Multiple functions of junctin and junctate, two distinct isoforms of aspartyl beta-hydroxylase

The single genomic locus, AbetaH-J-J, encodes three functionally distinct proteins aspartyl beta-hydroxylase, junctin and junctate by alternative splicing. Among these three proteins, junctin and junctate could play important roles in the regulation of intracellular Ca(2+) by regulating either Ca(2+) release from intracellular Ca(2+) stores or Ca(2+) influx in various biological processes. Here we review recent findings concerning the expressional regulations and the proposed functions of junctin and junctate.     

Molecular cloning, expression, functional characterization, chromosomal localization, and gene structure of junctate, a novel integral calcium binding protein of sarco(endo)plasmic reticulum membrane

Screening a cDNA library from human skeletal muscle and cardiac muscle with a cDNA probe derived from junctin led to the isolation of two groups of cDNA clones. The first group displayed a deduced amino acid sequence that is 84% identical to that of dog heart junctin, whereas the second group had a single open reading frame that encoded a polypeptide with a predicted mass of 33 kDa, whose first 78 NH(2)-terminal residues are identical to junctin whereas its COOH terminus domain is identical to aspartyl beta-hydroxylase, a member of the alpha-ketoglutarate-dependent dioxygenase family. We named the latter amino acid sequence junctate. Northern blot analysis indicates that junctate is expressed in a variety of human tissues including heart, pancreas, brain, lung, liver, kidney, and skeletal muscle. Fluorescence in situ hybridization analysis revealed that the genetic loci of junctin and junctate map to the same cytogenetic band on human chromosome 8. Analysis of intron/exon boundaries of the genomic BAC clones demonstrate that junctin, junctate, and aspartyl beta-hydroxylase result from alternative splicing of the same gene. The predicted lumenal portion of junctate is enriched in negatively charged residues and is able to bind calcium. Scatchard analysis of equilibrium (45)Ca(2+) binding in the presence of a physiological concentration of KCl demonstrate that junctate binds 21.0 mol of Ca(2+)/mol protein with a k(D) of 217 +/- 20 microm (n = 5). Tagging recombinant junctate with green fluorescent protein and expressing the chimeric polypeptide in COS-7-transfected cells indicates that junctate is located in endoplasmic reticulum membranes and that its presence increases the peak amplitude and transient calcium released by activation of surface membrane receptors coupled to InsP(3) receptor activation. Our study shows that alternative splicing of the same gene generates the following functionally distinct proteins: an enzyme (aspartyl beta-hydroxylase), a structural protein of SR (junctin), and a membrane-bound calcium binding protein (junctate).     

Increased Ca2+ storage capacity of the skeletal muscle sarcoplasmic reticulum of transgenic mice over-expressing membrane bound calcium binding protein junctate

Junctate is an integral sarco(endo)plasmic reticulum protein expressed in many tissues including heart and skeletal muscle. Because of its localization and biochemical characteristics, junctate is deemed to participate in the regulation of the intracellular Ca2+ concentration. However, its physiological function in muscle cells has not been investigated yet. In this study we examined the effects of junctate over-expression by generating a transgenic mouse model which over-expresses junctate in skeletal muscle. Our results demonstrate that junctate over-expression induced a significant increase in SR Ca2+ storage capacity which was paralleled by an increased 4-chloro-m-cresol and caffeine-induced Ca2+ release, whereas it did not affect SR Ca2+-dependent ATPase activity and SR Ca2+ loading rates. In addition, junctate over-expression did not affect the expression levels of SR Ca2+ binding proteins such as calsequestrin, calreticulin and sarcalumenin. These findings suggest that junctate over-expression is associated with an increase in the SR Ca2+ storage capacity and releasable Ca2+ content and support a physiological role for junctate in intracellular Ca2+ homeostasis.