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J. Biol. Chem., Vol. 279, Issue 23, 24297-24306, June 4, 2004

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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||, Tünde Kovács**, and Jocelyne Enouf

From the INSERM U.348, IFR6 Circulation Lariboisière, Hôpital Lariboisière, 8 Rue Guy Patin, 75475 Paris Cedex 10, France, the ^||Department of Physiology, University of Aarhus, Ole Worms Allé 160, DK-8000 Aarhus C, Denmark, and the ^**National Medical Center, Institute of Haematology and Immunology, H-1113 Budapest, Hungary

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 ([Ca²⁺]_c) and (ii) ER Ca²⁺ content ([Ca²⁺]_er); similar characteristics were shown for the following: (i) the effects of the SERCA inhibitor, thapsigargin, on Ca²⁺ release ([Ca²⁺]_Tg) and subsequent Ca²⁺ 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.

Received for publication, December 30, 2003 , and in revised form, March 17, 2004.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY460339.

^* This work was supported in part by the INSERM, by a grant from the Association Française Contre les Myopathies, France (to J. E.), by a grant from the Danish Medical Research Council, Denmark (to J. P. A.), and by Hungarian Academy of Sciences Grant OTKA T032766, Hungary (to T. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of fellowship from the Fondation pour la Recherche Médicale.

^¶ Both authors contributed equally to this work.

To whom correspondence should be addressed. Tel.: 33-1-53-20-37-91; Fax: 33-1-49-95-85-79; E-mail: jocelyne.enouf{at}larib.inserm.fr.

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