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.
Highlights
G-protein-coupled receptors (GPCRs)3 can be stimulated by diverse signals such as light, smell and taste, small molecules, peptides, and proteins
The P2Y receptor family is composed of currently eight members that are all GPCRs, termed P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14 [9]
All receptors were individually fused at their C termini with various color variants of the green fluorescent protein (GFP), and their internalization was monitored by confocal microscopy
Summary
Materials—ADP, ATP, UDP, UTP, 2-methylthio-ADP hexokinase, poly-D-lysine, and glucose were purchased from Sigma. HEK-293 cells were co-transfected with the indicated P2Y receptor and -arrestin-2-YFP construct. B, cells were co-transfected with GRK2, -arrestin-2-YFP, and P2Y6, P2Y11, or P2Y12 receptor tagged with CFP and studied for receptor internalization. Upon stimulation with the appropriate agonist, the four endogenous purine receptor agonists ADP, ATP, UDP, P2Y1, P2Y2, and P2Y4 receptors caused rapid translocation of and UTP (100 M) caused any change in the cytosolic localiza- -arrestin-2-YFP to the plasma membrane (Fig. 2A). The P2Y6, P2Y11, and P2Y12 receptors were clearly localized at the cell surface (Fig. 2A, right panels), they did tagged P2Y12 receptor [20], all receptors were functional with not result in -arrestin-2-YFP translocation to the plasma respect to downstream inositol phosphate signaling.
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