Lack of nucleotide-promoted second messenger signaling responses in 1321N1 cells expressing the proposed P2Y receptor, p2y7

A recently cloned G protein-coupled receptor (named the p2y7 receptor) with relatively low sequence identity to previously cloned P2Y receptors was proposed to be a member of this family of receptors on the basis of both a radioligand binding assay with [S-35]dATP alpha S and an inositol phosphate response to ATP in COS-7 cells transiently transfected with receptor cDNA. Previous work in our laboratory has shown that [S-35]dATP alpha S is not a general radioligand for the identification of P2Y receptors and that COS-7 cells express an endogenous P2Y receptor (P2Y(2)) that complicates the analysis of nucleotide-promoted inositol phosphate responses. Thus, data supporting inclusion of the p2y7 receptor in the P2Y family of receptors are equivocal. To determine unambiguously whether the p2y7 receptor is a P2Y receptor subtype, a p2y7 receptor bearing an epitope-tag at its NH2-terminus was expressed in 1321N1 cells and cell surface expression of the receptor was demonstrated by an intact cell-based ELISA. Cells shown to express epitope-tagged p2y7 receptors by ELISA were examined for their second messenger signaling properties in response to a range of nucleotides. ATP, UTP, ADP, UDP, and dATP alpha S had no effect on phospholipase C or adenylyl cyclase activities in cells expressing the p2y7 receptor. Experimental controls utilizing expression of other G protein-coupled receptors showed that 1321N1 cells displayed robust responses for each of these signaling pathways. These data, together with the low sequence identity of the p2y7 receptor to other P2Y receptors, indicate that the p2y7 is not a member of the P2Y family of signaling molecules. (C) 1997 Academic Press.