Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET)

Abstract

Heptahelical receptors that interact with heterotrimeric G proteins represent the largest family of proteins involved in signal transduction across biological membranes. Although these receptors generally were believed to be monomeric entities, a growing body of evidence suggests that they may form functionally relevant dimers. However, a definitive demonstration of the existence of G protein-coupled receptor (GPCR) dimers at the surface of living cells is still lacking. Here, using bioluminescence resonance energy transfer (BRET), as a protein-protein interaction assay in whole cells, we unambiguously demonstrate that the human beta(2)-adrenergic receptor (beta(2)AR) forms constitutive homodimers when expressed in HEK-293 cells. Receptor stimulation with the hydrophilic agonist isoproterenol led to an increase in the transfer of energy between beta(2)AR molecules genetically fused to the BRET donor (Renilla luciferase) and acceptor (green fluorescent protein), respectively, indicating that the agonist interacts with receptor dimers at the cell surface. Inhibition of receptor internalization did not prevent agonist-promoted BRET, demonstrating that it did not result from clustering of receptors within endosomes. The notion that receptor dimers exist at the cell surface was confirmed further by the observation that BS3, a cell-impermeable cross-linking agent, increased BRET between beta(2)AR molecules. The selectivity of the constitutive interaction was documented by demonstrating that no BRET occurred between the beta(2)AR and two other unrelated GPCR. In contrast, the well characterized agonist-dependent interaction between the beta(2)AR and the regulatory protein beta-arrestin could be monitored by BRET. Taken together, the data demonstrate that GPCR exist as functional dimers in vivo and that BRET-based assays can be used to study both constitutive and hormone-promoted selective protein-protein interactions.

Figure 1

Co-immunoprecipitation of β₂AR molecules bearing different immunological epitopes. c-myc-β₂AR-Rluc and β₂AR-YFP were expressed (lanes 2–4) or not (lane 1) in HEK-293 cells and immunoprecipitated with the agarose-conjugated anti-c-myc mAb (Santa Cruz Biotechnology). The immunoprecipitated proteins then were resolved by SDS/PAGE and immunoblotted with the polyclonal anti-GFP antibody (CLONTECH; this antibody also recognizes YFP). The occurrence of receptor dimerization is revealed by the fact that the YFP-tagged β₂AR is coimmunoprecipitated with the c-myc-tagged receptor by the anti-c-myc mAb (lane 4).

Figure 2

BRET in living HEK-293 cells expressing β₂AR-YFP and β₂AR-Rluc. (A) HEK-293 cells expressing β₂AR-YFP (asterisks), β₂AR-Rluc (■), or coexpressing β₂AR-YFP and β₂AR-Rluc (▴) or KAIB-YFP and KAIB-Rluc (○) were incubated with 5 μM coelenterazine h (Molecular Probes), and light-emission acquisition was performed immediately. All spectra were normalized as percentage of maximal emission. The spectra shown are representative of four independent experiments. (B) Luminescence and fluorescence signals were quantitated by using a BretCount (Packard), allowing the sequential integration of the signals detected in the 440- to 500-nm and 510- to 590-nm windows. The BRET ratio was defined as [(emission at 510–590) − (emission at 440–500) × Cf]/(emission at 440–500), where Cf corresponded to (emission at 510–590)/(emission at 440–500) for the β₂AR-Rluc expressed alone in the same experiments. Readings were started immediately after coelenterazine addition, and repeated measures were taken for ≈20 min. The data shown represent the mean ± SEM of four independent readings. (C) To assess the specificity of interaction, BRET ratios were measured in cells coexpressing β₂AR-Rluc and β₂-YFP together or individually with either CCR5-YFP and Mel1a-Rluc, respectively.

Figure 3

BRET as a sensor for biological processes in vivo. (A) Schematic representation of the Rluc-YFP fusion construct harboring a consensus cleavage site (DEVD) for caspase-3. (B) The BRET ratio measured in cells expressing the fusion protein treated or not with staurosporine and/or the caspase-3 inhibitor-I. The data shown represent the mean ± SEM of four independent readings.

Figure 4

Effect of chemical crosslinking on β₂AR dimerization. (A) A FLAG-tagged β₂AR was immunoprecipitated from cells treated or not for 30 min with the cell-impermeable chemical crosslinker BS3 by using a polyclonal anti-β₂AR antibody (Santa Cruz Biotechnology). Immunoprecipitates then were resolved by SDS/PAGE, and the receptor was detected by Western blot analysis by using the monoclonal anti-FLAG antibody (Sigma). (B) BRET ratio measured in cells expressing the indicated constructs. Cells were treated or not with BS3 for 30 min before the addition of coelenterazine. The data represent the mean ± SEM of four independent readings.

Figure 5

Effect of agonist treatment. BRET ratio measured in cells expressing β₂AR-Rluc and β₂AR-YFP after addition of increasing concentrations of the agonist isoproterenol in the presence (▾) or absence (■) of 10 μM of the β-adrenergic antagonist propranolol. The data represent the mean ± SEM of four independent readings that were analyzed by using a four-parameter logistic equation (GraphPad prism 2.01) and fixing the Hill coefficient to 1 (EC₅₀ = 30 nM). The data also were analyzed, allowing for variable Hill coefficients. In that case, the fitted Hill coefficient was found to be 0.64 and the EC₅₀ was 28 nM.

Figure 6

Effect of the inhibition of endocytosis on BRET. BRET ratio was measured in cells expressing the indicated constructs in the presence or absence of the β₂-adrenergic and CCR5 agonists isoproterenol (10 μM) and MIP-1α (0.2 μM). The effect of coexpressing the dominant negative mutant of dynamin (DynK44A) was also assessed. The data represent the mean ± SEM of four independent readings. Receptor expression levels were as follows: β₂AR-Rluc, 211 fmol/mg protein; β₂AR-Rluc and β₂AR-YFP, a total of 398 fmol/mg protein; β₂AR-Rluc and CCR5-YFP, 400 fmol/mg protein for the β₂AR and 456 fmol/mg protein for CCR5.

Figure 7

Agonist dependence of β-arrestin/β₂AR interactions assessed by BRET. BRET ratio measured in cells coexpressing β₂AR-Rluc and β₂-arrestin-YFP in the presence of increasing concentrations of the agonist isoproterenol (■). The open square represents the value observed in the absence of isoproterenol, and the open circle represents the value observed in cells expressing β₂AR-Rluc alone. The data represent the mean ± SEM of four independent readings that were analyzed by using a four-parameter logistic equation (GraphPad prism 2.01) and fixing the Hill coefficient to 1 (EC₅₀ = 0.45 nM). The data also were analyzed allowing for variable Hill coefficients. In this case, the fitted Hill coefficient was found to be 0.74 and the EC₅₀ was 0.43 nM.