Use of expression mutants and monoclonal antibodies to map the erythrocyte Ca2+ pump.
Deletion and truncation mutants of the human erythrocyte Ca2+ pump (hPMCA4b) were expressed in COS-1 cells. The reactivity patterns of these mutants with seven monoclonal antibodies were examined. Of the seven, six (JA9, JA3, 1G4, 4A4, 3E10 and 5F10) react from the cytoplasmic side. JA9 and JA3 reacted near the NH2 terminus and the COOH terminus of the molecule, respectively. 5F10 and 3E10 recognized portions of the large hydrophilic region in the middle of the protein. The epitopes of 1G4 and 4A4 were discontinuous and included residues from the long hydrophilic domain and residues between the proposed transmembrane domains M2 and M3. Antibody 1B10, which reacts from the extracellular side, recognized the COOH-terminal half of the molecule. These results show that the NH2 terminus, the COOH terminus, the region between M2 and M3, and the large hydrophilic region are all on the cytoplasmic side. This means that there are an even number of membrane crossings in both the NH2-terminal and the COOH-terminal halves. Between residues 75 and 300 there must be at least two membrane crossings, and there are at least two membrane crossings in the COOH-terminal half of the molecule.
Adamo
H P
HP
Department of Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, Minnesota 55905.
Caride
A J
AJ
Penniston
J T
JT
D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001666:Binding Sites, Antibody; D000252:Calcium-Transporting ATPases / Q000097:blood / Q000235:genetics* / Q000276:immunology; D002460:Cell Line; D002872:Chromosome Deletion; D000939:Epitopes / Q000032:analysis; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D008961:Models, Structural; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D015183:Restriction Mapping; D014162:Transfection
D000911:Antibodies, Monoclonal; D000939:Epitopes; D009838:Oligodeoxyribonucleotides; D000252:Calcium-Transporting ATPases