Characterization of P-2 purinergic receptors on human erythro leukemia cells

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-[(32)p]-AzPET-ADP was incorporated into three protein species with mobilities corresponding to Mr similar to 55 kDa (doublet) and similar to 43 kDa. Labeling of similar to 55 kDa proteins was specifically inhibited by ADP, while that of the similar to 43 kDa was inhibited specifically by UTP. Nucleotide sequence analysis of the positive clones obtained by screening the HEL cell cDNA Library with mouse P-2U cDNA revealed that the P-2U receptor from HEL cells is identical to the previously cloned human P-2U receptor. These experiments suggest that the HEL cells contain a P-2Y purinoceptor responding to ADP, in addition to a P-2U receptor and possibly also a third P2 purinoceptor with a unique agonist profile.