Abstract

The P2Y₁₁ 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_qfunction 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 > 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 (I_sc) (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 I_sc 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 I_sc but with low potency. The data support the BL localization of cP2Y₁₁ 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.