Probing the expression and function of the P2X7 purinoceptor with antibodies raised by genetic immunization.
The cytolytic P2X7 purinoceptor is widely expressed on leucocytes and has sparked interest because of its peculiar ability to induce a large nonselective membrane pore following treatment of cells with ecto-ATP. Antibodies raised against synthetic P2X7 peptides generally work well in Western-Blot analyses but fail to recognize the native protein on the cell surface. Genetic immunization is a useful technique to raise antibodies directed against proteins in native conformation. Using this technique we have generated highly specific polyclonal (rabbit) and monoclonal (rat) anti-P2X7 antibodies that readily detect mouse P2X7 on the surface of living cells by immunofluorescence analyses and flow cytometry. Binding of these antibodies to P2X7 is reduced within seconds after treatment of cells with ATP, suggesting that ligand binding induces a conformational shift and/or the shedding of P2X7. By site directed mutagenesis we have mutated three conserved arginine residues (R294A, R307A, R316A) in the extracellular loop of P2X7 near the second transmembrane region. Each of these mutations results in loss of ATP response. FACS and immunoblot analyses reveal that the R294A mutant is expressed at higher levels than wild-type P2X7 in transfected cells, whereas the R307A and R316A mutants are barely detectable because there is no or very little protein synthesis of these constructs. In accord with its resistance to ATP-induced activation the R294A mutant is not down-modulated from the cell surface by ATP-treatment.
Adriouch
Sahil
S
Institute of Immunology, University Hospital, 20246 Hamburg, Germany.
Dubberke
Gudrun
G
Diessenbacher
Philip
P
Rassendren
François
F
Seman
Michel
M
Haag
Friedrich
F
Koch-Nolte
Friedrich
F
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D002460:Cell Line; D018076:DNA, Complementary / Q000276:immunology; D006801:Humans; D007114:Immunization / Q000379:methods; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011817:Rabbits; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D058486:Receptors, Purinergic P2X7; D016415:Sequence Alignment; D014162:Transfection
D000906:Antibodies; D018076:DNA, Complementary; D008565:Membrane Proteins; C546468:P2RX7 protein, human; C546469:P2rx7 protein, mouse; C546470:P2rx7 protein, rat; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7