Three Novel Sarco/endoplasmic Reticulum Ca2+-ATPase (SERCA) 3 Isoforms

Abstract

Sarco/endoplasmic reticulum Ca 2؉ -ATPases (SERCAs) pump Ca 2؉ into the endoplasmic reticulum. Recently, three human SERCA3 (h3a-c) proteins and a previously unknown rat SERCA3 (r3b/c) mRNA have been described. Here, we (i) document two novel human SERCA3 splice variants h3d and h3e, (ii) provide data for the expression and mechanisms regulating the expression of all known SERCA3 variants (r3a, r3b/c, and h3a-e), and (iii) show functional characteristics of the SERCA3 isoforms. h3d and h3e are issued from the insertion of an additional penultimate exon 22 resulting in different carboxyl termini for these variants. Distinct distribution patterns of the SERCA3 gene products were observed in a series of cell lines of hematopoietic, epithelial, embryonic origin, and several cancerous types, as well as in panels of rat and human tissues. Hypertension and protein kinase C, calcineurin, or retinoic acid receptor signaling pathways were found to differently control rat and human splice variant expression, respectively. Stable overexpression of each variant was performed in human embryonic kidney 293 cells, and the SERCA3 isoforms were fully characterized. All SERCA3 isoforms were found to pump Ca 2؉ with similar affinities. However, they modulated the cytosolic Ca 2؉ concentration ([Ca 2؉ ] c ) and the endoplasmic reticulum Ca 2؉ content ([Ca 2؉ ] er ) in different manners. A newly generated polyclonal antibody and a pan-SERCA3 antibody proved the endogenous expression of the three novel SERCA3 proteins, h3d, h3e, and r3b/c. All these data suggest that the SERCA3 gene products have a more widespread role in cellular Ca 2؉ signaling than previously appreciated.

Effect of Hailey-Hailey Disease Mutations on the Function of a New Variant of Human Secretory Pathway Ca2+/Mn2+-ATPase (hSPCA1)

“…We have provided evidence for the existence of up to four distinct ATP2C1 splice variants in human keratinocytes, generated by alternative processing at the 3Ј-end of the ATP2C1 pre-mRNA. Similar to ATP2C1, internal 5Ј donor splice sites are involved in generating five alternatively spliced human ATP2A3 (SERCA3) transcripts (27,28) and four alternatively processed ATP2B1 (PMCA1) transcripts (29 -31) due to the alternative exclusion, inclusion, or partial inclusion of exons at the 3Ј-end of the gene. So far, no evidence has been provided for the existence of alternatively spliced transcripts in the S. cerevisiae or C. elegans SPCA orthologues.…”

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

“…Next, rats were found to be devoid of SERCA3b and SERCA3c mRNAs, which are replaced by a so-called SERCA3b/c isoform, which is due to an extension of exon 21 (23). Finally, we found that the insertion of an additional exon 22 in human SERCA3b and SERCA3c mRNAs led to SERCA3d and SERCA3e isoforms (24). Although the rationale for such a diversity of novel SERCA3 isoforms is still not understood, functional differences in the human SERCA3a, -3b, and -3c isoforms were recently pointed out, particularly one step of their catalytic cycle, the dephosphorylation of the E 2 P intermediate, varying with the length of the alternatively spliced C terminus (25).…”

“…24 for the IID8, PL/IM430, N89, and anti-hSERCA3b antibodies. For the h3f protein, nitrocellulose membranes were incubated with a 1:2000 dilution of the anti-h3f antibody, in Tris-buffered saline (pH 7.4), 5% non-fat milk, and 0.1% Tween 20 for 2 h at room temperature.…”

“…RNA Extraction and RT-PCR-Total RNA extraction from human platelets and cell lines was as described (24). For RT-PCR experiments, essentially identical protocols as in Ref.…”

“…Antibodies for Immunoblottings-For human SERCA2b (h2b), the Pan-h2 monoclonal antibody, IID8 (24), was used (BioMol, Plymouth Meeting, PA). For h3f, the Pan-h3 monoclonal antibody, PL/IM430, and polyclonal antibody, N89, directed against the N-terminal parts of SERCA3 proteins (21,26) and our recently developed isoform-specific h3b polyclonal antibody (21) were used.…”