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P2Y₁ Receptor Activation Elicits Its Partition out of Membrane Rafts and Its Rapid Internalization from Human Blood Vessels: Implications for Receptor Signaling

Centro de Regulación Celular y Patología, J.V. Luco, Instituto Milenio de Biología Fundamental y Aplicada (A.N., M.I.P., M.V.D., C.S.E., A.G., J.P.H.-T.), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Biológicas (A.N., M.I.P., M.V.D., C.S.E., J.P.H.-T.), and Departamento de Inmunología Clínica y Reumatología (A.G.), Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile

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.

Address correspondence to: Dr. J. Pablo Huidobro-Toro, Departamento de Ciencias Fisiológicas, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile, P.O. Box 114-D, Santiago 1, Chile. E-mail: jphuidobro{at}bio.puc.cl