Human platelets express the SERCA2-b isoform of Ca(2+)-transport ATPase.
Previous biochemical studies suggested that the human platelet Ca2+ATPase system may be cell-specific. To test this hypothesis, we first undertook the molecular cloning of Ca2+ATPase from human erythroleukaemia (HEL) cells, because this human cell line exhibits megakaryocytic features and expresses a Ca2+ATPase that cross-reacts with platelet Ca(2+)-ATPase. For this cloning, an HEL-cell cDNA library was screened with a rat cardiac Ca2+ATPase cDNA probe. The insert of the longest clone isolated was 3.9 kb and its sequence displayed a 100% identity with that of the non-muscle human Ca2+ATPase 2-b isoform, termed SERCA2-b (sarco-endoplasmic-reticulum Ca2+ATPase). The 3.9 kb cDNA covered a subtotal coding region and part of the 3' non-coding end of the SERCA2-b mRNA. It cross-hybridized with the 4 kb transcript species of cardiac SERCA2-a and with non-muscle SERCA2-b mRNAs, but not with fast-skeletal-muscle SERCA1 mRNA. We next confirmed that SERCA2-b was a component of the platelet Ca2+ATPase system because (1) the platelet clones isolated from a platelet cDNA library exhibited a 100% homology with HEL-cell cDNA; (2) SERCA2-b mRNA was amplified by PCR on total platelet RNA and (3) platelet Ca2+ATPase cross-reacted with a polyclonal SERCA2-b-specific antiserum. Platelets therefore contain a Ca2+ATPase definitely identified as the SERCA2-b isoform of Ca2+ATPase, thus eliminating the possibility that they only contain a single specific Ca2+ATPase.
Enouf
J
J
U348 INSERM, Hôpital Lariboisière, Paris, France.
Bredoux
R
R
Papp
B
B
Djaffar
I
I
Lompré
A M
AM
Kieffer
N
N
Gayet
O
O
Clemetson
K
K
Wuytack
F
F
Rosa
J P
JP
D000818:Animals; D000906:Antibodies; D001792:Blood Platelets / Q000201:enzymology*; D015153:Blotting, Western; D000252:Calcium-Transporting ATPases / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification; D004915:Leukemia, Erythroblastic, Acute / Q000201:enzymology; D009130:Muscle, Smooth / Q000201:enzymology; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification; D015183:Restriction Mapping; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D014407:Tumor Cells, Cultured
D000906:Antibodies; D007527:Isoenzymes; D011994:Recombinant Proteins; D000252:Calcium-Transporting ATPases