TY - JOUR T1 - P2Y<sub>1</sub> Receptor Activation Elicits Its Partition out of Membrane Rafts and Its Rapid Internalization from Human Blood Vessels: Implications for Receptor Signaling JF - Molecular Pharmacology JO - Mol Pharmacol SP - 1666 LP - 1677 DO - 10.1124/mol.108.048496 VL - 74 IS - 6 AU - Andrés Norambuena AU - M. Inés Poblete AU - M. Verónica Donoso AU - C. Sofía Espinoza AU - Alfonso González AU - J. Pablo Huidobro-Toro Y1 - 2008/12/01 UR - http://molpharm.aspetjournals.org/content/74/6/1666.abstract N2 - The nucleotide P2Y1 receptor (P2Y1R) is expressed in both the endothelial and vascular smooth muscle cells; however, its plasma membrane microregionalization and internalization in human tissues remain unknown. We report on the role of membrane rafts in P2Y1R signaling by using sodium carbonate or OptiPrep sucrose density gradients, Western blot analysis, reduction of tissue cholesterol content, and vasomotor assays of endothelium-denuded human chorionic arteries. In tissue extracts prepared either in sodium carbonate or OptiPrep, approximately 20 to 30% of the total P2Y1R mass consistently partitioned into raft fractions and correlated with vasomotor activity. Vessel treatment with methyl β-cyclodextrin reduced the raft partitioning of the P2Y1R and obliterated the P2Y1R-mediated contractions but not the vasomotor responses elicited by either serotonin or KCl. Perfusion of chorionic artery segments with 100 nM 2-methylthio ADP or 10 nM [[(1R,2R,3S,4R,5S)-4-[6-amino-2-(methylthio)-9H-purin-9-yl] 2,3dihydroxybicyclo[3.1.0]hex-1-yl]methyl] diphosphoric acid mono ester trisodium salt (MRS 2365), a selective P2Y1R agonist, not only displaced within 4 min the P2Y1R localization out of membrane rafts but also induced its subsequent internalization. 2′-Deoxy-N6-methyladenosine 3′,5′-bisphosphate tetrasodium salt (MRS 2179), a specific P2Y1R antagonist, did not cause a similar displacement but blocked the agonist-induced exit from rafts. Neither adenosine nor uridine triphosphate displaced the P2Y1R from the membrane raft, further evidencing the pharmacodynamics of the receptor-ligand interaction. Vascular reactivity assays showed fading of the ligand-induced vasoconstrictions, a finding that correlated with the P2Y1R exit from raft domains and internalization. These results demonstrate in intact human vascular smooth muscle the association of the P2Y1R to membrane rafts, highlighting the role of this microdomain in P2Y1R signaling. The American Society for Pharmacology and Experimental Therapeutics ER -