Calreticulin signaling in health and disease

Calreticulin is an endoplasmic reticulum Ca(2+) binding chaperone that has multiple functions inside and outside of the endoplasmic reticulum. It is involved in the quality control of newly synthesized proteins and glycoproteins, interacting with various other endoplasmic reticulum chaperones, specifically calnexin and ER protein of 57-kDa in the calreticulin/calnexin cycle. Calreticulin also plays a crucial role in regulating intracellular Ca(2+) homeostasis, associating calreticulin with a wide variety of signaling processes, such as cardiogenesis, adipocyte differentiation and cellular stress responses. The role of calreticulin outside of the endoplasmic reticulum is also extensive, including functions in wound healing and immunity. Therefore, calreticulin has important implications in health and disease. Signaling facts.     

Cardiac-specific expression of calcineurin reverses embryonic lethality in calreticulin-deficient mouse

Calreticulin is an endoplasmic reticulum resident Ca(2+)-binding chaperone. The importance of the protein is illustrated by embryonic lethality because of impaired cardiac development in calreticulin-deficient mice. The molecular details underlying this phenotype are not understood. In this study, we show that overexpression of activated calcineurin reverses the defect in cardiac development observed in calreticulin-deficient mice and rescues them from embryonic lethality. The surviving mice show no defect in cardiac development but exhibited growth retardation, hypoglycemia, increased levels of serum triacylglycerols, and cholesterol. Reversal of embryonic lethality because of calreticulin deficiency by activated calcineurin underscores the impact of the calreticulin-calcineurin functions on the Ca(2+)-dependent signaling cascade during early cardiac development. These findings show that calreticulin and calcineurin play fundamental roles in Ca(2+)-dependent pathways essential for normal cardiac development and explain the molecular basis for the rescue of calreticulin-deficient phenotype.     

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 sensitizes SERCA2a to oxidative stress

Calreticulin (CRT), a Ca(2+)-binding molecular chaperone in the endoplasmic reticulum, plays a vital role in cardiac physiology and pathology. Oxidative stress is a main cause of myocardiac disorder in the ischemic heart, but the function of CRT under oxidative stress is not fully understood. In this study, the effect of overexpression of CRT on sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) 2a under oxidative stress was examined using myocardiac H9c2 cells transfected with the CRT gene. The in vitro activity of SERCA2a and uptake of (45)Ca(2+) into isolated microsomes were suppressed by H(2)O(2) in CRT-overexpressing cells compared with controls. Moreover, SERCA2a protein was degraded via a proteasome-dependent pathway following the formation of a complex with CRT under the stress with H(2)O(2). Thus, we conclude that overexpression of CRT enhances the inactivation and degradation of SERCA2a in the cells under oxidative stress, suggesting some pathophysiological functions of CRT in Ca(2+) homeostasis of myocardiac disease.     

Calreticulin affects beta-catenin-associated pathways

Calreticulin, a Ca(2+) storage protein and chaperone in the endoplasmic reticulum, also modulates cell adhesiveness. Overexpression of calreticulin correlates with (i) increased cell adhesiveness, (ii) increased expression of N-cadherin and vinculin, and (iii) decreased protein phosphorylation on tyrosine. Among proteins that are dephosphorylated in cells that overexpress calreticulin is beta-catenin, a structural component of cadherin-dependent adhesion complexes, a member of the armadillo family of proteins and a part of the Wnt signaling pathway. We postulate that the changes in cell adhesiveness may be due to calreticulin-mediated effects on a signaling pathway from the endoplasmic reticulum, which impinges on the Wnt signaling pathway via the cadherin/catenin protein system and involves changes in the activity of protein-tyrosine kinases and/or phosphatases.     

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.     

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.     

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.     

Calcium storage in nonmuscle tissues: is the retina special?

It is now well established that calreticulin is a high capacity Ca(2+)-binding protein which is a major Ca2+ storage protein of the lumen of endoplasmic reticulum membranes in a wide variety of tissues with the exception of skeletal and cardiac muscles. However, in nervous tissue, confusion exists regarding the nature of the intracellular Ca2+ stores, as the organelle responsible for Ca2+ storage has been identified as the endoplasmic reticulum by some investigators and as the specialized organelle, calciosome by others. Calreticulin, calsequestrin, and calsequestrin-like proteins have all been, on different occasions, reported to be present in calciosomes. Cerebral and cerebellar tissues, moreover, have been shown to contain somewhat different systems of Ca(2+)-buffering proteins. In the present paper we discuss evidence that the Ca2+ storage systems of the retina may prove to be more complex than those of other neuronal tissues. Biochemical and immunocytochemical evidence indicates the presence of either an isoform of calreticulin or another protein that is antigenically similar to calreticulin, but of slightly higher molecular weight, in the endoplasmic reticulum of both neurons and Müller glia from rabbit neural retina. However, as retinal neurons express Purkinje cell markers, one may expect to observe the presence of calsequestrin in these cells as well. Secondly, antibodies against the onchocercal RAL-1 antigen recognize a protein sharing 62-65% amino acid sequence identity with calreticulin. The anti-RAL-1 antibodies show specificity for the retina. Whether or not the RAL-1 antigen is an active part of the Ca2+ storage systems of the retina remains to be verified.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Ca2+ homeostasis and apoptotic resistance of neuroendocrine-differentiated prostate cancer cells

Neuroendocrine (NE) differentiation is a hallmark of advanced, androgen-independent prostate cancer, for which there is no successful therapy. NE tumor cells are nonproliferating and escape apoptotic cell death; therefore, an understanding of the apoptotic status of the NE phenotype is imperative for the development of new therapies for prostate cancer. Here, we report for the first time on alterations in intracellular Ca(2+) homeostasis, which is a key factor in apoptosis, caused by NE differentiation of androgen-dependent prostate cancer epithelial cells. NE-differentiating regimens, either cAMP elevation or androgen deprivation, resulted in a reduced endoplasmic reticulum Ca(2+)-store content due to both SERCA 2b Ca(2+) ATPase and luminal Ca(2+) binding/storage chaperone calreticulin underexpression, and to a downregulated store-operated Ca(2+) current. NE-differentiated cells showed enhanced resistance to thapsigargin- and TNF-alpha-induced apoptosis, unrelated to antiapoptotic Bcl-2 protein overexpression. Our results suggest that targeting the key players determining Ca(2+) homeostasis in an attempt to enhance the proapoptotic potential of malignant cells may prove to be a useful strategy in the treatment of advanced prostate cancer.     

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.     

Enhanced calreticulin expression promotes calcium-dependent apoptosis in postnatal cardiomyocytes

Calreticulin (CRT) is one of the major Ca2+ binding chaperone proteins of the endoplasmic reticulum (ER) and an unusual luminal ER protein. Postnatally elevated expression of CRT leads to impaired development of the cardiac conductive system and may be responsible for the pathology of complete heart block. In this study, the molecular mechanisms that affect Ca2+-dependent signal cascades were investigated using CRT-overexpressing cardiomyocytes. In particular, we asked whether calreticulin plays a critical role in the activation of Ca2+-dependent apoptosis. In the cells overexpressing CRT, the intracellular calcium concentration was significantly increased and the activity of PKC and level of SECAR2a mRNA were reduced. Phosphorylation of Akt and ERKs decreased compared to control. In addition the activity of the anti-apoptotic factor, Bcl-2, was decreased and the activities of pro-apoptotic factor, Bax, p53 and caspase 8 were increased, leading to a dramatic augmentation of caspase 3 activity. Our results suggest that enhanced CRT expression in mature cardiomyocytes disrupts intracellular calcium regulation, leading to calcium-dependent apoptosis.