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Primary structure of rat plasma membrane Ca(2+)-ATPase isoform 4 and analysis of alternative splicing patterns at
1Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, OH 45267-0524.
Abstract:
We have determined the primary structure of the rat plasma membrane Ca(2+)-ATPase isoform 4 (PMCA4), and have analysed its mRNA tissue distribution and alternative splicing patterns at splice site A. Rat PMCA4 (rPMCA4) genomic clones were isolated and used to determine the coding sequences and intron/exon organization of the 5'-end of the gene, and the remaining coding sequence was determined from PCR-amplified cDNA fragments. Pairwise comparisons reveal that the amino acid sequence of rPMCA4 has diverged substantially from those of rPMCA isoforms 1, 2 and 3 (73-76% identity) and from that of human PMCA4 (87%). Despite the high degree of sequence divergence between the two species, comparisons of intron and untranslated mRNA sequences with the corresponding human sequences confirm the identity of this rat isoform as PMCA4. Northern blot studies demonstrate that the PMCA4 mRNA is expressed in all rat tissues examined except liver, with the highest levels in uterus and stomach. A combination of PCR analysis of alternative splicing patterns and sequence analysis of the gene demonstrate that a 36 nt exon at site A is included in PMCA4 mRNAs of most tissues but is largely excluded in heart and testis. Alternative splicing of both the 36 nt exon and a previously characterized 175 nt exon at splice site C, each of which can be either included or excluded in a highly tissue-specific manner, leads to the production of four different PMCA4 variants ranging in size from 1157 to 1203 amino acids.
Insights
Researchers detailed the rat plasma membrane Ca(2+)-ATPase isoform 4 (PMCA4) structure and gene expression. They found tissue-specific alternative splicing generates multiple PMCA4 variants in rats.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- The plasma membrane Ca(2+)-ATPase (PMCA) family regulates intracellular calcium levels.
- Understanding specific isoforms, like PMCA4, is crucial for cellular calcium homeostasis.
- Investigating the rat PMCA4 (rPMCA4) provides insights into mammalian calcium transport mechanisms.
Purpose of the Study:
- To determine the primary structure and analyze mRNA tissue distribution of rat PMCA4.
- To investigate alternative splicing patterns of rPMCA4 at splice site A.
- To compare rPMCA4 with other isoforms and its human counterpart.
Main Methods:
- Isolation of rat PMCA4 genomic clones.
- PCR amplification of cDNA fragments to determine coding sequences.
- Northern blot analysis for mRNA tissue distribution.
- PCR and sequence analysis for alternative splicing patterns.
Main Results:
- The amino acid sequence of rPMCA4 shows significant divergence from other rat PMCA isoforms but high identity (87%) with human PMCA4.
- PMCA4 mRNA is expressed in most rat tissues, with highest levels in uterus and stomach, but not in the liver.
- Alternative splicing at splice site A, involving a 36 nt exon, occurs in a tissue-specific manner, with exclusion in heart and testis.
- Combined splicing of exons at sites A and C generates four PMCA4 variants.
Conclusions:
- The primary structure of rPMCA4 has been elucidated.
- Tissue-specific expression and alternative splicing contribute to functional diversity of PMCA4.
- These findings enhance understanding of calcium regulation by PMCA4 in rats.