Cloning and Expression of a Human P2UNucleotide Receptor, a Target for Cystic Fibrosis Pharmacotherapy

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 91; no. 8; pp. 3275 - 3279
• Main Authors: C. Edward Parr, Daniel M. Sullivan, Anthony M. Paradiso, Eduardo R. Lazarowski, Laranell H. Burch, John C. Olsen, Laurie Erb, Gary A. Weisman, Richard C. Boucher, John T. Turner
• Format: Journal Article
• Language: Latin
• Published: National Academy of Sciences of the United States of America 12.04.1994
• ISSN: 0027-8424; 1091-6490

The Cl-secretory pathway that is defective in cystic fibrosis (CF) can be bypassed by an alternative pathway for Cl-transport that is activated by extracellular nucleotides. Accordingly, the P2receptor that mediates this effect is a therapeutic target for improving Cl-secretion in CF patients. In this paper, we report the sequence and functional expression of a cDNA cloned from human airway epithelial (CF/T43) cells that encodes a protein with properties of a P2Unucleotide receptor. With a retrovirus system, the human airway clone was stably expressed in 1321N1 astrocytoma cells, a human cell line unresponsive to extracellular nucleotides. Studies of inositol phosphate accumulation and intracellular Ca2+mobilization induced by extracellular nucleotides in 1321N1 cells expressing the receptor identified this clone as the target receptor in human airway epithelia. In addition, we independently isolated an identical cDNA from human colonic epithelial (HT-29) cells, indicating that this is the same P2Ureceptor that has been functionally identified in other human tissues. Expression of the human P2Ureceptor (HP2U) in 1321N1 cells revealed evidence for autocrine ATP release and stimulation of transduced receptors. Thus, HP2U expression in the 1321N1 cell line will be useful for studying autocrine regulatory mechanisms and in screening of potential therapeutic drugs.