Cloning, Expression, Signaling Mechanisms, and Membrane Targeting of P2Y₁₁Receptors in Madin Darby Canine Kidney Cells

Abstract

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.

P2Y11 Receptors Activate Adenylyl Cyclase and Contribute to Nucleotide-promoted cAMP Formation in MDCK-D1Cells

“…20 and data not shown). These findings suggested that the receptor responsible for the indo-insensitive effect is not the P2Y 1 receptor but instead is another receptor, perhaps the P2Y 11 receptor, which in MDCK-D 1 cells also responds to adenine bisphosphates (14,21).…”

“…Cells were used in assays at 60 -80% confluency. GFP-tagged cP2Y 11 receptor-overexpressing MDCK-D 1 cells were cultured from the stable cell line prepared by Zambon et al (14).…”

“…5 and 6). P2Y receptors in MDCK-D 1 cells modulate membrane potential and short circuit current, and the receptors regulate phospholipases, intracellular [Ca 2ϩ ], prostaglandin E 2 (PGE 2 ) formation, and cAMP accumulation (1,(7)(8)(9)(10)(11)(12)(13)(14).Cyclooxygenase (COX) inhibitors, such as indomethacin (indo), have proven useful for studying P2Y receptors in MDCK-D 1 cells (1, 12). Agonist-stimulated cAMP accumulation in MDCK-D 1 cells occurs via both indo-sensitive and -insensitive pathways.…”

Purinergic signalling in the kidney in health and disease

“…Lanthanum inhibits UTP-induced Ca 2+ mobilization in MDCK cells [164]. P2Y 1 , P2Y 2 and P2Y 11 receptor mRNA was shown to be expressed by MDCK cells, but the P2Y 2 receptor was dominant in mediating ATP activation of cAMP formation [155,295,373,427]. A later paper showed expression of P2Y 6 (as well as P2Y 1 , P2Y 2 and P2Y 11 ) receptors in MDCK cells [142].…”

A novel nucleotide receptor in Xenopus activates the cAMP second messenger pathway

“…In mammals, eight P2Y receptors have been cloned and proven to be nucleotide receptors, either activating phospholipase C (P2Y 1 , P2Y 2 , P2Y 4 and P2Y 6 ) or inhibiting adenylyl cyclase (P2Y 12 , P2Y 13 and P2Y 14 ). Uniquely, P2Y 11 activates both phospholipase C and adenylyl cyclase [2,3]. In humans P2Y 11 is selective for ATP [2] but in dogs it is selective for ADP [3].…”