Agonist-selective, receptor-specific interaction of human P2Y receptors with beta-arrestin-1 and -2

Abstract

Interaction of G-protein-coupled receptors with beta-arrestins is an important step in receptor desensitization and in triggering "alternative" signals. By means of confocal microscopy and fluorescence resonance energy transfer, we have investigated the internalization of the human P2Y receptors 1, 2, 4, 6, 11, and 12 and their interaction with beta-arrestin-1 and -2. Co-transfection of each individual P2Y receptor with beta-arrestin-1-GFP or beta-arrestin-2-YFP into HEK-293 cells and stimulation with the corresponding agonists resulted in a receptor-specific interaction pattern. The P2Y(1) receptor stimulated with ADP strongly translocated beta-arrestin-2-YFP, whereas only a slight translocation was observed for beta-arrestin-1-GFP. The P2Y(4) receptor exhibited equally strong translocation for beta-arrestin-1-GFP and beta-arrestin-2-YFP when stimulated with UTP. The P2Y(6), P2Y(11), and P2Y(12) receptor internalized only when GRK2 was additionally co-transfected, but beta-arrestin translocation was only visible for the P2Y(6) and P2Y(11) receptor. The P2Y(2) receptor showed a beta-arrestin translocation pattern that was dependent on the agonist used for stimulation. UTP translocated beta-arrestin-1-GFP and beta-arrestin-2-YFP equally well, whereas ATP translocated beta-arrestin-1-GFP to a much lower extent than beta-arrestin-2-YFP. The same agonist-dependent pattern was seen in fluorescence resonance energy transfer experiments between the fluorescently labeled P2Y(2) receptor and beta-arrestins. Thus, the P2Y(2) receptor would be classified as a class A receptor when stimulated with ATP or as a class B receptor when stimulated with UTP. The ligand-specific recruitment of beta-arrestins by ATP and UTP stimulation of P2Y(2) receptors was further found to result in differential stimulation of ERK phosphorylation. This suggests that the two different agonists induce distinct active states of this receptor that show differential interactions with beta-arrestins.

FIGURE 1.

Agonist-induced internalization of YFP-tagged P2Y_{1,2,4,6,11,12} receptors in HEK-293 cells. Cells were transfected with fluorescently tagged P2Y receptor constructs and studied for receptor internalization. A, left column represents cells prior to stimulation with the indicated agonist. The right column shows the same cells 15 min after exposure to the indicated agonist. A punctate pattern occurred for the P2Y₁, P2Y₂, and P2Y₄ receptor, whereas no punctate pattern was observed for the P2Y₆, P2Y₁₁, and P2Y₁₂ receptor tagged with YFP. All agonist concentrations were 100 μm final. Data are representative examples of at least three individual experiments. The experiments are added as supplemental movies 1-6. B, cells were co-transfected with GRK2 and P2Y₆, P2Y₁₁, or P2Y₁₂ receptor tagged with YFP and studied for receptor internalization. The left column represents cells prior to stimulation with the indicated agonist. The right column shows the same cells 15 min after exposure to the indicated agonist. Under these conditions a punctate pattern did occur for the P2Y₆, P2Y₁₁, and P2Y₁₂ receptor tagged with YFP. All agonist concentrations were 100 μm final. Data are representative examples of at least three individual experiments. The experiments are added as supplemental movies 7-9. White scale bars represent 10 μm.

FIGURE 2.

β-Arrestin-2 translocation induced by stimulation of P2Y_{1,2,4,6,11,12} receptor. HEK-293 cells were co-transfected with the indicated P2Y receptor and β-arrestin-2-YFP construct. A, left column represents cells prior to stimulation with the indicated agonist. The middle column shows the same cells 15 min after exposure to the indicated agonist. A clear β-arrestin-2 translocation was observed for the P2Y₁, P2Y₂, and P2Y₄ receptor, whereas no β-arrestin-2 translocation was observed for the P2Y₆, P2Y₁₁, and P2Y₁₂ receptor. The right column shows localization of the CFP-tagged receptor construct to demonstrate co-transfection of the cells that were studied. All agonist concentrations were 100 μm final. Data are representative examples of at least three individual experiments. B, cells were co-transfected with GRK2, β-arrestin-2-YFP, and P2Y₆, P2Y₁₁, or P2Y₁₂ receptor tagged with CFP and studied for receptor internalization. The left column represents cells prior to stimulation with the indicated agonist. The middle column shows the same cells 15 min after exposure to the indicated agonist. A clear β-arrestin-2 translocation was observed for the P2Y₆ and P2Y₁₁ receptor, whereas no β-arrestin-2 translocation was seen for the P2Y₁₂ receptor. The right column shows localization of the CFP-tagged receptor construct to demonstrate co-transfection of the cells that were studied. All agonist concentrations were 100 μm final. Data are representative examples of at least three individual experiments. White scale bars represent 10 μm.

FIGURE 3.

Quantification of β-arrestin translocation induced by the P2Y_{1,2,4,6,11,12} receptor. Data from experiments as described in Fig. 2 and supplemental Fig. 2 were quantified and corrected for photobleaching as described under “Experimental Procedures.” Cells were co-transfected with wild-type P2Y receptors, β-arrestin-1-GFP or β-arrestin-2-YFP, and GRK2 as indicated. Circles indicate β-arrestin-1-GFP translocation, and squares represent data for β-arrestin-2-YFP translocation. Data represent average data ± S.E. of 10-12 different cells from at least three different experiments.

FIGURE 4.

Effect of dynamin-K44A on agonist-induced internalization of YFP-tagged P2Y_{1,2,4,6,11,12} receptors in HEK-293 cells. Cells were transfected with fluorescently tagged P2Y receptor constructs and dynamin-K44A mutant and studied for receptor internalization. The left column represents cells prior to stimulation with the indicated agonist. The right column shows the same cells 15 min after exposure to the indicated agonist. No punctate pattern was observed for the P2Y₁, P2Y₄, P2Y₆, P2Y₁₁, and P2Y₁₂ receptor tagged with YFP in the presence of dynamin K44A, whereas a punctate pattern still occurred for the P2Y₂ receptor. All agonist concentrations were 100 μm final. Data are representative examples of at least three individual experiments. White scale bars represent 10 μm.

FIGURE 5.

Differential effects of the two endogenous P2Y₂ receptor ligands. A, quantification of β-arrestin translocation after ATP (left) or UTP exposure (right). Data were quantified and corrected for photobleaching as described under “Experimental Procedures.” Circles represent translocation of β-arrestin-1-GFP, and squares represent translocation of β-arrestin-2-YFP. Data are average data ± S.E. from 10 to 12 cells of at least three different experiments. B, data from 15-min time points after agonist-induced arrestin translocation of panel (Fig. 4, left and right) were recalculated as % translocation (100% minus remaining cytosolic fluorescence) and presented as sorted for β-arrestin-1 or -2 rather than agonist. UTP exhibited stronger translocation for β-arrestin-1 than ATP (p < 0.01), whereas ATP exhibited stronger translocation for β-arrestin-2 than UTP (p < 0.05). C, left, detection of interactions between P2Y₂-YFP and β-arrestin-1-Cerulean by FRET. Normalized FRET ratios (F_YFP/F_CFP) of single cells co-transfected with P2Y₂-YFP and β-arrestin-1-Cerulean (left panel) or P2Y₂-YFP and β-arrestin-2-CFP (right panel) are shown. The cells were constantly superfused with buffer or buffer supplemented with ATP or UTP as described. Sample traces are shown that are representative for seven cells in three different experiments. D, left, time dependence of ATP (100 μm)-stimulated ERK phosphorylation in HEK-293 cells stably expressing human P2Y₂-YFP receptor; right, time dependence of UTP (100 μm)-stimulated ERK phosphorylation in HEK-293 cells stably expressing human P2Y₂ receptor. The data shown are representative for six individual experiments with ATP and UTP done in parallel.