Identification, expression, function, and localization of a novel (sixth) isoform of the human sarco/endoplasmic reticulum Ca2+ATPase 3 gene

Régis Bobe, Raymonde Bredoux, Elisabeth Corvazier, Jens Peter Andersen, Johannes D. Clausen, Leonard Dode, T. Kovács, Jocelyne Enouf

Research output: Contribution to journalArticle

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Abstract

Understanding of Ca2+ signaling requires the knowledge of proteins involved in this process. Among these proteins are sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) that pump Ca2+ 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 Ca2+ concentration ([Ca 2+]c) and (ii) ER Ca2+ content ([Ca 2+]er); similar characteristics were shown for the following: (i) the effects of the SERCA inhibitor, thapsigargin, on Ca 2+ release ([Ca2+]Tg) and subsequent Ca 2+ entry ([Ca2+]e) and (ii) the low apparent Ca2+ affinity and the enhanced rate of dephosphorylation of the E2P 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 Ca2+ signaling than previously appreciated.

Original languageEnglish
Pages (from-to)24297-24306
Number of pages10
JournalJournal of Biological Chemistry
Volume279
Issue number23
DOIs
Publication statusPublished - Jun 4 2004

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Endoplasmic Reticulum
Adenosine Triphosphatases
Protein Isoforms
Genes
U937 Cells
Messenger RNA
Proteins
Sarcoplasmic Reticulum Calcium-Transporting ATPases
Amino Acids
Thapsigargin
Confocal microscopy
HEK293 Cells
RNA-Directed DNA Polymerase
Reverse Transcriptase Polymerase Chain Reaction
Fluorescence Microscopy
Recombinant Proteins
Confocal Microscopy
Rats
Exons
Pumps

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification, expression, function, and localization of a novel (sixth) isoform of the human sarco/endoplasmic reticulum Ca2+ATPase 3 gene. / Bobe, Régis; Bredoux, Raymonde; Corvazier, Elisabeth; Andersen, Jens Peter; Clausen, Johannes D.; Dode, Leonard; Kovács, T.; Enouf, Jocelyne.

In: Journal of Biological Chemistry, Vol. 279, No. 23, 04.06.2004, p. 24297-24306.

Research output: Contribution to journalArticle

Bobe, Régis ; Bredoux, Raymonde ; Corvazier, Elisabeth ; Andersen, Jens Peter ; Clausen, Johannes D. ; Dode, Leonard ; Kovács, T. ; Enouf, Jocelyne. / Identification, expression, function, and localization of a novel (sixth) isoform of the human sarco/endoplasmic reticulum Ca2+ATPase 3 gene. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 23. pp. 24297-24306.
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