Molecular Cloning, Expression, Functional Characterization, Chromosomal Localization, and Gene Structure of Junctate, a Novel Integral Calcium Binding Protein of Sarco(endo)plasmic Reticulum Membrane

Abstract

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 ␤-hydroxylase, a member of the ␣-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 ␤-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

Calcium cycling proteins in heart failure, cardiomyopathy and arrhythmias

“…Although triadin and junctin are the products of different genes, they exhibit intriguing structural and amino acid sequence similarities and play an important role in the regulation of Ca 2+ release from the RyR2. Junctate, an alternative splicing form of the same gene generating junctin and aspartyl β-hydroxylase, is a newly identified 33-kDa Ca 2+ binding protein in the integral SR membrane and three cardiac isoforms (junctate 1, 2 and 3) were cloned from the mouse heart (Treves et al, 2000;Hong et al, 2001). Crosstalk between L-type Ca 2+ channels on the sarcolemmal membrane and the RyR2 on the SR is a fundamental feature of excitation-contraction (EC) coupling in the heart.…”

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

“…During the past decade, several groups have focused their research on the proteomic structure of the longitudinal SR and terminal cisternae: in the present investigation we identified and characterized junctate a novel integral membrane protein of the SR/ER membranes . Junctate is a 33 kDa protein which is expressed in a variety of tissues including brain, kidney, liver and heart and to a lesser extent in skeletal muscle; it results from alternative splicing of the same gene which generates junctin and aspartyl-b-hydroxylase (Dinchuk et al, 2000;Treves et al, 2000;Hong et al, 2001). Junctin is an integral SR membrane protein that forms a quaternary complex with the RyRs, calsequestrin, and triadin and has been shown to participate in Ca 2þ release from heart and skeletal muscle (Jones et al, 1995;Zhang et al, 1997).…”

Absence of Post-translational Aspartyl β-Hydroxylation of Epidermal Growth Factor Domains in Mice Leads to Developmental Defects and an Increased Incidence of Intestinal Neoplasia

“…These studies yielded the surprising observation that two additional proteins are produced from this locus. One of these proteins, humbug (junctate), shares an identical NH 2 -terminal half of the protein with BAH, but lacks the entire 52-kDa COOH-terminal catalytic domain (6,7). Humbug encodes a type II membrane protein with a short putative amino-terminal cytoplasmic domain, a transmembrane domain, and a highly charged lumenal domain.…”

“…bug, was derived directly from the observation that these two proteins are encoded by the same locus as junctin and share significant coding regions (6,7). Direct proof of interaction of BAH and humbug with the ryanodine receptor has not been shown, but preliminary experiments suggest that overexpression of humbug in in vitro systems can alter Ca 2ϩ movement in cell culture systems (7).…”

“…Direct proof of interaction of BAH and humbug with the ryanodine receptor has not been shown, but preliminary experiments suggest that overexpression of humbug in in vitro systems can alter Ca 2ϩ movement in cell culture systems (7).…”