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J. Biol. Chem., Vol. 283, Issue 16, 11050-11063, April 18, 2008

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Constitutive and Agonist-induced Dimerizations of the P2Y₁ Receptor

RELATIONSHIP TO INTERNALIZATION AND SCAFFOLDING^*

Roy C. Y. Choi¹, Joseph Simon, Karl W. K. Tsim, and Eric A. Barnard²

From the Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, United Kingdom and the Department of Biology, Hong Kong University of Science and Technology, Hong Kong, China

In living cells, P2Y₁ receptor dimerization was quantitated by an improved version of fluorescence resonance energy transfer donor photobleaching analysis. 44% of the P2Y₁ receptors expressed in HEK293 cell membranes exist as dimers in the resting state, inducible by agonist exposure to give 85-100% dimerization. Monomer and constitutive dimers are fully active. Agonist-induced dimerization follows desensitization and is fully reversible upon withdrawal of agonist. Receptor dimers are required for internalization at 37 °C but are not sufficient; at 20 °C dimerization also occurs, but endocytosis is abolished. Removal of the C-terminal 19 amino acids abolished both dimerization and internalization, whereas full activation by agonists was retained up to a loss of 39 amino acids, confirming active monomers. This receptor is known to bind through its last four amino acids (DTSL) to a scaffolding protein, Na/H exchanger regulatory factor-2, which was endogenous here, and DTSL removal blocked constitutive dimerization specifically. Distinction should therefore be made between the following: 1) constitutive dimers tethered to a scaffolding protein, together with effector proteins, within a signaling micro-domain, and 2) free dimers in the cell membrane, which here are inducible by agonist exposure. For the class A G-protein-coupled receptors, we suggest that the percentages of free monomers, and in many cases of induced free dimers, commonly become artifactually increased; this would arise from an excess there of the receptor over its specific scaffold and from a lack of the native targeting of the receptor to that site.

Received for publication, November 12, 2007 , and in revised form, February 8, 2008.

^* This work was supported in part by Wellcome Trust Grant 081706 (to E. A. B.) and by the Council of Hong Kong Research Grants 6237/04M and 6419/05M (to K. W. K. T.). 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.

¹ Recipient of an International Postdoctoral Fellowship of the Royal Society.

² To whom correspondence should be addressed. Tel.: 44-1223-847-876; E-mail: eb247{at}cam.ac.uk.

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