Cloning, expression, signaling mechanisms, and membrane targeting of P2Y(11) receptors in Madin Darby canine kidney cells.
The P2Y(11) receptor is hypothesized to link to both G(s) and G(q), although this proposition is based on expression and separate assays of G(s) and G(q) function in different cell types [J Biol Chem 1997;272:31969-31973]. We have cloned and characterized a canine P2Y(11)-like (cP2Y(11)) receptor from cultured Madin Darby canine kidney (MDCK-D1) cells. When cP2Y(11) receptors are expressed in canine thymocyte (CF2Th) cells that normally lack functional purinergic responses, ADP beta S stimulates phosphatidylinositol (PI) hydrolysis, Ca(2+) 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 Ca(2+). The rank order of potency for stimulation of PI hydrolysis by cP2Y(11) receptors (adenosine 5'-(2-O-thio) diphosphate = 2-methylthio-ADP >/= 2-methylthio-ATP >> ADP > ATP) differs from that of hP2Y(11) receptors. Microscopic examination of MDCK-D1 cells expressing carboxyl-terminal green fluorescent protein (GFP)-tagged cP2Y(11) (cP2Y(11)-GFP) receptors indicates primarily basolateral (BL) targeting. BL addition of 200 microM ADP beta S to confluent monolayers of MDCK-D1 cells produces an increase in short circuit current (I(sc)) (11.6 +/- 1.6 microA/cm(2)) whereas apical addition of agonist has no effect, confirming targeting of functional endogenous P2Y(11) receptors to the BL surface. In contrast, when either cP2Y(11) or cP2Y(11)-GFP is overexpressed in MDCK-D1 cells, the sensitivity of I(sc) to BL agonist increases by nearly 2 orders of magnitude, as if receptor density normally limited agonist potency; moreover, apical addition of ADP beta S now produces an increase in I(sc) but with low potency. The data support the BL localization of cP2Y(11) receptors and receptor coupling to changes in I(sc) 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.
Zambon
A C
AC
The Biomedical Sciences Graduate Program, University of California at San Diego, La Jolla, California, USA.
Brunton
L L
LL
Barrett
K E
KE
Hughes
R J
RJ
Torres
B
B
Insel
P A
PA
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004285:Dogs; D005453:Fluorescence; D049452:Green Fluorescent Proteins; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000502:physiology*; D014018:Tissue Distribution
D008164:Luminescent Proteins; C109734:P2RY11 protein, human; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D049452:Green Fluorescent Proteins