Modulation of SERCA2 activity: regulated splicing and interaction with phospholamban.

Ca(2+)-uptake into intracellular stores is mediated by the sarco/endoplasmic reticulum Ca(2+)ATPases (SERCAs). This review deals first with the gene structural and the characterization of the tissue-specific SERCA2 transcript processing. Secondly, the two different protein isoforms and their regulation are described. Finally, this review ends with a discussion on the possible physiological role of the SERCA2 isoform diversity.

Sequence and spatial requirements for regulated muscle-specific processing of the sarco/endoplasmic reticulum Ca(2+)-ATPase 2 gene transcript.

Expression of the muscle-specific 2a isoform of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2) requires activation of an otherwise inefficient splicing process at the 3'-end of the primary gene transcript. The sequence and topology requirements for this regulated splicing event were studied in the BC3H1 myogenic cell line using a minigene containing the 3'-end of the SERCA2 gene. In undifferentiated BC3H1 cells, the splice process is made inefficient by the presence of a weak muscle-type 5'-donor site (5'D1) and a long terminal intron. Both optimizing the 5'D1 and decreasing the length of the muscle-specific intron, induced muscle-type splicing in undifferentiated myogenic cells. Moreover, the induction of muscle-type transcripts was only observed when two competing processing sites, the polyadenylation site (pAu) used in non-muscle cells and the second neuronal 5'-donor site (5'D2), were weak. Indeed, making 5'D2 consensus induced neuronal-type splicing in undifferentiated myocytes and prevented the appearance of muscle-type transcripts. Similarly, replacing the polyadenylation site (pAu) with a strong site almost completely inhibited muscle-type splicing after myogenic differentiation. We conclude that weak processing sites and a long terminal intron are required for tissue-dependent mRNA processing of the SERCA2 transcript.

The functional importance of the extreme C-terminal tail in the gene 2 organellar Ca(2+)-transport ATPase (SERCA2a/b).

Ca(2+)-uptake experiments in microsomal fractions from transfected COS-1 cells have revealed a functional difference between the non-muscle SERCA2b Ca2+ pump and its muscle-specific SERCA2a splice variant. Structurally, the two pumps differ only in their C-terminal tail. The last four amino acids of SERCA2a are replaced in SERCA2b by a 49-residue-long peptide chain containing a very hydrophobic stretch which could be an additional transmembrane segment. The functionally important subdomains in the SERCA2b tail were analysed by constructing three SERCA2b deletion mutants lacking 12, 31 or 49 amino acids. The mutants and the parental SERCA2 pumps were expressed in COS-1 cells and analysed for functional difference. SERCA2b had a twofold higher Ca2+ affinity, a twofold lower turnover rate and a 10-fold lower vanadate-sensitivity than SERCA2a and the mutants. Since each of the three truncated versions of SERCA2b acquire the characteristic properties of SERCA2a, it is concluded that the stretch of the last 12 residues of SERCA2b is of critical importance.

A sarco/endoplasmic reticulum Ca(2+)-ATPase 3-type Ca2+ pump is expressed in platelets, in lymphoid cells, and in mast cells.

An organellar-type of Ca2+ pump formerly detected by means of its phosphoprotein intermediate in platelets and in lymphoid cells, and which runs in acid gels at 97 kDa, is now characterized as sarco/endoplasmic reticulum Ca2+ATPase 3 (SERCA3). SERCA3 is co-expressed in these cells along with the housekeeping SERCA2b. This conclusion is based on the following observations. 1) Tryptic digestion the phosphoprotein intermediate of SERCA3 expressed in COS cells yields a phosphorylated fragment of about 80 kDa, which can be clearly distinguished from the 57-kDa fragments formed in the SERCA1 and SERCA2 pumps. This 80-kDa fragment comigrates with a similar phosphoprotein fragment previously observed in human platelets (Papp, B., Enyedi, A., Pászty, K., Kovács, T., Sarkadi, B., Gárdos, G., Wuytack, F., and Enouf, J. (1992) Biochem. J. 288, 297-302). 2) An antiserum directed against an NH2-terminal SERCA3-specific peptide (N89) reacts with SERCA3 expressed in COS cells and with the 97-kDa protein in rat platelets and the corresponding protein in human platelets. Likewise an antiserum against the rat SERCA3 terminus (C90) binds to SERCA3 expressed in COS cells and to the 97-kDa band in rat platelets, but it does not recognize the human platelet pump. In conformity with the predicted absence of the T1 tryptic cleavage site in SERCA3, the autophosphorylated aspartyl residue and the COOH-terminal epitope were co-localized on the 80-kDa fragment. 3) The co-expression of nearly equal levels of SERCA3 and SERCA2b messengers in human lymphoblastoid Jurkat cells and in proliferating rat mucosal mast cells was also demonstrated by reverse transcriptase polymerase chain reaction.

Ca(2+)-transport ATPases and their regulation in muscle and brain.

Eukaryotic cells express one or more isoforms of a sarco(endo)plasmic reticulum (SERCA) and of a plasma membrane (PMCA) Ca2+ pump. Both the SERCA and PMCA gene transcripts are subject to alternative processing in a differentiation stage-dependent and tissue-dependent manner. The Ca2+ pump isoforms thus generated may present different functional properties. This is exemplified by the SERCA2a and SERCA2b isoforms which differ in their Ca2+ sensitivity. Analysis of the cDNA structures for PMCA1 predicts protein isoforms with variant calmodulin- and phospholipid-binding domains. A comparative study of the tissue-specific mechanisms governing SERCA-PMCA transcript processing and a more detailed study of the functional implication of the PMCA pumps isoform diversity will be challenging subjects for future studies.

Functional difference between SERCA2a and SERCA2b Ca2+ pumps and their modulation by phospholamban.

COS 1 cells were transfected with full-length pig stomach sarcoplasmic/endoplasmic reticulum Ca2+ pump (SERCA)2a or SERCA2b cDNA. Ca2+ uptake by microsomes from transfected cells revealed that the Ca2+ affinity of the SERCA2b Ca2+ pump (K0.5 0.17 +/- 0.01 microM) was higher than that of the SERCA2a Ca2+ pump (K0.5 0.31 +/- 0.02 microM). Thapsigargin-sensitivity was found to be identical for the two isoforms. The Ca2+ affinity of both the SERCA2a and SERCA2b Ca2+ pumps was decreased by a factor of two when they were co-expressed with phospholamban.

Molecular cloning and sequencing of the plasma-membrane Ca2+ pump of pig smooth muscle.

cDNAs coding for the plasma-membrane Ca2+ pump have been isolated from a pig smooth-muscle cDNA library and sequenced. The open reading frame encodes a protein of 1220 amino acids, which corresponds to the one already described in a human teratoma cell line. We demonstrate here that this cDNA probably represents the only isoform of the plasma-membrane Ca2(+)-transport ATPase expressed in this smooth muscle. There is no evidence for the expression of any other plasma-membrane Ca2(+)-pump gene, or for the presence of other alternatively spliced isoforms. These results are in apparent contradiction to those obtained on protein levels which demonstrate the reaction of at least two different polypeptides with a panel of antibodies against the plasma-membrane ATPase. It is suggested that these two polypeptides could result from a post-translational modification of one single enzyme.

cDNA cloning and sequencing of phospholamban from pig stomach smooth muscle.

Phospholamban cDNA from pig stomach smooth muscle was cloned and sequenced. The 737-nucleotide-residue cDNA contained an open reading frame of 156 nucleotide residues encoding a peptide of 52 amino acid residues (Mr 6080). This peptide shares 100% sequence identity with dog cardiac-muscle phospholamban. It differs from rabbit cardiac-muscle and slow-twitch skeletal-muscle phospholamban only at position 2, which is a glutamic acid residue in rabbit phospholamban, but an aspartic acid residue in the pig smooth-muscle protein. Northern-blot analysis reveals the presence of several phospholamban mRNAs in smooth muscle, but a 900-nucleotide-residue and a 2800-nucleotide-residue transcript predominate.