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Use of expression mutants and monoclonal antibodies to map the erythrocyte Ca2+ pump
H P Adamo1, A J Caride, J T Penniston
1Department of Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, Minnesota 55905.
Abstract:
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.
Insights
Researchers mapped the human erythrocyte Ca2+ pump (hPMCA4b) structure using antibody binding on mutants. This revealed cytoplasmic accessibility of key domains, suggesting an even number of membrane crossings.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- The human erythrocyte Ca2+ pump (hPMCA4b) is crucial for calcium homeostasis.
- Understanding its membrane topology is essential for elucidating its function.
Purpose of the Study:
- To determine the topology of the hPMCA4b by mapping antibody epitopes on deletion and truncation mutants.
- To identify cytoplasmic and extracellular regions of the pump.
Main Methods:
- Expression of hPMCA4b deletion and truncation mutants in COS-1 cells.
- Analysis of reactivity patterns with seven specific monoclonal antibodies.
- Mapping of antibody epitopes to specific regions of the hPMCA4b protein.
Main Results:
- Six of seven antibodies reacted with mutants from the cytoplasmic side, recognizing the NH2 terminus, COOH terminus, and a large hydrophilic region.
- Two antibodies (1G4, 4A4) recognized discontinuous epitopes involving the hydrophilic domain and the M2-M3 transmembrane region.
- One antibody (1B10) reacted from the extracellular side, recognizing the COOH-terminal half.
Conclusions:
- The NH2 terminus, COOH terminus, M2-M3 region, and large hydrophilic region are located on the cytoplasmic side.
- This topology implies an even number of membrane crossings in both the NH2-terminal and COOH-terminal halves of hPMCA4b.
- At least two membrane crossings occur between residues 75-300 and in the COOH-terminal half.