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 P2Y11 receptors.
The recently cloned canine P2Y11 receptor (cP2Y11) and its human homolog (hP2Y11) were stably expressed in Chinese hamster ovary cells (CHO-K1) and 1321N1 human astrocytoma cells, and their agonist selectivities and coupling efficiencies to phospholipase C and adenylyl cyclase were assessed. Adenosine triphosphate nucleotides were much more potent and efficacious at the hP2Y11 receptor than their corresponding diphosphates in promoting both inositol phosphate and cyclic AMP accumulation. In contrast, adenosine diphosphate 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 minimized nucleotide degradation. Moreover, 2-methylthioadenosine-5'-triphosphate and 2-methylthioadenosine-5'-diphosphate were 58- and 75-fold more potent than ATP and ADP, respectively, at the cP2Y11 receptor compared with only 2- to 3-fold more potent at the hP2Y11 receptor. Mutational analysis revealed that the change of Arg-265, which is located at the juxtaposition of transmembrane domain 6 and the third extracellular loop in the hP2Y11 receptor, to glutamine in the cP2Y11 receptor is at least partly responsible for the diphosphate selectivity but not the increased sensitivity to 2-thioether-substituted adenine nucleotides at the canine receptor. These results imply a key role for a positively charged arginine residue in contributing to the recognition of extracellular nucleotides by the P2Y11 receptor and perhaps other P2Y receptors.
Qi
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AD
Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599-7365, USA.
Zambon
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Insel
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Nicholas
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RA
D000227:Adenine Nucleotides / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D004285:Dogs; D005973:Glutamine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007294:Inositol / Q000378:metabolism; D050356:Lipid Metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D014530:Uridine Diphosphate / Q000378:metabolism; D014544:Uridine Triphosphate / Q000378:metabolism
D000227:Adenine Nucleotides; D008565:Membrane Proteins; C109734:P2RY11 protein, human; D018048:Receptors, Purinergic P2; D005973:Glutamine; D007294:Inositol; D014530:Uridine Diphosphate; D001120:Arginine; D000242:Cyclic AMP; D014544:Uridine Triphosphate