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 .
We have investigated the nature of the nucleotide receptors on human erythro leukemia (HEL) cells, a cell line with some megakaryocytic properties, using a combination of pharmacological, photoaffinity labeling, and molecular biological techniques. Fura-2 loaded HEL cells responded to 2-methylthio ATP, ATP, 2-methylthio ADP, ADP and UTP with an increase in intracellular calcium. 2 Methylthio ADP was the most potent agonist. When external calcium was chelated with EDTA, calcium responses were observed indicating the mobilization of intracellular stores. These responses showed evidence of both homologous and heterologous receptor desensitization. In photoaffinity labeling experiments, beta-[32P]-AzPET-ADP was incorporated into three protein species with mobilities corresponding to M(r) approximately 55 kDa (doublet) and approximately 43 kDa. Labeling of approximately 55 kDa proteins was specifically inhibited by ADP, while that of the approximately 43 kDa was inhibited specifically by UTP. Nucleotide sequence analysis of the positive clones obtained by screening the HEL cell cDNA library with mouse P2U cDNA revealed that the P2U receptor from HEL cells is identical to the previously cloned human P2U receptor. These experiments suggest that the HEL cells contain a P2Y purinoceptor responding to ADP, in addition to a P2U receptor and possibly also a third P2 purinoceptor with a unique agonist profile.
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