Abstract

Platelet activation plays an essential role in thrombosis. ADP-induced platelet aggregation is mediated by two distinct G protein-coupled ADP receptors, Gq-linked P2Y₁, and Gi-linked P2T_AC, which has not been cloned. The cDNA encoding a novel G protein-coupled receptor, termed HORK3, was isolated. The HORK3gene and P2Y₁ gene were mapped to chromosome 3q21-q25. HORK3, when transfected in the rat glioma cell subline (C6–15), responded to 2-methylthio-ADP (2MeSADP) (EC₅₀ = 0.08 nM) and ADP (EC₅₀ = 42 nM) with inhibition of forskolin-stimulated cAMP accumulation. 2MeSADP (EC₅₀ = 1.3 nM) and ADP (EC₅₀ = 18 nM) also induced intracellular calcium mobilization in P2Y₁-expressing cells. These results show that HORK3 is a Gi/o-coupled receptor and that its natural ligand is ADP. AR-C69931 MX and 2MeSAMP, P2T_AC antagonists, selectively inhibited 2MeSADP-induced adenylyl cyclase inhibition in HORK3-expressing cells. On the other hand, A3P5PS, a P2Y₁ antagonist, blocked only 2MeSADP-induced calcium mobilization in P2Y₁-expressing cells. HORK3 mRNA was detected in human platelets and the expression level of HORK3 was equivalent to that of P2Y₁. These observations indicate that HORK3 has the characteristics of the proposed P2T_AC receptor. We have also determined that [³H]2MeSADP binds to cloned HORK3 and P2Y₁. Competition binding experiments revealed a similarity in the rank orders of potency of agonists and the selectivity of antagonists as obtained in the functional assay. These results support the view that P2Y₁ functions as a high-affinity ADP receptor and P2T_AC as a low-affinity ADP receptor in platelets.