UTP is a potent whole agonist at both the human P2Y4 (hP2Y4) and rat P2Y4 (rP2Y4) receptor. with that of the rP2Y4 receptor yielded a chimeric receptor that was activated fully by UTP and near fully by ATP albeit with lower potencies than those observed at the rP2Y4 receptor. These potencies were increased and ATP was converted to a full agonist by replacing both the NH2 terminus and EL2 in the hP2Y4 receptor with the corresponding regions from your rP2Y4 receptor. Mutational analysis of the five divergent amino acids in EL2 between the two receptors revealed that three amino acids Asn-177 Ile-183 and Leu-190 contribute to the capacity of EL2 to impart ATP agonism. Taken together these results suggest that the second extracellular loop and the NH2 terminus form a functional motif that plays a key role in determining whether ATP functions as an agonist or antagonist at mammalian P2Y4 receptors. Extracellular nucleotides elicit diverse physiological results by activating G protein-coupled P2Y receptors (1 2 Molecular cloning PB-22 and heterologous receptor appearance studies have resulted in the id and characterization of eight individual P2Y (hP2Y)1 receptor subtypes (hP2Y1 2 4 6 11 hP2Y1 hP2Y2 hP2Y4 hP2Y6 and hP2Y11 receptors screen 27-52% amino acidity identity and few via heterotrimeric G protein PB-22 from the Gq family members towards the activation of phospholipase C era of inositol phosphates and mobilization of intracellular Ca2+ shops (2-4). Furthermore to coupling to phospholipase C the horsepower2Y11 receptor also lovers to Gs to activate adenylyl cyclase and promotes cyclic AMP deposition (5-7). The lately discovered P2Y12 P2Y13 and P2Y14 receptors that are encoded on a brief portion of chromosome 3 possess high sequence identification with one another (40-48%) but talk about relatively little series identity (22-25%) using the various other horsepower2Y receptors. The P2Y12 receptor provides been proven to end up being the Gi-coupled receptor in platelets that alongside the P2Y1 receptor mediate ADP-promoted platelet aggregation SNX25 (8-11). P2Y13 and P2Y14 receptors may also be combined to Gi and so are turned on by ADP and UDP-glucose respectively (12 13 Distinctions in nucleotide selectivity have already been observed between types orthologues of P2Y receptors. Including the avian p2con3 and rat P2Y6 receptor are types homologues with ~65% identification that differ within their capability to mediate adenine nucleotide-promoted inositol phosphate deposition (14). Whereas UDP and UTP possess equivalent potencies at both receptors ADP and ATP PB-22 are somewhat more powerful and efficacious on the avian p2con3 receptor than on the rat P2Y6 receptor. Individual and canine P2Con11 receptors which talk about ~70% amino acidity identification (15) also differ within their ability to end up being turned on by adenine nucleotides. ATP nucleotides are stronger and efficacious than their matching diphosphate nucleotides on the individual P2Y11 receptor for advertising PB-22 of both inositol PB-22 phosphate and cAMP deposition whereas ADP nucleotides are somewhat more powerful than their matching triphosphates on the canine P2Y11 receptor (16). We’ve also observed an extraordinary difference in the nucleotide selectivities and agonism antagonism between rat and individual P2Y4 receptors which display 83% sequence identification (17). Under circumstances that reduced confounding factors such as for example nucleotide fat burning capacity bioconversion and endogenous nucleotide discharge UTP ATP diadenosine tetraphosphate ITP GTP CTP and XTP had been all complete agonists on the rP2Y4 receptor whereas just UTP GTP and ITP turned on the hP2Y4 receptor. Furthermore whereas ATP acted like a PB-22 potent full agonist in the rP2Y4 receptor it was a similarly potent competitive antagonist in the hP2Y4 receptor. The capacity of ATP to bind with relatively high affinity to both varieties homologues of the P2Y4 receptor but to act as an agonist at one receptor and an antagonist in the additional provides an ideal signaling system to pursue the structural basis of agonism antagonism. A series of hP2Y4/rP2Y4 receptor chimeras was constructed and we statement here that the second extracellular loop (EL2) of the P2Y4 receptor is definitely a major determinant of agonist antagonist activity of ATP whereas the NH2 terminus takes on a lesser part. Moreover 3 amino acids from EL2 of the hP2Y4 receptor when replaced with the related residues from your rP2Y4 receptor are adequate to convert ATP from an.