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Cellular Functions of EndoplasmicReticulum Chaperones Calreticulin,Calnexin, and Erp57

Karen Bedard,* Eva Szabo,{ Marek Michalak,* and Michal Opas{

*Membrane Protein Research Group and Department of Biochemistry,

University of Alberta, Edmonton, Alberta, Canada T6G 2H7{Department of Laboratory Medicine and Pathobiology,

University of Toronto, Toronto, Ontario, Canada M5S 1A8

Glycosylated proteins destined for the cell surface or to be secreted from the

cell are trafficked trough the endoplasmic reticulum during synthesis and folding.

Correct folding is determined in large part by the sequence of the protein, but it is

also assisted by interaction with enzymes and chaperones of the endoplasmic

reticulum. Calreticulin, calnexin, and ER57 are among the endoplasmic

chaperones that interact with partially folded glycoproteins and determine if the

proteins are to be released from the endoplasmic reticulum to be expressed, or

alternatively, if they are to be sent to the proteosome for degradation. Studies on

the effect of alterations in the expression and function of these proteins are

providing information about the importance of this quality control system, as well

as uncovering other important functions these proteins play outside of the

endoplasmic reticulum.

KEY WORDS: Adhesion, Calreticulin, Calnexin, ERp57, Protein folding,

Calcium homeostasis. � 2005 Elsevier Inc.

I. Introduction

Attaining the correct conformation is essential for the proper functioning

and expression of proteins. For surface and secreted proteins, which pass

through the endoplasmic reticulum (ER) during assembly, this folding

process is aided by interaction with chaperone proteins, which stabilize

International Review of Cytology, Vol. 245 91 0074-7696/05 $35.00Copyright 2005, Elsevier Inc. All rights reserved. DOI: 10.1016/S0074-7696(05)45004-4

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intermediate conformations allowing time to achieve the correct folding

structure and preventing aggregations. There is an interest in understanding

this process, as defects in folding and processing have been associated with a

number of diseases including cystic fibrosis, prion diseases, and Alzheimer’s.

Furthermore, while these ER chaperones are primarily identified for their

role in protein folding, they are also responsible for a number of other critical

functions, including regulation of calcium homeostasis, activation of specific

transcription factors, and oxidative stress, to name a few. The multifunction-

al nature and differential expression of these unique chaperones may have

relevance to a wide range of conditions, including cardiovascular develop-

ment and function, cancer and neurodegenerative conditions, metabolic

problems, and others. This review will focus on the function of ER chaper-

ones calreticulin, calnexin, and ERp57, and the consequences of their altered

expression and function.

II. Endoplasmic Reticulum and Chaperone Proteins

A. Endoplasmic Reticulum

The ER is a network of membrane‐bound tubules continuous with the

nuclear envelope and found throughout the cytoplasm. The lumen of ER is

a distinctly different environment from the rest of the cell, suited to perform

its functions of xenobiotic metabolism, phospholipid and steroid synthesis,

calcium sequestration, and the synthesis of membrane‐bound and secreted

proteins. As these surface and secreted proteins are transcribed, they are

translocated to the lumen of the ER, where they interact with chaperone

proteins, including Grp78, Grp94, protein disulfide isomerase (PDI),

PDI‐like proteins, calreticulin, calnexin, and ERp57. These chaperones assist

the newly formed protein in reaching its correct folding formation by stabi-

lizing intermediate forms, slowing the folding process, and preventing

misfolding and aggregations.

ER is also an important ion storage organelle. The ion concentrations in

the lumen of the ER resemble the extracellular environment, which may be

important in the synthesis of membrane surface and secreted proteins. The

reported calcium content of the ER ranges from the high micromolar to

millimolar range, orders of magnitude higher than 100 nM calcium con-

centrations found the cytosol. Release of calcium from the ER into the

cytosol leads to large increases in the cytoplasmic calcium concentration,

and is an important component of intracellular signaling. Numerous cyto-

plasmic transcription factors, phosphatases, enzymes, and channels are

sensitive to changes in calcium concentration modulator of their activity.

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The sarcoplasmic/endoplasmic calcium ATPase (SERCA) acts to transport

calcium from the cytosol into the ER, and maintain this concentration

gradient, while channels in the ER allow for stimulated release of calcium.

The high concentration of calcium in the ER, combined with the volume of

the cell that the ER can occupy, allows for the potential of calcium released

from the ER to reach toxic levels in the cell if its release is not controlled.

Calcium‐binding proteins within the lumen of the ER, including calreticulin

and calnexin (Baksh andMichalak, 1991; Tjoelker et al., 1994), act to further

regulate the amount of free versus bound calcium within the ER. This free

calcium concentration is important for controlling the amount of calcium

that can be released, as well as for regulating calcium‐dependent processes inthe ER, such as chaperone interactions with each other and with their

substrates. The binding of glycoproteins to calnexin is calcium dependent

(Capps and Zuniga, 1994; Le et al., 1994).

B. Chaperoning of N‐Glycosylated Proteins through ER

As proteins are translocated into the ER, the leader sequence is cleaved by

protease. This is followed by the addition of an oligosaccharide by a trans-

ferase. This addition of sugar can act to stabilize the protein, increase

its solubility (Drickamer and Taylor, 1998; Dwek, 1996; O’Connor and

Imperiali, 1996; Wormald and Dwek, 1999; Wormald et al., 2002), and assist

in trafficking the protein. Although mature glycoproteins have very hetero-

geneous N‐linked glycans, all glycoproteins go through similar trimming in

the ER, and acquire their final glycoprotein structure as they pass through

the Golgi. TwoN‐acetylglucosamines and nine mannoses with three terminal

glucose residues are assembled onto a core carbohydrate, which is then

transferred to asparagine residues of the nascent polypeptide chain. As

soon as the oligosaccharide is added, trimming begins. The three terminal

glucoses are trimmed by glucosidase I and II, and a terminal mannose is

trimmed by one or more ER mannosidases (Helenius and Aebi, 2004). Both

calreticulin and calnexin bind transiently to a newly synthesized glycoprotein

intermediate, which still contains one terminal glucose. This serves to prevent

aggregation, protect proteins from premature degradation, and ensure the

correct folding status of proteins before continuing in the intracellular traf-

ficking pathway. The binding of calreticulin and calnexin to their substrates

is mediated, at least in part, by a lectin site that recognizes the N‐linkedoligosaccharide processing intermediate Glc1Man9GlcNAc2 (Chen et al.,

1995; Helenius and Aebi, 2001). Both chaperones require the presence of

calcium (Chevet et al., 1999; Vassilakos et al., 1998) and both require the

terminal glucose to be present for the binding of the majority of their

substrates (Di Jeso et al., 2003; Hammond et al., 1994; Nauseef et al., 1995;

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Ou et al., 1993; Peterson et al., 1995; Rodan et al., 1996), there is evidence

that calnexin and calreticulin can recognize the polypeptide segments of

the glycoproteins as well (Arunachalam and Cresswell, 1995; Baksh et al.,

1995; Carreno et al., 1995; Ihara et al., 1999; Margolese et al., 1993; Nigam

et al., 1994; Rojiani et al., 1991; van Leeuwen and Kearse, 1996; Ware et al.,

1995; Zhang et al., 1995; Zapun et al., 1997). The binding of proteins to

calreticulin or calnexin is terminated by removal of the third glucose by

glucosidase II. Inhibition of glucose trimming may lead to accelerated deg-

radation (Moore et al., 1993; Saito et al., 1999) or delayed secretion (Kearse

et al., 1994; Lodish and Kong, 1984; Sasak et al., 1985). If the protein is not

correctly folded, it can be reglycosylated by UDP‐glucose:glycoprotein glu-

cosyltransferase (UGGT), and reassociate with calreticulin and calnexin. In

this model, a sensor of glycoprotein folding, UGGT, recognizes incompletely

folded proteins and reglucosylates them, allowing them to reassociate with

the ER chaperones (Rabouille and Spiro, 1992). This cycle of association

with the chaperone, glucosidase II trimming and release from the chaperone,

assessment of foldedness, and glucosylation if necessary by UGGT followed

by reassociation with the chaperones acts as a quality control mechanism

(Fig. 1).

C. ER Quality Control Proteins

Calreticulin is a soluble protein found within the lumen of the ER. It has

three domains, the compact globular N‐domain encompassing the first 200

residues that does not bind calcium, the extended arm formed by the hairpin

loop of the P‐domain encompassing residues 187–317, enriched in proline

residues, and binding calcium with low capacity, high affinity (�1 mol/mol

protein, K¼ 10 mM), and finally the carboxy‐terminal C domain that encom-

passes 310–401 and binds calcium with high capacity, low affinity

(�18 mol/mol protein, K ¼ 2 mM) (Parodi, 2000). The primary function

ascribed to calreticulin has been as a chaperone protein where it binds to

newly synthesized glycoproteins, preventing aggregation and allowing the

proteins to attain their correct folding conformation. Calreticulin also has an

important role in calcium regulation.

Although calreticulin is found primarily in the ER (Fig. 2A), the protein

has also been detected on the cell surface and circulating outside of the cell

(Johnson et al., 2001). The function of this extra‐ER calreticulin is not clear,

but its presence in these locations may have implications in some autoim-

mune diseases (Eggleton, 2003). Adverse drug reactions with circulating

antibodies to calreticulin have also been reported (Murphy‐Ullrich, 2001;

Nair et al., 1999). In addition to these locations calreticulin was also detected

with specific antibodies in the nucleus of some cells, such as the nucleus of

94 BEDARD ET AL.

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UNCORRECTEDPROOFFIG. 1 The quality control cycle. (A). As glycoproteins are translocated into the endoplasmic reticulum (ER), an

oligosaccharide is added. (B). Two of the glucose moieties are cleaved off by glucosidase enzymes. This allows the protein to

interact with the chaperones calreticulin (CRT) and calnexin (CNX). (C). The terminal glucose is cleaved and the protein

dissociates fromthe chaperone. (D) If theproteinhasnot yet reached theappropriate folding conformation, aglucosemolecule

is added back to the oligosaccharide and the protein can go through another round of association with the chaperones

calreticulin and calnexin.

95

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squamous carcinoma cells in response to ionizing radiation (Ramsamooj

et al., 1995) and in the nucleus of dexamethasone‐treated LM cells (Roderick

et al., 1997), but evidence contrary to these findings indicated that the nuclear

localization of calreticulin was an artifact of immunostaining (Michalak

et al., 1996). However, calreticulin is clearly present in the nuclear structures

throughout early human development. It is not present intranuclearly, in-

stead it appears to be enriched in the nuclear envelope of human oocytes and

embryos (Balakier et al., 2002). The nuclear envelope functions as a calcium

store continuous with the ER, thus calcium‐mediated events have been

implicated in a variety of nuclear activities, including modulation of chro-

matin structure and function, gene expression, DNA synthesis, nucleocyto-

plasmic transport, and changes in nuclear architecture (Ashby and Tepikin,

2001; Bachs et al., 1992; Berridge et al., 2000; Petersen et al., 1998; Santella

FIG. 2 Distribution of calreticulin (CRT, A), calnexin (CNX, B) and ERp57 (C) in mouse

fibroblasts, detected by immunofluorescence labeling and visualization by confocal microscopy.

All proteins localize to the ER.

AU1

AU2

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and Carafoli, 1997). Regulation of gene expression seems to be capable

of differentiating between nuclear and cytoplasmic calcium signaling

(Hardingham and Bading, 1999; Hardingham et al., 1997). Therefore, the

presence of the calcium‐binding chaperones (including sometimes calnexin)

within nuclear structures of human oocytes and embryos might also be

crucial for similar nuclear activities. There is also evidence that calreticulin

can be found in the cytoplasm (Baksh and Michalak, 1991; Jethmalani et al.,

1997; Macias et al., 2003; Yoon et al., 2000). Calreticulin has also been found

in the Golgi where it may associate with endomannosidase. This may play a

role in calreticulin–substrate dissociation (Holaska et al., 2001).

Calnexin is a type I membrane protein composed of a luminal domain

highly homolgous to calreticulin and a cytoplasmic domain of 88 amino

acids. Calnexin shares 42–78% sequence identity with calreticulin (Spiro

et al., 1996), with the highest homology in the proline‐rich extended arm of

the high‐affinity calcium‐binding P‐domain (Baksh and Michalak, 1991;

Wada et al., 1991). Calnexin acts to prevent export of incorrectly or incom-

pletely folded proteins (Jackson et al., 1994; Rajagopalan et al., 1994;

Tjoelker et al., 1994). The protein also helps to prevent rapid degradation

(Jackson et al., 1994; Kearse et al., 1994; Rajagopalan and Brenner, 1994).

Calreticulin and calnexin exhibit prolonged interaction with mutant glyco-

proteins that fail to fold (Helenius, 1994; Moore et al., 1993; Nauseef et al.,

1995; Peterson et al., 1995). Prolonged interaction with calnexin results in the

substrate being directed to the proteosome for degradation (Jakob et al.,

1998; Otteken and Moss, 1996). Misfolded proteins, but not proteins under-

going productive folding, are extracted from calnexin by the a‐mannosidase

I‐like protein EDEM (Liu et al., 1999). EDEM does not interact with

calreticulin (Molinari et al., 2003).

Although calnexin lacks the high capacity calcium‐binding domain, it has

been reported that the phosphorylation status of the cytoplasmic tail can

regulate calcium through an interaction with SERCA2B (Oda et al., 2003;

Roderick et al., 2000). Like calreticulin, calnexin is predominantly located in

the ER (Fig. 2B), but it has also been identified at the cell surface of a number

of cells (Schrag et al., 2001). What role calnexin plays at the cell surface is not

clear, but again, circulating autoantibodies to calnexin have been found in

patients with autoimmune diseases. Calnexin has not been found in the

cytoplasm or nucleus, unlike calreticulin. On the other hand, it appears

that in mammalian somatic cells, calnexin and calreticulin are always

expressed in an identical pattern within the ER. The heterogeneity in com-

partmentalization of calnexin and calreticulin is, however, evident during

development. In human oocytes the two chaperones calnexin and calreticulin

are nonuniformly distributed (Balakier et al., 2002). Even though both are

localized to the cortex region of the oocyte, their distribution in the region

differs; calnexin has an interesting trilaminar arrangement, while calreticulin

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is found predominantly in the outer edge of the cortex (Fig. 3). During cell

division there is a dynamic process in effect, since the localization of calnexin

changes from the trilaminar distribution of calnexin in the germinal vesicles

to a single layer of patches in metaphase I/metaphase II oocytes (Fig. 3). The

differential distribution may reflect their functional differences. Calnexin has

a chaperoning function, while calreticulin acts as a chaperone as well as a

calcium‐storage protein affecting many different cellular functions.

ERp57 is a protein disulfide isomerase ortholog that forms complexes with

both calreticulin and calnexin (Okazaki et al., 2000). Like its ortholog, PDI,

ERp57 assists in disulfide bond formation, however, ERp57 performs this

function for glycosylated proteins. Both the association and the release of

substrates with ERp57 are controlled by the glycosylation status of the

proteins (Elliott et al., 1997; Van der Wal et al., 1998; Zapun et al., 1998).

Like PDI, ERp57 has a modular domain formed by a, b, b0, a0, and c

domains. As in PDI, the a and a0 domains contain the thioredoxin domains

Cys‐Xaa‐Xaa‐Cys. In PDI the b domains determine substrate binding, while

in ERp57, the equivalent domains are responsible for the interaction with

calreticulin and calnexin (Molinari and Helenius, 1999). The c domain com-

FIG. 3 Human oocyte showing distribution of calreticulin (CRT) (A and B) and calnexin

(CNX) (C and D) and during development. The heterogeneity in distribution of calnexin and

calreticulin can be observed in the nondividing oocyte (A and C) and in the oocyte that is going

through mitotic division (B and D).

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prises the carboxy‐terminus. Mapping of interaction sites indicates that

ERp57 will interact with calreticulin and calnexin through association with

the proline‐rich P‐domain arm of the chaperones. ERp57 has also been

shown to regulate the redox status of the luminal face of SERCA2b,

providing dynamic control over ER calcium (Russell et al., 2004). So far

exclusive localization of ERp57 has been to the ER (Fig. 2C).

D. Importance of the Components of the QualityControl Cycle

The importance of the components involved in the synthesis and quality

control of glycosylated proteins is revealed through the effects of deficits in

these components. Mice with inactivated N‐acetlyglucosamine transferase I

die at mid‐gestation age. Patients with mutations in the glucosyltransferase

involved in the synthesis of the oligosaccharide suffer from severe abnorm-

alities including psychomotor retardation and seizures (Li and Camacho,

2004). In yeast, UDP‐Glc:glycoprotein glucosyltransferase is essential for

Schizosaccharomyces pombe viability under conditions of extreme ER stress

(Stanley and Ioffe, 1995). Calreticulin‐deficient mice die mid‐gestationally(Westphal et al., 2003). Calnexin‐deficient mice are viable but with a

pronounced ataxic phenotype (Fanchiotti et al., 1998).

The absence of calreticulin is lethal to the developing mouse embryo.

Calreticulin‐deficient mice display defects in the developing heart (Mesaeli

et al., 1999) and 16% displayed exencephaly (brain outside of skull) (Denzel

et al., 2002). Examination of the expression of the calreticulin gene in the

developing embryo shows little expression in most tissue, but strong expres-

sion in the heart, liver, and in some central nervous system (CNS) tissue at

the stage of development when calreticulin deficiency is lethal. Calreticulin‐deficient mice are ‘‘rescued’’ by a cardiac‐specific constitutively active form

of calcineurin (Mesaeli et al., 1999). The ‘‘rescued’’ mouse is by no means

completely normal, however, with a reduced body size and problems with

lipid regulation, among other things. This may be related to the high level of

expression of calreticulin in adipose tissue. The rescue mouse, along with

further in vitro studies, did however indicate that an important function of

calreticulin is its role as an upstream regulator of calcineurin (Rauch et al.,

2000).

Just as the absence of calreticulin is lethal during embryonic devel-

opment, where its expression in the heart is normally high, overexpression

of calreticulin leads to heart problems after birth, when the expression of

calreticulin is normally down‐regulated. Mice overexpressing calreticulin

in the heart experience complete and sudden heart block after birth (Guo

et al., 2002).

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The absence of calnexin leads to a very different phenotype. The mice are

viable, but with a reduced survivability. They are smaller in size than their

wild‐type littermates. They display an abnormal gait and appear to have

reduced limb coordination (Lynch and Michalak, 2003). The mice are viable

with no histological evidence of cardiovascular defects or changes in cardio-

vascular functional parameters (K. Bedard and M. Michalak, unpublished).

Neurologically, there is a reduction in the number of large myelinated nerve

fibers (Nakamura et al., 2001), which may account for the motor defect. No

difference was observed in the total expression of a number of proteins

chaperoned by calnexin (Denzel et al., 2002).

The cardiovascular phenotype in the calreticulin embryo and neurological

phenotype in the calnexin embryo correlate with the pattern of gene expres-

sion. Calreticulin expression in the developing embryo is low in the CNS but

high in the heart. The opposite pattern is observed for calnexin expression

(K. Bedard, unpublished), suggesting the need for two similar proteins may

relate to their expression patterns.

A striking feature revealed by these studies is that calreticulin and calnexin

are unable to compensate for the loss of each other, therefore suggesting

unique and nonoverlapping functions (Denzel et al., 2002; Mesaeli et al.,

1999; Nakamura et al., 2001). One function of calreticulin that cannot be

compensated by calnexin is its role in modulation of calcium homeostasis

(Denzel et al., 2002; Knee et al., 2003) We created viable crt� and cnx�/� cell

lines indicating that in mammalian cell culture calreticulin and calnexin (and

the calreticulin/calnexin cycle) are not essential for cell survival (Arnaudeau

et al., 2002; Nakamura et al., 2001). Other studies support this idea, for

example, Saccharomyces cervisiae lacks most of the calnexin/calreticulin

components (Mesaeli et al., 1999). Deletion of glucosidase II in mammalian

cells and glucosidase II and UGGT, which are key components of the

calreticulin/calnexin cycle, in S. pombe has no consequences on cellular

function (Scott and Dawson, 1995). Yet, calnexin deficiency is lethal in

S. pombe (Parlati et al., 1995). In Dictyostelium and Caenorhabditis elegans

calnexin and calreticulin deficiency is not lethal but it affects phagocytosis in

Dictyostelium (D’Alessio et al., 1999) and promotes necrotic cell death in

C. elegans (Parlati et al., 1995). In summary, these findings support our

hypothesis that calreticulin and calnexin are multifunctional proteins. The

molecular chaperone function of calreticulin and calnexin may only partially

explain phenotypes of cnx�/� and crt�/� mice.

E. Need for Two Similar Chaperone Proteins

ERp57 is homologous with PDI and calreticulin is homologous with cal-

nexin. The existence of such homologous proteins in structure and function

may seem redundant. However, there are important differences between

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these and other chaperones. ERp57 tends to catalyze the rearrangement of

disulfide bonds in glycosylated proteins while PDI handles nonglycosylated

proteins. Calreticulin is a soluble luminal protein, while calnexin is bound to

the membrane. This may lead to differences in the substrates with which each

interacts. For example, there are a large number of substrates that have been

demonstrated to interact with both calreticulin and calnexin. However, for

many substrates, interaction has been reported for only one or the other, and

in a few cases, selectivity for one over the other has been observed (Table I).

Even among the substrates that are able to interact with either chaperone,

there may be differences in the stage of folding at which the substrate

interacts with each chaperone (Cresswell, 2000; Hebert et al., 1996; Muller‐Taubenberger et al., 2001; Xu et al., 2001). Although physical interactions

with either substrate may be possible, the relevance of those interactions may

be affected by the relative abundance of each chaperone in a given tissue or at

a specific stage of development. This is an area that has not been fully

explored, but there are changes in the transcriptional activation of calreticu-

lin and calnexin in various tissues through development from embryo to

adult. Further, there are differences in the relative protein expression of

calreticulin and calnexin in adult tissues. Finally, the requirement of two

seemingly similar proteins may be related to unique functions of one or both

proteins outside of the quality control process. This is well documented by

gene knockout results.

III. Nonchaperone Functions of the Quality ControlCycle Components

The additional functions of the components of the glycosylation and quality

control cycle have been elucidated by genetic manipulation of the proteins

involved. In calreticulin‐deficient cells, there is acceleration of protein fold-

ing, but quality control is impaired (Guo et al., 2002; Knee et al., 2003;

Molinari et al., 2004). Substrate interaction with calnexin is reduced and the

accumulation of unfolded proteins leads to the triggering of an unfolded

protein response (Diedrich et al., 2001). Depletion of calreticulin accelerates

viral glycoprotein maturation, with only a small decrease in folding efficien-

cy. Similarly, depletion of calnexin had little effect on the maturation of

many viral proteins. Only when both were depleted was a large decrease in

ER quality control observed (Sadasivan et al., 1996). From the whole animal

studies, the depletion of these chaperones has made it clear that these

proteins are very important. However, cell culture studies, and the fact

that the embryos or mice developed to the extent that they did, also make

it clear that chaperoning is not required for the expression of surface

and secreted glycoproteins. These ER chaperones not only have roles in

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TABLE I

Proteins Known to Be Chaperoned by Calreticulin, Calnexin, or Both Chaperones

Calreticulin Calnexin Both

Myeloperoxidaseb Pmp‐22c,d MHC class Ie

Myelin proteolipid

protein f

Type I and III IP3Rc,g Influenza HAh

Integrin a‐chaini VSV G‐proteinc,h Tyrosinasej

MHC class II a and bk Glut Il

NMDA subunit NR1m Thromboglobulinn

Naþ, Kþ‐ATPasek Thyrotropin receptoro

a‐Antitrypsinp Meprin Ag

Integrin b‐chainr SERTs

AMPA receptort von Willebrand factoru

Nicotinic acetylcholine

receptorvTransferrinw

a‐Fetoproteinw

HIV envelope protein

gp160x

aMHC, major histocompatibility complex; IP3R, inositol 1,4,5‐trisphosphate receptor; VSV

¼ vesicular stomatitis virus; NMDA ¼ N‐methyl‐D‐aspartate; SERT ¼ serotonin transporter;

AMPA ¼ a‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate.bNauseef et al. (1995).cDoes not interact with calreticulin.dDickson et al. (2002).eBouvier, M. (2003). Accessory proteins and the assembly of human class I MHC molecules:

A molecular and structural perspective. Mol. Immunol. 39, 697–706.fGudz, T. I., Schneider, T. E., Haas, T. A., and Macklin, W. B. (2002). Myelin proteolipid

protein forms a complex with integrins and may participate in integrin receptor signaling in

oligodendrocytes. J. Neurosci. 22, 7398–7407.gJoseph, S. K., Boehning, D., Bokkala, S., Watkins, R., and Widjaja, J. (1999). Biosynthesis

of inositol trisphosphate receptors: Selective association with the molecular chaperone calnexin.

Biochem. J. 342(Pt. 1), 153–161.hPeterson et al. (1995).iKwon, M. S., Park, C. S., Choi, K., Ahnn, J., Kim, J. I., Eom, S. H., Kaufman, S. J., and

Song, W. K. (2000). Calreticulin couples calcium release and calcium influx in integrin‐mediated

calcium signaling. Mol. Biol. Cell 11, 1433–1443.jHalaban et al. (2000).kArunachalam and Cresswell (1995).lOliver, J. D., Hresko, R. C., Mueckler, M., and High, S. (1996). The glut 1 glucose

transporter interacts with calnexin and calreticulin. J. Biol. Chem. 271, 13691–13696.mHughes, P. D., Wilson, W. R., and Leslie, S. W. (2001). Effect of gestational ethanol

exposure on the NMDA receptor complex in rat forebrain: From gene transcription to cell

surface. Brain Res. Dev. Brain Res. 129, 135–145.nDi Jeso et al. (2003).

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regulating protein folding, but also calcium homeostasis, cell adhesion,

cancer, apoptosis, oxidative stress, mitochondrial function, phagocytosis,

and gene transcription.

Calreticulin also plays an important role in control of calcium homeosta-

sis. Calreticulin‐deficient cells display impaired agonist‐stimulated calcium

release from ER stores by bradykinin, which may be in part due to a failure

of the bradykinin to interact with its surface receptor and increase inositol

1,4,5‐triphosphate (InsP3) (Knee et al., 2003). Calreticulin‐deficient cells alsolack the transient rise in calcium from outside the cell that normally accom-

panies engagement of integrins during cell adhesion (Molinari et al., 2004).

However, there are conflicting reports on the effect of calreticulin on ER

calcium storage. Calreticulin‐deficient cells have a reduced ER calcium stor-

age, (Coppolino et al., 1997; Nakamura et al., 2001). Cells overexpressing

calreticulin had higher levels of ER calcium and a larger release of calcium

leading to larger cytosolic calcium levels. The mitochondrial calcium re-

sponse was shorter. There was no difference in calcium response in calnexin

overexpressing cells (Nakamura et al., 2001). Some studies have found no

effect of calreticulin deficiency on the amount of thapsigargin or InsP3-

sensitive ER stored calcium (Arnaudeau et al., 2002; Opas et al., 1996).

Cell shape, adhesion, and motility are controlled by a variety of pathways,

many of them calcium regulated. Alterations in the level of expression of

calreticulin indeed affect all the aforementioned cell properties (Fadel et al.,

1999, 2001; Opas et al., 1996). Calreticulin‐deficient cells have impaired

adhesion (Coppolino et al., 1997; Liu et al., 1994). It has been suggested

that this may be mediated by direct interaction between calreticulin and

oSiffroi‐Fernandez, S., Giraud, A., Lanet, J., and Franc, J. L. (2002). Association of the

thyrotropin receptor with calnexin, calreticulin and BiP. Effects on the maturation of the

receptor. Eur. J. Biochem. 269, 4930–4937.pLe et al. (1994).qTsukuba et al. (2002).rLenter and Vestweber (1994).sTate, C. G., Whiteley, E., and Betenbaugh, M. J. (1999). Molecular chaperones stimulate

the functional expression of the cocaine‐sensitive serotonin transporter. J. Biol. Chem. 274,

17551–17558.tRubio, M. E., and Wenthold, R. J. (1999). Calnexin and the immunoglobulin binding

protein (BiP) coimmunoprecipitate with AMPA receptors. J. Neurochem. 73, 942–948.uAllen et al. (2001).vChang, W., Gelman, M. S., and Prives, J. M. N. (1997). Calnexin‐dependent enhancement

of nicotinic acetylcholine receptor assembly and surface expression. J. Biol. Chem. 272, 28925–

28932.wWada, I., Imai, S., Kai, M., Sakane, F., and Kanoh, H. (1995). Chaperone function of

calreticulin when expressed in the endoplasmic reticulum as the membrane‐anchored and

soluble forms. J. Biol. Chem. 270, 20298–20304.xOtteken and Moss (1996).

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KxGFFKR sequence of a‐integrins (Coppolino et al., 1995; Dedhar, 1994).

Consequently, to functionally affect integrins clustered in focal contacts,

calreticulin should be present in the cytoplasm, but there is no direct evidence

for this. Thus, calreticulin‐modulated changes in cell adhesiveness have to be

correlated with up‐regulation of adhesion‐specific proteins. Overexpression

of calreticulin increases both cell‐to‐substratum and cell‐to‐cell adhesiveness,and establishes vinculin‐rich cell‐to‐cell junctions by increasing overall vin-

culin levels in cells (Opas et al., 1996). Thus the adhesion‐related effects of

differential expression of calreticulin are vinculin mediated and the absence

from focal contacts or cytoplasm indicates that, in vivo, the adhesion‐relatedfunctions are performed from within the ER lumen. Also, overexpressed

cytoplasmically targeted GFP‐calreticulin did not localize in focal contacts

(M. Opas, unpublished data). Furthermore, targeting of calreticulin to the

cytoplasm either by microinjection or by expression of a leaderless calreticu-

lin had no effect on cell morphology, cytoskeleton, or cell adhesion

(M. Opas, unpublished data). Leung‐Hagesteijn et al. (1994) reported colo-

calization of antibody‐clustered integrins with calreticulin at the cytoplasmic

surface of carcinoma cells, but in normal cells no such colocalization was

found. Hence, it can be concluded that cytoplasmic calreticulin is both not

detectable and nonfunctional in terms of regulating cell adhesion. Most

importantly, studies show that both transcriptional activation by steroid

receptors and cell adhesion in vivo are affected only by the full‐length,ER‐targeted form of calreticulin but not by a truncated, cytosolically tar-

geted mutant protein (Fadel et al., 1999; Michalak et al., 1996; Opas et al.,

1996). A more recent report from Dedhar’s group postulates that calreticulin

may be involved in integrin‐dependent Ca signaling rather than direct regu-

lation of integrin activity (Coppolino et al., 1997). While this observation

requires further investigation, it is not inconsistent with the hypothesis that

calreticulin may function in adhesion as a ‘‘signaling’’ molecule from within

the ER lumen (Michalak et al., 2002; Papp et al., 2003).

Overexpression of calreticulin also increases N‐cadherin levels and

decreases tyrosine phosphorylation of cellular proteins, such as b‐catenin(Fig. 4) (Fadel et al., 2001). b‐Catenin is a component of the cadherin‐mediated adhesion complexes and is also part of the Wnt signaling pathway

(Hutzfeld, 1999). Calreticulin from the ER influences tyrosine phosphoryla-

tion of b‐catenin but not its expression; b‐catenin is underphosphorylated in

calreticulin overexpressor cells, but protein and mRNA levels stay the same

FIG. 4 Calreticulin influences a number of adhesion‐related pathways, such as cadherin/b‐catenin, calmodulin/CaMK II, ERK, and PI3K pathways and steroid receptor‐mediated

pathways. It can also influence nuclear trafficking. TSP, thrombospondin; PAX, paxillin; FAK,

focal adhesion kinase; CaM, calmodulin; CRT, calreticulin; SOC, store‐operated channel.

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in the cells (Fadel et al., 2001). Calreticulin can also affect cell adhesion

through the calmodulin and calcium‐mediated kinase (CaMK) II pathway.

Inhibition of calmodulin or CaMK II rescued the calreticulin under‐expressor phenotype by increased spreading (over 3‐fold) and increased

paxillin and focal adhesion kinase (FAK) phosphorylation and protein levels

(E. Szabo, unpublished data). FAK is a regulatory molecule that binds to

paxillin and in turn paxillin provides docking sites for FAK, src, and vinculin

targeting them to the focal contacts (Burridge and Chrzanowska‐Wodnicka,

1996; Turner, 1998). Paxillin levels are elevated when calreticulin is under-

expressed, which indicates that calreticulin influences paxillin levels, suggest-

ing that calreticulin function in the case of this focal adhesion protein is as a

chaperone (Fig. 4) (E. Szabo, unpublished data). Fibronectin levels are also

regulated by differential calreticulin levels. Overexpression of calreticulin

leads to increased fibronectin protein levels and fibronectin deposition,

explaining the increased spreading observed when calreticulin is overex-

pressed; the converse is observed for calreticulin underexpressing cells.

When calmodulin and the CaMK II pathway are inhibited, fibronectin

level and deposition increase and the calreticulin underexpressing cell pheno-

type is rescued (E. Szabo, unpublished data). When the calreticulin under-

expressor cells were plated on fibronectin‐coated substrata spreading of the

cells was induced. The cells overcame their poorly adhesive phenotype by

induction of many tensin‐rich fibrillar adhesions, thus compensating for

the paucity of vinculin in these cells. The calreticulin overexpressing cells

form vinculin‐rich focal contacts as opposed to tensin‐rich adhesions, since

vinculin levels are elevated in these cells (Fadel et al., submitted).

Extracellular calreticulin is involved in cellular adhesion and migration,

but its role is unclear, since it does not possess a transmembrane domain.

Extracellular calreticulin is a C1q coreceptor (Ghiran et al., 2003; McGreal

and Gasque, 2001), which complexes with CD91 on phagocytes for apoptotic

cell ingestion (Basu et al., 2001; Ogden et al., 2001; Vandivier et al., 2002), has

antithrombotic effects (Dai et al., 1997; Kuwabara et al., 1995), inhibits

melanoma cell spreading (White et al., 1995; Zhu et al., 1997), and inhibits

angiogenesis (Pike et al., 1998, 1999); however, the mechanisms behind these

phenomena are unknown. It was recently shown that thrombospondin med-

iates the disassembly of focal contacts by interacting with cell surface calre-

ticulin (Fig. 4) (Goicoechea et al., 2000). The thrombospondin‐binding site

was mapped to the N‐domain of calreticulin (Goicoechea et al., 2002) and

biochemical evidence indicates the presence of a calreticulin complex with

low‐density lipoprotein receptor‐related protein at the cell surface (Orr et al.,

2003). Thrombospondin stimulates focal adhesion disassembly and motility

through the heparin‐binding domain, hep I, which binds to the calreticulin

and leads to phosphoinositide 3‐kinase (PI3K) activation, and stimulation of

extracellular signal‐regulated kinase (ERK) and Gi protein systems (Fig. 4)

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(Orr et al., 2002, 2003). The sources of extracellular calreticulin have been

subject to speculation (Eggleton et al., 1997); nevertheless the serum level

of calreticulin in patients with systemic lupus erythematosus averages

4.44 mg/ml versus 0.42 mg/ml in control sera (Fukazawa, 1994).

Another indirect way that calreticulin may influence cell adhesion is

through its affects on the transmembrane influx of calcium via store‐operatedcalcium channels (SOCs), conceivably by controlling InsP3‐releasable calci-

um from the ER (Bastianutto et al., 1995; Fasolato et al., 1998; Mery et al.,

1996; Xu et al., 2000). Calreticulin also affects the function of SERCA2b and

the InsP3R (Camacho and Lechleiter, 1995; Jouaville et al., 1995), both of

which may be structurally coupled to SOCs (Lockwich et al., 2001). The

structural relationship between the SR/ER calcium release channels and

SOCs of the plasma membrane has been a matter of controversy, however,

substantial evidence points to structural coupling between the two channels

(Putney, 1999). In view of the heterogeneity of the SR/ER (Meldolesi and

Pozzan, 1998; Petersen et al., 2001), it is intuitive that not all of the ER is

coupled to the plasma membrane, however, morphological data supporting

this notion are scarce. Trp (a putative SOC component) reportedly associates

with InsP3R, SERCA, and caveolin in caveolar calcium signaling complexes

(Lockwich et al., 2000, 2001). Calreticulin was shown to coimmunoprecipi-

tate with caveolin (Darby et al., 2000). Caveolin coclusters with a1 integrinsand its down‐regulation inhibits a1 integrin‐mediated adhesion to fibronectin

(Wei et al., 1999). Furthermore, tyrosine phosphorylated caveolin has been

localized to focal contacts (Volonte et al., 2001). Finally, in fibroblasts, the

InsP3R was localized to focal contacts (Sugiyama et al., 2000) and pilot

TIRFM data showed localization of InsP3Rs to a subset of focal contacts

(M. Opas, unpublished data).

Although calreticulin mechanism(s) of action are still elusive, it is conceiv-

able that the affects observed on focal adhesion may be due to calreticulin

effects on multiple signaling pathways, which include cadherin/vinculin pro-

tein system, changes in tyrosine kinases and phosphatases, interaction with

the Wnt pathway, direct affect on the calmodulin/CaMK II pathway, inter-

action with InsP3 receptor‐mediated signaling, and steroid receptors on the

cell surface (Fig. 4).

Recent studies implicated that calreticulin functions in nucleocytoplasmic

transport, but it is not actually localized to the nucleus. Immunogold labeling

indicated that calreticulin is localized to the ER reticulum and to the nuclear

envelope (Huh and Yoo, 2003). Importantly, the nuclear envelope is contin-

uous with the ER and calcium‐mediated events have been implicated in

nucleocytoplasmic transport (Ashby and Tepikin, 2001; Bachs et al., 1992;

Berridge et al., 2000; Petersen et al., 1998; Santella and Carafoli, 1997).

Hence, it can be deduced that calreticulin would have a function in these

calcium‐mediated events. Calreticulin has been shown to be involved in

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regulation of nuclear export and import of NFAT3 (Mesaeli et al., 1999) and

MEF2C (Li et al., 2002). Calreticulin has also been shown to be involved in

nuclear localization of p53. In calreticulin‐null cells p53 localization to the

nucleus and apoptosis rate are greatly reduced, indicating that calreticulin is

needed for p53 nuclear localization and for proper apoptotic function

(Mesaeli and Phillipson, 2004). It has been shown in vitro and in hetrokaryon

fusion assays that nuclear export of glucocorticoid receptors is mediated

through direct contact between calreticulin and the receptor DNA‐bindingdomain; moreover, the export of glucocorticoid receptors to the cytoplasm is

compromised in calreticulin‐deficient cells (Holaska et al., 2001, 2002).

Calnexin expression has also been linked to adhesion. Integrins associate

during their synthesis with calnexin (Coppolino et al., 1997). Adhesion of

cells stimulates expression of adhesion‐related proteins, including calnexin

(Opas et al., 1996). Breast cancer cells grown in suspension express less

calnexin compared to cells as a monolayer, where adhesion is possible

(Lenter and Vestweber, 1994). Expression of adhesion molecules CD44 and

LFA‐1 is lower in cells lacking calnexin (Lam et al., 2001).

Changes in cell adhesion properties are extremely important in the pro-

gression, invasion, and metastasis of cancerous tumors. The importance of

calreticulin and calnexin in the expression and function of adhesion proteins

may therefore make these chaperone proteins important players in cancer.

On the one hand, to be released to metastasize, the adhesion must be reduced.

On the other, to invade the new target, adhesions must be formed between

the circulating cancerous cell and the invaded tissue. Metastasis of tumors is

responsible for the vast majority of deaths associated with cancer. Calreti-

culin influences cell adhesion. Calreticulin is also strongly induced in colon

cancer, where it is found in the nuclear matrix (Yeates and Powis, 1997).

Abnormalities in the expression and functional activity of cell adhesion

molecules are implicated in the development and progression of the majority

of colorectal cancers (Malyguine et al., 1998). Similarly, calnexin, which can

also influence adhesion, is increased in the progressive stages of breast cancer

(Brunagel et al., 2003). On the other hand, calnexin expression is decreased in

metastatic stages of melanoma compared to primary stages (Buda and

Pignatelli, 2004). Calnexin expression is also decreased in colon and breast

cancer cells grown in suspension compared to those adhered to a plate (Li

et al., 2001). This is consistent with the idea that calreticulin and calnexin

expression can influence adhesion and thus affect tumor invasion and metas-

tasis. Extracellular calreticulin may be especially important in affecting

adhesive phenotype by contributing to deadhesion or, in other words, main-

tenance of the intermediate adhesive state of a cell (Murphy‐Ullrich, 2001)

(Fig. 4).

One of the ways the body protects itself from developing cancers is to

initiate apoptosis in cells with abnormalities. The tumor suppressor protein

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p53 is a transcription factor involved in inducing apoptosis in potentially

damaged cells. p53 induces expression of genes that contain the p53 binding

site and represses those that do not. Calreticulin‐deficient cells had a reduced

level of p53, and a reduced ability to induce p53 in response to DNA damage

(Dissemond et al., 2004). This is consistent with the finding that overexpres-

sion of calreticulin results in an increased release of cytochrome c from the

mitochondria and an increased sensitivity to thapsigargin‐ and staurosporin‐induced apoptosis, while cells deficient in calreticulin had a decrease in

cytochrome c release and caspase 3 activity, and were more resistant to

apoptosis (Yeates and Powis, 1997). Overexpression of calnexin had no effect

on drug‐induced apoptosis (Mesaeli and Phillipson, 2004). Calnexin‐deficientcells, however, are relatively resistant to apoptosis (Nakamura et al., 2000).

Oxidative stress can lead to either apoptotic or necrotic cell death. Reac-

tive oxygen species (ROS) are produced normally during the production of

ATP and are managed effectively by antioxidant systems such as glutathione,

superoxide dismutase, catalase, and other cellular components. However,

excessive generation of ROS, or impaired protective mechanisms, can lead

to toxicity. ROS are thought to play an important role in a wide range of

pathologies including Alzheimer’s disease, Parkinson’s disease, stroke, and

aging. Oxidative stress can occur from sustained ER stress (Nakamura et al.,

2000). Calnexin‐ and calreticulin‐deficient cells show evidence of existing in a

state of sustained ER stress, and further showed an impaired ability to

respond to further ER stress by inducing protective chaperones such as

GRP78. Oxidative stress from ER stress can derive from two sources, the

oxidative process of bond formation as the ER deals with accumulated

proteins, and also through mitochondrial reactive oxygen species (ROS)

production (Zuppini et al., 2002). The lumen of the ER has an oxidizing

environment with a reduced glutathione (GSH) to glutathione disulfide

(GSSG) ratio of 2:1 compared to 30:1 to 100:1 found in the cytosol (Haynes

et al., 2004). Disruption of the ER luminal environment therefore can alter

the oxidative state of the cell. The mitochondrial contribution of ROS after

ER stress could be either due to depletion of GSH due to the demand of the

ER, thereby reducing the antioxidant capacity of the cell, or alternatively by

signaling from the ER to the mitochondria (Haynes et al., 2004). ER stress

has been shown to impact on mitochondrial‐associated proteins (Noiva,

1999). Another mechanism by which calnexin and calreticulin can alter the

level of oxidative stress is through their influence on the production of

protective proteins. Tyrosinase is an enzyme associated with the production

of the protein melanin. Mutations in tyrosinase lead to the prolonged inter-

action of the enzymes with calnexin and calreticulin, and results in albinism

(Merad‐Boudia et al., 1998). Abnormal calnexin association with tyrosinase‐related protein can also lead to increased sensitivity to oxidative stress (Hori

et al., 2002). It is further hypothesized that calnexin may be involved in

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signaling to increase production of oxidative stress relief proteins (Halaban

et al., 2000).

Phagocytosis involves the uptake of microorganisms, damaged or dead

cells, cell debris, or insoluble circulating particles. It involves attraction of

phagocytic cells, adhesion, then protrusion of the membrane to encompass

and take in the particle. In macrophages, ER chaperones including calreti-

culin and calnexin were targeted to the phagocytic cup (Jimbow et al., 2001).

The results do not indicate whether the phagocytosis is dependent on these

chaperones or if they just mark the proximity of the ER to the phagocytic

cup. However, in Dictyostelium, the only microorganism known to contain

both calnexin and calreticulin, gene replacement of both of these genes led to

an impairment in phagocytic ability. Immunofluorescence demonstrated that

the ER comes in direct contact with the phagocytic cup (Vinayagamoorthy

and Rajakumar, 1996).

The main role of calnexin appears not to be related to assisting in the

expression of proteins, as calnexin‐deficient mice and cells appear to have

normal protein expression. Rather the literature suggests that its main func-

tion is binding to and retaining mutant proteins and directing them for

degradation. Examples of mutations in proteins leading to their prolonged

association with calnexin include mutations in the von Willebrand factor

resulting in a bleeding disorder (Muller‐Taubenberger et al., 2001), muta-

tions in the peripheral myelin protein pmp‐22 resulting in neuropathy

(Winrow et al., 1995), and mutations in tyrosinase resulting in albinism

(Allen et al., 2001).

Degradation of proteins involves communication from the ER to the

cytosolically located proteosome. It is possible mutations in membrane

spanning proteins may be detected in the cytosol, but many proteins synthe-

sized in the ER lack a membrane spanning domain. The cytoplasmic tail of

calnexin may provide communication about defective proteins from the ER

to the cytoplasm. Mutation of the secretory protein a1‐antitrypsin leads to its

retention in the ER associated with calnexin, followed by polyubiquitination

of calnexin (Dickson et al., 2002). Similar observations were made for the

mutations in the secretory protein meprin (Halaban et al., 2000).

A clinically significant example of calnexin retaining mutant proteins is

the �F508 mutation of the CFTR chloride channel responsible for 70%

of clinical cases of cystic fibrosis. In this disease, the deletion of an amino

acid leads to a decreased expression of the chloride channel at the cell surface,

as the channel is retained in the ER bound to calnexin. Interestingly, in

in vitro studies where the channel is permitted to move to the surface

by altering glycogen content (Qu et al., 1996), or temperature (Tsukuba

et al., 2002), the channel has some chloride flux activity. This opens the

possibility that alteration in calnexin function may have potential therapeutic

benefits.

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There are other examples in which mutant proteins are retained in the ER

by calnexin. Mutations in cartilage oligomeric matrix protein (COMP) result

in early osteoarthritis. This COMP mutation results in retention of the

protein in the ER, and an increased expression of calnexin, suggesting the

mutated substrate may be retained by calnexin (Sato et al., 1996). There is a

prolonged interaction between calnexin and the ER‐retained mutated vaso-

pressin receptor, one of many mutations involved in nephrogenic diabetes

insipidus (Denning et al., 1992). Mutations in lysozyme enzymes can be

associated with systemic amyloidosis, a condition in which protein deposits

form in tissues from proteolytic fragments of serum amyloid. In calnexin

disrupted S. cerevisiae, the secretion of a mutant amyloidogenic lysozyme

was increased, while the mutant enzyme was retained by calnexin in wild‐type S. cerevisiae (Morello et al., 2001; Vranka et al., 2001). Amyloids can

be formed not only from deposits of serum amyloid fragments, but may be

made up of a number of plasma proteins, which have been transformed from

soluble proteins into insoluble fibrils. These formations occur in a number of

diseases including Alzheimer’s and prion diseases.

Calnexin appears to play a role in prion diseases. Prion proteins are N‐glycosolated proteins found abundantly in the brain, and normally transported

to the surface. In prion disease the prion protein accumulates in a different

physical state with reduced solubility and protease susceptibility. The disease

form of the prion protein has been shown to coimmunoprecipitate with

calnexin and to be retained in the ER.

IV. Concluding Remarks

The presence of each of the components involved in the processing of glycosy-

lated proteins through the ER is essential for the normal development of a

healthy animal. However, it is remarkable, given the large and diverse group of

surface and secreted proteins handled by this pathway, that development pro-

ceeds to the extent that it does. Studies on calreticulin have revealed that it is

involved in a wide variety of cellular functions outside of its role as a chaperone

(Johnson et al., 2001), and that these, and not protein folding, may be the

essential functions of this ER protein. Indeed, calreticulin affects important cell

functions such as adhesion via regulation of expression of proteins important in

adhesion, as well as via its effects on intracellular signaling pathways (Fig. 4).

Consequently, calreticulin knockout yields a lethal phenotype. The primary

function of calnexin, it appears, is in recognizing and retaining defective pro-

teins, but it is not required for the synthesis and expression of proteins. This is

supported by the finding that the absence of calnexin, a chaperone with fewer of

these extra functions, has a comparatively mild phenotype.

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Acknowledgments

This work was supported by grants from the CIHR (to M.M. and M.O.) and from the Heart

and Stroke Foundations of Ontario (to M.O.). M.M. is a CIHR Senior Investigator. M.O. is a

member of the Heart & Stroke/Richard Lewar Centre of Excellence.

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