Human P2Y2 receptor polymorphism: identification and pharmacological characterization of two allelic variants

1. In the process of cloning the human P2Y2 receptor in order to establish 1321N1 cell lines expressing this receptor, we detected a gene polymorphism characterized by an arginine 334 to cysteine 334 transition. 2. The frequency distribution of the polymorphism was studied in a European population. We observed that 66% of the tested persons are homozygotes R/R, 29% are heterozygotes R/C and 5% are homozygotes C/C. The frequency of the R allele was 0.8 versus 0.2 for the C allele. 3. We stably expressed each form of the human P2Y2 receptor into 1321N1 cells and isolated clones by limiting dilution. The effects of nucleotides and antagonists on inositol trisphosphate accumulation and cyclic AMP formation were compared between the two cell lines. 4. The time-courses of inositol trisphosphate accumulation as well as concentration-response curves characterizing the effects of UTP, ATP, AP4A and ATP gamma S were mostly similar, except for slight kinetic differences (slower time-course with the 334C form). 5. The sensitivity to pertussis toxin of inositol trisphosphates accumulation was critically dependent on the agonist concentration and stimulation duration, suggesting the involvement of a Gi.0 protein during the early stimulation by low nucleotide concentrations. No inhibition of cyclic AMP accumulation could be detected. These properties were observed with both polymorphic receptors.