The P2Y family of G-protein coupled receptors are activated by adenine and uridine di- and triphosphate nucleotides and nucleotide sugars and are implicated in a wide range of important human physiologies. Difficulty studying these receptors and in their successful manipulation as therapeutic targets has historically derived from a lack of available pharmacological tools that discriminate among members of the P2Y receptor family. The studies described here focus on the P2Y₁ receptor, a key mediator of ADP-induced platelet aggregation. Based on the structure of the recently synthesized, high-affinity P2Y₁ receptor-selective antagonist, 2-iodo-N ⁶-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate (MRS2500), we undertook the development of a high-specific radioactivity radioligand for the P2Y₁ receptor, suitable for the study of endogenous receptors in mammalian tissues and cell lines. Using an enzymatic phosphorylation reaction, we successfully generated [³²P]MRS2500 with a specific activity of 9120 Ci/mmol. The selectivity and affinity of [³²P]MRS2500 for the P2Y₁ receptor were confirmed in radioligand binding assays with Sf9 insect cell membranes overexpressing recombinant P2Y₁ receptors. The utility of [³²P]MRS2500 for the study of endogenous P2Y₁ receptors was examined using washed human platelets and membranes prepared from various tissues of the adult rat.

We applied this high-specific radioactivity radioligand to observe surface expression of P2Y₁ receptor binding sites in human platelets and MDCK(II) epithelial cells following incubation with P2Y₁ receptor agonists. In human platelets, the rapid, agonist-promoted desensitization of the P2Y₁ receptor observed after incubation with the selective agonist (N)-methanocarba-2-methylthioadenosine-diphosphate (MRS2365) also occurs for the G_q-coupled 5-HT_2A receptor after incubation of platelets with 5-hydroxytryptamine (5-HT). The rapid, agonist-promoted desensitization of the P2Y₁ receptor of platelets was accompanied by a modest decrease (< 20%) in the number of surface [ ³²P]MRS2500 binding sites and only a partial recovery of P2Y ₁ receptor responsiveness after removal of the selective agonist MRS2365. Platelets, therefore, appear to employ a unique mechanism for prolonged termination of P2Y₁ receptor signaling in which desensitized receptors are maintained at the cell surface, unable to respond to subsequent agonist stimulation. In intact MDCK(II) cells overexpressing recombinant P2Y₁ receptors, incubation with 2MeSADP was followed by a 50% loss of surface [³²P]MRS2500 binding sites that was agonist-concentration dependent and required the formation of clathrin-coated pits. Mutagenesis studies indicated that this rapid, agonist-promoted loss of surface binding sites requires two serine residues, Ser352 and Ser354, in the receptor carboxyl terminus. The findings presented here indicate that P2Y₁ receptor cell surface expression is regulated in an agonist-dependent manner that differs in at least two cell types and suggests an important role for phosphorylation in agonist-dependent desensitization and internalization of the P2Y₁ receptor.