4.6 Article

Constitutive and agonist-induced dimerizations of the P2Y1 receptor -: Relationship to internalization and scaffolding

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JOURNAL OF BIOLOGICAL CHEMISTRY
卷 283, 期 16, 页码 11050-11063

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AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M709266200

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  1. Wellcome Trust [081706, 076139] Funding Source: Medline

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In living cells, P2Y(1) receptor dimerization was quantitated by an improved version of fluorescence resonance energy transfer donor photobleaching analysis. 44% of the P2Y(1) 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 degrees C but are not sufficient; at 20 degrees 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.

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