FUNCTIONAL EXPRESSION OF A P-2T ADP RECEPTOR IN XENOPUS OOCYTES INJECTED WITH MEGAKARYOCYTE (CMK-11-5) RNA

Since the P-2T purinergic (ADP) receptor is unique to the megakaryocyt ic/platelet lineage, cells of this lineage were screened for the relat ive effects of ADP and ATP in intracellular Ca2+ levels. Like platelet s, CMK 11-5 cells responded with an increase in intracellular Ca2+ mob ilization in response to ADP but not to ATP or adenosine. In contrast, both nucleotides increased intracellular Ca2+ mobilization in the meg akaryoblastic cell lines MO7E and Meg-01, indicating that they contain P-2Y receptors or a mixed complement of purinergic receptors. Pharmac ological responsiveness of CMK 11-5 cells to nucleotides paralleled th ose of platelets, in which ADP and ADP-alpha-S are active as agonists and ATP and ATP-alpha-S are inactive as agonists but act as antagonist s. [H-3]ADP and S-35-ATP-alpha-S bound to CMK 11-5 cells at a high-aff inity site (K-d1 and K-i1, 262 and 125 nmol/L, respectively) and a low -affinity site (K-d2 and K-i2, 10100 and 5400 nmol/L, respectively) wi th 2x10(6) to 6x10(6) sites per cell. ADP bound at both sites was comp eted with ADP, ATP, and ATP-alpha-S with affinities in a rank order si milar to that found for platelets (ATP-alpha-S approximate to ATP appr oximate to ADP greater than or equal to ADP-beta-S approximate to aden osine), suggesting the presence of a PZT receptor on CMK 11-5 cells. P hotoaffinity labeling of intact CMK 11-5 cells with S-35-ATP-alpha-S r esulted in the labeling of the alpha-subunit of GP IIb as found with p latelets, although this was confirmed to be independent of ADP recepto rs. After RNA from CMK 11-5 cells was microinjected into Xenopus oocyt es, only ADP and ADP-alpha-S stimulated Ca-45(2+) efflux, which was no t observed with ATP, 2-methylthio-ATP, alpha,beta-methylene-ATP, ATP-g amma-S, ATP-alpha-S, or adenosine. In addition, incubation of RNA-inje cted oocytes with ATP or ATP-alpha-S but not adenosine blocked the Ca- 45(2+) response to ADP. These experiments demonstrate that a nascent r eceptor 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 o ocytes.