Monitoring the expression of purinoceptors and nucleotide-metabolizing ecto-enzymes with antibodies directed against proteins in native conformation

Following their release from cells, ATP and NAD, the universal currencies of energy metabolism, function as extracellular signalling molecules. Mammalian cells express numerous purinoceptors, i.e., the nucleotide-gated P2X ion channels and the G-protein-coupled P2Y receptors. Signalling through purinoceptors is controlled by nucleotide-metabolizing ecto-enzymes, which regulate the availability of extracellular nucleotides. These enzymes include ecto-nucleoside triphosphate diphosphohydrolases (ENTPD, CD39 family) and ecto-nucleotide pyrophosphatase/phosphodiesterases (ENPP, CD203 family). Investigation of these receptors and enzymes has been hampered by the lack of available antibodies, especially ones that recognize these proteins in their native conformation. This study reports the use of genetic immunization to generate such antibodies against P2X(1), P2X(4), P2X(7), ENTPD1, ENPTD2, ENPTD5, ENPTD6, ENPP2, ENPP3, ENPP4, ENPP5, and ENPP6. Genetic immunization ensures expression of the native protein by the cells of the immunized animal and yields antibodies directed against proteins in native conformation (ADAPINCs). Such antibodies are especially useful for immunofluorescence and immunoprecipitation analyses, whereas antibodies against synthetic peptides usually function well only in Western-blot analyses. Here we illustrate the utility of the new antibodies to monitor the cell surface expression of and to purify some key players of purinergic signalling.