Allosteric interactions across native adenosine-A3 receptor homodimers: quantification using single-cell ligand-binding kinetics

Abstract

A growing awareness indicates that many G-protein-coupled receptors (GPCRs) exist as homodimers, but the extent of the cooperativity across the dimer interface has been largely unexplored. Here, measurement of the dissociation kinetics of a fluorescent agonist (ABA-X-BY630) from the human A(1) or A(3) adenosine receptors expressed in CHO-K1 cells has provided evidence for highly cooperative interactions between protomers of the A(3)-receptor dimer in single living cells. In the absence of competitive ligands, the dissociation rate constants of ABA-X-BY630 from A(1) and A(3) receptors were 1.45 ± 0.05 and 0.57 ± 0.07 min(-1), respectively. At the A(3) receptor, this could be markedly increased by both orthosteric agonists and antagonists [15-, 9-, and 19-fold for xanthine amine congener (XAC), 5'-(N-ethyl carboxamido)adenosine (NECA), and adenosine, respectively] and reduced by coexpression of a nonbinding (N250A) A(3)-receptor mutant. The changes in ABA-X-BY630 dissociation were much lower at the A(1) receptor (1.5-, 1.4-, and 1.5-fold). Analysis of the pEC(50) values of XAC, NECA, and adenosine for the ABA-X-BY630-occupied A(3)-receptor dimer yielded values of 6.0 ± 0.1, 5.9 ± 0.1, and 5.2 ± 0.1, respectively. This study provides new insight into the spatial and temporal specificity of drug action that can be provided by allosteric modulation across a GPCR homodimeric interface.

Figure 1.

The 2-drug model of constitutive GPCR homodimerization. A) Within the 2-drug model of constitutive GPCR homodimerization, ligand A and ligand B can bind to the first and/or second protomer in the homodimer, R-R. Binding of ligand A and ligand B is defined by their affinity for the free GPCR homodimer, K_a and K_b, respectively, and the cooperativity factors α, β, and γ. B, C) Influence of γ = 1 (B) and γ = 0.001 (C) on the equilibrium binding properties A and B. D) Influence of γ = 0.01 on the dissociation of A in the presence of increasing concentrations of B. [Ligand B] = ● 0 nM, ○ 10 nM, ▴ 30 nM, ▵ 100 nM, ▾ 300 nM; k_a+ = 5 × 10⁷ M⁻¹ s⁻¹; k_a− = 0.5 s⁻¹; k_b+ = 5 × 10⁷ M⁻¹ s⁻¹; k_b− = 0.5 s⁻¹; α = 1; β = 1; R_tot = 1 × 10⁻⁹ M.

Figure 2.

Cell surface A₃-AR homomeric interactions. A) CHO cells transiently expressing A₃-AR^YFP. B) CHO cells transiently expressing A₃-AR^N-YFP and A₃-AR^C-YFP. Images are single confocal equatorial slices, representative of those obtained from 3 wells in each of 4 separate transfections.

Figure 3.

Enhanced dissociation of a fluorescent adenosine derivative from the A₃-AR in the presence of a competitive ligand. Binding of 30 nM ABA-X-BY630 to CHO-A₃ cells after 0, 20 and 40 s of infinite dilution in the absence (A) or presence (B) of 1 μM XAC. Rate of perfusion was maintained at >12 complete fluid exchanges/min.

Figure 4.

Competitive ligands significantly enhance the dissociation of 30 nM ABA-X-BY630 from the A₃-AR. A–C) ABA-X-BY630 (30 nM) dissociation kinetics from CHO-A₃ cells in the absence (●) or presence of XAC (A; ○ 10 nM, ▾ 100 nM, ▿ 1 μM, ▴ 10 μM); NECA (B; ○ 100 nM, ▾ 1 μM, ▿ 10 μM, ▴ 100 μM); or adenosine (C; ▾ 1 μM, ▿ 10 μM, ▴ 100 μM). D–F) ABA-X-BY630 (30 nM) dissociation kinetics from CHO-A₁ cells in the absence (●) or presence (○) of XAC (D; 10 μM), NECA (E; 100 μM) or adenosine (F; 100 μM). Rate of perfusion was maintained at >12 complete fluid exchanges/min. Specific binding data was normalized as a percentage of fluorescent intensity at t =0 and globally analyzed according to a monophasic exponential decay. Data points are expressed as means ± se from 3–11 separate experiments; each replicate represents the average of 10 cells.

Figure 5.

Concentration-k_off relationship of ABA-X-BY630 dissociation in the absence and presence of competitive ligands at the A₃-AR and A₁-AR. Dissociation rate k_off of 30 nM ABA-X-BY630 from CHO-A₃ (A) and CHO-A₁ cells (B) in the absence (□) or presence of XAC (■), NECA (○) and adenosine (●). Values for k_off were derived from global analysis of dissociation kinetic data to a monophasic exponential decay.). Rate of perfusion was maintained at >12 complete fluid exchanges/min. Data points are expressed as means ± se from 3–11 separate experiments; each replicate represents the average of 10 cells.

Figure 6.

Competitive ligands inhibit the binding of 30 nM ABA-X-BY630 to the A₃-AR. A–C) ABA-X-BY630 (30 nM) association kinetics at CHO-A₃ cells in the absence (●) or presence of XAC (A; ○ 10 nM, ▴ 100 nM, ▵ 1 μM); NECA (B; ○ 10 nM, ▴ 100 nM, ▵ 1 μM, ▾ 10 μM); or adenosine (C; ▵ 1 μM, ▴ 10 μM). D) ABA-X-BY630 (30 nM) specific binding at 2 min association in the presence of XAC (■), NECA (○) and adenosine (●). Rate of perfusion was maintained at >12 complete fluid exchanges/min. Data points are expressed as means ± se from 2 to 11 separate experiments; each replicate represents the average of 10 cells.

Figure 7.

Cell surface expression of A₃-AR fusions. Expression of A₃-AR^GFP (A; green channel) and A₃(N250A)-AR^YFP (B; red channel) in CHO cells. Images are single confocal equatorial slices.

Figure 8.

Nonbinding A₃(N250A)-AR can disrupt cooperative interactions between ABA-X-BY630 and NECA at the A₃-AR. A) Expression of A₃-AR^GFP (green channel) and A₃-AR^YFP (red channel) in CHO cells. B, C) Influence of A₃-AR^GFP/A₃-AR^YFP fluorescence ratio (red, >10; blue, 1–9; black, 0.1–0.9) on dissociation of 30 nM ABA-X-BY630 in the absence (B) and presence (C) of 1 μM NECA. D) Dissociation rate of 30 nM ABA-X-BY630 in the absence (open bars) and presence (solid bars) of 1 μM NECA; cells are grouped according to their A₃-AR^GFP/A₃-AR^YFP fluorescence ratio. E) Expression of A₃-AR^GFP (green channel) and A₃(N250A)-AR^YFP (red channel) in CHO cells. F, G) Influence of A₃-AR^GFP/A₃(N250A)-AR^YFP fluorescence ratio (red, >10; blue, 1–9; black, 0.1–0.9) on dissociation of 30 nM ABA-X-BY630 in the absence (F) and presence (G) of 1 μM NECA. H) Dissociation rate of 30 nM ABA-X-BY630 in the absence (open bars) and presence (solid bars) of 1 μM NECA; cells are grouped according to their A₃-AR^GFP/A₃(N250A)-AR^YFP fluorescence ratio. Rate of perfusion was maintained at >12 complete fluid exchanges/min. Data points are expressed as means ± se from 5 to 38 cells from ≥3 separate experiments.

Figure 9.

A kinetic 2-drug model of GPCR homodimerization can fit experimental data. Parameter estimation methods were applied to the kinetic 2-drug model of GPCR homodimerization fitting the association (A) and dissociation (B) of 30 nM ABA-X-BY630 in the presence of increasing concentrations of XAC and the association (C) and dissociation (D) of 30 nM ABA-X-BY630 in the presence of increasing concentrations of NECA. Data points are expressed as the mean from 3 to 11 separate experiments; each replicate represents the average of 10 cells.