Coordination between ecto-nucleotidases and purinergic receptor-mediated signaling

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Extracellular ATP produces diverse physiological effects by activating multiple P2 receptors. Although P2 receptor activation is terminated by hydrolysis of ATP by ecto-nucleotidases, this metabolic step results in adenosine (Ade) production, thereby initiating the P1 receptor activation. We demonstrated that extracellular ATP stimulates A_<2A> and A_<2B> receptor through a rapid and localized Ade production on membrane surface. Furthermore, ecto-alkaline phosphatase (eALP) was found to act as ecto-nucleotidases. Heterologous expression of A_<2A> and A_<2B> receptors in Xenopus oocyte revealed that ATP can stimulate each P1 receptor as rapidly as direct stimulation with Ade in an ecto-nucleotidase-dependent manner. The ecto-nucleotidase-dependent P1 receptor activation by ATP was resistant to elimination of Ade by Ade deaminase (ADA). Analysis of extracellular [^3H]ATP metabolism in NG108-15 cells and Xenopus oocyte showed that the metabolically generated [^3H]Ade was retained on the membrane surface even in the presence of ADA. When NG108-15 cells were transfected with eALP-GFP fusion protein, eALP-GFP was localized on membrane surface and co-internalized with A_<2A> receptors upon prolonged A_<2A> receptor activation. These findings suggest that the close association of P1 receptors to ecto-nucleotidases may comprise a functional receptor for ATP, and some physiological responses to ATP would occur through this mechanism.