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.