A highly active 120-kDa truncated mutant of the plasma membrane Ca2+ pump.
A truncated mutant (hPMCA4b(ct120)) of the plasma membrane Ca2+ pump was expressed in COS-1 cells. The full-length pump (hPMCA4b) consisted of 1205 residues, and the mutant lacked 120 residues (including the 28-residue calmodulin-binding inhibitory domain) at the COOH terminus. To characterize this construct, Ca2+ transport was determined in a microsomal fraction. Phosphate was added to increase Ca2+ uptake, and specificity was enhanced by adding thapsigargin to inhibit the endoplasmic reticulum Ca2+ pump. The mutant showed a similar level of expression as hPMCA4b and a Ca2+ affinity and Ca2+ transport activity about equal to that of hPMCA4b when hPMCA4b was activated. Addition of the synthetic peptide C28R2, corresponding to the calmodulin binding region of the pump, inhibited the mutant and restored the non-activated state of the enzyme. In these respects, the truncated mutant acted like hPMCA4b, when hPMCA4b had been proteolyzed to cleave a bond between the calmodulin binding region and the NH2-terminal portion of the molecule. This indicates that the effects of proteolysis are due to removal of the COOH terminus and not to rearrangement of the two fragments. Since the truncated mutant was fully active and its tryptic digestion resulted in the appearance of the expected 81- and 76-kDa active fragments, we concluded that the COOH-terminal portion which is missing cannot be important in synthesis or proper folding of the enzyme.
Department of Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, Minnesota 55905.
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Enyedi
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Verma
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Filoteo
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JT
Penniston
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology*; D002147:Calmodulin / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D007668:Kidney; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016296:Mutagenesis; D016133:Polymerase Chain Reaction / Q000379:methods; D015183:Restriction Mapping; D017384:Sequence Deletion; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D014162:Transfection
D002147:Calmodulin; D013729:Terpenes; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D002118:Calcium