EXPRESSION OF 2 ISOFORMS OF THE 3RD SARCO ENDOPLASMIC RETICULUM CA(2+)ATPASE (SERCA3) IN PLATELETS - POSSIBLE RECOGNITION OF THE SERCA3B ISOFORM BY THE PL/IM430 MONOCLONAL-ANTIBODY/

Human platelets express several sarco/endoplasmic reticulum Ca2+ ATPas e (SERCA) isoenzymes: SERCA2b of 100 kDa apparent molecular mass and t wo distinct enzymes of 97 kDa, one of them identified as being the SER CA3a isoform. The molecular identity of the third enzyme specifically recognized by the PL/IM430 monoclonal antibody has remained elusive. F irst, the study of the 3'-end part of platelet SERCA3 mRNA, by means o f RT-PCR amplification using sets of primers covering the N-3 to N (ul timate) exons of the human SERCA3 sequence, revealed the presence of t wo distinct mRNA sequences, SERCA3a and a longer variant. Second, this additional sequence was identified as SERCA3b and found to refer to t he insertion of a new exon of 73 bp, located at bp 349 from the beginn ing of the intronic sequence, linking the penultimate (N-1) exon to th e last exon (N) of the human SERCA3 gene. Third, a relationship betwee n the expression of this SERCA3b mRNA and the PL/IM430 recognizable SE RCA protein was observed. SERCA3b mRNA was found to be absent in epith elial HeLa cells not recognized by the PL/IM430 antibody and the expre ssion of this SERCA3b RNA species correlated with that of the SERCA pr otein recognized by PL/IM430 which was down-modulated in the platelet precursor megakaryocytic CHRF 288-11 cell line as well as upon in vitr o Iymphocyte activation. Taken together, these results strongly suppor t the notion of the presence of the SERCA3b protein in human cells by showing SERCA3b mRNA in platelets and the fact that the protein corres ponding to this mRNA species is very likely the 97 kDa protein recogni zed by the PL/IM430 antibody. (C) 1998 Federation of European Biochemi cal Societies.