Calreticulin, a multifunctional Ca2+ binding chaperone of the endoplasmic reticulum.

Calreticulin is a ubiquitous endoplasmic reticulum Ca2+ binding chaperone. The protein has been implicated in a variety of diverse functions. Calreticulin is a lectin-like chaperone and, together with calnexin, it plays an important role in quality control during protein synthesis, folding, and posttranslational modification. Calreticulin binds Ca2+ and affects cellular Ca2+ homeostasis. The protein increases the Ca2+ storage capacity of the endoplasmic reticulum and modulates the function of endoplasmic reticulum Ca2+-ATPase. Calreticulin also plays a role in the control of cell adhesion and steroid-sensitive gene expression. Recently, the protein has been identified and characterized in higher plants but its precise role in plant cells awaits further investigation.     

Effects of diabetes mellitus on salivary secretion and its composition in the human

Calreticulin is a Ca2+ binding/storage chaperone resident protein of the endoplasmic reticulum. This protein plays a key role in the calreticulin/calnexin cycle and the quality control pathways in the endoplasmic reticulum. Calreticulin deficiency is lethal due to impaired cardiac development. However, over-expression of the protein in developing and postnatal heart leads to bradycardia, complete heart block and sudden death. Ultrastructural evidence indicates that the deficiency associated with the absence of calreticulin in the heart may be due to a defect in the development of the contractile apparatus and/or a defect in development of the conductive system as well as a metabolic abnormality. Collectively, we postulate that calreticulin and endoplasmic reticulum plays an important role in cardiac development and postnatal pathologies.     

Calreticulin in cardiac development and pathology

Calreticulin is a Ca(2+) binding/storage chaperone resident in the lumen of endoplasmic reticulum (ER). The protein is an important component of the calreticulin/calnexin cycle and the quality control pathways in the ER. In mice, calreticulin deficiency is lethal due to impaired cardiac development. This is not surprising because the protein is expressed at high level at early stages of cardiac development. Overexpression of the protein in developing and postnatal heart leads to bradycardia, complete heart block and sudden death. Recent studies on calreticulin-deficient and transgenic mice revealed that the protein is a key upstream regulator of calcineurin-dependent pathways during cardiac development. Calreticulin and ER may play important role in cardiac development and postnatal pathologies.     

Regulation of expression and intracellular distribution of calreticulin, a major calcium binding protein of nonmuscle cells

In the present study we have demonstrated the presence of calreticulin, a major Ca(2+)-sequestering protein of nonmuscle cells, in a variety of cell types in tissue culture. The protein localizes to the endoplasmic reticulum in most cell types and also to the nuclear envelope or nucleoli-like structures in some cell types. Calreticulin is enriched in the rough endoplasmic reticulum, suggesting a possible involvement in protein synthesis. Calreticulin terminates with the KDEL-COOH sequence, which is likely responsible for its endoplasmic reticulum localization. Unlike some other KDEL proteins, calreticulin expression is neither heat-shock nor Ca(2+)-shock dependent. Using a variety of metabolic inhibitors, we have shown that the pool of calreticulin in L6 cells has a relatively slow turnover and a stable intracellular distribution. In proliferating muscle cells in culture (both L6 and human skeletal muscle) calreticulin is present in the endoplasmic reticulum, and additional intranuclear staining is observed. When fusion of the L6 cells is inhibited with either a high serum concentration or TGF-beta or TPA, the nucleolar staining by anticalreticulin antibodies is diminished, although the presence of calreticulin in the endoplasmic reticulum remains unchanged. In contrast, in differentiated (i.e., fused) muscle cells neither intranuclear nor intracellular staining for calreticulin is present. We conclude, therefore, that calreticulin is abundant in the endoplasmic reticulum in proliferating myoblasts, while it is present in only small amounts in sarcoplasmic reticulum membranes in terminally differentiated myotubes. We propose a model for the domain structure of calreticulin that may explain the differential subcellular distribution of this protein. Because of its widespread distribution in nonmuscle tissues, we postulate that calreticulin is a multifunctional protein that plays an important role in Ca(2+) sequestering and thus that it is the nonmuscle analog of calsequestrin.     

Overexpression of calreticulin modulates protein kinase B/Akt signaling to promote apoptosis during cardiac differentiation of cardiomyoblast H9c2 cells

Calreticulin is a Ca(2+)-binding molecular chaperone of the lumen of the endoplasmic reticulum. Calreticulin has been shown to be essential for cardiac and neural development in mice, but the mechanism by which it functions in cell differentiation is not fully understood. To examine the role of calreticulin in cardiac differentiation, the calreticulin gene was introduced into rat cardiomyoblast H9c2 cells, and the effect of calreticulin overexpression on cardiac differentiation was examined. Upon culture in a differentiation medium containing fetal calf serum (1%) and retinoic acid (10 nm), cells transfected with the calreticulin gene were highly susceptible to apoptosis compared with controls. In the gene-transfected cells, protein kinase B/Akt signaling was significantly suppressed during differentiation. Furthermore, protein phosphatase 2A, a Ser/Thr protein phosphatase, was significantly up-regulated, implying suppression of Akt signaling due to dephosphorylation of Akt by the up-regulated protein phosphatase 2A via regulation of Ca(2+) homeostasis. Thus, overexpression of calreticulin promotes differentiation-dependent apoptosis in H9c2 cells by suppressing the Akt signaling pathway. These findings indicate a novel mechanism by which cytoplasmic Akt signaling is modulated to cause apoptosis by a resident protein of the endoplasmic reticulum, calreticulin.     

Identification of calreticulin as a ligand of GABARAP by phage display screening of a peptide library

4-Aminobutyrate type A (GABA(A)) receptor-associated protein (GABARAP) is a ubiquitin-like modifier implicated in the intracellular trafficking of GABA(A) receptors, and belongs to a family of proteins involved in intracellular vesicular transport processes, such as autophagy and intra-Golgi transport. In this article, it is demonstrated that calreticulin is a high affinity ligand of GABARAP. Calreticulin, although best known for its functions as a Ca(2+) -dependent chaperone and a Ca(2+) -buffering protein in the endoplasmic reticulum, is also localized to the cytosol and exerts a variety of extra-endoplasmic reticulum functions. By phage display screening of a randomized peptide library, peptides that specifically bind GABARAP were identified. Their amino acid sequences allowed us to identify calreticulin as a potential GABARAP binding protein. GABARAP binding to calreticulin was confirmed by pull-down experiments with brain lysate and colocalization studies in N2a cells. Calreticulin and GABARAP interact with a dissociation constant K(d) = 64 nm and a mean lifetime of the complex of 20 min. Thus, the interaction between GABARAP and calreticulin is the strongest so far reported for each protein.     

Calreticulin and calnexin in the endoplasmic reticulum are important for phagocytosis.

Calreticulin and calnexin are Ca2+-binding proteins with chaperone activity in the endoplasmic reticulum. These proteins have been eliminated by gene replacement in Dictyostelium, the only microorganism known to harbor both proteins; family members in Dictyostelium are located at the base of phylogenetic trees. A dramatic decline in the rate of phagocytosis was observed in double mutants lacking calreticulin and calnexin, whereas only mild changes occurred in single mutants. Dictyostelium cells are professional phagocytes, capable of internalizing particles by a sequence of activities: adhesion of the particle to the cell surface, actin-dependent outgrowth of a phagocytic cup, and separation of the phagosome from the plasma membrane. In the double-null mutants, particles still adhered to the cell surface, but the outgrowth of phagocytic cups was compromised. Green fluorescent protein-tagged calreticulin and calnexin, expressed in wild-type cells, revealed a direct link of the endoplasmic reticulum to the phagocytic cup enclosing a particle, such that the Ca2+ storage capacity of calreticulin and calnexin might directly modulate activities of the actin system during particle uptake.     

The ins and outs of calreticulin: from the ER lumen to the extracellular space

Calreticulin was first isolated 26 years ago. Since its discovery as a minor Ca(2+)-binding protein of the sarcoplasmic reticulum, the appreciation of its importance has grown, and it is now recognized to be a multifunctional protein, most abundant in the endoplasmic reticulum (ER). The protein has well-recognized physiological roles in the ER as a molecular chaperone and Ca(2+)-signalling molecule. However, it has also been found in other membrane-bound organelles, at the cell surface and in the extracellular environment, where it has recently been shown to exert a number of physiological and pathological effects. Here, we will focus on these less-well-characterized functions of calreticulin.     

Calreticulin in the heart

Calreticulin is a Ca²⁺ binding/storage chaperone resident protein of the endoplasmic reticulum. This protein plays a key role in the calreticulin/calnexin cycle and the quality control pathways in the endoplasmic reticulum. Calreticulin deficiency is lethal due to impaired cardiac development. However, over-expression of the protein in developing and postnatal heart leads to bradycardia, complete heart block and sudden death. Ultrastructural evidence indicates that the deficiency associated with the absence of calreticulin in the heart may be due to a defect in the development of the contractile apparatus and/or a defect in development of the conductive system as well as a metabolic abnormality. Collectively, we postulate that calreticulin and endoplasmic reticulum plays an important role in cardiac development and postnatal pathologies. (Mol Cell Biochem 263: 137–142, 2004)     

Expression and purification of mammalian calreticulin in Pichia pastoris

Calreticulin is a 46-kDa Ca(2+)-binding chaperone of the endoplasmic reticulum membranes. The protein binds Ca(2+) with high capacity, affects intracellular Ca(2+) homeostasis, and functions as a lectin-like chaperone. In this study, we describe expression and purification procedures for the isolation of recombinant rabbit calreticulin. The calreticulin was expressed in Pichia pastoris and purified to homogeneity by DEAE-Sepharose and Resource Q FPLC chromatography. The protein was not retained in the endoplasmic reticulum of Pichia pastoris but instead it was secreted into the external media. The purification procedures reported here for recombinant calreticulin yield homogeneous preparations of the protein by SDS-PAGE and mass spectroscopy analysis. Purified calreticulin was identified by its NH(2)-terminal amino acid sequences, by its Ca(2+) binding, and by its reactivity with anti-calreticulin antibodies. The protein contained one disulfide bond between (88)Cys and (120)Cys. CD spectral analysis and Ca(2+)-binding properties of the recombinant protein indicated that it was correctly folded.     

Calreticulin, a Ca2+-binding chaperone of the endoplasmic reticulum

Calreticulin is a 46-kDa Ca2+-binding chaperone found across a diverse range of species. The protein is involved in the regulation of intracellular Ca2+ homeostasis and endoplasmic reticulum (ER) Ca2+ storage capacity. Calreticulin is also an important molecular chaperone involved in "quality control" within secretory pathways. The protein contains structurally and functionally unique domains with specialized functions. Studies on calreticulin knockout mice indicate that the protein is essential in early cardiac development. The protein also plays an important role in autoimmunity and cancer.     

The evolutionary history of calreticulin and calnexin genes in green plants

Calreticulin and calnexin are Ca²⁺-binding chaperones localized in the endoplasmic reticulum of eukaryotes acting in glycoprotein folding quality control and Ca²⁺ homeostasis. The evolutionary histories of calreticulin and calnexin gene families were inferred by comprehensive phylogenetic analyses using 18 completed genomes and ESTs covering the major green plants groups, from green algae to angiosperms. Calreticulin and calnexin possibly share a common origin, and both proteins are present along all green plants lineages. The calreticulin founder gene within green plants duplicated in early tracheophytes leading to two possible groups of orthologs with specialized functions, followed by lineage-specific gene duplications in spermatophytes. Calnexin founder gene in land plants was inherited from basal green algae during evolution in a very conservative copy number. A comprehensive classification in possible groups of orthologs and a catalog of calreticulin and calnexin genes from green plants are provided.     

Calreticulin, Ca2+-binding chaperon of the endoplasmic reticulum

The endoplasmic reticulum (ER) plays a vital role in many cellular processes, including Ca2+ storage and release. Calreticulin is a Ca2+-binding chaperon residing in ER. The protein is a key component of the quality control pathways in ER. In the ER lumen, calreticulin performs two major functions, works as a chaperon and regulates Ca2+ homeostasis. In cardiac muscle, calreticulin plays an important role in cardiac development and pathology.     

The use of calnexin and calreticulin by cellular and viral glycoproteins

Calnexin and calreticulin are homologous lectin chaperones that assist maturation of cellular and viral glycoproteins in the mammalian endoplasmic reticulum. Calnexin and calreticulin share the same specificity for monoglucosylated protein-bound N-glycans but associate with a distinct set of newly synthesized polypeptides. We report here that most calnexin substrates do not associate with calreticulin even upon selective calnexin inactivation, while BiP associates more abundantly with nascent polypeptides under these conditions. Calreticulin associated more abundantly with orphan calnexin substrates only in infected cells and preferentially with polypeptides of viral origin, showing stronger dependence of model viral glycoproteins on endoplasmic reticulum lectins. This may explain why inactivation of the calnexin cycle affects viral replication and infectivity but not viability of mammalian cells.     

Functional relationship between calreticulin, calnexin, and the endoplasmic reticulum luminal domain of calnexin

Calnexin is a membrane protein of the endoplasmic reticulum (ER) that functions as a molecular chaperone and as a component of the ER quality control machinery. Calreticulin, a soluble analog of calnexin, is thought to possess similar functions, but these have not been directly demonstrated in vivo. Both proteins contain a lectin site that directs their association with newly synthesized glycoproteins. Although many glycoproteins bind to both calnexin and calreticulin, there are differences in the spectrum of glycoproteins that each binds. Using a Drosophila expression system and the mouse class I histocompatibility molecule as a model glycoprotein, we found that calreticulin does possess apparent chaperone and quality control functions, enhancing class I folding and subunit assembly, stabilizing subunits, and impeding export of assembly intermediates from the ER. Indeed, the functions of calnexin and calreticulin were largely interchangeable. We also determined that a soluble form of calnexin (residues 1-387) can functionally replace its membrane-bound counterpart. However, when calnexin was expressed as a soluble protein in L cells, the pattern of associated glycoproteins changed to resemble that of calreticulin. Conversely, membrane-anchored calreticulin bound to a similar set of glycoproteins as calnexin. Therefore, the different topological environments of calnexin and calreticulin are important in determining their distinct substrate specificities.     

Calreticulin: from Ca2+ binding to control of gene expression

Calreticulin is a highly conserved Ca(2+)-binding/storage protein of the endoplasmic reticulum (ER). Recently, it has been shown to play a role in the control of gene expression by interacting with the DNA-binding domain of various steroid receptors. How does this ER protein gain access to the nuclear steroid receptors? We propose that calreticulin undergoes unique intracellular trafficking that allows it to colocalize with and bind to steroid receptors.     

Calreticulin, and not calsequestrin, is the major calcium binding protein of smooth muscle sarcoplasmic reticulum and liver endoplasmic reticulum

The distribution of calsequestrin and calreticulin in smooth muscle and non-muscle tissues was investigated. Immunoblots of endoplasmic reticulum proteins probed with anti-calreticulin and anti-calsequestrin antibodies revealed that only calreticulin is present in the rat liver endoplasmic reticulum. Membrane fractions isolated from uterine smooth muscle, which are enriched in sarcoplasmic reticulum, contain a protein band which is immunoreactive with anti-calreticulin but not with anti-calsequestrin antibodies. The presence of calreticulin in these membrane fractions was further confirmed by 45Ca2+ overlay and "Stains-All" techniques. Calreticulin was also localized to smooth muscle sarcoplasmic reticulum by the indirect immunofluorescence staining of smooth muscle cells with anti-calreticulin antibodies. Furthermore, both liver and uterine smooth muscle were found to contain high levels of mRNA encoding calreticulin, whereas no mRNA encoding calsequestrin was detected. We have employed an ammonium sulfate precipitation followed by Mono Q fast protein liquid chromatography, as a method by which calsequestrin and calreticulin can be isolated from whole tissue homogenates, and by which they can be clearly resolved from one another, even where present in the same tissue. Calreticulin was isolated from rabbit and bovine liver, rabbit brain, rabbit and porcine uterus, and bovine pancreas and was identified by its amino-terminal amino acid sequence. Calsequestrin cannot be detected in preparations from whole liver tissue, and only very small amounts of calsequestrin are detectable in ammonium sulfate extracts of uterine smooth muscle. We conclude that calreticulin, and not calsequestrin, is a major Ca2+ binding protein in liver endoplasmic reticulum and in uterine smooth muscle sarcoplasmic reticulum. Calsequestrin and calreticulin may perform parallel functions in the lumen of the sarcoplasmic and endoplasmic reticulum.