An arginine/glutamine difference at the juxtaposition of transmembrane domain 6 and the third extracellular loop contributes to the markedly different nucleotide selectivities of human and canine P2Y(11) receptors

The recently cloned canine P2Y(11) receptor (cP2Y(11)) and its human homolo g (hP2Y(11)) were stably expressed in Chinese hamster ovary cells (CHO-K1) and 1321N1 human astrocytoma cells, and their agonist selectivities and cou pling efficiencies to phospholipase C and adenylyl cyclase were assessed. A denosine triphosphate nucleotides were much more potent and efficacious at the hP2Y(11) receptor than their corresponding diphosphates in promoting bo th inositol phosphate and cyclic AMP accumulation. In contrast, adenosine d iphosphate nucleotides were considerably more potent at the cP2Y11 receptor than their corresponding triphosphate analogs. The tri- versus diphosphate specificity of the two receptors was further confirmed in studies using Ca 2+ mobilization as a measure of receptor activation under conditions that m inimized nucleotide degradation. Moreover, 2-methylthioadenosine-5'-triphos phate and 2-methylthioadenosine-5'-diphosphate were 58- and 75-fold more po tent than ATP and ADP, respectively, at the cP2Y(11) receptor compared with only 2- to 3-fold more potent at the hP2Y(11) receptor. Mutational analysi s revealed that the change of Arg-265, which is located at the juxtapositio n of transmembrane domain 6 and the third extracellular loop in the hP2Y(11 ) receptor, to glutamine in the cP2Y(11) receptor is at least partly respon sible for the diphosphate selectivity but not the increased sensitivity to 2-thioether-substituted adenine nucleotides at the canine receptor. These r esults imply a key role for a positively charged arginine residue in contri buting to the recognition of extracellular nucleotides by the P2Y(11) recep tor and perhaps other P2Y receptors.