Use of expression mutants and monoclonal antibodies to map the erythrocyte Ca2+ pump.

  title={Use of expression mutants and monoclonal antibodies to map the erythrocyte Ca2+ pump.},
  author={Hugo P Adamo and Ariel J. Caride and John T. Penniston},
  journal={The Journal of biological chemistry},
  volume={267 20},
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… 
Epitope Mapping by Deletion Mutants Reveals the Transmembrane Topology of the Plasma Membrane Ca2+ Pump
It is demonstrated that the plasma membrane CaZ+ pump, like all the other P-type ATPases examined so far, has a cytoplasmically oriented NH2-terminus and that each half contains at least one pair of transmembrane crossings.
Deletions in the Acidic Lipid-binding Region of the Plasma Membrane Ca2+ Pump
Results suggest that deletion of residues 296–349 recreates a form of PMCA similar to that resulting from the binding of acidic lipids at the AL region, which acts as an acidic lipid-binding inhibitory domain capable of adjusting the Ca2+ affinity of the PMCA to the lipid composition of the membrane.
Deletions in the AL region of the h4xb plasma membrane Ca2+ pump
The results show that alterations in the region of the alternative splicing site A change the to Ca2+ of the human isoform 4 of the PMCA.
Deletion of Amino Acid Residues 18-75 Inactivates the Plasma Membrane Ca2+ Pump*
Results suggest that the segment of amino acids 18-75 is essential for the activity of the plasma membrane Ca2+ pump.
Amino Acid Residues 18–75 Are Essential for Expression of an Active Plasma Membrane Ca2+ Pump a
Results show that Caz+ transport activity of microsomes from cells expressing the hPMCA4b(d18-75) (ct120) protein was not significantly different, suggesting that deletion of residues 18-75 neither impeded the insertion in the membrane nor extensively affected the folding of the mutant protein.
The role of the Ca2+ binding ligand Asn879 in the function of the plasma membrane Ca2+ pump.
A mutant PMCA in which the Asn879 was substituted by Asp was constructed and the mutation nearly eliminated the ability of the enzyme to hydrolyze pNPP which is maximal in the absence of Ca(2+) revealing a major effect of the mutation on the Ca( 2+)-independent reactions of the transport cycle.
A Comparative Functional Analysis of Plasma Membrane Ca2+ Pump Isoforms in Intact Cells*
The results indicate that the availability of calmodulin may not be critical for the modulation of PMCA pumps in vivo and thatPMCA4CII(4a) was as effective as PMCA4CI(4b), even if its affinity for calmodoxin is much lower.
A Mutant of the Plasma Membrane Ca2+ Pump Highly Sensitive to Inhibition by Mg2+a
Despite its lower activity, the apparent affinity for Ca2+ of the V674P (ct 120) enzyme was at least as high as that of hPMCA4b (ct120), indicating that the lower activity of theVh74P (CT120) enzyme could not be accounted for by a reduction in the affinity forCa2+ at the transport site.
Substitution of the N-terminal segment of the plasma membrane Ca pump isoform 4 by that of isoform 1 results in a fully functional chimeric enzyme.
Results demonstrate that for activity the h4 Ca pump requires a specific amino acid sequence at its N-terminus, and the essential elements for a fully active enzyme can be provided by the N-Terminal segment of isoform h1 despite the variability.
A Signal for Endoplasmic Reticulum Retention Located at the Carboxyl Terminus of the Plasma Membrane Ca2+-ATPase Isoform 4CI (*)
The results suggest that the sequence Glu1067-Arg1087 encodes a masked signal for ER retention and for the degradation of the protein.