P2Y receptors are a class of G protein-coupled receptors activated primarily by ATP, UTP, and UDP. Five mammalian P2Y receptors have been cloned so far including P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11. P2Y1, P2Y2, and P2Y6 couple to the activation of phospholipase C, whereas P2Y4 and P2Y11 couple to the activation of both phospholipase C and the adenylyl cyclase pathways. Additional ADP receptors linked to G␣ i have been described but have not yet been cloned. SP1999 is an orphan G protein-coupled receptor, which is highly expressed in brain, spinal cord, and blood platelets. In the present study, we demonstrate that SP1999 is a G␣ icoupled receptor that is potently activated by ADP. In an effort to identify ligands for SP1999, fractionated rat spinal cord extracts were assayed for Ca 2؉ mobilization activity against Chinese hamster ovary cells transiently transfected with SP1999 and chimeric G␣ subunits (G␣ q/i ). A substance that selectively activated SP1999-transfected cells was identified and purified through a series of chromatographic steps. Mass spectral analysis of the purified material definitively identified it as ADP. ADP was subsequently shown to inhibit forskolin-stimulated adenylyl cyclase activity through selective activation of SP1999 with an EC 50 of 60 nM. Other nucleotides were able to activate SP1999 with a rank order of potency 2-MeS-ATP ‫؍‬ 2-MeS-ADP > ADP ‫؍‬ adenosine 5-O-2-(thio-)diphosphate > 2-Cl-ATP > adenosine 5-O-(thiotriphosphate). Thus, SP1999 is a novel, G␣ i -linked receptor for ADP.Purine and pyrimidine nucleotides are known to modulate a variety of physiological functions by interaction with two types of cell surface receptors: P2X and P2Y receptors (1, 2). P2X receptors are ligand-gated ion-channels, whereas P2Y receptors are G protein-coupled receptors (GPCRs).

We have identified an orphan G protein-coupled receptor, SP174, that shares a high degree of homology with the recently described ADP receptor P2Y 12 . mRNA for SP174 is abundant in the brain and in cells of the immune system. In the present study, we demonstrate that SP174 is also a receptor for ADP, which is coupled to G␣i. ADP potently stimulates SP174 with an EC 50 of 60 nM, and other related nucleotides are active as well, with a rank order of potency 2-methylthio-ADP tetrasodium ϭ adenosine 5Ј-O-2-(thio)diphosphate ϭ 2-methylthio-ATP tetrasodium Ͼ ADP Ͼ AP3A ϾATP Ͼ IDP. This pharmacological profile is similar to that for P2Y 12 . We have also identified the murine homolog of SP174, which exhibits 75% homology to the human receptor. ADP is also a potent agonist at the murine receptor, and its pharmacological profile is similar to its human counterpart, but ADP and related nucleotides are more potent at the murine receptor than the human receptor. In keeping with the general nomenclature for the purinergic receptors, we propose designating this novel receptor P2Y 13 .P2Y receptors are G protein-coupled receptors that respond to the presence of extracellular nucleotides. Both purine and pyrimidine nucleotides can modulate a variety of physiological functions by interaction with P2Y receptors (Harden et al., 1995;Burnstock, 1997;Ralevic and Burnstock, 1998). Six mammalian P2Y receptors have been cloned so far, including P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , and more recently, P2Y 12 (Burnstock, 1997;Communi et al., 1997;Ralevic and Burnstock, 1998;Hollopeter et al., 2001;Zhang et al., 2001). P2Y 1 , P2Y 2 , and P2Y 6 couple to the activation of phospholipase C (PLC); P2Y 11 couples to the activation of both PLC and the adenylyl cyclase pathways, whereas human P2Y 4 couples to adenylyl cyclase pathways at the early stage and PLC at a later stage (Communi et al., 1996;Ralevic and Burnstock, 1998). P2Y 1 is selectively activated by ADP with ATP, being either a partial agonist or an antagonist; P2Y 2 is activated equipotently by ATP and UTP; human P2Y 11 is selectively activated by ATP; human P2Y 6 is selectively activated by UDP, whereas rat P2Y 6 is selectively activated by UTP; and human P2Y 4 is activated selectively by UTP, whereas rat and murine P2Y 4 are activated equipotently by ATP and UTP (Harden et al., 1995;Burnstock, 1997;Ralevic and Burnstock, 1998). In contrast, the P2Y 12 receptor recently described by us and Hollopeter et al. (2001) is potently activated by ADP and is coupled to the inhibition of adenylyl cyclase activity through the G␣i class of G proteins.Analysis of the expression profile of P2Y 12 receptor mRNA revealed that it is expressed at high levels in platelets, in addition to brain tissue. The data presented by Hollopeter et al. (2001) and the analysis of platelet function in P2Y 12 null mice by Foster et al. (2001) clearly indicate that P2Y 12 represents the long sought-after platelet ADP receptor. Furthermore, these studies reveal that P2Y 12 is the molecular target of th...

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