Functional Expression of a P 2T ADP Receptor in Xenopus Oocytes Injected With Megakaryocyte (CMK 11-5) RNA

Abstract

Abstract Since the P 2T purinergic (ADP) receptor is unique to the megakaryocytic/platelet lineage, cells of this lineage were screened for the relative effects of ADP and ATP in intracellular Ca 2+ levels. Like platelets, CMK 11-5 cells responded with an increase in intracellular Ca 2+ mobilization in response to ADP but not to ATP or adenosine. In contrast, both nucleotides increased intracellular Ca 2+ mobilization in the megakaryoblastic cell lines MO7E and Meg-01, indicating that they contain P 2Y receptors or a mixed complement of purinergic receptors. Pharmacological responsiveness of CMK 11-5 cells to nucleotides paralleled those of platelets, in which ADP and ADP-α-S are active as agonists and ATP and ATP-α-S are inactive as agonists but act as antagonists. [ 3 H]ADP and 35 S-ATP-α-S bound to CMK 11-5 cells at a high-affinity site ( K d1 and K i1 , 262 and 125 nmol/L, respectively) and a low-affinity site ( K d2 and K i2 , 10 100 and 5400 nmol/L, respectively) with 2×10 6 to 6×10 6 sites per cell. ADP bound at both sites was competed with ADP, ATP, and ATP-α-S with affinities in a rank order similar to that found for platelets (ATP-α-S≈ATP≈ADP≥ADP-β-S≈adenosine), suggesting the presence of a P 2T receptor on CMK 11-5 cells. Photoaffinity labeling of intact CMK 11-5 cells with 35 S-ATP-α-S resulted in the labeling of the α-subunit of GP IIb as found with platelets, although this was confirmed to be independent of ADP receptors. After RNA from CMK 11-5 cells was microinjected into Xenopus oocytes, only ADP and ADP-α-S stimulated 45 Ca 2+ efflux, which was not observed with ATP, 2-methylthio-ATP, α,β-methylene-ATP, ATP-γ-S, ATP-α-S, or adenosine. In addition, incubation of RNA-injected oocytes with ATP or ATP-α-S but not adenosine blocked the 45 Ca 2+ response to ADP. These experiments demonstrate that a nascent receptor that responded specifically to ADP but not to other P 1 , P 2Y , P 2X , and P 2U agonists was expressed in functional form on Xenopus oocytes.