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J. Biol. Chem., Vol. 283, Issue 45, 30933-30941, November 7, 2008

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Agonist-selective, Receptor-specific Interaction of Human P_2Y Receptors with β-Arrestin-1 and -2^*

From the Institute for Pharmacology and Toxicology, Versbacher Strasse 9, D-97078 Wuerzburg, Germany

Interaction of G-protein-coupled receptors with β-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 β-arrestin-1 and -2. Co-transfection of each individual P2Y receptor with β-arrestin-1-GFP or β-arrestin-2-YFP into HEK-293 cells and stimulation with the corresponding agonists resulted in a receptor-specific interaction pattern. The P2Y₁ receptor stimulated with ADP strongly translocated β-arrestin-2-YFP, whereas only a slight translocation was observed for β-arrestin-1-GFP. The P2Y₄ receptor exhibited equally strong translocation for β-arrestin-1-GFP and β-arrestin-2-YFP when stimulated with UTP. The P2Y₆, P2Y₁₁, and P2Y₁₂ receptor internalized only when GRK2 was additionally co-transfected, but β-arrestin translocation was only visible for the P2Y₆ and P2Y₁₁ receptor. The P2Y₂ receptor showed a β-arrestin translocation pattern that was dependent on the agonist used for stimulation. UTP translocated β-arrestin-1-GFP and β-arrestin-2-YFP equally well, whereas ATP translocated β-arrestin-1-GFP to a much lower extent than β-arrestin-2-YFP. The same agonist-dependent pattern was seen in fluorescence resonance energy transfer experiments between the fluorescently labeled P2Y₂ receptor and β-arrestins. Thus, the P2Y₂ 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 β-arrestins by ATP and UTP stimulation of P2Y₂ 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 β-arrestins.

Received for publication, February 22, 2008 , and in revised form, July 25, 2008.

^* This work was supported by the Deutsche Forschungsgemeinschaft Grant "Sonderforschungsbereich" SFB487 "Regulatory Membrane Proteins," by the Fonds der Chemischen Industrie, and by the Ernst-Jung Award for Medicine. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S5 and Movies 1-9.

² Present address: University of Reading, School of Pharmacy, Whiteknights, P. O. Box 228, Reading, Berkshire RG6 6AJ, United Kingdom.

¹ To whom correspondence should be addressed. E-mail: c.Hoffmann{at}toxi.uni-wuerzburg.de.

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