-Rluc and {beta}-arrestin-2-YFP was found to be biphasic, with a high potency close to the one revealed in the absence of {beta}3AR (35.6 ± 5.6 nM) and a second potency that was significantly right-shifted (EC50, 26,680 ± 3192 nM). The high potency component is not statistically different from the one observed in cells expressing the {beta}2AR alone and thus most likely reflects the potency of the {beta}2AR homo-oligomer to recruit {beta}-arrestin-2. The presence of a {beta}2AR homo-oligomer component indicates that the amount of {beta}3AR expressed in this transient expression system used was not sufficient to saturate all the {beta}2AR. When expressed alone, the {beta}3AR cannot recruit {beta}-arrestin-2 (48) (data not shown). Thus, the low potency component of the {beta}-arrestin-2 recruitment detected in the biphasic curve probably corresponds to the recruitment of {beta}-arrestin-2 to the {beta}2/3AR hetero-oligomer, consistent with the hypothesis that the hetero-oligomer has a decreased ability to recruit {beta}-arrestin-2.

The correlation between the reduced {beta}2AR endocytosis and the reduced ability of the {beta}2/3AR hetero-oligomer to recruit {beta}-arrestin was further investigated in an additional cell system. For this purpose, COS-1 cells expressing {beta}2AR-Rluc and {beta}-arrestin-2-YFP in the presence or absence of excess {beta}3AR were used to measure the agonist-promoted {beta}2AR endocytosis and {beta}-arrestin recruitment, in parallel, in the same cells. In these experiments, COS-1 cells expressed 1.4 ± 0.4 pmol/mg of {beta}2AR-Rluc and 6.2 ± 1.6 pmol/mg {beta}3AR, resulting in a {beta}3AR/{beta}2AR ratio of 4.4 ± 0.8. As shown in Fig. 5A and in agreement with the results obtained in the HEK293-{beta}2/3AR cells, the robust ISO-promoted internalization of {beta}2AR observed in cells expressing the {beta}2AR was inhibited by 95 ± 7.8% in cell co-expressing an excess of {beta}3AR. This confirms that the dominant-negative effect of the {beta}3AR on {beta}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 {beta}-arrestin recruitment in cells co-expressing the {beta}3AR (Fig. 5B). Taken together, these data support the hypothesis that the dominant-negative effect of the {beta}3AR on {beta}2AR endocytosis could result from a reduced ability of the hetero-oligomer to recruit {beta}-arrestin efficiently.



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FIG. 5.
ISO-promoted {beta}-arrestin-2 recruitment and {beta}2/3AR hetero-oligomer endocytosis in COS-1 cells. COS-1 cells transiently expressing {beta}2AR-Rluc and {beta}-arrestin-2-YFP in the presence or absence of co-expressed {beta}3AR were stimulated or not for 30 min with ISO (1 µM). A, ISO-promoted endocytosis of the {beta}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 {beta}-arrestin-2-YFP recruitment by {beta}2AR-Rluc was monitored by BRET1 following the addition of coelenterazine H to cells derived form the transfection experiments described in A. The {beta}-arrestin-2-YFP/{beta}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 {beta}3AR. The expression of the receptors was determined by radioligand binding assays and found to be 1.4 ± 0.4 pmol/mg for {beta}2AR-Rluc and 6.2 ± 1.6 pmol/mg for {beta}3AR.

 
ISO-induced Desensitization of Agonist-promoted AC Activity in HEK293-{beta}2/3AR Cells—Because of the central role played by {beta}-arrestin in the agonist-promoted desensitization of the {beta}2AR, we next examined the ability of the {beta}2/3AR hetero-oligomer to undergo ISO-promoted desensitization. For this purpose, ISO-promoted AC activity was assessed in cell membranes derived from HEK293-{beta}2AR or HEK293-{beta}2/3AR cells pretreated or not with ISO for 1 h. As shown in Fig. 6, ISO stimulated AC with almost identical potency in membranes derived from naive HEK293-{beta}2AR and HEK-{beta}2/3AR cells (EC50: {beta}2AR, 32.5 ± 4.2 nM; {beta}2/3AR, 29.2 ± 3.5 nM). The agonist-promoted desensitization of the {beta}2AR can be readily seen by the significant decrease in ISO potency observed in membranes derived from HEK293-{beta}2AR cells pretreated with ISO (EC50, 7560 ± 137 nM). In the case of the HEK293-{beta}2/3AR cells, ISO pretreatment led to a complex dose-response that was best fitted to a two component curve. The first component displayed an EC50 (39.4 ± 4.8 nM) almost identical to that obtained in membranes derived from untreated cells, whereas the EC50 of the second component was significantly right-shifted to a value similar to that obtained in the desensitized {beta}2AR-expressing membranes (EC50, 12,470 ± 1730 nM). Because {beta}3AR does not undergo agonist-promoted desensitization (24, 26), and no {beta}2AR homo-oligomer exists in the HEK293-{beta}2/3AR cells, we conclude that the first component of the curve represents the non-desensitized {beta}3AR, whereas the second component corresponds to the desensitized {beta}2/3AR hetero-oligomer. These data therefore suggest that despite its inefficient recruitment of {beta}-arrestin, the {beta}2/3AR hetero-oligomer can undergo agonist-promoted desensitization, indicating the contribution of {beta}-arrestin-independent mechanism(s).



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FIG. 6.
ISO-promoted desensitization of the {beta}2/3AR hetero-oligomer. HEK293-{beta}2AR (triangle) or HEK293-{beta}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.

 
{beta}2/3AR-mediated Activation of ERK1/2—Several studies (4952) have suggested that the activation of the ERK1/2 signaling pathway by {beta}2AR involves {beta}-arrestin recruitment and {beta}2AR endocytosis. Particularly relevant to the present study is the observation that hetero-oligomerization between {beta}2AR and either {beta}1AR or {kappa}-opioid receptors blocked both {beta}2AR endocytosis and {beta}2AR-mediated ERK1/2 activation (15, 16). To determine whether the inhibition of {beta}2AR endocytosis resulting from its hetero-oligomerization with {beta}3AR also blocked its ability to activate ERK1/2, the agonist-stimulated ERK1/2 activity was assessed. As shown in Fig. 7, ISO promoted ERK1/2 phosphorylation in HEK293-{beta}2/3AR cells as well as in cells expressing {beta}2AR or {beta}3AR alone. To determine whether the {beta}2/3AR hetero-oligomer contributed to the response or if it could be entirely attributed to the {beta}3AR, cells were stimulated with the {beta}2AR-selective agonist FEN. Fifty nM FEN activated ERK1/2 in HEK293-{beta}2/3AR cells but not in cells expressing {beta}3AR alone, indicating that {beta}2/3AR hetero-oligomers are able to activate ERK1/2.



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FIG. 7.
Receptor-stimulated ERK1/2 activation. HEK293-{beta}2AR, HEK293-{beta}2/3AR, and HEK293-{beta}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.

 
{beta}2AR and {beta}3AR-stimulated ERK1/2 activity was previously shown to be PTX-sensitive, suggesting a role for Gi/o in this pathway (30, 31). To determine whether this is also a property of the {beta}2/3AR hetero-oligomer, the effect of PTX was investigated. In cells expressing the {beta}2AR or {beta}3AR alone, PTX significantly decreased the agonist-stimulated ERK1/2 activation (Fig. 7), confirming previous reports. In contrast, ERK1/2 activity stimulated by the selective {beta}2AR agonist FEN was found to be resistant to PTX treatment in HEK293-{beta}2/3AR cells (Fig. 7), indicating that the {beta}2/3AR activates ERK1/2 in a Gi/o-independent manner. This PTX resistance cannot be attributed to a cell-specific difference such as those recently found among various HEK293 cell clones (53) because the ISO-stimulated ERK1/2 activity (resulting from the {beta}3AR homo-oligomers) was found to be PTX-sensitive in the same HEK293-{beta}2/3AR cell clone (Fig. 7). Together, these results indicate that the {beta}2/3AR hetero-oligomer activates the ERK1/2 through a signaling cascade that is independent of the activation of Gi/o.

Effect of PTX on FEN-mediated cAMP Accumulation in Cells Expressing {beta}2AR, {beta}3AR, or {beta}2/3AR—To determine whether the lack of Gi/o dependence for the {beta}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 {beta}2AR-, {beta}3AR-, and {beta}2/3AR-expressing cells, indicating that it alleviated an inhibitory influence of Gi/o on the receptor-mediated responses. However, cAMP production induced by a concentration of FEN (50 nM), which activates only the {beta}2AR, was PTX-sensitive in HEK293-{beta}2AR but not in HEK293-{beta}2/3AR cells, whereas {beta}3AR-expressing cells did not respond at all (Fig. 8). Thus, in contrast to {beta}2AR or {beta}3AR, {beta}2/3AR-stimulated AC activity is not limited by a concomitant activation of Gi/o proteins, confirming that the hetero-oligomer has a distinct G protein coupling pattern.


    DISCUSSION
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
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 DISCUSSION
 REFERENCES
 
Resonance energy transfer approaches similar to the BRET assay used in the present study are be