title: Constitutive and agonist-induced dimerizations of the P2Y1 receptor: relationship to internalization and scaffolding.
abstract: 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.
authors.affiliation: Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, United Kingdom.
authors.forename: Roy C Y
authors.initials: RC
authors.lastname: Choi
authors.forename: Joseph
authors.initials: J
authors.lastname: Simon
authors.forename: Karl W K
authors.initials: KW
authors.lastname: Tsim
authors.forename: Eric A
authors.initials: EA
authors.lastname: Barnard
mesh_terms: D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008954:Models, Biological; D009711:Nucleotides / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000737:chemistry*; D058472:Receptors, Purinergic P2Y1; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D009711:Nucleotides; C546382:P2RY1 protein, human; D010750:Phosphoproteins; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor