Expression of two isoforms of the third sarco/endoplasmic reticulum Ca²⁺ATPase (SERCA3) in platelets. Possible recognition of the SERCA3b isoform by the PL/IM430 monoclonal antibody¹

Abstract

Human platelets express several sarco/endoplasmic reticulum Ca P+ ATPase (SERCA) isoenzymes: SERCA2b of 100 kDa apparent molecular mass and two distinct enzymes of 97 kDa, one of them identified as being the SERCA3a isoform. The molecular identity of the third enzyme specifically recognized by the PL/IM430 monoclonal antibody has remained elusive. First, the study of the 3P-end part of platelet SERCA3 mRNA, by means of RT-PCR amplification using sets of primers covering the N33 to N (ultimate) exons of the human SERCA3 sequence, revealed the presence of two distinct mRNA sequences, SERCA3a and a longer variant. Second, this additional sequence was identified as SERCA3b and found to refer to the insertion of a new exon of 73 bp, located at bp 349 from the beginning of the intronic sequence, linking the penultimate (N31) exon to the last exon (N) of the human SERCA3 gene. Third, a relationship between the expression of this SERCA3b mRNA and the PL/ IM430 recognizable SERCA protein was observed. SERCA3b mRNA was found to be absent in epithelial HeLa cells not recognized by the PL/IM430 antibody and the expression of this SERCA3b RNA species correlated with that of the SERCA protein recognized by PL/IM430 which was down-modulated in the platelet precursor megakaryocytic CHRF 288-11 cell line as well as upon in vitro lymphocyte activation. Taken together, these results strongly support the notion of the presence of the SERCA3b protein in human cells by showing SERCA3b mRNA in platelets and the fact that the protein corresponding to this mRNA species is very likely the 97 kDa protein recognized by the PL/IM430 antibody.z 1998 Federation of European Biochemical Societies.

Biogenesis of endoplasmic reticulum proteins involved in Ca2+ signalling during megakaryocytic differentiation: an in vitro study

“…Moreover, unfortunately, no information can be expected from rat or mouse, because of their striking differences at least in the nature of their platelet SERCA3 [22,23] and levels of SERCA2b-protein expression [57]. However, it was recently found that the temporal patterns of Ca# + signals depend critically on the SERCA2 isoforms [58] or on the set of InsP $ -R subtypes [59].…”

“…Although some controversy exists concerning the expression of InsP $ -Rs, human platelets were reported to express either InsP $ -RI and II or InsP $ -RI and III. About SERCAs, platelets account for the first cells shown to simultaneously express the ubiquitous SERCA2b [18] and SERCA3a proteins [19][20][21] as well as SERCA3b [22] and SERCA3c [23] mRNAs.…”

“…The monoclonal PL\IM430 and IID8 antibodies were used to visualize SERCA3 and SERCA2b isoforms, respectively [20][21][22]32]. As controls of platelet maturation and ER proteins, the β subunit of integrin α IIb β $ , GPIIIa and calreticulin were visualized using specific polyclonal antibodies.…”

“…The panel is representative of three independent experiments. and 3c mRNAs [22,23]. We performed reverse transcription followed by comparative specific PCR on total RNA isolated from control and PMA-treated MEG 01 cells ( Figure 5).…”

Basal Transcription of the Mouse Sarco(endo)plasmic Reticulum Ca2+-ATPase Type 3 Gene in Endothelial Cells Is Controlled by Ets-1 and Sp1

“…At 14 days post-coitum it disappears from the heart tube and is present in the dorsal aorta and as small foci in the liver (10). Several groups (12)(13)(14)(15) 2 have recently identified multiple SERCA3 isoforms generated by alternative splicing, and the respective functions of these isoforms are still unknown.…”