CLONING AND EXPRESSION OF A HUMAN-P(2U) NUCLEOTIDE RECEPTOR, A TARGETFOR CYSTIC-FIBROSIS PHARMACOTHERAPY

The Cl- secretory pathway that is defective in cystic fibrosis (CF) ca n be bypassed by an alternative pathway for Cl- transport that is acti vated by extracellular nucleotides. Accordingly, the P2 receptor that mediates this effect is a therapeutic target for improving Cl- secreti on in CF patients. In this paper, we report the sequence and functiona l expression of a cDNA cloned from human airway epithelial (CF/T43) ce lls that encodes a protein with properties of a P2U nucleotide recepto r. With a retrovirus system, the human airway clone was stably express ed in 1321N1 astrocytoma cells, a human cell line unresponsive to extr acellular nucleotides. Studies of inositol phosphate accumulation and intracellular Ca2+ mobilization induced by extracellular nucleotides i n 1321N1 cells expressing the receptor identified this done as the tar get 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 P2U receptor that has been function ally identified in other human tissues. Expression of the human P2U re ceptor (HP2U) in 1321N1 cells revealed evidence for autocrine ATP rele ase 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.