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 P2 receptor 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 P2U nucleotide 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 P2U receptor that has been functionally identified in other human tissues. Expression of the human P2U receptor (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.

The P2Y2 nucleotide receptor (P2Y2R) contains the integrin-binding domain arginine-glycine-aspartic acid (RGD) in its first extracellular loop, raising the possibility that this G protein–coupled receptor interacts directly with an integrin. Binding of a peptide corresponding to the first extracellular loop of the P2Y2R to K562 erythroleukemia cells was inhibited by antibodies against αVβ3/β5 integrins and the integrin-associated thrombospondin receptor, CD47. Immunofluorescence of cells transfected with epitope-tagged P2Y2Rs indicated that αV integrins colocalized 10-fold better with the wild-type P2Y2R than with a mutant P2Y2R in which the RGD sequence was replaced with RGE. Compared with the wild-type P2Y2R, the RGE mutant required 1,000-fold higher agonist concentrations to phosphorylate focal adhesion kinase, activate extracellular signal–regulated kinases, and initiate the PLC-dependent mobilization of intracellular Ca2+. Furthermore, an anti-αV integrin antibody partially inhibited these signaling events mediated by the wild-type P2Y2R. Pertussis toxin, an inhibitor of Gi/o proteins, partially inhibited Ca2+ mobilization mediated by the wild-type P2Y2R, but not by the RGE mutant, suggesting that the RGD sequence is required for P2Y2R-mediated activation of Go, but not Gq. Since CD47 has been shown to associate directly with Gi/o family proteins, these results suggest that interactions between P2Y2Rs, integrins, and CD47 may be important for coupling the P2Y2R to Go.

Background-Extracellular uridine 5Ј-triphosphate (UTP) induces mitogenic activation of smooth muscle cells (SMCs) through binding to P2Y 2 nucleotide receptors. P2Y 2 receptor mRNA is upregulated in intimal lesions of rat aorta, but it is unclear how this G-protein-coupled receptor contributes to development of intimal hyperplasia. Methods and Results-This study used a silicone collar placed around rabbit carotid arteries to induce vascular injury and intimal thickening. Collar placement caused rapid upregulation of P2Y 2 receptor mRNA in medial SMCs before appearance of neointima. Fura-2 digital imaging of single SMCs was used to measure changes in myoplasmic calcium concentration (Ca m ) in response to P2Y receptor agonists. In contrast to UDP, activation by UTP or adenosine 5Ј-triphosphate (ATP) greatly increased Ca m , which indicates upregulation of functional P2Y 2 receptors at which UTP and ATP are equipotent agonists. The number of responsive cells was significantly greater for freshly dispersed SMCs from collared arteries than for controls. Perivascular infusion of UTP (100 mol/L) within the collar significantly enhanced neointimal development. Intimas that resulted from UTP exposure were infiltrated by macrophages. Moreover, increased expression of osteopontin occurred in response to in situ application of UTP. ATP or UTP also stimulated osteopontin expression in cultured SMCs in a dose-dependent manner. Furthermore, P2Y 2 antisense oligonucleotide inhibited osteopontin expression induced by UTP. Conclusions-These findings indicate for the first time a role for the UTP/ATP receptor, P2Y 2 , in development of intimal hyperplasia associated with atherosclerosis and restenosis.

Extracellular ATP and ADP mediate diverse physiological responses in mammalian cells, in part through the activation of G protein-coupled P 2 purinoceptors. The cloning and expression of cDNAs encoding several P 2 purinoceptor subtypes have enabled rapid advances in our understanding of the structural and functional properties of these receptors. The current report describes the isolation of a gene from a human genomic library that encodes a protein with the greatest similarity to the human P 2U purinoceptor, a subtype that is distinguished by its ability to be activated by uridine nucleotides as well as adenine nucleotides. When expressed in a mammalian cell line, this novel receptor is activated specifically by UTP and UDP but not by ATP and ADP. Activation of this uridine nucleotide receptor resulted in increased inositol phosphate formation and calcium mobilization. Fluorescence in situ hybridization revealed that the gene encoding the uridine nucleotide receptor is located in region q13 of the X chromosome. Dendrogram analysis of the G protein-coupled P 2 purinoceptors and the uridine nucleotide receptor indicates that these receptors belong to a family that may be more aptly named nucleotide receptors.Nucleotides comprise one of the most recently recognized groups of compounds that function as extracellular signaling molecules (1). Among the nucleotide family, ATP is the only established neurotransmitter/hormone. Recent articles have reported the cloning of the cDNAs and the predicted amino acid sequences for a variety of P 2 purinoceptors, including the G protein-coupled P 2U receptor, where UTP and ATP are equally efficacious, and the P 2Y receptors, where ATP, but not UTP, is active (reviewed in Ref. 2). Pharmacological and structural data indicate that the G protein-coupled P 2 purinoceptors consist of several related but clearly distinct subtypes within the GPCR 1 superfamily. We report herein the isolation of a human gene, intronless within its coding region, that encodes a protein with a predicted structure characteristic of GPCRs and a sequence most similar to the P 2U purinoceptor. The results of functional studies indicate that the expressed protein encoded by the newly cloned DNA is a uridine nucleotide receptor. (CRL9078) were purchased from the American Type Culture Collection (Rockville, MD). G418 (Geneticin) and the BioNick labeling kit were purchased from Life Technologies, Inc. All other chemicals were purchased from Sigma. PCR and Genomic Library Screening-Human genomic DNA was amplified by PCR using degenerate oligonucleotides based on the sequences encoding transmembrane domains 3 (P1, 5Ј-GT(G/C)AT-GAG(T/C)G(T/C)(G/A/C)GACCG(C/A)TA) and 7 (P2, 5Ј-GGGGTT(G/ C)AGGCA(G/C)(G/C)(T/A)GTT)that are conserved between the opioidand somatostatin-like GPCRs 7 and 8 (3), the opioid receptors, and the somatostatin receptors. The PCR conditions were as follows: denaturation at 94°C for 3 min, annealing at 42°C for 2 min, and extension at 72°C for 3 min, for 30 cycles, followed by a 7-min extension at...