Identification, expression, function, and localization of a novel (sixth) isoform of the human sarco/endoplasmic reticulum Ca2+ATPase 3 gene.
Understanding of Ca(2+) signaling requires the knowledge of proteins involved in this process. Among these proteins are sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCAs) that pump Ca(2+) into the endoplasmic reticulum (ER). Recently, the human SERCA3 gene was shown to give rise to five isoforms (SERCA3a-e (h3a-h3e)). Here we demonstrate the existence of an additional new member, termed SERCA3f (h3f). By reverse transcriptase-PCR using monocytic U937 cell RNA, h3f mRNA was found to exclude the antepenultimate exon 21. h3f mRNA expression appeared as a human-specific splice variant. It was not found in rats or mice. h3f mRNA gave rise to an h3f protein differing in its C terminus from h3a-h3e. Of particular interest, h3f diverged in the first amino acids after the first splice site but presented the same last 21 amino acids as h3b. Consequently, we further investigated the structure-function-location relationships of the h3b and h3f isoforms. Comparative functional study of h3b and h3f recombinant proteins in intact HEK-293 cells and in fractionated membranes showed the following distinct characteristics: (i) resting cytosolic Ca(2+) concentration ([Ca(2+)](c)) and (ii) ER Ca(2+) content ([Ca(2+)](er)); similar characteristics were shown for the following: (i) the effects of the SERCA inhibitor, thapsigargin, on Ca(2+) release ([Ca(2+)](Tg)) and subsequent Ca(2+) entry ([Ca(2+)](e)) and (ii) the low apparent Ca(2+) affinity and the enhanced rate of dephosphorylation of the E(2)P phosphoenzyme intermediate. Subcellular location of h3b and h3f by immunofluorescence and/or confocal microscopy using the h3b- and h3f-specific polyclonal and the pan-h3 monoclonal (PL/IM430) antibodies suggested overlapping but distinct ER location. The endogenous expression of h3f protein was also proved in U937 cells. Altogether these data suggest that the SERCA3 isoforms have a more widespread role in cellular Ca(2+) signaling than previously appreciated.
Bobe
Régis
R
INSERM U.348, IFR6 Circulation Lariboisière, Hôpital Lariboisière, 8 Rue Guy Patin, 75475 Paris Cedex 10, France.
Bredoux
Raymonde
R
Corvazier
Elisabeth
E
Andersen
Jens Peter
JP
Clausen
Johannes D
JD
Dode
Leonard
L
Kovács
Tünde
T
Enouf
Jocelyne
J
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000096:biosynthesis / Q000737:chemistry* / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005091:Exons; D018922:HL-60 Cells; D006801:Humans; D015151:Immunoblotting; D019169:Jurkat Cells; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection; D020298:U937 Cells
D000911:Antibodies, Monoclonal; C506951:Atp2a3 protein, rat; D018076:DNA, Complementary; D020033:Protein Isoforms; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012313:RNA; D019284:Thapsigargin; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506949:ATP2A3 protein, human; C506950:Atp2a3 protein, mouse; D000252:Calcium-Transporting ATPases; D002118:Calcium