Identification, Expression, Function, and Localization of a Novel (Sixth) Isoform of the Human Sarco/Endoplasmic Reticulum Ca2+ATPase 3 Gene

Abstract

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. 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.Cell Ca 2ϩ signaling is a dynamic oscillatory process regulating a variety of important cellular functions such as secretion, contraction, metabolism, neuronal plasticity, and gene transcription (1). Accordingly, Ca 2ϩ signaling is strictly controlled in space, time, and amplitude. This very tight control is necessary to enable cells to extract relevant information from the Ca 2ϩ signal but also implies that disturbances in the intracellular Ca 2ϩ signaling can lead to a plethora of consequences.Understanding Ca 2ϩ signaling requires the basic knowledge of structures involved in this process. Among these structures are the Ca 2ϩ transport proteins inserted in the membranes of the endoplasmic reticulum (ER), 1 the intracellular reservoir of Ca 2ϩ ions. These include Ca 2ϩ channels (ryanodine and inositol 1,4,5-trisphosphate receptor channels) and Ca 2ϩ pumps (sarco/endoplasmic reticulum Ca 2ϩ -ATPases (SERCAs)). Although much data are available on the role of the channels in the elaboration of the Ca 2ϩ signal, little is known concerning how the signal may be modified by expression of SERCAs with different functional properties.In recent years, enormous advances in our understanding of SERCA structure-function relationships were made, including new insights regarding their three-dimensional structure (2, 3). Furthermore, relevant physiological functions of SERCAs have been defined through their potential role in various human diseases (4-6). However, some of the results contrast with those obtained in mice using SERCA1-3 knock ...

Determination of apparent calcium affinity for endogenously expressed human sarco(endo)plasmic reticulum calcium-ATPase isoform SERCA3

“…However, the lack of an inhibitory antibody specific to SERCA3 prevented validation of these data (note that PL/IM430 is highly specific to only human SERCA3). Nonetheless, because of the conserved nature of SERCA3 amino acid sequence among many species (human, mouse, rat, rabbit, dog, chicken, and cow) and because all six human SERCA3 isoforms display lower apparent affinity for Ca 2ϩ (8,37), it is highly likely that SERCA3 pumps also display low apparent Ca 2ϩ affinity in these animal species. Indeed, studies with recombinant rat and rabbit SERCA3 proteins have shown that those enzymes display lower apparent affinity for Ca 2ϩ as expected (15,44).…”

“…1) Recombinant SERCA3a displayed lower apparent affinity for Ca 2ϩ when assayed for Ca 2ϩ -ATPase activity and E-P formation (K 0.5 ϳ1.1 M vs. ϳ0.3 M for SERCA2b) than other SERCA isoforms (34). More recently, with use of recombinant proteins expressed heterologously, it was shown that all six SERCA3 isoforms display similarly lower apparent affinity for Ca 2ϩ (8,37). 2) SERCA3 differed from other isoforms, in that its pH optimum was 7.2-7.4, whereas that for SERCA1 and SERCA2 pumps was 6.8 -7.0.…”

“…The ATP2A3 gene is alternatively spliced to generate six different transcripts encoding the isoforms SERCA3a, SERCA3b, SERCA3c, SERCA3d, SERCA3e, and SERCA3f, which differ in the length and sequence of their COOHterminal ends (8). SERCA3 is expressed abundantly in only a select number of adult tissue and cell types, predominantly of nonmuscle origin.…”

Functional Comparison between Secretory Pathway Ca2+/Mn2+-ATPase (SPCA) 1 and Sarcoplasmic Reticulum Ca2+-ATPase (SERCA) 1 Isoforms by Steady-state and Transient Kinetic Analyses

“…The methods used for the analysis of the catalytic cycle in steady-state and transient-kinetic conditions have been previously established from studies with SERCA1a mutants (31,32) and used very recently to characterize SERCA2 (33) and SERCA3 isoforms (34,35) as well as Darier disease (SERCA2b) mutants (33). These methods were directly applicable to expressed human SPCA1 enzymes.…”

Impaired megakaryocytopoiesis in type 2B von Willebrand disease with severe thrombocytopenia

“…These were essentially performed according to published procedures. 23,24 The primers used to amplify mRNA for PMCA1b, PMCA4b, SERCA2b, SERCA3a-to 3c, and InsP3-R1 to -R3 were detailed previously by us. 23,26,27 PCR was performed acording to Martin et al 23 where a touchdown-PCR was performed using 10 cycles with annealing temperature decrements from 65°C to 55°C.…”