Functional expression of a P2T ADP receptor in Xenopus oocytes injected with megakaryocyte (CMK 11-5) RNA.
Since the P2T 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 Ca2+ levels. Like platelets, CMK 11-5 cells responded with an increase in intracellular Ca2+ mobilization in response to ADP but not to ATP or adenosine. In contrast, both nucleotides increased intracellular Ca2+ mobilization in the megakaryoblastic cell lines MO7E and Meg-01, indicating that they contain P2Y 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-alpha-S are active as agonists and ATP and ATP-alpha-S are inactive as agonists but act as antagonists. [3H]ADP and 35S-ATP-alpha-S bound to CMK 11-5 cells at a high-affinity site (Kd1 and Ki1, 262 and 125 nmol/L, respectively) and a low-affinity site (Kd2 and Ki2, 10,100 and 5400 nmol/L, respectively) with 2 x 10(6) to 6 x 10(6) sites per cell. ADP bound at both sites was competed with ADP, ATP, and ATP-alpha-S with affinities in a rank order similar to that found for platelets (ATP-alpha-S approximately ATP approximately ADP > or = ADP-beta-S approximately adenosine), suggesting the presence of a P2T receptor on CMK 11-5 cells. Photoaffinity labeling of intact CMK 11-5 cells with 35S-ATP-alpha-S resulted in the labeling of the alpha-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-alpha-S stimulated 45Ca2+ efflux, which was not observed with ATP, 2-methylthio-ATP, alpha, beta-methylene-ATP, ATP-gamma-S, ATP-alpha-S, or adenosine. In addition, incubation of RNA-injected oocytes with ATP or ATP-alpha-S but not adenosine blocked the 45Ca2+ response to ADP. These experiments demonstrate that a nascent receptor that responded specifically to ADP but not to other P1, P2Y, P2X, and P2U agonists was expressed in functional form on Xenopus oocytes.
Greco
N J
NJ
Platelet Biology Department, American Red Cross, Jerome Holland Laboratory, Rockville, Md 20855, USA. greco@usa.redcross.org
D000241:Adenosine / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000502:physiology; D000255:Adenosine Triphosphate / Q000502:physiology; D000345:Affinity Labels; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D015870:Gene Expression; D006801:Humans; D008533:Megakaryocytes / Q000378:metabolism*; D008845:Microinjections; D009865:Oocytes; D018048:Receptors, Purinergic P2 / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D014982:Xenopus laevis
D000345:Affinity Labels; D018048:Receptors, Purinergic P2; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000241:Adenosine; D002118:Calcium