Plasma Membrane Calcium Pump Isoform Research Articles

Molecular cloning of a plasma membrane calcium pump from human osteoblasts.

The osteoblast plays a critical role in bone formation, bone remodelling, bone matrix formation, and matrix calcification. To better understand the process of osteoblast-controlled bone formation, we determined the structure and isoform types of the plasma membrane calcium pump from normal human osteoblasts. A complementary DNA library from normal human osteoblasts was screened for plasma membrane calcium pump clones. Sequencing and analysis of cDNA clones revealed the presence of a 3986 base pair cDNA that encoded a 1220 amino acid protein that was similar to the human plasma membrane calcium pump isoform 1. Polyadenylated RNA from human osteoblast cells contains bands of RNA approximately 5050 and 6750 bases long. Reverse transcription of polyadenylated RNA from human osteoblasts followed by amplification of the RNA-DNA duplex with calcium pump isoform-specific primers revealed the presence of isoforms 1 and 2 of the calcium pump. Isoform 4 was not detected. We conclude that normal adult human osteoblasts contain a plasma membrane calcium pump that is similar to the human plasma membrane calcium pump isoform 1. It is likely that this pump plays an important role in the cell biology of the human osteoblast.

MRNAs for plasma membrane calcium pump isoforms differing in their regulatory domain are generated by alternative splicing that involves two internal donor sites in a single exon.

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.