- Email: [email protected]
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in a tightly membrane-associated form in PC12 cells. J. Biol. Chem. 267, 4110–4118 Burger, K. (2000) Greasing membrane fusion and fission machineries. Traffic 1, 605–613 Ikonen, E. and Simons, K. (1998) Protein and lipid sorting from the trans-Golgi network to the plasma membrane in polarized cells. Semin. Cell Dev. Biol. 9, 503–509 Martin-Belmonte, F. et al. (2000) Thyroglobulin is selected as luminal protein cargo for apical transport via detergent-resistant membranes in epithelial cells. J. Biol. Chem. 275, 41074–41081 Wang, Y. et al. (2000) Cholesterol is required for the formation of immature secretory granules from the trans-Golgi network. Traffic 1, 952–962 Blasquez, M. et al. (2000) Involvement of the membrane lipid bilayer in sorting prohormone convertase 2 into the regulated secretory pathway. Biochem. J. 349, 843–852 Palmer, D.J. and Christie, D.L. (1992) Identification of molecular aggregates containing glycoproteins III, J, K (carboxypeptidase H) and H (Kex2-related proteases) in the soluble and membrane fractions of adrenal medullary chromaffin granules. J. Biol. Chem. 267, 19806–19812 Dhanvantari, S. and Loh, Y.P. (2000) Lipid raft association of carboxypeptidase E is necessary for its function as a regulated secretory pathway sorting receptor. J. Biol. Chem. 275, 29887–29893 Arvan, P. et al. (1991) Protein discharge from immature secretory granules displays both regulated and constitutive characteristics. J. Biol. Chem. 266, 14171–14174 Dittié, A.S. et al. (1996) The AP-1 adaptor complex binds to immature secretory granules from PC12
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cells, and is regulated by ADP-ribosylation factor. J. Cell Biol. 132, 523–536 Kuliawat, R. et al. (1997) Differential sorting of lysosomal enzymes out of the regulated secretory pathway in pancreatic β-cells. J. Cell Biol. 137, 595–608 Klumperman, J. et al. (1998) Mannose 6phosphate receptors are sorted from immature secretory granules via adaptor protein AP-1, clathrin, and syntaxin 6-positive vesicles. J. Cell Biol. 141, 359–371 Dittié, A.S. et al. (1997) Interaction of furin in immature secretory granules from neuroendocrine cells with the AP-1 adaptor complex is modulated by casein kinase II phosphorylation. EMBO J. 16, 4859–4870 Dittié, A.S. et al. (1999) Differential distribution of mannose-6-phosphate receptors and furin in immature secretory granules. J. Cell Sci. 112, 3955–3966 Varlamov, O. et al. (1999) Localization of metallocarboxypeptidase D in AtT-20 cells. Potential role in prohormone processing. J. Biol. Chem. 274, 14759–14767 Eng, F.J. et al. (1999) Sequences within the cytoplasmic domain of Gp180/carboxypeptidase D mediate localization to the trans-Golgi network. Mol. Biol. Cell 10, 35–46 Fernandez, C.J. et al. (1997) Distinct molecular events during secretory granule biogenesis revealed by sensitivities to brefeldin A. Mol. Biol. Cell 8, 2171–2185 De Lisle, R.C. and Bansal, R. (1996) Brefeldin A inhibits the constitutive-like secretion of a sulfated protein in pancreatic acinar cells. Eur. J. Cell Biol. 71, 62–71
45 Turner, M.D. and Arvan, P. (2000) Protein traffic from the secretory pathway to the endosomal system in pancreatic β-cells. J. Biol. Chem. 275, 14025–14030 46 Tooze, S.A. (1991) Biogenesis of secretory granules. Implications arising from the immature secretory granule in the regulated pathway of secretion. FEBS Lett. 285, 220–224 47 Urbé, S. et al. (1998) Homotypic fusion of immature secretory granules during maturation in a cell-free assay. J. Cell Biol. 143, 1831–1844 48 Jahn, R. and Sudhof, T.C. (1999) Membrane fusion and exocytosis. Annu. Rev. Biochem. 68, 863–911 49 Gerona, R.R.L. et al. (2000) The C terminus of SNAP25 is essential for Ca2+-dependent binding of synaptotagmin to SNARE complexes. J. Biol. Chem. 275, 6328–6336 50 Davis, A.F. et al. (1999) Kinetics of synaptotagmin responses to Ca2+ and assembly with the core SNARE complex onto membranes. Neuron 24, 363–376 [published erratum appears in Neuron 24, 1049 (1999)] 51 Eaton, B.A. et al. (2000) Biogenesis of regulated exocytotic carriers in neuroendocrine cells. J. Neurosci. 20, 7334–7344 52 Austin, C. et al. (2000) Direct and GTP-dependent interaction of ADP-ribosylation factor 1 with clathrin adaptor protein AP-1 on immature secretory granules. J. Biol. Chem. 275, 21862–21869 53 Wan, L. et al. (1998) PACS-1 defines a novel gene family of cytosolic sorting proteins required for trans-Golgi network localization. Cell 94, 205–216
The ins and outs of calreticulin: from the ER lumen to the extracellular space Steven Johnson, Marek Michalak, Michal Opas and Paul Eggleton Calreticulin was first isolated 26 years ago. Since its discovery as a minor Ca2++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 Ca2++-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.
In 1974, a high-affinity Ca2+-binding protein was isolated from the sarcoplasmic reticulum (SR) of rabbit muscle and given the name HACBP (for: highaffinity calcium-binding protein)1. As it proved difficult to purify and was only a minor component of the SR, interest dwindled until 1989 when the cDNA encoding this Ca2+-binding protein was isolated
simultaneously by the groups of Koch and Michalak2,3. Examination of the amino acid sequence of the HACBP revealed the presence of the endoplasmic reticulum (ER) retrieval sequence KDEL (Ref. 4) at the C-terminus of the protein. Further analysis revealed that HACBP was in fact a major Ca2+-binding protein in the ER lumen5 and, in order to acknowledge both its Ca2+-binding properties and its localization to the ER lumen, HACBP was renamed calreticulin (CRT). In the 11 years since the rabbit and mouse cDNAs were isolated, numerous CRT cDNAs have been isolated from diverse genera, including mammals6, insects7, nematodes8, protozoa9 and plants10. There is a remarkable conservation of both the genomic organization and the amino acid sequence of CRT throughout evolution, indicating that it has an
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Table 1. Effects of altered cellular expression of calreticulina CRT cellular expression level
Refs
Calreticulin upregulation Increased Ca2+ storage capacity of ER
14
Modulation of cell adhesiveness
19,36
Modulation of store-operated Ca2+ influx
14
Increased sensitivity to apoptosis
–b
Modulation of steroid-sensitive gene expression
18,19
Appearance of surface CRT
45
Modulation of SERCA2 function
32
Calreticulin deficiency Embryonic lethal at E14.5
28,29
Impaired cardiac development
28
Changes in cell adhesiveness
29,34
Increased resistance to apoptosis
76
Accumulation of misfolded proteins
–b
Modulation of
Ca2+-dependent
gene transcription
28
Inhibition of agonist-dependent Ca2+ release from
28
ER stores aAbbreviations:
CRT, calreticulin; E, embryonic day; ER, endoplasmic reticulum; SERCA, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase.
bI.
Steven Johnson* Paul Eggleton MRC Immunochemistry Unit, University of Oxford, Oxford, UK OX1 3QU. *e-mail: [email protected] Marek Michalak CIHR Group in Molecular Biology of Membrane Proteins, Dept of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. Michal Opas Dept of Anatomy and Cell Biology, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
Ahsan, R. Knee and M. Michalak (unpublished).
important role in cellular function. The human protein consists of a 17-residue hydrophobic ER signal sequence, followed by the 400 amino acids of the mature protein. Analysis of the primary sequence of the protein has led to it being divided into three domains2. The N-terminal 180 residues are termed the N-domain and are predicted to form a globular β-sheet structure. Residues 181–290 are rich in proline (referred to as the P-domain) and contain two sets of repeated amino acid sequences unique to CRT and its molecular homologues calnexin and calmegin11. The P-domain binds to Ca2+ and acts as the high-affinity (Kd = 1 µM) Ca2+-binding site of CRT12. The C-terminal 110 amino acids of the protein (termed the C-domain) are rich in the acidic residues aspartic acid and glutamic acid and bind to Ca2+ with low affinity (Kd = 2 mM) but high capacity (20–30 moles Ca2+ mole–1 protein)12. Initial studies on CRT concentrated on the Ca2+-binding properties of the protein and its ability to modulate Ca2+ homeostasis13,14. However, interest in CRT intensified during the 1990s, a time when there was a massive increase in the number of functions with which CRT was associated and the cellular locations at which it was seen. Proposed functions for CRT range from chaperoning in the ER15 to anti-thrombotic effects at the cell surface16, and from the regulation of Ca2+ signalling17 to the modulation of gene expression18,19 and cellular adhesion20 (Box 1). Altered expression of CRT also has profound effects on many cellular functions (Table 1). Here is where the controversy associated with CRT lies; how can this one protein play a role in so many important cellular functions, and where within the cell does it carry out these functions? It is accepted that the http://tcb.trends.com
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majority of cellular CRT is located in the ER, where it plays important roles in molecular chaperoning and Ca2+ signalling (Fig. 1). Earlier reports indicated that CRT might also function in the cytoplasm and/or in the nucleus as a modulator of integrin function and steroidsensitive gene expression18,19. However, the protein has not been found in the cytoplasm, and there is no plausible explanation for how it could relocate from the ER lumen into the cytoplasm77. Reports on the nuclear localization of CRT are likely artifacts of immunostaining21, and therefore these aspects of the cellular localization of CRT will not be discussed here. However, there is considerable evidence to indicate that CRT is found outside the cell, although how the protein relocates from the ER to the outside of the cell remains unclear. In addition, immune responses against human and parasitic forms of CRT have been detected in autoimmune patient sera, making extracellular CRT of clinical importance. The purpose of this review, therefore, will be to delineate the functions of CRT by following the protein on a journey out of the cell, beginning with its recognized physiological functions in the ER, including the important lessons learned from recent gene-deletion studies, through to the known pathological effects of extracellular CRT. Excellent review articles have recently been published on the structure and chaperone function of CRT15,22. Hence, we intend to consider here the less well-characterized functions of CRT, including cell-surface and extracellular events. Molecular chaperoning – the story unfolds then refolds
Many cellular and extracellular proteins are folded in the ER by a specific set of chaperones, which is now accepted to include CRT and its membrane-bound homologue calnexin (CNX). CRT and CNX participate in a well-characterized cycle of binding and release to newly folded proteins and glycoproteins via a Glc1Man7–9GlcNAc2 carbohydrate ligand (reviewed in Ref. 15) or through direct interaction with misfolded polypeptides (Fig. 1). However, although it is now generally accepted that CRT is an important chaperone, two questions still need to be answered: first, exactly what directs unfolded glycoproteins to bind to CRT and, second, does CRT bind to carbohydrate only or does it also bind directly to polypeptides? The answer to the first question is dependent on the position of the carbohydrate in the protein, both in the primary sequence and the tertiary structure. Elegant studies on the interactions of chaperones with viral glycoproteins [influenza virus haemagglutinin (HA), vesicular stomatitis virus glycoprotein (VSV-G) and Semliki forest virus (SFV) spike proteins p62 and E1] demonstrated that proteins that fold in the ER make use of chaperones in distinct combinations and different orders23. HA and SFV p62 both bind to CRT or CNX, and this interaction is followed by the recruitment of ERp57, a thiol reductase that facilitates correct disulfide bond formation. VSV-G and SFV E1, by contrast, bind first to BiP (immunoglobulin binding
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Box 1. Selected cellular functions attributed to calreticulin Adhesion • Acrosome function and sperm motility • Activates integrins • Affects cell migration • Control of cellular adhesiveness • Inhibits angiogenesis • Initiates cell spreading • Regulates expression of vinculin • Upregulates expression of N-CAM • Wound healing Blood function • Anti-thrombotic activity • Autoantigen • Binds to complement C1q (C1q receptor?) • Component of lytic granules in CTLs and NKs • Component of tick saliva • Inhibits perforin-dependent killing • Interacts with perforin • Modulates platelet activation Development • Affects cardiac development • Affects neuronal development • Essential for mouse embryogenesis • Induces complete cardiac block • Oocyte fertilization • Regulates bone cell function ER functions • ‘Ca2+ sensor’ in the ER lumen • Binds to Mg2+-ATP • Ca2+ binding and storage • ER chaperone • Essential for glycoprotein maturation • Important for MHC class I assembly • Modulates inositol-(1,4,5)-trisphosphatedependent Ca2+ release
protein), a chaperone that recognizes hydrophobic polypeptide sequences, followed by the recruitment of protein disulfide isomerase (PDI), another ER-lumenal thiol reductase. The major difference between these viral glycoproteins is in the position of their glycosylation in the primary sequence; HA and SFV p62 are both glycosylated in their first 50 N-terminal residues, whereas VSV-G and SFV E1 are glycosylated more C-terminally. It appears, therefore, that ER chaperones compete for binding sites on a newly synthesized polypeptide. Interestingly, it has recently been demonstrated that the distinct differences in the spectrum of glycoproteins that CRT and CNX bind to can be explained by their topological environment – that is, by the fact that CRT is soluble, whereas CNX is membrane bound24. A question that has yet to be fully answered under in vivo conditions is whether the chaperone activity http://tcb.trends.com
• • •
Modulates SERCA2b function Regulation of store-operated Ca2+ influx Zn2+ binding and storage
Gene expression • Androgen-sensitive gene in prostate cancer • Control of Rubella virus replication • Control of steroid-sensitive gene expression • Marker of viral infection • Modulates vitamin D3 signal transduction • Participates in host response to tumor Others • Affects phosphotyrosine level • Important for cellular proliferation • Increases sensitivity to apoptosis • Induces NO formation in endothelial cells • Intracellular iron transport • Longterm memory molecule in Aplysia • Mediates mitogenic effects of fibrinogen • Stress protein Abbreviations: CTL, cytotoxic T-lymphocyte; ER endoplasmic reticulum; MHC, major histocompatibility complex; N-CAM, neural cell-adhesion molecule; NK, natural killer cell; NO, nitric oxide; SERCA, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase. For further details, see review articles in Refs a–d and references therein.
References a Eggleton, P. et al. (1997) Clinical relevance of calreticulin in systemic lupus erythematosus. Lupus 6, 564–571 b Crofts, A.J. and Denecke, J. (1998) Calreticulin and calnexin in plants. Trends Plant Sci. 3, 396–399 c Nakhasi, H.L. et al. (1998) Implications of calreticulin function in parasite biology. Parasitol. Today 14, 157–160 d Michalak, M. et al. (1999) Calreticulin: one protein, one gene, many functions. Biochem. J. 344, 281–292
of CRT is due purely to its carbohydrate binding (lectin) activity or whether there is an additional role for CRT as a classical chaperone – that is, that CRT also possesses a site capable of binding to polypeptide segments of unfolded glycoproteins. It has been suggested that CRT functions only through its lectin activity15 or through protein–protein interactions alone25. However, the most interesting model of the chaperone function of CRT incorporates both of these activities and hence is referred to as the ‘dualbinding model’26. The in vitro assays previously used to characterize the functions of HSP90, HSP70, HSP60 and members of the small heat-shock protein families of chaperones have demonstrated that, in addition to its lectin function, CRT can bind to polypeptides and distinguish between native and non-native conformations of glycoproteins. The ‘dualbinding model’ therefore proposes that CRT binds to
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Endoplasmic reticulum
Cytoplasm Ca2+-ATPase
G
Ca2+
Ca2+
Ca2+
Ca2+
G
InsP3 receptor
CRT
G
G
NH2
Calreticulin (CRT)
CNX
PDI
Glycoprotein
ERp57
BiP
Ribosome TRENDS in Cell Biology
Fig. 1. Model summarizing the functions of calreticulin (CRT) in the lumen of the endoplasmic reticulum (ER). CRT and calnexin (CNX) act as molecular chaperones to newly synthesized and unfolded proteins and glycoproteins (shown in blue). Competition occurs between the CRT/CNX pathway and the classical BiP pathway, depending on the position of glycosylation in the glycoprotein. CRT also acts as a major Ca2+ store of the ER and influences the activity of both the inositol (1,4,5)-trisphosphate (InsP3) receptor and the sarcoplasmic/endoplasmic reticulum Ca2+ATPase (SERCA2), thereby modulating Ca2+ homeostasis and signalling.
unfolded glycoprotein through interactions with both carbohydrate and exposed hydrophobic polypeptide sequences26. However, as discussed previously, proteins that do not have glycosylation sites within their first 50 amino acid sequence can enter the BiP/PDI pathway in vivo23. Interestingly, the misfolded-protein-binding capabilities of CRT appear to be affected by Ca2+, Zn2+ and ATP, suggesting that CRT undergoes different conformational changes upon binding to these small molecules. This finding is further supported by experiments involving the proteolytic digestion of CRT in the presence and absence of these molecules27. Although there have been a large number of publications describing the chaperoning function of CRT, the model of its chaperone function detailed above (Fig. 1) is based on studies of only a limited number of substrates. Further investigations will be required to fully appreciate this important function of CRT and CNX. Ca2++ homeostasis and signalling: lessons from deletion of the gene encoding CRT
A major recent insight into the functions of CRT has undoubtedly come from the development of CRT-deficient cell lines and mice28,29. The CRT gene deletion is in fact the first reported mouse knockout of an ER-lumenal protein. Given the plethora of functions with which CRT has been linked, it was anticipated that the protein would be essential for organism survival. Indeed, the most prominent http://tcb.trends.com
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feature of the knockout is that it is an embryonic lethal mutation. The crt–/– embryos were viable until 14.5 days into gestation28,29. Initial gross morphological and histological examination of crt–/– embryos revealed several prominent defects, in the heart and body wall28,29. The most obvious defect in embryos surviving to 18.5 days is the failure to absorb the umbilical hernia (omphalocele)28. The most likely cause of embryonic lethality, however, is cardiac failure as embryos at 12.5, 14.5 and 18 days all displayed signs of cardiac pathology characterized by thinner ventricular walls with deep, intertrabecular recesses. This indicates that CRT must play a role during cardiac development28. CRT is expressed at a low level in the mature heart28. However, using transgenic mice expressing a green-fluorescent protein (GFP) reporter gene under the control of the CRT promoter, it was demonstrated that CRT is upregulated in the heart during the middle stages of embryogenesis, from 9.5 to 14.5 days28, while, at later stages (18 days) and after birth, CRT is expressed at negligible levels. This gives strong support to the aforementioned notion of the importance of CRT in cardiac development28. It seems that cardiomyocytes must be sensitive to changes in the abundance of CRT and that it is physiologically important to downregulate the expression of CRT after birth because elevated expression of CRT in newborn hearts is associated with severe cardiac pathology and death (K. Nakamura and M. Michalak, unpublished). Recent experiments indicate that the cardiac transcription factor Nkx2.5 activates expression of the gene encoding CRT in the heart, and the transcription factor COUP-TF1 might be involved in its postnatal suppression30. It is clear from the CRT gene-knockout studies, therefore, that the protein plays an important role in the development of the heart, but it is still not clear which of its many proposed functions is responsible for mediating this role. The most likely mechanism is through the ability of CRT to modulate cellular Ca2+ signalling. CRT is important in the regulation of Ca2+ homeostasis, and it is capable of influencing sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2) function and Ca2+ release from the ER mediated by the inositol (1,4,5)-trisphosphate receptor28,31,32. These functions might be modulated by Ca2+ binding to the high-capacity C-domain of CRT or to the highaffinity P-domain, which has been implicated in many interactions of CRT, possibly through its lectin activity33. At present, most available data suggest that the pathology observed in CRTdeficient mice relates directly to the role of CRT in modulation of Ca2+ homeostasis28,29. Alternatively, in its role as a chaperone15, CRT might affect the proper folding, posttranslational modification, assembly and/or intracellular trafficking of many membrane proteins involved in control of Ca2+
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Focal adhesion disassembly
Extracellular CRT acts at the cell surface
Secretory vesicles Cytotoxic T cell granule
Golgi network
Regulation of apoptosis
(b)
(a) Decreased level of Tyr phosphorylation Endoplasmic reticulum
Increased vinculin protein
Increased CRT level influences gene expression of other proteins
Increased cell adhesion
Nucleus
Calreticulin in the secretory pathway
Calreticulin (CRT)
Granzyme B
Thrombospondin
Glycoprotein
Integrin
Perforin TRENDS in Cell Biology
Fig. 2. Calreticulin (CRT) in the secretory pathway and its effects on cell adhesion. CRT is found on the surface of some types of cells under certain conditions, and there are several possible ways in which the protein might relocate from the endoplasmic reticulum (ER) to the cell surface. Both ER-resident and cell-surface CRT might affect cell adhesiveness through multiple pathways. (a) CRT has been shown to shuttle between the ER and the Golgi and might eventually proceed through the secretory pathway to the cell surface, sometimes still associated with glycoproteins. (b) CRT colocalizes with lytic perforin to the lysosome-like cytotoxic T-lymphocyte (CTL) secretory granules, where it might prevent organelle autolysis. Release of granule content in the activated CTL might be one mechanism for extracellular targeting of CRT. Dashed arrows correspond to uncharacterized effects of CRT.
homeostasis. Finally, considering the similarity in cardiac phenotype between CRT knockout mice and several mutations ablating cell-adhesion proteins, the defective heart morphogenesis might be attributed in part to a fault in cell communication and supracellular organization, which might involve altered cell adhesiveness29. Cellular adhesion: the gene-deletion story continues
The development of an embryonic stem (ES) cell line lacking CRT suggested that, at the single cell level, a lack of CRT had less severe consequences as this cell line proved to be viable and had normal growth rates34. The CRT-deficient ES cells were, however, severely impaired in their ability to adhere to fibronectin and laminin through cell-surface integrins and also in integrin-mediated extracellular Ca2+ influx. CRT appears to affect cell–substratum adhesion that depends on formation of focal adhesions20. Opinion is still divided as to whether these effects are mediated by CRT directly contacting the integrins through their cytoplasmic tails or through signalling from within the ER. However, there is a lack of supporting evidence pinpointing the presence of CRT in the cytosol despite the ability of http://tcb.trends.com
CRT to bind to integrin tails in vitro35. There is now evidence that CRT can modulate the cytosolic levels of molecules associated with adhesion, such as vinculin36, from within the ER and hence alter formation of focal adhesions (Fig. 2). Furthermore, CRT also has a drastic effect on the total cellular levels of phosphotyrosine, again from within the ER20. A correlation between cell adhesiveness and the overall intracellular phosphotyrosine level has been well established37, and it is the ability of a cell to form focal adhesions that is affected38. This, therefore, might be a mechanism by which CRT affects cell adhesiveness through modulation of phosphotyrosine signalling pathways. Yet another possibility, which has emerged more recently, is that CRT might affect cellular adhesion from the cell surface, which we will describe later.
A major question that has puzzled biochemists and cell biologists is how CRT moves from the ER to the outside of the cell (Fig. 2). Although CRT contains the ER-retrieval sequence KDEL at its C-terminus, the protein has been identified on the surface of a wide variety of cell types, including endothelial cells39 and neuronal cells40, where it has been implicated in a variety of functions. The important issue of how CRT escapes ER retention and is translocated to the cell surface remains to be answered, although there is now good evidence localizing CRT to the secretory pathway from studies on plant cells41, B16 mouse melanoma cells42, rat hepatocytes43, Vero cells44 and also the neuronal cell line NG-108-1545. In the neuronal cell line, a number of ER-resident proteins containing the KDEL sequence have been identified at the cell surface by surface biotinylation, including CRT and PDI40. Strikingly, the transport of these proteins is inhibited upon treatment of the cells with brefeldin A, an inhibitor of ER–Golgi trafficking, therefore supporting the notion that they are transported through the secretory pathway. Several theories could explain the transport of ER proteins to the cell surface. The proteins might be expressed in different isoforms that do not contain the ER-retrieval sequence. In support of this idea, CRT on the surface of NG-108-15 cells is not recognized by antibodies against KDEL40; however, this could be because the KDEL receptor is transported to the surface in complex with CRT, as was observed in B16 cells42. Another possibility is that CRT might be proteolytically processed by ER-lumenal proteases to a form missing the KDEL ER-retrieval sequence, given that, under ER-lumenal Ca2+ concentrations, the C-domain of the protein containing the KDEL amino acid sequence is susceptible to proteolysis27. Saturation of the ER-retention machinery could also play a role46. This could be especially relevant in studies where CRT expression is upregulated, such as in
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Endothelial cell
(b)
Activation of human neutrophilis
CRT Nitric oxide produced Endoplasmic reticulum
Serine proteases
(c) Vasostatin
Release of CRT during cell stress • Apoptosis • Necrosis • T-cell secretion
(a)
C1q
Nucleus
Blocks complement activation
Inhibits endothelial cell proliferation and prevents angiogenesis, ultimately reducing tumor formation
Antibodies raised against host and parasitic forms of CRT
Release from parasites (e.g. hookworms)
Calreticulin (CRT)
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Fig. 3. Roles of extracellular calreticulin (CRT) in disease. Once CRT is released from the cell by either active secretory processes or cell death, it can have various effects on cell functions. (a) The protein becomes a target for both cell-mediated and innate immune responses, and parasites might exploit the anti-thrombotic and complement-inhibiting characteristics of CRT to suppress host defence actions. (b) Surface-bound CRT on endothelial cells can provoke inflammatory events, for example stimulation of nitric oxide production. (c) An N-terminal fragment of CRT called vasostatin plays an active role in preventing angiogenesis and tumor growth.
activated T-cells, where the protein is targeted to the cytotoxic T-lymphocyte (CTL) granules47. Interaction between a CTL and its target cells stimulates release of granule contents, including CRT, into the extracellular space, providing one mechanism for extracellular targeting of the protein. Finally, protein glycosylation might play a role as experiments carried out in Chinese hamster ovary cells have shown that CRT becomes glycosylated under conditions of stress, resulting in the redistribution of the protein within the cell48. Calreticulin at the cell surface
The role of CRT at the cell surface is unclear. CRT does not possess a transmembrane domain, but it clearly orchestrates a number of cellular events from the cell surface, including cellular adhesion and migration. CRT might modulate cell adhesion from within the cell through an interaction with integrin tails or through regulation of focal-adhesionassociated proteins as well as modulation of cytosolic phosphotyrosine levels. Another possibility is that CRT can modulate cell adhesion from outside the cell. CRT has been demonstrated to bind to the extracellular matrix proteins Bb fibrinogen49 and laminin50, and it has been reported that cell-surface CRT can complex with integrins42,51. Antibodies against CRT can prevent spreading of B16 cells50 and block neurite formation in differentiating NG-108-15 http://tcb.trends.com
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cells45. Recent work on endothelial cells has added yet another twist to the already complicated involvement of CRT in cell adhesion as it has been shown that thrombospondin-induced disassembly of focal adhesions is mediated by cell-surface CRT39. An intriguing role for CRT in cell adhesion has been implicated from the recent discovery that CRT, or fragments of the protein, inhibits angiogenesis and suppresses tumor growth52. Tumor growth and metastasis formation are dependent on an adequate blood supply, and therefore the generation of new blood vessels (angiogenesis) is essential for the progression of a tumor. CRT disrupts this process by specifically inhibiting the proliferation of endothelial cells53 (Fig. 3). Thus CRT has now become of intense interest in the quest for cancer therapies54. An alternative function of cell-surface CRT might be in regulating responses of the immune system. CRT is present in the CTL granules, where it has been proposed to prevent perforin from forming pores in the granule membrane, either by Ca2+-chelation55 or direct interactions with perforin56. However, recent work has suggested that CRT has a more active role in preventing autolysis of the lymphocyte by binding directly to the cell surface57. Experiments performed on erythrocytes showed that CRT bound to the membrane, where it prevented the insertion of perforin and hence prevented cell lysis. Furthermore, inhibition of lysis was neither dependent on a direct interaction between CRT and perforin nor on the ability of CRT to chelate Ca2+. The ability of CRT to bind directly to membranes has also been demonstrated in endothelial cells, where thrombosis was inhibited by the stimulation of nitric oxide production16. Finally, cell-surface CRT has been implicated in the upregulation of the immune system. An anti-microbial peptide, which activates neutrophils, has been shown to act through interactions with cell-surface CRT58. Extracellular calreticulin
As well as playing a vital role in cellular function, CRT has also been implicated in a number of pathological processes (Fig. 3). Autoantibodies against CRT have been found in approximately 40% of systemic lupus erythematosus (SLE) patients, and a number of patients with secondary Sjögrens syndrome59. Interestingly, SLE patients also raise autoantibodies against other ER chaperones such as grp9460. CRT is known to associate with antigens of the Ro/SS-A complex, a soluble ribonucleoprotein complex consisting of at least four protein components and four small cytoplasmic RNA components61. Autoantibodies are generated against all elements of the complex, although there is evidence that CRT is antigenic in its own right62. Other than in SLE, antibodies to CRT have been detected in patients with rheumatoid arthritis63, celiac disease64, complete congenital heart block65 and halothane hepatitis66. It appears, however, that CRT
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Acknowledgements The experimental work of the authors has been supported by the Arthritis Research Campaign (PE grant E0521), Biotechnology and Biological Sciences Research Council (S.J.), the Canadian Institutes of Health Research (M.M., M.O.) and the Heart and Stroke Foundations of Alberta (M.M.) and Ontario (M.O.). Marek Michalak is a CIHR Senior Scientist and an AHFMR Medical Scientist.
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is more than just an autoantigen. In SLE models, CRT has been demonstrated to contribute to the progression of an autoantibody response by ‘epitope spreading’67, and CRT has also been demonstrated to bind to the RNA of viruses such as Rubella virus that are capable of generating an autoimmune response68. A major contributing factor to autoimmune diseases such as SLE is the failure to clear immune complexes, a process largely mediated by the first component of the classical pathway of complement, C1q. The importance of this mechanism is highlighted by the fact that patients who lack C1q almost always develop active SLE69. Significantly, it has been demonstrated that CRT can bind to C1q70 and, furthermore, can compete with antibody for binding to C1q and inhibition of C1q-dependent hemolysis. Extracellular CRT might therefore contribute to the progression of autoimmune disease by preventing immune complex clearance. This activity of CRT might have been hijacked by various parasites as a mechanism for evading the immune system. Several parasites such as Onchocerca volvulus71, Necator americanus72 and the ixodid tick Amblyomma americanum73 have been shown to secrete CRT homologues, which might prevent the complement system from acting against the parasite. Extracellular CRT is, therefore, implicated in the progression of a number of diseases, through several potential mechanisms. Given that CRT has been detected on the surface of cells, an obvious question is: when does extracellular CRT become a problem? The answer to this is not known. Clearly, the release of CRT from necrotic cells is a contributing factor for the protein to become immunoreactive74, but there is also evidence demonstrating that the release of CRT and the other Ro/SS-A antigens from apoptotic blebs plays a role in autoantibody production75. Another factor, which might also be important, is the nature of the interaction of CRT with the cell surface. As yet, this interaction has not been defined, but there is evidence that the C-terminal domain
References 1 Ostwald, T.J. and MacLennan, D.H. (1974) Isolation of a high affinity calcium binding protein from sarcoplasmic reticulum. J. Biol. Chem. 249, 974–979 2 Smith, M.J. and Koch, G.L. (1989) Multiple zones in the sequence of calreticulin (CRP55, calregulin, HACBP), a major calcium binding ER/SR protein. EMBO J. 8, 3581–3586 3 Fliegel, L. et al. (1989) Molecular cloning of the high affinity calcium-binding protein (calreticulin) of skeletal muscle sarcoplasmic reticulum. J. Biol. Chem. 264, 21522–21528 4 Pelham, H.R.B. (1989) Control of exit from the endoplasmic reticulum. Annu. Rev. Cell Biol. 5, 1–23 5 Opas, M. et al. (1991) Regulation of expression and intracellular distribution of calreticulin, a major calcium binding protein of nonmuscle cells. J. Cell Physiol. 149, 160–171 6 McCauliffe, D.P. et al. (1990) Molecular cloning, expression, and chromosome 19 localization of a human Ro/SS-A autoantigen. J. Clin. Invest. 85, 1379–1391 http://tcb.trends.com
of CRT is involved16,57. It is interesting to note, therefore, that digestion of CRT with elastase and cathepsin G – the major proteases released during inflammation – results in the C-terminus of the protein being cleaved off, leaving a stable N-terminal fragment59. This could potentially provide a mechanism for the release of CRT into the circulation, where it has pathological effects. Concluding remarks
Calreticulin is now accepted to be a multifunctional, multi-compartmental protein. Here, we have attempted to summarize not only its wellcharacterized roles in the ER but also some of its less-well-understood roles outside this organelle. A major leap forward in the field of CRT research has come with the development of CRT-deficient mice, which have demonstrated the importance of CRT in organogenesis, particularly the development of the heart28,29. Furthermore, these studies have highlighted some of the limitations of investigating protein function purely at a cellular, rather than organism, level as such a role had not been considered previously because of the low level of expression of CRT in the mature heart. Hopefully, further characterization of the CRTdeficient mice will provide a more-detailed insight into the roles of CRT in developing organisms. In addition, such studies might reveal whether CRT has an important role in pathological conditions. For example, autoantibodies against CRT have been identified in patients suffering from complete congenital heart block65, and transgenic mice overexpressing CRT in the heart also show congenital heart block (K. Nakamura and M. Michalak, unpublished). Therefore, by using a combination of approaches, including cellular biology, gene deletion and clinical investigation, we can begin to unravel the mysteries that have surrounded CRT for so long.
7 Smith, M.J. (1992) Nucleotide sequence of a Drosophila melanogaster gene encoding a calreticulin homologue. DNA Seq. 3, 247–250 8 Smith, M.J. (1992) A C. elegans gene encodes a protein homologous to mammalian calreticulin. DNA Seq. 2, 235–240 9 Joshi, M. et al. (1996) Isolation and characterization of Leishmania donovani calreticulin gene and its conservation of the RNA binding activity. Mol. Biochem. Parasitol. 81, 53–64 10 Denecke, J. et al. (1995) The tobacco homolog of mammalian calreticulin is present in protein complexes in vivo. Plant Cell 7, 391–406 11 Watanabe, D. et al. (1994) Molecular cloning of a novel Ca2+-binding protein (calmegin) specifically expressed during male meiotic germ cell development. J. Biol. Chem. 269, 7744–7749 12 Baksh, S. and Michalak, M. (1991) Expression of calreticulin in Escherichia coli and identification of its Ca2+ binding domains. J. Biol. Chem. 266, 21458–21465 13 Van Delden, C. et al. (1992) Purification of an inositol 1,4,5-trisphosphate-binding calreticulincontaining intracellular compartment of HL-60
cells. Biochem. J. 281, 651–656 14 Mery, L. et al. (1996) Overexpression of calreticulin increases intracellular Ca2+ storage and decreases store-operated Ca2+ influx. J. Biol. Chem. 271, 9332–9339 15 Helenius, A. et al. (1997) Calnexin, calreticulin and the folding of glycoproteins. Trends Cell Biol. 7, 193–200 16 Kuwabara, K. et al. (1995) Calreticulin, an antithrombotic agent which binds to vitamin K-dependent coagulation factors, stimulates endothelial nitric oxide production, and limits thrombosis in canine coronary arteries. J. Biol. Chem. 270, 8179–8187 17 Llewelyn Roderick, H. et al. (1998) Role of calreticulin in regulating intracellular Ca2+ storage and capacitative Ca2+ entry in HeLa cells. Cell Calcium 24, 253–262 18 Burns, K. et al. (1994) Modulation of gene expression by calreticulin binding to the glucocorticoid receptor. Nature 367, 476–480 19 Dedhar, S. (1994) Novel functions for calreticulin: interaction with integrins and
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41 Borisjuk, N. et al. (1998) Calreticulin expression in plant cells: developmental regulation, tissue specificity and intracellular distribution. Planta 206, 504–514 42 Zhu, Q. et al. (1997) Calreticulin–integrin bidirectional signaling complex. Biochem. Biophys. Res. Commun. 232, 354–358 43 Zuber, C. et al. (2000) Golgi apparatus immunolocalization of endomannosidase suggests post-endoplasmic reticulum glucose trimming: implications for quality control. Mol. Biol. Cell 11, 4227–4240 44 Day, P.J. et al. An interaction between ricin and calreticulin that may have implications for toxin trafficking. J. Biol. Chem. (in press) 45 Xiao, G. et al. (1999) Calreticulin is transported to the surface of NG108-15 cells where it forms surface patches and is partially degraded in an acidic compartment. J. Neurosci. Res. 58, 652–662 46 Crofts, A.J. et al. (1999) Saturation of the endoplasmic reticulum retention machinery reveals anterograde bulk flow. Plant Cell 11, 2233–2248 47 Burns, K. et al. (1992) Calreticulin in T-lymphocytes. Identification of calreticulin in T-lymphocytes and demonstration that activation of T cells correlates with increased levels of calreticulin mRNA and protein. J. Biol. Chem. 267, 19039–19042 48 Jethmalani, S.M. et al. (1997) Intracellular distribution of heat-induced stress glycoproteins. J. Cell. Biochem. 66, 98–111 49 Gray, A.J. et al. (1995) The mitogenic effects of the B beta chain of fibrinogen are mediated through cell surface calreticulin. J. Biol. Chem. 270, 26602–26606 50 White, T.K. et al. (1995) Cell surface calreticulin is a putative mannoside lectin which triggers mouse melanoma cell spreading. J. Biol. Chem. 270, 15926–15929 51 Kwon, M.S. et al. (2000) Calreticulin couples calcium release and calcium influx in integrinmediated calcium signaling. Mol. Biol. Cell 11, 1433–1443 52 Pike, S.E. et al. (1998) Vasostatin, a calreticulin fragment, inhibits angiogenesis and suppresses tumor growth. J. Exp. Med. 188, 2349–2356 53 Pike, S.E. et al. (1999) Calreticulin and calreticulin fragments are endothelial cell inhibitors that suppress tumor growth. Blood 94, 2461–2468 54 Yao, L. et al. (2000) Effective targeting of tumor vasculature by the angiogenesis inhibitors vasostatin and interleukin-12. Blood 96, 1900–1905 55 Dupuis, M. et al. (1993) The calcium-binding protein calreticulin is a major constituent of lytic granules in cytolytic T lymphocytes. J. Exp. Med. 177, 1–7 56 Andrin, C. et al. (1998) Interaction between a Ca2+-binding protein calreticulin and perforin, a component of the cytotoxic T-cell granules. Biochemistry 37, 10386–10394 57 Fraser, S.A. et al. (2000) Perforin lytic activity is controlled by calreticulin. J. Immunol. 164, 4150–4155 58 Cho, J.H. et al. (1999) Activation of human neutrophils by a synthetic anti-microbial peptide, KLKLLLLLKLK-NH2, via cell surface calreticulin. Eur. J. Biochem. 266, 878–885 59 Eggleton, P. et al. (2000) Fine specificity of autoantibodies to calreticulin: epitope mapping and characterization. Clin. Exp. Immunol. 120, 384–391
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60 Boehm, J. et al. (1994) Systemic lupus erythematosus is associated with increased autoantibody titers against calreticulin and grp94, but calreticulin is not the Ro/SS-A antigen. Eur. J. Clin. Invest. 24, 248–257 61 Cheng, S.T. et al. (1996) Calreticulin binds hYRNA and the 52-kDa polypeptide component of the Ro/SS-A ribonucleoprotein autoantigen. J. Immunol. 156, 4484–4491 62 Rokeach, L.A. et al. (1991) Characterization of the autoantigen calreticulin. J. Immunol. 147, 3031–3039 63 Verreck, F.A. et al. (1995) DR4Dw4/DR53 molecules contain a peptide from the autoantigen calreticulin. Tissue Antigens 45, 270–275 64 Tuckova, L. et al. (1997) Anti-gliadin antibodies in patients with celiac disease cross-react with enterocytes and human calreticulin. Clin. Immunol. Immunopathol. 85, 289–296 65 Orth, T. et al. (1996) Complete congenital heart block is associated with increased autoantibody titers against calreticulin. Eur. J. Clin. Invest. 26, 205–215 66 Gut, J. et al. (1993) Mechanisms of halothane toxicity: novel insights. Pharmacol. Ther. 58, 133–155 67 Kinoshita, G. et al. (1998) Spreading of the immune response from 52 kDa Ro and 60 kDa Ro to calreticulin in experimental autoimmunity. Lupus 7, 7–11 68 Singh, N.K. et al. (1994) Identification of calreticulin as a rubella virus RNA binding protein. Proc. Natl. Acad. Sci. U. S. A. 91, 12770–12774 69 Walport, M.J. et al. (1998) C1q and systemic lupus erythematosus. Immunobiology 199, 265–285 70 Kovacs, H. et al. (1998) Evidence that C1q binds specifically to CH2-like immunoglobulin gamma motifs present in the autoantigen calreticulin and interferes with complement activation. Biochemistry 37, 17865–17874 71 McCauliffe, D.P. et al. (1990) A human Ro/SS-A autoantigen is the homologue of calreticulin and is highly homologous with onchocercal RAL-1 antigen and an aplysia ‘memory molecule’. J. Clin. Invest. 86, 332–335 72 Pritchard, D.I. et al. (1999) A hookworm allergen which strongly resembles calreticulin. Parasite Immunol. 21, 439–450 73 Sanders, M.L. et al. (1998) Antibody to a cDNAderived calreticulin protein from Amblyomma americanum as a biomarker of tick exposure in humans. Am. J. Trop. Med. Hyg. 59, 279–285 74 Basu, S. et al. (2000) Necrotic but not apoptotic cell death releases heat shock proteins, which deliver a partial maturation signal to dendritic cells and activate the NF-κB pathway. Int. Immunol. 12, 1539–1546 75 Casciola Rosen, L.A. et al. (1994) Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes. J. Exp. Med. 179, 1317–1330 76 Nakamura, K. et al. (2000) Changes in endoplasmic reticulum luminal environment affect cell sensitivity to apoptosis. J. Cell Biol. 150, 731–740 77 Note added in proof: A recent paper does in fact give evidence localizing CRT to the cytoplasm, and the authors propose a role in nuclear transport [Holaska, J.M. et al. (2001) Calreticulin is a receptor for nuclear export. J. Cell Biol. 152, 127–140]
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in a tightly membrane-associated form in PC12 cells. J. Biol. Chem. 267, 4110–4118 Burger, K. (2000) Greasing membrane fusion and fission machineries. Traffic 1, 605–613 Ikonen, E. and Simons, K. (1998) Protein and lipid sorting from the trans-Golgi network to the plasma membrane in polarized cells. Semin. Cell Dev. Biol. 9, 503–509 Martin-Belmonte, F. et al. (2000) Thyroglobulin is selected as luminal protein cargo for apical transport via detergent-resistant membranes in epithelial cells. J. Biol. Chem. 275, 41074–41081 Wang, Y. et al. (2000) Cholesterol is required for the formation of immature secretory granules from the trans-Golgi network. Traffic 1, 952–962 Blasquez, M. et al. (2000) Involvement of the membrane lipid bilayer in sorting prohormone convertase 2 into the regulated secretory pathway. Biochem. J. 349, 843–852 Palmer, D.J. and Christie, D.L. (1992) Identification of molecular aggregates containing glycoproteins III, J, K (carboxypeptidase H) and H (Kex2-related proteases) in the soluble and membrane fractions of adrenal medullary chromaffin granules. J. Biol. Chem. 267, 19806–19812 Dhanvantari, S. and Loh, Y.P. (2000) Lipid raft association of carboxypeptidase E is necessary for its function as a regulated secretory pathway sorting receptor. J. Biol. Chem. 275, 29887–29893 Arvan, P. et al. (1991) Protein discharge from immature secretory granules displays both regulated and constitutive characteristics. J. Biol. Chem. 266, 14171–14174 Dittié, A.S. et al. (1996) The AP-1 adaptor complex binds to immature secretory granules from PC12
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45 Turner, M.D. and Arvan, P. (2000) Protein traffic from the secretory pathway to the endosomal system in pancreatic β-cells. J. Biol. Chem. 275, 14025–14030 46 Tooze, S.A. (1991) Biogenesis of secretory granules. Implications arising from the immature secretory granule in the regulated pathway of secretion. FEBS Lett. 285, 220–224 47 Urbé, S. et al. (1998) Homotypic fusion of immature secretory granules during maturation in a cell-free assay. J. Cell Biol. 143, 1831–1844 48 Jahn, R. and Sudhof, T.C. (1999) Membrane fusion and exocytosis. Annu. Rev. Biochem. 68, 863–911 49 Gerona, R.R.L. et al. (2000) The C terminus of SNAP25 is essential for Ca2+-dependent binding of synaptotagmin to SNARE complexes. J. Biol. Chem. 275, 6328–6336 50 Davis, A.F. et al. (1999) Kinetics of synaptotagmin responses to Ca2+ and assembly with the core SNARE complex onto membranes. Neuron 24, 363–376 [published erratum appears in Neuron 24, 1049 (1999)] 51 Eaton, B.A. et al. (2000) Biogenesis of regulated exocytotic carriers in neuroendocrine cells. J. Neurosci. 20, 7334–7344 52 Austin, C. et al. (2000) Direct and GTP-dependent interaction of ADP-ribosylation factor 1 with clathrin adaptor protein AP-1 on immature secretory granules. J. Biol. Chem. 275, 21862–21869 53 Wan, L. et al. (1998) PACS-1 defines a novel gene family of cytosolic sorting proteins required for trans-Golgi network localization. Cell 94, 205–216
The ins and outs of calreticulin: from the ER lumen to the extracellular space Steven Johnson, Marek Michalak, Michal Opas and Paul Eggleton Calreticulin was first isolated 26 years ago. Since its discovery as a minor Ca2++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 Ca2++-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.
In 1974, a high-affinity Ca2+-binding protein was isolated from the sarcoplasmic reticulum (SR) of rabbit muscle and given the name HACBP (for: highaffinity calcium-binding protein)1. As it proved difficult to purify and was only a minor component of the SR, interest dwindled until 1989 when the cDNA encoding this Ca2+-binding protein was isolated
simultaneously by the groups of Koch and Michalak2,3. Examination of the amino acid sequence of the HACBP revealed the presence of the endoplasmic reticulum (ER) retrieval sequence KDEL (Ref. 4) at the C-terminus of the protein. Further analysis revealed that HACBP was in fact a major Ca2+-binding protein in the ER lumen5 and, in order to acknowledge both its Ca2+-binding properties and its localization to the ER lumen, HACBP was renamed calreticulin (CRT). In the 11 years since the rabbit and mouse cDNAs were isolated, numerous CRT cDNAs have been isolated from diverse genera, including mammals6, insects7, nematodes8, protozoa9 and plants10. There is a remarkable conservation of both the genomic organization and the amino acid sequence of CRT throughout evolution, indicating that it has an
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Table 1. Effects of altered cellular expression of calreticulina CRT cellular expression level
Refs
Calreticulin upregulation Increased Ca2+ storage capacity of ER
14
Modulation of cell adhesiveness
19,36
Modulation of store-operated Ca2+ influx
14
Increased sensitivity to apoptosis
–b
Modulation of steroid-sensitive gene expression
18,19
Appearance of surface CRT
45
Modulation of SERCA2 function
32
Calreticulin deficiency Embryonic lethal at E14.5
28,29
Impaired cardiac development
28
Changes in cell adhesiveness
29,34
Increased resistance to apoptosis
76
Accumulation of misfolded proteins
–b
Modulation of
Ca2+-dependent
gene transcription
28
Inhibition of agonist-dependent Ca2+ release from
28
ER stores aAbbreviations:
CRT, calreticulin; E, embryonic day; ER, endoplasmic reticulum; SERCA, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase.
bI.
Steven Johnson* Paul Eggleton MRC Immunochemistry Unit, University of Oxford, Oxford, UK OX1 3QU. *e-mail: [email protected] Marek Michalak CIHR Group in Molecular Biology of Membrane Proteins, Dept of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. Michal Opas Dept of Anatomy and Cell Biology, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
Ahsan, R. Knee and M. Michalak (unpublished).
important role in cellular function. The human protein consists of a 17-residue hydrophobic ER signal sequence, followed by the 400 amino acids of the mature protein. Analysis of the primary sequence of the protein has led to it being divided into three domains2. The N-terminal 180 residues are termed the N-domain and are predicted to form a globular β-sheet structure. Residues 181–290 are rich in proline (referred to as the P-domain) and contain two sets of repeated amino acid sequences unique to CRT and its molecular homologues calnexin and calmegin11. The P-domain binds to Ca2+ and acts as the high-affinity (Kd = 1 µM) Ca2+-binding site of CRT12. The C-terminal 110 amino acids of the protein (termed the C-domain) are rich in the acidic residues aspartic acid and glutamic acid and bind to Ca2+ with low affinity (Kd = 2 mM) but high capacity (20–30 moles Ca2+ mole–1 protein)12. Initial studies on CRT concentrated on the Ca2+-binding properties of the protein and its ability to modulate Ca2+ homeostasis13,14. However, interest in CRT intensified during the 1990s, a time when there was a massive increase in the number of functions with which CRT was associated and the cellular locations at which it was seen. Proposed functions for CRT range from chaperoning in the ER15 to anti-thrombotic effects at the cell surface16, and from the regulation of Ca2+ signalling17 to the modulation of gene expression18,19 and cellular adhesion20 (Box 1). Altered expression of CRT also has profound effects on many cellular functions (Table 1). Here is where the controversy associated with CRT lies; how can this one protein play a role in so many important cellular functions, and where within the cell does it carry out these functions? It is accepted that the http://tcb.trends.com
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majority of cellular CRT is located in the ER, where it plays important roles in molecular chaperoning and Ca2+ signalling (Fig. 1). Earlier reports indicated that CRT might also function in the cytoplasm and/or in the nucleus as a modulator of integrin function and steroidsensitive gene expression18,19. However, the protein has not been found in the cytoplasm, and there is no plausible explanation for how it could relocate from the ER lumen into the cytoplasm77. Reports on the nuclear localization of CRT are likely artifacts of immunostaining21, and therefore these aspects of the cellular localization of CRT will not be discussed here. However, there is considerable evidence to indicate that CRT is found outside the cell, although how the protein relocates from the ER to the outside of the cell remains unclear. In addition, immune responses against human and parasitic forms of CRT have been detected in autoimmune patient sera, making extracellular CRT of clinical importance. The purpose of this review, therefore, will be to delineate the functions of CRT by following the protein on a journey out of the cell, beginning with its recognized physiological functions in the ER, including the important lessons learned from recent gene-deletion studies, through to the known pathological effects of extracellular CRT. Excellent review articles have recently been published on the structure and chaperone function of CRT15,22. Hence, we intend to consider here the less well-characterized functions of CRT, including cell-surface and extracellular events. Molecular chaperoning – the story unfolds then refolds
Many cellular and extracellular proteins are folded in the ER by a specific set of chaperones, which is now accepted to include CRT and its membrane-bound homologue calnexin (CNX). CRT and CNX participate in a well-characterized cycle of binding and release to newly folded proteins and glycoproteins via a Glc1Man7–9GlcNAc2 carbohydrate ligand (reviewed in Ref. 15) or through direct interaction with misfolded polypeptides (Fig. 1). However, although it is now generally accepted that CRT is an important chaperone, two questions still need to be answered: first, exactly what directs unfolded glycoproteins to bind to CRT and, second, does CRT bind to carbohydrate only or does it also bind directly to polypeptides? The answer to the first question is dependent on the position of the carbohydrate in the protein, both in the primary sequence and the tertiary structure. Elegant studies on the interactions of chaperones with viral glycoproteins [influenza virus haemagglutinin (HA), vesicular stomatitis virus glycoprotein (VSV-G) and Semliki forest virus (SFV) spike proteins p62 and E1] demonstrated that proteins that fold in the ER make use of chaperones in distinct combinations and different orders23. HA and SFV p62 both bind to CRT or CNX, and this interaction is followed by the recruitment of ERp57, a thiol reductase that facilitates correct disulfide bond formation. VSV-G and SFV E1, by contrast, bind first to BiP (immunoglobulin binding
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Box 1. Selected cellular functions attributed to calreticulin Adhesion • Acrosome function and sperm motility • Activates integrins • Affects cell migration • Control of cellular adhesiveness • Inhibits angiogenesis • Initiates cell spreading • Regulates expression of vinculin • Upregulates expression of N-CAM • Wound healing Blood function • Anti-thrombotic activity • Autoantigen • Binds to complement C1q (C1q receptor?) • Component of lytic granules in CTLs and NKs • Component of tick saliva • Inhibits perforin-dependent killing • Interacts with perforin • Modulates platelet activation Development • Affects cardiac development • Affects neuronal development • Essential for mouse embryogenesis • Induces complete cardiac block • Oocyte fertilization • Regulates bone cell function ER functions • ‘Ca2+ sensor’ in the ER lumen • Binds to Mg2+-ATP • Ca2+ binding and storage • ER chaperone • Essential for glycoprotein maturation • Important for MHC class I assembly • Modulates inositol-(1,4,5)-trisphosphatedependent Ca2+ release
protein), a chaperone that recognizes hydrophobic polypeptide sequences, followed by the recruitment of protein disulfide isomerase (PDI), another ER-lumenal thiol reductase. The major difference between these viral glycoproteins is in the position of their glycosylation in the primary sequence; HA and SFV p62 are both glycosylated in their first 50 N-terminal residues, whereas VSV-G and SFV E1 are glycosylated more C-terminally. It appears, therefore, that ER chaperones compete for binding sites on a newly synthesized polypeptide. Interestingly, it has recently been demonstrated that the distinct differences in the spectrum of glycoproteins that CRT and CNX bind to can be explained by their topological environment – that is, by the fact that CRT is soluble, whereas CNX is membrane bound24. A question that has yet to be fully answered under in vivo conditions is whether the chaperone activity http://tcb.trends.com
• • •
Modulates SERCA2b function Regulation of store-operated Ca2+ influx Zn2+ binding and storage
Gene expression • Androgen-sensitive gene in prostate cancer • Control of Rubella virus replication • Control of steroid-sensitive gene expression • Marker of viral infection • Modulates vitamin D3 signal transduction • Participates in host response to tumor Others • Affects phosphotyrosine level • Important for cellular proliferation • Increases sensitivity to apoptosis • Induces NO formation in endothelial cells • Intracellular iron transport • Longterm memory molecule in Aplysia • Mediates mitogenic effects of fibrinogen • Stress protein Abbreviations: CTL, cytotoxic T-lymphocyte; ER endoplasmic reticulum; MHC, major histocompatibility complex; N-CAM, neural cell-adhesion molecule; NK, natural killer cell; NO, nitric oxide; SERCA, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase. For further details, see review articles in Refs a–d and references therein.
References a Eggleton, P. et al. (1997) Clinical relevance of calreticulin in systemic lupus erythematosus. Lupus 6, 564–571 b Crofts, A.J. and Denecke, J. (1998) Calreticulin and calnexin in plants. Trends Plant Sci. 3, 396–399 c Nakhasi, H.L. et al. (1998) Implications of calreticulin function in parasite biology. Parasitol. Today 14, 157–160 d Michalak, M. et al. (1999) Calreticulin: one protein, one gene, many functions. Biochem. J. 344, 281–292
of CRT is due purely to its carbohydrate binding (lectin) activity or whether there is an additional role for CRT as a classical chaperone – that is, that CRT also possesses a site capable of binding to polypeptide segments of unfolded glycoproteins. It has been suggested that CRT functions only through its lectin activity15 or through protein–protein interactions alone25. However, the most interesting model of the chaperone function of CRT incorporates both of these activities and hence is referred to as the ‘dualbinding model’26. The in vitro assays previously used to characterize the functions of HSP90, HSP70, HSP60 and members of the small heat-shock protein families of chaperones have demonstrated that, in addition to its lectin function, CRT can bind to polypeptides and distinguish between native and non-native conformations of glycoproteins. The ‘dualbinding model’ therefore proposes that CRT binds to
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Fig. 1. Model summarizing the functions of calreticulin (CRT) in the lumen of the endoplasmic reticulum (ER). CRT and calnexin (CNX) act as molecular chaperones to newly synthesized and unfolded proteins and glycoproteins (shown in blue). Competition occurs between the CRT/CNX pathway and the classical BiP pathway, depending on the position of glycosylation in the glycoprotein. CRT also acts as a major Ca2+ store of the ER and influences the activity of both the inositol (1,4,5)-trisphosphate (InsP3) receptor and the sarcoplasmic/endoplasmic reticulum Ca2+ATPase (SERCA2), thereby modulating Ca2+ homeostasis and signalling.
unfolded glycoprotein through interactions with both carbohydrate and exposed hydrophobic polypeptide sequences26. However, as discussed previously, proteins that do not have glycosylation sites within their first 50 amino acid sequence can enter the BiP/PDI pathway in vivo23. Interestingly, the misfolded-protein-binding capabilities of CRT appear to be affected by Ca2+, Zn2+ and ATP, suggesting that CRT undergoes different conformational changes upon binding to these small molecules. This finding is further supported by experiments involving the proteolytic digestion of CRT in the presence and absence of these molecules27. Although there have been a large number of publications describing the chaperoning function of CRT, the model of its chaperone function detailed above (Fig. 1) is based on studies of only a limited number of substrates. Further investigations will be required to fully appreciate this important function of CRT and CNX. Ca2++ homeostasis and signalling: lessons from deletion of the gene encoding CRT
A major recent insight into the functions of CRT has undoubtedly come from the development of CRT-deficient cell lines and mice28,29. The CRT gene deletion is in fact the first reported mouse knockout of an ER-lumenal protein. Given the plethora of functions with which CRT has been linked, it was anticipated that the protein would be essential for organism survival. Indeed, the most prominent http://tcb.trends.com
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feature of the knockout is that it is an embryonic lethal mutation. The crt–/– embryos were viable until 14.5 days into gestation28,29. Initial gross morphological and histological examination of crt–/– embryos revealed several prominent defects, in the heart and body wall28,29. The most obvious defect in embryos surviving to 18.5 days is the failure to absorb the umbilical hernia (omphalocele)28. The most likely cause of embryonic lethality, however, is cardiac failure as embryos at 12.5, 14.5 and 18 days all displayed signs of cardiac pathology characterized by thinner ventricular walls with deep, intertrabecular recesses. This indicates that CRT must play a role during cardiac development28. CRT is expressed at a low level in the mature heart28. However, using transgenic mice expressing a green-fluorescent protein (GFP) reporter gene under the control of the CRT promoter, it was demonstrated that CRT is upregulated in the heart during the middle stages of embryogenesis, from 9.5 to 14.5 days28, while, at later stages (18 days) and after birth, CRT is expressed at negligible levels. This gives strong support to the aforementioned notion of the importance of CRT in cardiac development28. It seems that cardiomyocytes must be sensitive to changes in the abundance of CRT and that it is physiologically important to downregulate the expression of CRT after birth because elevated expression of CRT in newborn hearts is associated with severe cardiac pathology and death (K. Nakamura and M. Michalak, unpublished). Recent experiments indicate that the cardiac transcription factor Nkx2.5 activates expression of the gene encoding CRT in the heart, and the transcription factor COUP-TF1 might be involved in its postnatal suppression30. It is clear from the CRT gene-knockout studies, therefore, that the protein plays an important role in the development of the heart, but it is still not clear which of its many proposed functions is responsible for mediating this role. The most likely mechanism is through the ability of CRT to modulate cellular Ca2+ signalling. CRT is important in the regulation of Ca2+ homeostasis, and it is capable of influencing sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2) function and Ca2+ release from the ER mediated by the inositol (1,4,5)-trisphosphate receptor28,31,32. These functions might be modulated by Ca2+ binding to the high-capacity C-domain of CRT or to the highaffinity P-domain, which has been implicated in many interactions of CRT, possibly through its lectin activity33. At present, most available data suggest that the pathology observed in CRTdeficient mice relates directly to the role of CRT in modulation of Ca2+ homeostasis28,29. Alternatively, in its role as a chaperone15, CRT might affect the proper folding, posttranslational modification, assembly and/or intracellular trafficking of many membrane proteins involved in control of Ca2+
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Focal adhesion disassembly
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Fig. 2. Calreticulin (CRT) in the secretory pathway and its effects on cell adhesion. CRT is found on the surface of some types of cells under certain conditions, and there are several possible ways in which the protein might relocate from the endoplasmic reticulum (ER) to the cell surface. Both ER-resident and cell-surface CRT might affect cell adhesiveness through multiple pathways. (a) CRT has been shown to shuttle between the ER and the Golgi and might eventually proceed through the secretory pathway to the cell surface, sometimes still associated with glycoproteins. (b) CRT colocalizes with lytic perforin to the lysosome-like cytotoxic T-lymphocyte (CTL) secretory granules, where it might prevent organelle autolysis. Release of granule content in the activated CTL might be one mechanism for extracellular targeting of CRT. Dashed arrows correspond to uncharacterized effects of CRT.
homeostasis. Finally, considering the similarity in cardiac phenotype between CRT knockout mice and several mutations ablating cell-adhesion proteins, the defective heart morphogenesis might be attributed in part to a fault in cell communication and supracellular organization, which might involve altered cell adhesiveness29. Cellular adhesion: the gene-deletion story continues
The development of an embryonic stem (ES) cell line lacking CRT suggested that, at the single cell level, a lack of CRT had less severe consequences as this cell line proved to be viable and had normal growth rates34. The CRT-deficient ES cells were, however, severely impaired in their ability to adhere to fibronectin and laminin through cell-surface integrins and also in integrin-mediated extracellular Ca2+ influx. CRT appears to affect cell–substratum adhesion that depends on formation of focal adhesions20. Opinion is still divided as to whether these effects are mediated by CRT directly contacting the integrins through their cytoplasmic tails or through signalling from within the ER. However, there is a lack of supporting evidence pinpointing the presence of CRT in the cytosol despite the ability of http://tcb.trends.com
CRT to bind to integrin tails in vitro35. There is now evidence that CRT can modulate the cytosolic levels of molecules associated with adhesion, such as vinculin36, from within the ER and hence alter formation of focal adhesions (Fig. 2). Furthermore, CRT also has a drastic effect on the total cellular levels of phosphotyrosine, again from within the ER20. A correlation between cell adhesiveness and the overall intracellular phosphotyrosine level has been well established37, and it is the ability of a cell to form focal adhesions that is affected38. This, therefore, might be a mechanism by which CRT affects cell adhesiveness through modulation of phosphotyrosine signalling pathways. Yet another possibility, which has emerged more recently, is that CRT might affect cellular adhesion from the cell surface, which we will describe later.
A major question that has puzzled biochemists and cell biologists is how CRT moves from the ER to the outside of the cell (Fig. 2). Although CRT contains the ER-retrieval sequence KDEL at its C-terminus, the protein has been identified on the surface of a wide variety of cell types, including endothelial cells39 and neuronal cells40, where it has been implicated in a variety of functions. The important issue of how CRT escapes ER retention and is translocated to the cell surface remains to be answered, although there is now good evidence localizing CRT to the secretory pathway from studies on plant cells41, B16 mouse melanoma cells42, rat hepatocytes43, Vero cells44 and also the neuronal cell line NG-108-1545. In the neuronal cell line, a number of ER-resident proteins containing the KDEL sequence have been identified at the cell surface by surface biotinylation, including CRT and PDI40. Strikingly, the transport of these proteins is inhibited upon treatment of the cells with brefeldin A, an inhibitor of ER–Golgi trafficking, therefore supporting the notion that they are transported through the secretory pathway. Several theories could explain the transport of ER proteins to the cell surface. The proteins might be expressed in different isoforms that do not contain the ER-retrieval sequence. In support of this idea, CRT on the surface of NG-108-15 cells is not recognized by antibodies against KDEL40; however, this could be because the KDEL receptor is transported to the surface in complex with CRT, as was observed in B16 cells42. Another possibility is that CRT might be proteolytically processed by ER-lumenal proteases to a form missing the KDEL ER-retrieval sequence, given that, under ER-lumenal Ca2+ concentrations, the C-domain of the protein containing the KDEL amino acid sequence is susceptible to proteolysis27. Saturation of the ER-retention machinery could also play a role46. This could be especially relevant in studies where CRT expression is upregulated, such as in
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Endothelial cell
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Inhibits endothelial cell proliferation and prevents angiogenesis, ultimately reducing tumor formation
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Fig. 3. Roles of extracellular calreticulin (CRT) in disease. Once CRT is released from the cell by either active secretory processes or cell death, it can have various effects on cell functions. (a) The protein becomes a target for both cell-mediated and innate immune responses, and parasites might exploit the anti-thrombotic and complement-inhibiting characteristics of CRT to suppress host defence actions. (b) Surface-bound CRT on endothelial cells can provoke inflammatory events, for example stimulation of nitric oxide production. (c) An N-terminal fragment of CRT called vasostatin plays an active role in preventing angiogenesis and tumor growth.
activated T-cells, where the protein is targeted to the cytotoxic T-lymphocyte (CTL) granules47. Interaction between a CTL and its target cells stimulates release of granule contents, including CRT, into the extracellular space, providing one mechanism for extracellular targeting of the protein. Finally, protein glycosylation might play a role as experiments carried out in Chinese hamster ovary cells have shown that CRT becomes glycosylated under conditions of stress, resulting in the redistribution of the protein within the cell48. Calreticulin at the cell surface
The role of CRT at the cell surface is unclear. CRT does not possess a transmembrane domain, but it clearly orchestrates a number of cellular events from the cell surface, including cellular adhesion and migration. CRT might modulate cell adhesion from within the cell through an interaction with integrin tails or through regulation of focal-adhesionassociated proteins as well as modulation of cytosolic phosphotyrosine levels. Another possibility is that CRT can modulate cell adhesion from outside the cell. CRT has been demonstrated to bind to the extracellular matrix proteins Bb fibrinogen49 and laminin50, and it has been reported that cell-surface CRT can complex with integrins42,51. Antibodies against CRT can prevent spreading of B16 cells50 and block neurite formation in differentiating NG-108-15 http://tcb.trends.com
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cells45. Recent work on endothelial cells has added yet another twist to the already complicated involvement of CRT in cell adhesion as it has been shown that thrombospondin-induced disassembly of focal adhesions is mediated by cell-surface CRT39. An intriguing role for CRT in cell adhesion has been implicated from the recent discovery that CRT, or fragments of the protein, inhibits angiogenesis and suppresses tumor growth52. Tumor growth and metastasis formation are dependent on an adequate blood supply, and therefore the generation of new blood vessels (angiogenesis) is essential for the progression of a tumor. CRT disrupts this process by specifically inhibiting the proliferation of endothelial cells53 (Fig. 3). Thus CRT has now become of intense interest in the quest for cancer therapies54. An alternative function of cell-surface CRT might be in regulating responses of the immune system. CRT is present in the CTL granules, where it has been proposed to prevent perforin from forming pores in the granule membrane, either by Ca2+-chelation55 or direct interactions with perforin56. However, recent work has suggested that CRT has a more active role in preventing autolysis of the lymphocyte by binding directly to the cell surface57. Experiments performed on erythrocytes showed that CRT bound to the membrane, where it prevented the insertion of perforin and hence prevented cell lysis. Furthermore, inhibition of lysis was neither dependent on a direct interaction between CRT and perforin nor on the ability of CRT to chelate Ca2+. The ability of CRT to bind directly to membranes has also been demonstrated in endothelial cells, where thrombosis was inhibited by the stimulation of nitric oxide production16. Finally, cell-surface CRT has been implicated in the upregulation of the immune system. An anti-microbial peptide, which activates neutrophils, has been shown to act through interactions with cell-surface CRT58. Extracellular calreticulin
As well as playing a vital role in cellular function, CRT has also been implicated in a number of pathological processes (Fig. 3). Autoantibodies against CRT have been found in approximately 40% of systemic lupus erythematosus (SLE) patients, and a number of patients with secondary Sjögrens syndrome59. Interestingly, SLE patients also raise autoantibodies against other ER chaperones such as grp9460. CRT is known to associate with antigens of the Ro/SS-A complex, a soluble ribonucleoprotein complex consisting of at least four protein components and four small cytoplasmic RNA components61. Autoantibodies are generated against all elements of the complex, although there is evidence that CRT is antigenic in its own right62. Other than in SLE, antibodies to CRT have been detected in patients with rheumatoid arthritis63, celiac disease64, complete congenital heart block65 and halothane hepatitis66. It appears, however, that CRT
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Acknowledgements The experimental work of the authors has been supported by the Arthritis Research Campaign (PE grant E0521), Biotechnology and Biological Sciences Research Council (S.J.), the Canadian Institutes of Health Research (M.M., M.O.) and the Heart and Stroke Foundations of Alberta (M.M.) and Ontario (M.O.). Marek Michalak is a CIHR Senior Scientist and an AHFMR Medical Scientist.
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is more than just an autoantigen. In SLE models, CRT has been demonstrated to contribute to the progression of an autoantibody response by ‘epitope spreading’67, and CRT has also been demonstrated to bind to the RNA of viruses such as Rubella virus that are capable of generating an autoimmune response68. A major contributing factor to autoimmune diseases such as SLE is the failure to clear immune complexes, a process largely mediated by the first component of the classical pathway of complement, C1q. The importance of this mechanism is highlighted by the fact that patients who lack C1q almost always develop active SLE69. Significantly, it has been demonstrated that CRT can bind to C1q70 and, furthermore, can compete with antibody for binding to C1q and inhibition of C1q-dependent hemolysis. Extracellular CRT might therefore contribute to the progression of autoimmune disease by preventing immune complex clearance. This activity of CRT might have been hijacked by various parasites as a mechanism for evading the immune system. Several parasites such as Onchocerca volvulus71, Necator americanus72 and the ixodid tick Amblyomma americanum73 have been shown to secrete CRT homologues, which might prevent the complement system from acting against the parasite. Extracellular CRT is, therefore, implicated in the progression of a number of diseases, through several potential mechanisms. Given that CRT has been detected on the surface of cells, an obvious question is: when does extracellular CRT become a problem? The answer to this is not known. Clearly, the release of CRT from necrotic cells is a contributing factor for the protein to become immunoreactive74, but there is also evidence demonstrating that the release of CRT and the other Ro/SS-A antigens from apoptotic blebs plays a role in autoantibody production75. Another factor, which might also be important, is the nature of the interaction of CRT with the cell surface. As yet, this interaction has not been defined, but there is evidence that the C-terminal domain
References 1 Ostwald, T.J. and MacLennan, D.H. (1974) Isolation of a high affinity calcium binding protein from sarcoplasmic reticulum. J. Biol. Chem. 249, 974–979 2 Smith, M.J. and Koch, G.L. (1989) Multiple zones in the sequence of calreticulin (CRP55, calregulin, HACBP), a major calcium binding ER/SR protein. EMBO J. 8, 3581–3586 3 Fliegel, L. et al. (1989) Molecular cloning of the high affinity calcium-binding protein (calreticulin) of skeletal muscle sarcoplasmic reticulum. J. Biol. Chem. 264, 21522–21528 4 Pelham, H.R.B. (1989) Control of exit from the endoplasmic reticulum. Annu. Rev. Cell Biol. 5, 1–23 5 Opas, M. et al. (1991) Regulation of expression and intracellular distribution of calreticulin, a major calcium binding protein of nonmuscle cells. J. Cell Physiol. 149, 160–171 6 McCauliffe, D.P. et al. (1990) Molecular cloning, expression, and chromosome 19 localization of a human Ro/SS-A autoantigen. J. Clin. Invest. 85, 1379–1391 http://tcb.trends.com
of CRT is involved16,57. It is interesting to note, therefore, that digestion of CRT with elastase and cathepsin G – the major proteases released during inflammation – results in the C-terminus of the protein being cleaved off, leaving a stable N-terminal fragment59. This could potentially provide a mechanism for the release of CRT into the circulation, where it has pathological effects. Concluding remarks
Calreticulin is now accepted to be a multifunctional, multi-compartmental protein. Here, we have attempted to summarize not only its wellcharacterized roles in the ER but also some of its less-well-understood roles outside this organelle. A major leap forward in the field of CRT research has come with the development of CRT-deficient mice, which have demonstrated the importance of CRT in organogenesis, particularly the development of the heart28,29. Furthermore, these studies have highlighted some of the limitations of investigating protein function purely at a cellular, rather than organism, level as such a role had not been considered previously because of the low level of expression of CRT in the mature heart. Hopefully, further characterization of the CRTdeficient mice will provide a more-detailed insight into the roles of CRT in developing organisms. In addition, such studies might reveal whether CRT has an important role in pathological conditions. For example, autoantibodies against CRT have been identified in patients suffering from complete congenital heart block65, and transgenic mice overexpressing CRT in the heart also show congenital heart block (K. Nakamura and M. Michalak, unpublished). Therefore, by using a combination of approaches, including cellular biology, gene deletion and clinical investigation, we can begin to unravel the mysteries that have surrounded CRT for so long.
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cells. Biochem. J. 281, 651–656 14 Mery, L. et al. (1996) Overexpression of calreticulin increases intracellular Ca2+ storage and decreases store-operated Ca2+ influx. J. Biol. Chem. 271, 9332–9339 15 Helenius, A. et al. (1997) Calnexin, calreticulin and the folding of glycoproteins. Trends Cell Biol. 7, 193–200 16 Kuwabara, K. et al. (1995) Calreticulin, an antithrombotic agent which binds to vitamin K-dependent coagulation factors, stimulates endothelial nitric oxide production, and limits thrombosis in canine coronary arteries. J. Biol. Chem. 270, 8179–8187 17 Llewelyn Roderick, H. et al. (1998) Role of calreticulin in regulating intracellular Ca2+ storage and capacitative Ca2+ entry in HeLa cells. Cell Calcium 24, 253–262 18 Burns, K. et al. (1994) Modulation of gene expression by calreticulin binding to the glucocorticoid receptor. Nature 367, 476–480 19 Dedhar, S. (1994) Novel functions for calreticulin: interaction with integrins and
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