Molecular Cloning of a Novel P2 Purinoceptor from Human Erythroleukemia Cells

Abstract

Screening of a human erythroleukemia cell cDNA library with radiolabeled chicken P2Y 3 cDNA at low stringency revealed a cDNA clone encoding a novel G protein-coupled receptor with homology to P2 purinoceptors. This receptor, designated P2Y 7 , has 352 amino acids and shares 23-30% amino acid identity with the P2Y 1 -P2Y 6 purinoceptors. The P2Y 7 cDNA was transiently expressed in COS-7 cells: binding studies thereon showed a very high affinity for ATP (37 ؎ 6 nM), much less for UTP and ADP (ϳ1300 nM), and a novel rank order of affinities in the binding series studied of 8 nucleotides and suramin. The P2Y 7 receptor sequence appears to denote a different subfamily from that of all the other known P2Y purinoceptors, with only a few of their characteristic sequence motifs shared. The P2Y 7 receptor mRNA is abundantly present in the human heart and the skeletal muscle, moderately in the brain and liver, but not in the other tissues tested. The P2Y 7 receptor mRNA was also abundantly present in the rat heart and cultured neonatal rat cardiomyocytes. The P2Y 7 receptor is functionally coupled to phospholipase C in COS-7 cells transiently expressing this receptor. The P2Y 7 gene was shown to be localized to human chromosome 14. We have thus cloned a unique member of the P2Y purinoceptor family which probably plays a role in the regulation of cardiac muscle contraction.The widespread occurrence of metabotropic receptors for extracellular ATP has long been inferred from physiological and pharmacological evidence (1). A number of such G proteincoupled ATP receptors have been characterized and a consensus on their nomenclature has termed all of these as P2Y purinoceptors (to be individually named P2Y 1 to P2Y n ), regardless of previous terminology such as P 2U or P 2T for subclasses thereof (2). The first such receptors to be characterized by DNA cloning and expression were the P2Y 1 receptor (where UTP is inactive) (3) and the P2Y 2 receptor (ATP and UTP are equally active) (4). True species homologues (or orthologues) of P2Y 1 have since been obtained, e.g. bovine (5) and human (6), and of P2Y 2 , e.g. from human airway epithelium (7) or human erythroleukemia (HEL) 1 cells (8). Further types identified by cloning have been the P2Y 3 receptor (UDP Ͼ ADP Ͼ ATP) (9) and P2Y 4 (UTP Ͼ Ͼ ATP, and more strongly related to P2Y 2 ) (10, 11). Further novel P2Y receptors have recently been identified from their cDNAs from chicken activated T lymphocytes (12) and rat vascular smooth muscle cells (13) and designated P2Y 5 and P2Y 6 receptors. Previously we have demonstrated at least three P2 purinoceptors on the hematopoietic cell line, HEL cells, by intracellular calcium mobilization and by photoaffinity labeling (8). Here we report the molecular cloning and characterization of one of these, a novel P2 purinergic receptor designated P2Y 7 .

Towards a revised nomenclature for P1 and P2 receptors

“…In the case of the p2y5 (Ref. 20) and P2Y 7 receptors 21 , where the sequence homology of the two proteins to previously identified members of the P2Y receptor family is low, we anticipate further findings in this area ( Table 2).…”

Coactivation of two different G protein-coupled receptors is essential for ADP-induced platelet aggregation

“…ARL 66096 was a gift from Astra Research Laboratories (Loughborough, U.K.; formerly Fisons). All other materials and methods used have been described (8,10,11).…”

Identification of a Novel Human Eicosanoid Receptor Coupled to Gi/o

“…5-HPETE can be reduced by lipid peroxidases such as glutathione peroxidase to 5-HETE (25,26), which is finally oxidized to 5-oxo-ETE by 5(S)-hydroxyeicosanoid-specific dehydrogenase (27). Eicosanoid receptors recognizing prostaglandins D 2 , E 2 , and F 2␣ ; prostacyclin; thromboxane A 2 ; and leukotriene B 4 ; respectively, have been cloned and vigorously analyzed because of the importance of their implicated biological functions such as inflammation, blood clotting, and control of vascular tone (11,24,28). All of these eicosanoid receptors are categorized to GPCR.…”