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

• Published in: Molecular pharmacology 2008-12, Vol.74 (6), p.1666-1677
• Main Authors: Norambuena, Andrés, Poblete, M Inés, Donoso, M Verónica, Espinoza, C Sofía, González, Alfonso, Huidobro-Toro, J Pablo
• Format: Article
• Language: English
• Subjects: Blood Vessels - metabolism; Blood Vessels - physiology; Female; Humans; In Vitro Techniques; Membrane Microdomains - metabolism; Muscle Contraction; Muscle, Smooth, Vascular - metabolism; Muscle, Smooth, Vascular - physiology; Placenta - blood supply; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2 - physiology; Receptors, Purinergic P2Y1; Signal Transduction
• ISSN: 0026-895X; 1521-0111
• DOI: 10.1124/mol.108.048496

The nucleotide P2Y 1 receptor (P2Y 1 R) 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 P2Y 1 R 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 P2Y 1 R mass consistently partitioned into raft fractions and correlated with vasomotor activity. Vessel treatment with methyl β-cyclodextrin reduced the raft partitioning of the P2Y 1 R and obliterated the P2Y 1 R-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 [[(1 R ,2 R ,3 S ,4 R ,5 S )-4-[6-amino-2-(methylthio)-9 H -purin-9-yl] 2,3dihydroxybicyclo[3.1.0]hex-1-yl]methyl] diphosphoric acid mono ester trisodium salt (MRS 2365), a selective P2Y 1 R agonist, not only displaced within 4 min the P2Y 1 R localization out of membrane rafts but also induced its subsequent internalization. 2′-Deoxy- N 6 -methyladenosine 3′,5′-bisphosphate tetrasodium salt (MRS 2179), a specific P2Y 1 R antagonist, did not cause a similar displacement but blocked the agonist-induced exit from rafts. Neither adenosine nor uridine triphosphate displaced the P2Y 1 R 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 P2Y 1 R exit from raft domains and internalization. These results demonstrate in intact human vascular smooth muscle the association of the P2Y 1 R to membrane rafts, highlighting the role of this microdomain in P2Y 1 R signaling.