TY - JOUR T1 - Cloning, Expression, Signaling Mechanisms, and Membrane Targeting of P2Y<sub>11</sub> Receptors in Madin Darby Canine Kidney Cells JF - Molecular Pharmacology JO - Mol Pharmacol SP - 26 LP - 35 DO - 10.1124/mol.60.1.26 VL - 60 IS - 1 AU - Alexander C. Zambon AU - Laurence L. Brunton AU - Kim E. Barrett AU - Richard J. Hughes AU - Brian Torres AU - Paul A. Insel Y1 - 2001/07/01 UR - http://molpharm.aspetjournals.org/content/60/1/26.abstract N2 - The P2Y11 receptor is hypothesized to link to both Gs and Gq, although this proposition is based on expression and separate assays of Gs and Gqfunction in different cell types [J Biol Chem1997;272:31969–31973]. We have cloned and characterized a canine P2Y11-like (cP2Y11) receptor from cultured Madin Darby canine kidney (MDCK-D1) cells. When cP2Y11 receptors are expressed in canine thymocyte (CF2Th) cells that normally lack functional purinergic responses, ADPβS stimulates phosphatidylinositol (PI) hydrolysis, Ca2+ mobilization, and cAMP accumulation. Pharmacologic analysis indicates that the stimulation of cAMP production is direct and not a result of eicosanoid synthesis, activation of PKC, or elevation of cell Ca2+. The rank order of potency for stimulation of PI hydrolysis by cP2Y11 receptors (adenosine 5′-(2-O-thio) diphosphate = 2-methylthio-ADP ≥ 2-methylthio-ATP ≫ ADP &gt; ATP) differs from that of hP2Y11 receptors. Microscopic examination of MDCK-D1 cells expressing carboxyl-terminal green fluorescent protein (GFP)-tagged cP2Y11(cP2Y11-GFP) receptors indicates primarily basolateral (BL) targeting. BL addition of 200 μM ADPβS to confluent monolayers of MDCK-D1 cells produces an increase in short circuit current (Isc) (11.6 ± 1.6 μA/cm2) whereas apical addition of agonist has no effect, confirming targeting of functional endogenous P2Y11 receptors to the BL surface. In contrast, when either cP2Y11 or cP2Y11-GFP is overexpressed in MDCK-D1 cells, the sensitivity of Isc to BL agonist increases by nearly 2 orders of magnitude, as if receptor density normally limited agonist potency; moreover, apical addition of ADPβS now produces an increase in Isc but with low potency. The data support the BL localization of cP2Y11 receptors and receptor coupling to changes in Isc in MDCK-D1 cells except in cases in which receptors are overexpressed; receptor overexpression leads to altered sensitivities and sites of coupling to physiologic responses. GPCRG protein-coupled receptorMDCK-D1Madin-Darby canine kidney cellsPIphosphatidylinositideAAarachidonic acid and metabolitesIscshort-circuit currentAMacetoxymethyl esterRTreverse transcriptasePCRpolymerase chain reactionDMEMDulbecco's modified Eagle's mediumTCAtrichloroacetic acidIPinositol phosphates2 MT2-methylthioADPβSadenosine 5′-(2-O-thio) diphosphateGFPgreen fluorescent proteinBAPTA1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid ER -