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mRNAs for plasma membrane calcium pump isoforms differing in their regulatory domain are generated by alternative
E E Strehler1, M A Strehler-Page, G Vogel
1Laboratory for Biochemistry, Swiss Federal Institute of Technology, Zurich.
Abstract:
cDNA clones coding for human plasma membrane Ca2+ pump isoforms have been isolated from a fetal skeletal muscle cDNA library. Compared with the sequence of a teratoma cDNA-encoded pump these clones specify isoforms that contain either 29- or 38-amino acid insertions within the calmodulin-binding region. Replacement of two basic arginine residues by an aspartic acid and a glutamine residue could influence the binding of calmodulin to these isoforms. RNase mapping shows that RNA species containing the 29-residue-encoding insertion are particularly abundant in skeletal muscles. The sequences coding for the insertions are present on a single 154-base-pair exon, as demonstrated by an analysis of the corresponding genomic region, and they are included in their respective mRNAs by alternative splicing involving the differential usage of two internal "cryptic" donor splice sites in the presence of a nearby canonical one. Inclusion of the complete 154-base-pair exon results in an mRNA coding for a pump protein with a shorter C-terminal amino acid sequence that lacks a consensus site for phosphorylation by the cAMP-dependent kinase. Exclusion, inclusion, or partial inclusion of the same exon can thus lead to the production of four different mRNAs from a single gene. When expressed as protein, these mRNAs encode Ca2+ pump isoforms that differ in their C-terminal regulatory domains.
Insights
Researchers identified human plasma membrane calcium pump (Ca2+) isoforms with unique insertions in skeletal muscle. Alternative splicing of a single exon generates multiple Ca2+ pump variants, impacting calmodulin binding and C-terminal regulation.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Human plasma membrane Ca2+ pumps (SERCA) are crucial for calcium homeostasis.
- Isoforms of these pumps exhibit tissue-specific expression and regulatory differences.
Purpose of the Study:
- To identify and characterize novel human plasma membrane Ca2+ pump isoforms.
- To investigate the molecular mechanisms underlying isoform diversity in skeletal muscle.
Main Methods:
- cDNA library screening from fetal skeletal muscle.
- Sequence analysis of isolated cDNA clones.
- RNase mapping to determine RNA abundance.
- Genomic region analysis to identify exon-intron structure.
- Alternative splicing analysis.
Main Results:
- Isolated cDNA clones encode Ca2+ pump isoforms with 29- or 38-amino acid insertions in the calmodulin-binding region.
- These insertions involve amino acid substitutions that may affect calmodulin binding.
- RNA species with the 29-residue insertion are abundant in skeletal muscle.
- A single 154-base-pair exon is alternatively spliced, leading to four distinct mRNA variants.
- These variants encode Ca2+ pump isoforms with differing C-terminal regulatory domains, including phosphorylation sites.
Conclusions:
- Alternative splicing of a single exon generates diverse human plasma membrane Ca2+ pump isoforms in skeletal muscle.
- These isoforms exhibit variations in their calmodulin-binding and C-terminal regulatory regions.
- The identified isoforms contribute to the complex regulation of calcium transport in muscle tissue.