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• W2057764503 abstract "Extracellular nucleotides transmit signals into the cells through the P2 family of cell surface receptors. These receptors are amply expressed in human blood vessels and participate in vascular tone control; however, their signaling mechanisms remain unknown. Here we show that in smooth muscle cells of isolated human chorionic arteries, the activation of the P2Y2 receptor (P2Y2R) induces not only its partition into membrane rafts but also its rapid internalization. Cholesterol depletion with methyl-β-cyclodextrin reduced the association of the agonist-activated receptor into membrane rafts but did not affect either the UTP-mediated vasoconstrictions or the vasomotor responses elicited by both serotonin and KCl. Ex vivo perfusion of human chorionic artery segments with 1–10 μm UTP, a selective P2Y2R agonist, displaced the P2Y2R localization into membrane rafts within 1 min, a process preceded by the activation of both RhoA and Rac1 GTPases. AG1478, a selective and potent inhibitor of the epidermal growth factor receptor tyrosine kinase activity, not only blocked the UTP-induced vasomotor activity but also abrogated both RhoA and Rac1 activation, the P2Y2R association with membrane rafts, and its internalization. Altogether, these results show for the first time that the plasma membrane distribution of the P2Y2R is transregulated by the epidermal growth factor receptor, revealing an unsuspected functional interplay that controls both the membrane distribution and the vasomotor activity of the P2Y2R in intact human blood vessels. Extracellular nucleotides transmit signals into the cells through the P2 family of cell surface receptors. These receptors are amply expressed in human blood vessels and participate in vascular tone control; however, their signaling mechanisms remain unknown. Here we show that in smooth muscle cells of isolated human chorionic arteries, the activation of the P2Y2 receptor (P2Y2R) induces not only its partition into membrane rafts but also its rapid internalization. Cholesterol depletion with methyl-β-cyclodextrin reduced the association of the agonist-activated receptor into membrane rafts but did not affect either the UTP-mediated vasoconstrictions or the vasomotor responses elicited by both serotonin and KCl. Ex vivo perfusion of human chorionic artery segments with 1–10 μm UTP, a selective P2Y2R agonist, displaced the P2Y2R localization into membrane rafts within 1 min, a process preceded by the activation of both RhoA and Rac1 GTPases. AG1478, a selective and potent inhibitor of the epidermal growth factor receptor tyrosine kinase activity, not only blocked the UTP-induced vasomotor activity but also abrogated both RhoA and Rac1 activation, the P2Y2R association with membrane rafts, and its internalization. Altogether, these results show for the first time that the plasma membrane distribution of the P2Y2R is transregulated by the epidermal growth factor receptor, revealing an unsuspected functional interplay that controls both the membrane distribution and the vasomotor activity of the P2Y2R in intact human blood vessels. The plasma membrane (PM) 4The abbreviations and trivial name used are: PMplasma membraneP2YRP2Y receptorP2Y2RP2Y2 receptorP2Y1RP2Y1 receptorMβCDmethyl-β-cyclodextrinGPCRG protein-coupled receptorHCAhuman chorionic arterySMCsmooth muscle cell(s)EGFepidermal growth factorEGFRepidermal growth factor receptor5-HTserotoninAAarachidonic acidERKextracellular signal-regulated kinaseGSTglutathione S-transferaseMAPKmitogen-activated protein kinaseMRS 2365[[(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 monoester trisodium salt. nucleotide receptors of the P2Y (P2YR) family are all Gq or Gi protein-coupled receptors (GPCRs); this family is composed of eight members (P2YR1, P2YR2, P2YR4, P2YR6, and P2YR11–14) (1North R.A. Physiol. Rev. 2002; 82: 1013-1067Crossref PubMed Scopus (2490) Google Scholar). The P2YRs are ubiquitous and amply expressed in the brain and other organs (2Burnstock G. Physiol. Rev. 2007; 87: 659-797Crossref PubMed Scopus (1290) Google Scholar). They are activated by either purines, such as ADP/ATP, or pyrimidines, including UTP, UDP, CTP, or UDP-glucose (3Burnstock G. Trends Pharmacol. Sci. 2006; 27: 166-176Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar). Moreover, P2YRs are pharmacological targets in processes like platelet aggregation and treatment of cystic fibrosis (4Burnstock G. Pharmacol. Rev. 2006; 58: 58-86Crossref PubMed Scopus (527) Google Scholar, 5Abbracchio M.P. Burnstock G. Boeynaems J.M. Barnard E.A. Boyer J.L. Kennedy C. Knight G.E. Fumagalli M. Gachet C. Jacobson K.A. Weisman G.A. Pharmacol. Rev. 2006; 58: 281-341Crossref PubMed Scopus (1061) Google Scholar). plasma membrane P2Y receptor P2Y2 receptor P2Y1 receptor methyl-β-cyclodextrin G protein-coupled receptor human chorionic artery smooth muscle cell(s) epidermal growth factor epidermal growth factor receptor serotonin arachidonic acid extracellular signal-regulated kinase glutathione S-transferase mitogen-activated protein kinase [[(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 monoester trisodium salt. Both ADP and UTP have been recently recognized as signals involved in the contractility of the human vascular wall through the activation of P2Y1/2Rs (6Buvinic S. Briones R. Huidobro-Toro J.P. Br. J. Pharmacol. 2002; 136: 847-856Crossref PubMed Scopus (87) Google Scholar, 7Buvinic S. Poblete M.I. Donoso M.V. Delpiano A.M. Briones R. Miranda R. Huidobro-Toro J.P. J. Physiol. 2006; 573: 427-443Crossref PubMed Scopus (26) Google Scholar). In particular, the activation of P2Y1/2Rs by ADP, ATP, or UTP stimulates smooth muscle contraction in human chorionic arteries (HCA), whereas these nucleotides relax microvessels in the placental cotyledon through the release of nitric oxide (7Buvinic S. Poblete M.I. Donoso M.V. Delpiano A.M. Briones R. Miranda R. Huidobro-Toro J.P. J. Physiol. 2006; 573: 427-443Crossref PubMed Scopus (26) Google Scholar). Notwithstanding its contribution to vascular smooth muscle tone, little is known about its cellular mechanisms. Microregionalization is a common feature of cell signaling. Clathrin-coated pits, caveolae, and membrane rafts contain high concentrations of signaling molecules. An array of GPCR has also been identified in caveolae or rafts (reviewed in Ref. 8Allen J.A. Halverson-Tamboli R.A. Rasenick M.M. Nat. Rev. Neurosci. 2007; 8: 128-140Crossref PubMed Scopus (681) Google Scholar). In many cases, the receptor microregionalization is sensitive to ligand stimulation, altering the receptor clustering in or out of membrane rafts. We recently showed that in HCA, the P2Y1R-mediated vasocontractile activity depends on its association with membrane rafts (9Norambuena A. Poblete M.I. Donoso M.V. Espinoza C.S. González A. Huidobro-Toro J.P. Mol. Pharmacol. 2008; 74: 1666-1677Crossref PubMed Scopus (15) Google Scholar). Although membrane rafts have been involved in signal transduction mediated by UTP (10Sugawara Y. Nishii H. Takahashi T. Yamauchi J. Mizuno N. Tago K. Itoh H. Cell. Signal. 2007; 19: 1301-1308Crossref PubMed Scopus (50) Google Scholar), neither the P2Y2R cell membrane distribution nor the molecular mechanisms involved have been clarified. Membrane rafts are a dynamic assembly of cholesterol, glycosphingolipids, and proteins, such as caveolin, flotillins, Src family kinases, and glycosylphosphatidylinositol-linked proteins (11Simons K. Toomre D. Nat. Rev. Mol. Cell Biol. 2000; 1: 31-39Crossref PubMed Scopus (5187) Google Scholar, 12Brown D.A. London E. J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2065) Google Scholar). Cholesterol is a vital and major component of membrane rafts; it is known that its extraction with MβCD causes a loss in identifiable caveolae (13Hailstones D. Sleer L.S. Parton R.G. Stanley K.K. J. Lipid Res. 1998; 39: 369-379Abstract Full Text Full Text PDF PubMed Google Scholar, 14Westermann M. Steiniger F. Richter W. Histochem. Cell Biol. 2005; 123: 613-620Crossref PubMed Scopus (55) Google Scholar). The actin cytoskeleton also appears to play a role. Actin network disruption by latrunculin B, a toxin isolated from the red sponge Negombata magnifica (formerly Latrunculia magnifica), results in loss of the caveolae-F actin association (15Mundy D.I. Machleidt T. Ying Y.S. Anderson R.G. Bloom G.S. J. Cell Sci. 2002; 115: 4327-4339Crossref PubMed Scopus (261) Google Scholar). Filamin, an actin cross-linking protein, is one of the proteins identified as ligand of caveolin-1 (16Stahlhut M. van Deurs B. Mol. Biol. Cell. 2000; 11: 325-337Crossref PubMed Scopus (266) Google Scholar). Interestingly, activation of the P2Y2R induces its association with filamin and actin reorganization in aortic SMC (17Yu N. Erb L. Shivaji R. Weisman G.A. Seye C.I. Circ. Res. 2008; 102: 581-588Crossref PubMed Scopus (54) Google Scholar). Because of its increasing role in signal transduction, the mechanisms that govern organization of these microdomains are a matter of intense research and debate. Interestingly, recent data show that epidermal growth factor (EGF) induces the coalescence of different membrane rafts (18Hofman E.G. Ruonala M.O. Bader A.N. van den Heuvel D. Voortman J. Roovers R.C. Verkleij A.J. Gerritsen H.C. van Bergen En Henegouwen P.M. J. Cell Sci. 2008; 121: 2519-2528Crossref PubMed Scopus (119) Google Scholar), making it likely that the EGF·EGF receptor (EGFR) complex also transregulates signaling pathway(s) mediated by several other physiological inputs. Connected to the role of P2Y1/2Rs in cell proliferation, cell migration, and vascular tone control (6Buvinic S. Briones R. Huidobro-Toro J.P. Br. J. Pharmacol. 2002; 136: 847-856Crossref PubMed Scopus (87) Google Scholar, 7Buvinic S. Poblete M.I. Donoso M.V. Delpiano A.M. Briones R. Miranda R. Huidobro-Toro J.P. J. Physiol. 2006; 573: 427-443Crossref PubMed Scopus (26) Google Scholar, 9Norambuena A. Poblete M.I. Donoso M.V. Espinoza C.S. González A. Huidobro-Toro J.P. Mol. Pharmacol. 2008; 74: 1666-1677Crossref PubMed Scopus (15) Google Scholar, 19Liu J. Liao Z. Camden J. Griffin K.D. Garrad R.C. Santiago-Pérez L.I. González F.A. Seye C.I. Weisman G.A. Erb L. J. Biol. Chem. 2004; 279: 8212-8218Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 20Morris J.B. Pham T.M. Kenney B. Sheppard K.E. Woodcock E.A. J. Biol. Chem. 2004; 279: 8740-8746Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 21Schafer R. Sedehizade F. Welte T. Reiser G. Am. J. Physiol. Lung Cell Mol. Physiol. 2003; 285: L376-L385Crossref PubMed Scopus (105) Google Scholar, 22Soltoff S.P. Avraham H. Avraham S. Cantley L.C. J. Biol. Chem. 1998; 273: 2653-2660Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 23Wagstaff S.C. Bowler W.B. Gallagher J.A. Hipskind R.A. Carcinogenesis. 2000; 21: 2175-2181Crossref PubMed Scopus (83) Google Scholar), EGF is also a key regulator in a variety of cellular systems. It binds to EGFR, triggering tyrosine kinase activity that results in the activation of several signaling proteins, such Ras, phosphatidylinositol 3-kinase, and phospholipase Cγ pathways, leading to mitogenic events (24Hollenberg M.D. Mol. Cell Biochem. 1995; 149: 77-85Crossref PubMed Scopus (37) Google Scholar, 25Jorissen R.N. Walker F. Pouliot N. Garrett T.P. Ward C.W. Burgess A.W. Exp. Cell Res. 2003; 284: 31-53Crossref PubMed Scopus (1274) Google Scholar). The EGFR also plays a role in vascular physiology. EGF induces contractility of vascular and gastric smooth muscles (24Hollenberg M.D. Mol. Cell Biochem. 1995; 149: 77-85Crossref PubMed Scopus (37) Google Scholar, 26Berk B.C. Brock T.A. Webb R.C. Taubman M.B. Atkinson W.J. Gimbrone Jr., M.A. Alexander R.W. J. Clin. Invest. 1985; 75: 1083-1086Crossref PubMed Scopus (168) Google Scholar, 27Muramatsu I. Hollenberg M.D. Lederis K. Can J. Physiol. Pharmacol. 1985; 63: 994-999Crossref PubMed Scopus (55) Google Scholar). Moreover, activation of receptors for angiotensin and catecholamines results in transactivation of the EGFR, most probably by activation of metalloproteinases and release of EGFR ligands, such as HB-EGF (28Hao L. Du M. Lopez-Campistrous A. Fernandez-Patron C. Circ. Res. 2004; 94: 68-76Crossref PubMed Scopus (158) Google Scholar), a process associated with smooth muscle cell growth and contraction, respectively (29Zhang H. Chalothorn D. Jackson L.F. Lee D.C. Faber J.E. Circ. Res. 2004; 95: 989-997Crossref PubMed Scopus (90) Google Scholar, 30Thomas W.G. Brandenburger Y. Autelitano D.J. Pham T. Qian H. Hannan R.D. Circ. Res. 2002; 90: 135-142Crossref PubMed Scopus (162) Google Scholar). To date, the P2Y1R and P2Y2R are the only nucleotide receptors clearly demonstrated to transactivate the EGFR (19Liu J. Liao Z. Camden J. Griffin K.D. Garrad R.C. Santiago-Pérez L.I. González F.A. Seye C.I. Weisman G.A. Erb L. J. Biol. Chem. 2004; 279: 8212-8218Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 20Morris J.B. Pham T.M. Kenney B. Sheppard K.E. Woodcock E.A. J. Biol. Chem. 2004; 279: 8740-8746Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 21Schafer R. Sedehizade F. Welte T. Reiser G. Am. J. Physiol. Lung Cell Mol. Physiol. 2003; 285: L376-L385Crossref PubMed Scopus (105) Google Scholar, 22Soltoff S.P. Avraham H. Avraham S. Cantley L.C. J. Biol. Chem. 1998; 273: 2653-2660Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 23Wagstaff S.C. Bowler W.B. Gallagher J.A. Hipskind R.A. Carcinogenesis. 2000; 21: 2175-2181Crossref PubMed Scopus (83) Google Scholar, 31Buvinic S. Bravo-Zehnder M. Boyer J.L. Huidobro-Toro J.P. González A. J. Cell Sci. 2007; 120: 4289-4301Crossref PubMed Scopus (43) Google Scholar). Thus, it becomes possible to hypothesize that EGFR acts downstream of the P2Y2R signaling pathway to regulate nucleotide-induced vasoconstriction. The transactivation of the EGFR activity by GPCRs is a complex molecular process that governs many aspects of the cell fate (32Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1501) Google Scholar, 33Hackel P.O. Zwick E. Prenzel N. Ullrich A. Curr. Opin. Cell Biol. 1999; 11: 184-189Crossref PubMed Scopus (546) Google Scholar), nevertheless, the question of whether EGFR in turn transregulates GPCR activity in intact human tissues is a challenging issue that remains to be addressed (34Chen Y. Long H. Wu Z. Jiang X. Ma L. Mol. Biol. Cell. 2008; 19: 2973-2983Crossref PubMed Google Scholar). Here we report that in the SMC of HCA, the P2Y2R is not associated with membrane rafts; however, selective receptor activation with 1 μm UTP results in its rapid partition to raft domains. Stimulation with higher UTP concentrations further caused the internalization of the P2Y2R, a finding concomitant with the fading of the UTP-evoked contractions in these human vessels. Membrane raft disruption by tissue cholesterol depletion resulted in reduction of the receptor mass associated with these microdomains, a procedure that nonetheless did not modify the UTP-induced vasomotor responses. Remarkably, partition of the P2Y2R into rafts and its internalization were found to depend on the activation of the EGFR and the actin cytoskeleton, implying RhoA and Rac1 GTPase activity. The vasomotor action elicited by P2Y2R activation was also dependent on the EGFR tyrosine kinase activity. These results show for the first time that membrane distribution and the vasomotor activity of the P2Y2R are processes transregulated by the EGFR, revealing an unprecedented functional interplay between the P2Y2R and the EGFR in smooth muscle cells. ATP trisodium salt, ADP disodium salt, UTP trisodium salt, UDP disodium salt, uridine, serotonin (5-HT) as the hydrochloride salt, MβCD, and antiproteases were purchased from Sigma. MRS 2365 as the trisodium salt was purchased from Tocris Bioscience (Ellisville, MO). AG1478, latrunculin B, and PD98059 were purchased from Calbiochem. Percoll was from Amersham Biosciences. For the preparation of buffers, we only used analytical grade reagents, which were obtained from Merck. Full-term placentas from normal pregnancies delivered vaginally or by caesarean section were derived from the maternity ward associated with the Department of Obstetrics and Gynecology of the School of Medicine at the P. Catholic University Clinical Hospital, Santiago. The ethics committees from the School of Medicine and the Faculty of Biology approved the experimental protocols using human tissues; the guidelines for the handling of human materials were strictly adhered to. Appropriate informed consent was obtained as requested by the School of Medicine Ethics Committee. Concerted actions with other investigators in our Department allowed a more complete study of this organ; whereas we dissected chorionic blood vessels, other colleagues used the cord or the body of the placentas for independent protocols. The corresponding ethical regulations were strictly adhered to. At least 60 placentas were used in the experiments reported in this study. Full-term placentas were transported to the laboratory within 5–15 min of childbirth; immediately thereafter, segments of superficial HCA were carefully dissected from the main body to perform the protocols. For some experiments, segments conserved an intact endothelium, whereas in other experiments, the vessel segments were manually denuded of the endothelial cell layer by gently rubbing the internal vessel surface with a cotton swab; this procedure was described to eliminate the internal endothelial layer without damaging the adjacent smooth muscle layer (35Valdecantos P. Briones R. Moya P. Germain A. Huidobro-Toro J.P. Placenta. 2003; 24: 17-26Crossref PubMed Scopus (31) Google Scholar). To perform other protocols, as will be specified separately, HCA vessel segments were perfused with one of the nucleotide receptor agonists for 1, 2, or 4 min; immediately thereafter, these vessel segments were placed in liquid nitrogen until further tissue processing. Functional assays were also performed to assess the biological activity of the P2Y2R upon challenge with selective nucleotide agonists or the removal of the tissue cholesterol. Each of these protocols will be detailed below. Detergent-free purification of membrane rafts and plasma membrane-enriched fraction isolation from HCA were done according to the methods described by Song et al. (36Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (921) Google Scholar) and Smart et al. (37Smart E.J. Ying Y.S. Mineo C. Anderson R.G. Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 10104-10108Crossref PubMed Scopus (676) Google Scholar), respectively, and modified as we described previously (9Norambuena A. Poblete M.I. Donoso M.V. Espinoza C.S. González A. Huidobro-Toro J.P. Mol. Pharmacol. 2008; 74: 1666-1677Crossref PubMed Scopus (15) Google Scholar). Antibodies for the human P2Y1R were generated and characterized previously (7Buvinic S. Poblete M.I. Donoso M.V. Delpiano A.M. Briones R. Miranda R. Huidobro-Toro J.P. J. Physiol. 2006; 573: 427-443Crossref PubMed Scopus (26) Google Scholar). Antibodies against P2Y2R, P2X1R, RhoA, Cdc42, caveolin-3, flotillin-1, Gq, phospho-ERK, and Na+/K+ ATPase (β-subunit) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against Rac were from Cytoskeleton, Inc. (Denver, CO), and anti-ERK antibodies (pan-ERK) from BD Biosciences Pharmingen. Proteins were separated by SDS-PAGE on 10% acrylamide gels and transferred to polyvinylidene difluoride membranes. These membranes were incubated with the specific antibodies to detect the indicated proteins and visualized using horseradish peroxidase-conjugated secondary antibody and the ECL detection system (SuperSignal® West Femto, Pierce). Immunoblots were digitized in a VISTA-T630 UMax scanner driven by Adobe Photoshop CS (Adobe Systems, Mountain View, CA); quantitative analysis was done with the ImageJ software (National Institutes of Health). A 3–5-cm segment from the second bifurcation of the principal superficial chorionic artery was carefully dissected from the placental body. The tissue was immediately denuded of the endothelial cell layer as described previously (9Norambuena A. Poblete M.I. Donoso M.V. Espinoza C.S. González A. Huidobro-Toro J.P. Mol. Pharmacol. 2008; 74: 1666-1677Crossref PubMed Scopus (15) Google Scholar). Immediately thereafter, one end of the vessel was cannulated with PE 190 tubing and perfused with 95% O2, 5% CO2 gassed Krebs-Ringer buffer maintained at 37 °C at a flow of 4 ml/min; the vessels were placed inside a 1.5-ml Eppendorf tube, keeping humid and warm the external surface of the tissue. In this way, the buffer bathed the inside and outside of the vessel during the ex vivo perfusion procedure. After an equilibrium period of 15–20 min, the tissues were perfused with 1 μm UTP for 4 min. Upon completion of the perfusion procedure, the tissues were rapidly dismounted from the perfusion set and immediately immersed in liquid nitrogen until tissue processing for fractionation and sucrose gradient application as detailed above. Control experiments performed without freezing the tissue showed that these procedures did not alter the membrane raft localization of the P2Y2R and control proteins. To test the agonist specificity of the putative receptors, we next perfused separated artery segments for 4 min with one of the following nucleotides: 10 nm MRS 2365, 100 nm ADP, 1 μm UTP, or 1 μm uridine. Each of these studies was repeated in 3–4 separate vessels obtained from independent placentas. To examine whether the receptor microregionalization was altered by agonist activation and to assess the timing required for the P2Y2R to incorporate into raft domains, additional experiments were performed by perfusing 1 μm UTP for either 1, 2, or 4 min; immediately thereafter, blood vessels were immersed into liquid nitrogen for sucrose gradient centrifugation. To test the participation of actin cytoskeleton and the involvement of the EGFR on P2Y2R raft translocation, we perfused 100 nm latrunculin B or 100 nm AG1478 during 30 min. Next, UTP was added to a final concentration of 1 μm and perfused for another 4 min. The vessels were dismounted and processed as described previously. All these experiments were performed in Krebs-Ringer solutions gassed with 95% O2, 5% CO2 at 37 °C; protocols were repeated in at least 3–4 independent placentas. Parallel bioassays determined the course of the vasocontractile activity ensued by agonist application and the viability of the tissues; these protocols will be detailed below. Extraction and quantification of tissue cholesterol were done exactly as we described previously (9Norambuena A. Poblete M.I. Donoso M.V. Espinoza C.S. González A. Huidobro-Toro J.P. Mol. Pharmacol. 2008; 74: 1666-1677Crossref PubMed Scopus (15) Google Scholar). Intact segments of HCA were dissected from surrounding tissues; 0.3–0.5-cm width rings were carefully prepared for bioassays as previously detailed (9Norambuena A. Poblete M.I. Donoso M.V. Espinoza C.S. González A. Huidobro-Toro J.P. Mol. Pharmacol. 2008; 74: 1666-1677Crossref PubMed Scopus (15) Google Scholar). Muscular contractions were evoked with 1–2-min applications of increasing UTP concentrations; once the maximal contraction was reached, the agonists were rapidly rinsed, avoiding desensitization of the motor responses. To assess whether removal of the membrane cholesterol influenced the vasomotor responses elicited by UTP, the next series of experiments examined the effect of tissue incubation with 10 mm MβCD. These experiments were performed exactly as we described previously (9Norambuena A. Poblete M.I. Donoso M.V. Espinoza C.S. González A. Huidobro-Toro J.P. Mol. Pharmacol. 2008; 74: 1666-1677Crossref PubMed Scopus (15) Google Scholar). Once the rings reached the maximal contraction evoked by 70 mm KCl, 1 μm UTP was added to the tissue bath for less than 2 min to avoid tissue desensitization. Next, tissues were exposed to 10 mm MβCD for 90 min, after which the challenge with UTP was repeated. Results evaluated the contractile responses evoked by each vasomotor agonist before and after treatment with MβCD. In analogy to the observation of the loss of the vasomotor response following 10–100 μm UTP applications, an additional set of experiments analyzed the PM distribution of the P2Y2R in HCA segments perfused ex vivo with either 1, 10, or 100 μm UTP. The procedure for the PM assay of the P2Y2R was described above. To test the effects of AG1478 on the receptor internalization, we perfused Krebs-Ringer solution gassed with 95% O2, 5% CO2 at 37 °C containing 100 nm AG1478 for 30 min. Then UTP was added to a final concentration of 10 μm and perfused for another 4 min. RhoA activation was studied using as substrate the RhoA binding domain of rhoketin. Rac1 and Cdc42 were studied using as substrate the Pak1 binding domain. Both were contained in a glutathione S-transferase fusion proteins (GST-RhoA binding domain and GST-Pak1 binding domain) kindly provided by Dr. Keith Burridge (University of North Carolina) (38Waterman-Storer C.M. Worthylake R.A. Liu B.P. Burridge K. Salmon E.D. Nat. Cell Biol. 1999; 1: 45-50Crossref PubMed Scopus (402) Google Scholar). Briefly, HCA segments were perfused with Krebs-Ringer solution gassed with 95% O2, 5% CO2 at 37 °C and a flow rate of 4 ml min−1 containing 1 μm UTP for 4 min. HCA segments were ground in a cold slab in the presence of pull-down buffer (50 mm Tris, pH 7.6, 0.5 mm MgCl2, 500 mm NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate, 10 μg/ml each of aprotinin and leupeptin, and 1 mm phenylmethylsulfonyl fluoride). The extracts were centrifuged at 14,000 rpm for 10 min to eliminate debris. The supernatant was sequentially incubated for 1 h with 30 μg of either GST-RhoA binding domain or GST-Pak1 binding domain coupled to glutathione-Sepharose beads to precipitate RhoA-GTP or Rac1-GTP (38Waterman-Storer C.M. Worthylake R.A. Liu B.P. Burridge K. Salmon E.D. Nat. Cell Biol. 1999; 1: 45-50Crossref PubMed Scopus (402) Google Scholar). Total RhoA, Rac1, and Cdc42 present in 30 μg of whole cell lysates used for loading controls and pulled down RhoA-GTP, Rac1-GTP, and Cdc42-GTP were detected by immunoblot using the respective monoclonal antibodies. To analyze the time dependence of GTPase activation mediated by UTP, HCA segments were perfused with 1 μm UTP for 0.5 and 1 min. Agonist perfusion and pull-down assays were done as described above. Similarly, studies were performed to evaluate the UTP concentration dependence for the GTPases activation. For this purpose, HCA segments were perfused with 1, 10, and 100 μm UTP for 4 min. Finally, to test the effects of AG1478 on the GTPase activity, we perfused Krebs Ringer solution gassed with 95% O2, 5% CO2 at 37 °C containing 100 nm AG1478 for 30 min. Then UTP was added to a final concentration of 1 μm and perfused for another 4 min. The vessels were dismounted and processed as described above. Nucleotide-induced isometric vasomotor responses were quantified as the tension developed by each agonist in intact vessel rings or vessels denuded of the endothelial cell layer. The motor responses were quantified as the tension force that was expressed in g of tension developed; results were generally normalized agonist the standard of 70 mm KCl used at the beginning of each bioassay. At least 4–6 separate rings were examined per agonist examined; the rings were derived each time from separate placentas. Statistical analysis used Student’s t test when appropriate or Dunnett’s tables for multiple comparisons with a single control. In all cases, a p value less than 0.05 was considered significant. Membrane rafts enriched in glycosphingolipids and cholesterol are currently isolated by flotation in sucrose gradients as low density membranes. Analysis of the gradient’s fractions by Western blot indicated that the inactive P2Y2R did not associate with membrane rafts, as evidenced by its co-migration with the β subunit of Na+/K+ ATPase (n = 10), a non-membrane raft protein. As membrane raft markers, we showed that either flotillin-1 or caveolin-3, the muscle-caveolin variant (Fig. 1A), were consistently found floating in low density fractions containing 15–25% sucrose. In addition, protein Gq, the signaling partner of the P2Y2R (39Erb L. Liao Z. Seye C.I. Weisman G.A. Pflugers Arch. 2006; 452: 552-562Crossref PubMed Scopus (203) Google Scholar), was also partially localized in low density fractions (Fig. 1A). Densitometric analysis of the bands indicated that 100% (n = 10) of the total P2Y2R mass was excluded from the raft fractions (Fig. 1B). We also examined the distribution of two other nucleotide receptors. As much as 50 and 30% of the P2X1R and P2Y1R, respectively, were also found in low density fractions (Fig. 1C) (9Norambuena A. Poblete M.I. Donoso M.V. Espinoza C.S. González A. Huidobro-Toro J.P. Mol. Pharmacol. 2008; 74: 1666-1677Crossref PubMed Scopus (15) Google Scholar). Similar distribution of the P2Y2R was observed in parallel experiments performed using the OptiPrep™ procedure (9Norambuena A. Poblete M.I. Donoso M.V. Espinoza C.S. González A. Huidobro-Toro J.P. Mol. Pharmacol. 2008; 74: 1666-1677Crossref PubMed Scopus (15) Google Scholar). These results suggest that inactive P2Y2R does not have the properties for being targeted to membrane rafts. Because GPCR activation may induce in and out translocations from membrane rafts (40Chini B. Parenti M. J. Mol. Endocrinol. 2004; 32: 325-338Crossref PubMed Scopus (311) Google Scholar, 41Ostrom R.S. Insel P.A." @default.
• W2057764503 created "2016-06-24" @default.
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• W2057764503 date "2010-01-01" @default.
• W2057764503 modified "2023-09-28" @default.
• W2057764503 title "UTP Controls Cell Surface Distribution and Vasomotor Activity of the Human P2Y2 Receptor through an Epidermal Growth Factor Receptor-transregulated Mechanism" @default.
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• W2057764503 doi "https://doi.org/10.1074/jbc.m109.081166" @default.
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