Calreticulin: Endoplasmic reticulum Ca2+ gatekeeper

Abstract Endoplasmic reticulum (ER) luminal Ca2+ is vital for the function of the ER and regulates many cellular processes. Calreticulin is a highly conserved, ER‐resident Ca2+ binding protein and lectin‐like chaperone. Over four decades of studying calreticulin demonstrate that this protein plays a crucial role in maintaining Ca2+ supply under different physiological conditions, in managing access to Ca2+ and how Ca2+ is used depending on the environmental events and in making sure that Ca2+ is not misused. Calreticulin plays a role of ER luminal Ca2+ sensor to manage Ca2+‐dependent ER luminal events including maintaining interaction with its partners, Ca2+ handling molecules, substrates and stress sensors. The protein is strategically positioned in the lumen of the ER from where the protein manages access to and distribution of Ca2+ for many cellular Ca2+‐signalling events. The importance of calreticulin Ca2+ pool extends beyond the ER and includes influence of cellular processes involved in many aspects of cellular pathophysiology. Abnormal handling of the ER Ca2+ contributes to many pathologies from heart failure to neurodegeneration and metabolic diseases.


| THE C ALRE TICULIN G ENE
Two calreticulin genes (calreticulin-1 and calreticulin-2) have been identified in human, pig, rat and mouse. 52,53Plants have two distinct groups of calreticulin: calreticulin-1/calreticulin-2 and calreticulin-3 group encoded by three calreticulin genes. 37,54,55Plant calreticulin genes are not discussed in this review.The calreticulin-2 gene is expressed specifically in the testis and is silent in other tissues. 52The function of CALR-2 gene has yet to be determined and it is also not discussed here.The human calreticulin gene (CALR-1) is ubiquitously expressed, consists of 9 exons and spans approximately 5.8 kb and is localized in chromosome 19 (Figure 1). 56The nucleotide sequences of the human and mouse genes show greater than 70% identity, indicating a strong evolutionary conservation. 56,57Consequently, the regulation of the calreticulin gene, the amino acid sequences and function of the calreticulin protein are also highly conserved.The human and mouse calreticulin promoter contains several binding sites for transcription factors including tissue specific factors.Direct regulation of expression of the calreticulin gene by some of these factors has been confirmed experimentally including Nkx2.5, MEF2C, GATA6, PPARα, PPARγ, COUP-TF1 and Evi-1 factors. 58The CALR gene, along with several other genes, is also activated by environmental stress including depletion of intracellular Ca 2+ stores in vitro and in vivo. 57creased abundance of calreticulin under environmental stress conditions is a part of the ER stress coping response in attempt to build up ER Ca 2+ capacity necessary for restoration of ER homeostasis.
Except for their unique C-terminal domains, calreticulin and calnexin, a type 1 integral ER membrane chaperone (discussed below), share many structural features. 17,59However, the human calnexin gene (CANX) consist of 15 exons (33 kb) encoding 592 amino acid residue polypeptide and it is located on chromosome 5. 59,60 Furthermore, phylogenetic analysis indicates that calreticulin and calnexin existed as separate proteins derived from independent clades. 61It is important to realize that calnexin is anchored to the ER membrane by transmembrane domain with limited mobility in the ER 59 whereas calreticulin is a resident ER luminal protein with high Ca 2+ binding capacity free to move in the ER lumen. 17

| THE C ALRE TI CULIN PROTEIN
Calreticulin is made up of 417 amino acids, including a 17 residue N-terminal signal sequence directing the protein to the ER and a C-terminal KDEL ER retrieval signal (Figures 1 and 2).The protein is composed of three structural and functional domains.4][75] Many chaperones are regulated by ATP to facilitate chaperone-client interactions. 76,77As such, ATP also binds to calreticulin [78][79][80] and calnexin, 81,82 resulting in conformational changes.
The P-domain of calreticulin forms an extended arm which contains a binding site for the thiol oxidoreductase PDIA3 (also known as ERp57), 83,84 PDIA9 (also known as ERp29) 85 and cyclophilin B (Figure 2). 86,87The interaction between calreticulin and PDIA3 supports protein folding in the ER. 209][90] Originally, the N-and P-domains were considered as the core of the chaperone unit of calreticulin. 91Structural analyses of calreticulin by Kalle Gehring's group showed that a part of the Cdomain (from P 301 to E 363 ) also contributes to a carbohydrate binding pocket of the protein. 38,62Recombinant calreticulin P-domain purified from E. coli binds Ca 2+ with high affinity (K d < 1 μM) but at low capacity (1 mol of Ca 2+ /mol of protein). 60,64,91,92Analysis of the crystal structures of the N-and P-domains of calreticulin identified a Ca 2+ ion coordinated by amino acids Asp328, Gln26, Lys62 and Lys64 62 corresponding to the high-affinity Ca 2+ binding side identified for calreticulin.This Ca 2+ remains tightly bound to the protein and coordinates the calreticulin structure.The loss of Ca 2+ binding to the high-affinity site is expected to result in a drastic conformational change in calreticulin.
The C-domain of calreticulin, which contains five clusters of acidic amino acid residues, is the Ca 2+ binding unit of the protein and binds Ca 2+ with low affinity (K d = 2 mM) but at high capacity (20-30 mol of Ca 2+ /mol of protein). 60,91The unstructured Cterminal Ca 2+ binding domain terminates with a KDEL amino acid sequence that is responsible for ER retrieval of the protein.The C-domain of calreticulin is responsible for maintaining 50% of the total cellular Ca 2+ within the ER. 91,935][96] The beauty of calreticulin is that each of the calreticulin domains perform specialized Ca 2+ -dependent functions in the ER: the N-and P-domains playing a role of lectin-like chaperone, the P-domain providing site for Ca 2+ -dependent docking of folding enzymes and the C-domain being necessary for the high-capacity Ca 2+ binding role of the protein (discussed below).

| Three-dimensional structure of calreticulin
One important advance in our understanding of the structure of calreticulin was the elucidation of the crystal structure of the luminal domain of calnexin.Calnexin shares similarity with calreticulin's amino acid sequence encoding N-and P-domains. 59,73,97,98mbined with solving the structure of the P-domain of calreticulin by NMR technique, 64 the 3D structure provided for the first-time structural information on calreticulin (reviewed in 38 ).
For a decade, many attempts were undertaken to obtain a crystal structure of calreticulin.These efforts failed likely due to the flexible nature of the extended P-domain arm and unstructured Cdomain.A major breakthrough came from Kalle Gehring's group who was successful in solving the three-dimensional structure of recombinant calreticulin expressed without a larger portion of the flexible C-domain. 38,62This was followed by determination of the crystal structure of the globular domain of the calreticulin 63 and Cryo-EM studies of the human MHC-I peptide loading complex. 65A large portion of the C-domain (>30 residues) remains missing in these structures due to being highly disordered.Isolated C-domain of calreticulin remains disorder at low Ca 2+ concentration but shows a more compact conformation at the high Ca 2+ concentration. 99,100This likely contributes to a Ca 2+ sensor-like function of calreticulin. 99Recent elegant studies on structural properties of calreticulin mutants associated with MNP indicate that the C-domain is partially α-helical and it plays a role in CALRdel52 mutant interaction with the TpoR. 101Considering the high concentration of calreticulin in the ER lumen, the highly flexible (and mobile) Ca 2+ binding C-domain of calreticulin and extended P-domain arm of the protein are designed to protect the ER luminal environment against self-aggregation (crystallization) of the protein.Ca 2+ binding to calreticulin also contributes to preventing protein aggregation by prompting conformational changes sensitive to ER Ca 2+ fluctuations. 102The presence of the highly flexible protein regions and the unstructured low-affinity Ca 2+ binding segments are likely a common protective mechanism for preventing self-aggregation of other ERresident proteins that are highly concentrated in the ER lumen (100 mg protein/ml representing over 3% of all human proteins [Human Protein Atlas; https://www.proteinatl as.org/human prote ome/subce llula r/endop lasmi c+retic ulum#:~:text=246%20pro teins %20in%20the %20end oplas mic,a%20cel l%20to%20cel l%20var iation]).

F I G U R E 1
Human calreticulin gene and protein.The human calreticulin gene (CALR-1) is localized to chromosome 19 (19p13.13)and consist of 9 exons over 5.8 kb.The calreticulin protein consists of a signal peptide (red), N-terminal globular domain (green), central prolinerich P-domain (orange) and C-terminal Ca 2+ binding C-domain (blue).Repeats A (amino acid sequence PXXIX DPD AXK PED WDE [red boxes]) and B (amino acid sequence GXWXPPXIXNPXYX [yellow boxes]) are indicated in the P-domain.The location of the disulphide bond between Cys105-Cys137 and free Cys163 in the P-domain is indicated.CALR, calreticulin; PDI, protein disulphide isomerase; a.a, amino acid.
Based on available structural information, it is now possible to build a three-dimensional model of calreticulin with recognizable functionally important regions and identification of specific amino acid residues necessary for these functions (Figure 2).The carbohydrate recognition site encompasses amino acid residues 18-203 in the N-domain and residues Pro301 to Glu363 in the C-domain. 38,62te specific mutation analysis also identified a disulphide bridge between Cys105 and Cys137, and Lys111 as important in the binding of carbohydrate moieties by calreticulin. 38,62,75Notably, substitution of Cys137Ala, Cys105Ala and Trp319Ala alter chaperone function in vitro. 66These mutations change the flexibility of the calreticulin backbone and the secondary structure of the N-domain due to new interdomain contacts between the P-domain, globular N-domain and parts of the C-domain. 70The Lys111Ala substitution impairs calreticulin-carbohydrate interactions, 75 and Cys105 and Cys137 are involved in contacts with carbohydrate. 62Cys105, Cys137, Asp135 and Trp319 were identified important in peptide binding to calreticulin. 63Although four His residues within the N-domain of calreticulin have been proposed to coordinate zinc binding, [103][104][105] only His42 is accessible and together with Asp118, Asp121, His123 and Asp125 it forms a zinc binding site. 62However, the importance of these residues in the binding of zinc needs to be experimentally tested.Biochemical and biophysical analyses of several calreticulin mutants identified additional structural features of the protein, including conserved clusters of surface exposed amino acid residues important for maintaining structural stability of the protein. 71r172 and Asp187 are crucial for maintaining the native structure of the protein, while Tyr172 engages the free Cys163 residue to support the thermal stability of calreticulin.71 Amino acid residues Glu255, Glu256, Asp248, Glu260, Trp261 (Figure 2) at the tip of the hairpin-like structure of the P-domain are the site of binding of PDIA3, a folding enzyme [66][67][68][69] and PDIA9, a protein involved biosynthesis and trafficking of secretory and transmembrane proteins (Figure 2).106,107 Cyclophilin B, a member of the family of peptidyl cis/trans protein isomerases (PPI), important for collagen folding, 108 also binds to the tip of the P-domain of calreticulin (Figure 2).85 PDIA1 was the first oxidoreductase identified as interacting with calreticulin 109 and PDIA1 binding to calreticulin affects luminal ER redox conditions upon Ca 2+ depletion, 110  Considering the three-dimensional arrangement of the P-domain as a long arm extending from the globular, carbohydrate binding domain of calreticulin, the tip of the P-domain offers a perfect docking place for folding enzymes to associate with calreticulin to accelerate protein folding.The breakthrough advances in structural studies of calreticulin revealed a distinctive 3D architecture of the protein domains and helped to understand how calreticulin domains perform Ca 2+ -dependent specialized functions; from Ca 2+ binding, storage and control of Ca 2+ -dependent interactions with folding enzymes to lectin-like chaperone function.

| Phenotypes associated with known calreticulin gene variants
[96] More than 60 different variants leading to a shift of the reading frame in calreticulin exon 9 (encoding the Ca 2+ binding C-domain of the protein) have been identified in more than 30% of MPN patients. 94,95,111,112The two most common variants are a 52 base pair deletion (Type 1/CALRdel52 frequency 53%) and a 5 base pair insertion (Type 2/CALRins5 frequency 32%).These shift of the reading frame mutations result in expression of a novel C-terminal amino acid in the calreticulin protein (Figure 3).8][119][120][121] Furthermore, calreticulin mutants secreted from malignant cells inhibit phagocytosis of dying cancer cells by dendritic cells resulting in immunosuppressive effects. 122portantly, the frameshift mutations of calreticulin render the Cterminus of the protein with positively charged, affecting the Ca 2+ capacity to the protein. 115Comparison of CALRdel52 to CALRins5 (Figure 3) indicates that while CALRdel52 has all the negative charges of the calreticulin C-terminus converted to positive charges, there is only a ~ 50% change in charge in the CALRins5 variant. 94,95,112The loss of Ca 2+ binding sites on the CALRdel52 mutant (not to CALRins5) results in reduced ER Ca 2+ storage capacity and activation of UPR. 123ere are also a numerous somatic mutations in the calreticulin gene identified in variety of cancers (https://www.cbioportal.org/resul ts/mutat ions?case_set_id=all&gene_list=CALR&cancer_study_ list=5c8a7 d55e4 b0461 11fee 2296).These mutations are scattered across all regions of the protein but their functional consequences and their role in cancer biology are not known.
Recently, several variants in the calreticulin gene have been identified in sudden unexpected death patients 71 (Figure 4) although their contribution to the sudden unexpected death remains to be established.Intriguingly, these mutations are identified in different regions of calreticulin including the globular N-domain, P-domain and Ca 2+ binding C-terminal domain (Figure 4).These mutations are predicted to affect protein stability and Ca 2+ binding.Sudden unexpected death is frequently associated with heart failure due to altered Ca 2+ handling by cardiomyocytes [124][125][126] and impaired Ca 2+ binding to calreticulin variants is expected to contribute to the sudden unexpected death phenotype (see below for a role of calreticulin Ca 2+ pool in cardiac pathophysiology).

| THE ENDOPL A S MI C RE TICULUM: C ALRE TICULIN ' S ' DOMICILE'
8][129][130][131] The ER is a major component of the cellular reticular network (CRN) that includes the Golgi apparatus, lysosomes, peroxisomes, components of the endocytic pathway, as well as the nuclear envelope, 19,34,128 and occupies about 50% of the total CRN membrane area. 19,34,128The ER is the site for the synthesis of many proteins and lipids, structural components of biological membranes.1][142][143][144] Consequently, both calreticulin and Ca 2+ , being centrally located in the ER, have the ability to control major cellular functions far beyond the ER.
The main intracellular site for Ca 2+ storage and signalling is the ER. 12 ER luminal Ca 2+ modulates many ER functions including protein synthesis and folding, protein quality control, chaperonechaperone/folding enzymes and chaperone-client interactions, and ER stress coping responses including the unfolded protein response (UPR). 12In addition to the high content of Ca 2+ , the ER is well equipped with many resident proteins that monitor and assess cellular needs, respond to changes in cellular and organellar homeostasis, perform adjustments to homeostasis, eliminate misfolded proteins and maintain its own healthy homeostasis for immediate stress/emergency responses.Just like a hospital 'emergency room (ER)', the ER with its extraordinarily wide spectrum of associated functions and the ability to deal with many cellular emergencies.Calreticulin lives in the crowded ER 'emergency room' where it has the responsibility of supplying and managing Ca 2+ -dependent events.
There are instances when calreticulin is found outside the cell (reviewed in 30 ).[150][151][152] Extracellular calreticulin plays a protective role in the central nervous system as brain cells release native calreticulin under ER stress conditions. 153,154Furthermore, extracellular calreticulin (or the Cdomain fragment of the protein) binds to blood-clotting factors and inhibits injury-induced restenosis. 155lreticulin has been linked to many cancers (reviewed by Fucikova et al. 28 ).The Peter Henson group 156 discovered that extracellular calreticulin binds to and activates the LDL receptor-related protein (LRP) on phagocytic cell to prevent activation of the 'don't eat me' signal.
Obeid et al. 157 reported that in mice extracellular calreticulin generates 'eat me' signal for phagocytic cells.9][160] Thus, extracellular calreticulin promotes the uptake of cell by professional phagocytes and initiates of anticancer immunity.Many years ago, it was found that individuals infected with Trypanosoma cruzi presented with a low incidence of cancer compared to non-infected individuals. 161This was many years before calreticulin was discovered.
3][164][165][166][167][168][169] Both T. cruzi calreticulin and host extracellular calreticulin likely affect the immune response in cancer by similar mechanisms.The pool of calreticulin (ER versus extracellular and/or Ca 2+ handling by calreticulin) responsible for the beneficial or detrimental role in cancer remains to be determined.Importantly, it remains to be established whether and how calreticulin finds a way to exit the ER and travels all the way to the cell surface to influence pathophysiology.This may be facilitated by various stress stimuli, such as inflammation and hypoxia 45  from broken or apoptotic cells.The protein may also leave the ER bound to secreted proteins or newly synthesized cell surface receptors. 170,171Regardless, it is inevitable that the departure of calreticulin from the ER (its domicile) alters cellular Ca 2+ handling.

| C ALRE TI CULIN , THE CHAPERONE
72][173][174][175][176][177][178][179] The calreticulin/calnexin cycle is central to the protein quality control machinery, which disposes of misfolded secretory proteins before they can exit the ER. 20,180,181Since calreticulin is an ER lumen-resident protein, it is unconstrained and can move freely within the ER lumen.In contrast calnexin movement is restricted in the ER because it is an integral ER membrane protein anchored to the membrane via a transmembrane helix followed by large cytoplasmic domain (Figure 5). 59The chaperone function of calreticulin and calnexin ER luminal domain is interchangeable but the spectrum of glycoproteins that calreticulin or calnexin bind is determined by topological environment, that is whether they are attached to a membrane (calnexin) or are free in the ER lumen (calreticulin). 182Regardless, both proteins require the presence of Ca 2+ (supplied by calreticulin) to perform their role of chaperones. 60,664][185] Calreticulin forms heterodimeric complexes with PDIA3, a thiol oxidoreductase, and together they assist in the biogenesis of MHC class I. Calreticulin is also a structural component of the peptide loading complex, which consists of the HC-β2m heterodimer, calreticulin, and the additional components tapasin, transporter associated with antigen processing (TAP), and Bap31. 173,186,1879 and their interaction with PDIA3 and tapasin is also reduced. 188

| C ALRE TI CULIN , THE C A 2+ HANDLING PROTEIN
Ca 2+ is an important signalling molecule that has huge influence over nearly all cellular functions including gene expression, stress coping responses, motility, cell adhesion, muscle contraction, protein secretion, proliferation, apoptosis, cell metabolism and fertilization. 12,13e success or failure to control Ca 2+ homeostatic/signalling mechanisms have life or death consequences for the cell and the organism.Formation of intracellular Ca 2+ stores, the source of immediately available Ca 2+ to facilitate Ca 2+ signalling and Ca 2+ -dependent communication between intracellular organelles, is a decisive advantage making cells less dependent of the extracellular Ca 2+ .In the ER, calreticulin is intimately involved in integrating and coordinating many Ca 2+ -dependent pathways in virtually all cellular compartments. 137,191What distinguishes various cells and tissues are the specific mechanisms that regulate the initial extracellular Ca 2+ influx and the intracellular Ca 2+ mobilization.Importantly, these mechanisms are dependent on Ca 2+ stores and Ca 2+ storage/binding proteins.Calreticulin functions as a Ca 2+ storage protein and, in turn, Ca 2+ is dependent on calreticulin for maximal retention in the ER against continues flow of molecules and ions down the secretory pathway.Sometimes calreticulin can be found in intracellular organelles other than the ER where it manages Ca 2+ to support specialized functions and heterogeneity of the CRN.

| ER and Ca 2+ import, export and storage
2][193][194][195][196] The wide range of free ER luminal Ca 2+ concentrations is interpreted as being due to methodological challenges associated with measuring organellar ion concentrations.However, it is also possible that this reflects the dynamic nature of Ca 2+ concentration changes in the ER lumen driven by cellular Ca 2+ needs and environmental conditions.The total Ca 2+ concentration in the cytoplasm is also high (mM range), but in contrast to the ER lumen, the cytoplasmic free Ca 2+ is maintained at <100 nM. 197Once released from calreticulin, Ca 2+ can move much easier in the ER lumen, 198,199 but in the cytosol Ca 2+ is maintained under strict control, where it is bound to many high-affinity Ca 2+ binding proteins and cytoskeletal components.In the cytosol Ca 2+ functions as universal signalling molecule that regulates many cellular processes, including cell proliferation, metabolism and apoptosis. 51,197In the lumen of ER, Ca 2+ also plays a signalling role in regulating protein synthesis, folding, posttranslational modification and trafficking. 109,191,200,201ce accessed from calreticulin, Ca 2+ is released from the ER (ER Ca 2+ export) via the inositol 1,4,5-trisphosphate receptor (InsP 3 R)/ Ca 2+ channel or/and the ryanodine receptor/Ca 2+ channel (RyR) (Figure 6). 12,13,202,203The depletion of ER Ca 2+ triggers Ca 2+ entry from the extracellular space by store-operated Ca 2+ entry (SOCE), which plays a major role supplying Ca 2+ for refilling of the ER Ca 2+ store (ER Ca 2+ import) (Figure 6). 12,192,204,2055][206][207][208] In the plasma membrane the two main Ca 2+ influx channels are the ORAI and transient receptor potential channels (TRPC) that function in a STIM1-dependent way. 209Upon ER Ca 2+ depletion (dissociation of Ca 2+ from calreticulin), STIM1 clusters move towards the plasma membrane containing ORAI1 and TRPC and form membrane contacts to activate SOCE. 139,205,208,209The sarco-endoplasmic reticulum Ca 2+ ATPase (SERCA) pumps Ca 2+ from the cytosol into the ER for storage bound to calreticulin (ER Ca 2+ import) (Figure 6). 12,13The calreticulin mutant expressed in MPN cells causes abnormal regulation of SOCE 206 accounting for dysregulated Ca 2+ fluxes in MPN cells. 119It is not surprising cells expressing MPN variants of calreticulin have increased cytosolic Ca 2+ concentrations. 111er 50% of Ca 2+ stored in the ER is bound to calreticulin (ER Ca 2+ storage). 18,34,93,210In addition, the ER lumen contains other Ca 2+ binding-resident chaperones (GRP94, BiP/GRP78) and folding enzymes 18,19 but these proteins cannot substitute for calreticulin as a Ca 2+ source as they bind less Ca 2+ and are engaged in other specialized activities in the ER.However, calreticulin can hold large amounts of Ca 2+ ('calreticulin Ca 2+ pool') and depending on cellular needs Ca 2+ F I G U R E 6 ER, calreticulin's 'domicile'.Calreticulin is an ER-resident Ca 2+ binding/storage protein.Ca 2+ can be easily and rapidly released from calreticulin to other Ca 2+ handling proteins.Ca 2+ managed by calreticulin plays a signalling role and supports many protein-protein, chaperone-client interactions, as well as impacting stress responses and regulation of the UPR (stress coping response).In cells STIM1/ ORAI1/TRPC together with SERCA are responsible for Ca 2+ import to the ER whereas InsP 3 R and RyR play a role in Ca 2+ export from the ER.There are cases when calreticulin can be found outside the ER but how calreticulin exits the ER remains to be established.ATF6, activating transcription factor 6; Calr, calreticulin; Canx, calnexin; CypB, peptidylprolyl isomerase B; ER, endoplasmic reticulum; InsP 3 R, inositol trisphosphate receptor/Ca 2+ channel; IRE1α, inositol-requiring enzyme 1α; ORAI1, Ca 2+ release-activated Ca 2+ channel protein 1; PERK, protein kinase RNA-like endoplasmic reticulum kinase; PDI, protein disulphide isomerase RyR, ryanodine receptor/Ca 2+ channel; SERCA; sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase; SOCE, store-operated Ca 2+ entry; STIM1, stromal interaction molecule 1; UPR, unfolded protein response; TRPC, transient receptor potential channel.can be easily and rapidly released from the protein to other Ca 2+ handling proteins making calreticulin an ideal protein for regulating the activities of Ca 2+ -dependent cellular processes. 18,19,211,212For example, increase in the abundance of calreticulin Ca 2+ pool is vital for repetitive InsP 3 -induced Ca 2+ waves (Ca 2+ export). 213Calreticulin also regulates SERCA2b function (Ca 2+ import) either directly 214 or via complex formation with PDIA3. 215Increased expression of calreticulin (increased calreticulin ER Ca 2+ pool) has inhibitory effect on SOCE (Ca 2+ import). 216,217Furthermore, Ca 2+ entry from the extracellular space via SOCE (Ca 2+ import) is modulated by PDIA3 binding to STIM1 218,219 and calreticulin interacts with PDIA3 in a Ca 2+ -and calreticulin conformation-dependent manner. 102,200Importantly, depending on the environmental conditions, Ca 2+ is released from calreticulin when the protein undergoes conformational changes and 'senses' fluctuations in the ER luminal Ca 2+ concentration due to active Ca 2+ export/import and/or when calreticulin interacts with client proteins, chaperones, folding enzymes or Ca 2+ handling proteins such as InsP 3 R, STIM1 or SERCA.
The ER is heterogeneous with respect to the distribution of Ca 2+ handling proteins (Ca 2+ pumps, Ca 2+ release channels) 220 and calreticulin delivers Ca 2+ needed to maintain the functional heterogeneity of the ER Ca 2+ access to Ca 2+ handling proteins and organelles in the CRN.Finally, by handling the ER luminal Ca 2+ , calreticulin prevents Ca 2+ misuse that results in altered cell shape, cell motility, cell proliferation, membrane damage, mitochondrial calcification, impaired energy metabolism, improper folding of proteins and cell death.

| Calreticulin, Ca 2+ and ER stress
The ER plays a central role in managing cellular stress via mobilization of ER stress-coping responses, such as the UPR.The UPR is comprised of three signalling arms controlled by ER-associated integral membrane stress sensor proteins: the ER kinase dsRNAactivated protein kinase-like ER kinase (PERK), activating transcription factor 6 (ATF6) and the serine/threonine-protein kinase/ endoribonuclease inositol-requiring enzyme 1α (IRE1α) (Figure 6).These stress sensors bind the ER lumen chaperone BiP/GRP78 and under stress conditions BiP/GRP78 dissociates from IRE1α, PERK and ATF6 resulting in activation of UPR.There are two isoforms of ATF6, ATF6α and ATF6β, with ATF6α playing a primary role in the UPR.Interestingly, calreticulin gene is a target of ATF6β in the central nervous system. 221,222The ATF6β-dependent regulation of expression of calreticulin plays crucial role in neuronal survival under ER stress by improving intracellular Ca 2+ homeostasis. 221,222is further supports a notion that UPR signalling is designed to increase the expression of protein chaperones including calreticulin resulting in increased ER Ca 2+ capacity. 57,221,222Concomitant with this is increase in a number of ER-mitochondrial contact sites, 223,224 promoting transfer of Ca 2+ accumulated in the ER due to increase in calreticulin Ca 2+ pool, to mitochondria in exchange for ATP. 223,224lreticulin role in maintaining high ER Ca 2+ capacity is, therefore, vital in supporting energy production especially during times of cellular stress.Upregulation of the calreticulin gene expression at the time of cellular stress is a clever strategy to maintain high ER Ca 2+ capacity which is central for successful UPR coping responses.

| Calreticulin versus calsequestrin
In the skeletal and cardiac muscle, the majority of the ER membrane is organized into highly specialized membrane network referred to as sarcoplasmic reticulum (SR) responsible for handling Ca 2+ for the muscle contraction and relaxation.Functionally, muscle cells, just like any other cell, also contain conventional ER membranes equipped with and responsible for common cellular functions. 26][227] There are two isoforms of calsequestrin encoded by different genes, namely CASQ1 and CASQ2. 225The N-terminal region of calsequestrin contains three domains with high structural similarity to the thioredoxin fold found in PDI-like proteins (Figure 7), 225,228 and is distinct from the 3D structure of the N + P-domain of calreticulin (Figure 7).Although the N-terminal domains of calreticulin and calsequestrin are unique to each protein, both calreticulin and calsequestrin share amino acid sequence similarities in their C-terminal domains responsible for high-capacity Ca 2+ binding (Figure 7).Calsequestrin is designed to store Ca 2+ in the junctional SR and plays a limited role confined to support of muscle excitation-contraction coupling. 225The protein localization in muscle cells is restricted to the junctional SR due to oligomerization 225,229,230 and binding to the junctional SR proteins including RyR, triadin, junctin, IRE1α. 225In contrast, calreticulin is strategically positioned in the lumen of the ER in all cells (including muscle cells) from where the protein manages access to and distribution of Ca 2+ for not just one but many cellular Ca 2+ -signalling events.Thus, muscle cells differ from many cells because they express two high-capacity Ca 2+ binding proteins (calreticulin and calsequestrin) and, consequently, muscle cells contain two large and distinct pools of organellar Ca 2+ : calreticulin Ca 2+ pool and calsequestrin Ca 2+ pool (discussed below).

| C ALRE TI CULIN C A 2+ P OOL : LE SSON S FROM THE HE ART
Another major advance in calreticulin research was the creation of the calreticulin-deficient mouse model. 2,93This was the first animal model with targeted inactivation of a gene encoding an ER luminal protein.In mice, calreticulin deficiency is lethal at embryonic day 18.5 due to impaired development of the ventricular wall and septum. 93,231,232This was initially unexpected and showed the importance of calreticulin in cardiac function and development.
Two decades of studying calreticulin in the heart very well illustrate and exemplify the concept of co-dependence between calreticulin and Ca 2+ .

| Loss of calreticulin Ca 2+ pool in the heart
Cardiac development is a well-controlled molecular and morphogenetic event where Ca 2+ plays an important signalling role that affects many transcriptional processes during cardiogenesis. 233,234Any perturbations of Ca 2+ -dependent signalling pathways can have devastating consequences in the form of congenital heart disease. 233e whole body inactivation of calreticulin gene in mice is embryonic lethal at E18.5. 93,231In vitro and in vivo biochemical, cell biological and animal studies of calreticulin indicate that Ca 2+ handling by calreticulin is the key cause of embryonic lethality of calreticulin-deficient mice. 93,235Since Ca 2+ -dependent transcriptional mechanisms play crucial roles during cardiac development and pathology, 233,234,236,237 we tested whether loss of the calreticulin Ca 2+ pool in cardiomyocytes is responsible for embryonic lethality in the absence of calreticulin.This idea was validated by the creation of a 'rescue' mouse model with cardiac-specific expression of a constitutively active form of calcineurin. 235,238Calcineurin is a strictly Ca 2+ -dependent serine/threonine phosphatase that regulates the activity of transcription factor NF-AT. Increased expression of calcineurin in cardiomyocytes results in cardiac hypertrophy. 239stained Ca 2+ release from the ER is required to activate calcineurin phosphatase activity, 240 but deletion of the C-terminal domain of calcineurin produces a constitutively active phosphatase even in the absence of Ca 2+ release from the ER. 241Expression of constitutively active calcineurin in calreticulin-deficient cardiomyocytes rescued Calr −/− mice from embryonic lethality 235 indicating an essential role for the calreticulin Ca 2+ pool in Ca 2+ -dependent transcriptional events during cardiac development. 232,235,238,242,243This remarkable finding illustrates a key role of calreticulin in managing the Ca 2+ pool required in regulating Ca 2+ -dependent transcriptional pathways.Additional studies demonstrated that calreticulin similarly plays a role in the transcription of genes regulated by glucocorticoid receptor and other nuclear hormone receptors, [244][245][246][247][248] extending calreticulin influence beyond the ER and impacting on many cellular functions.
CALRdel52, the calreticulin gene variant associated with MPN has all the negative charges of the calreticulin Ca 2+ binding Cdomain converted to positive charges thus influencing the ER calreticulin Ca 2+ pool. 94,95Not surprisingly, replacement of the wild-type calreticulin gene by knock-in of the CALRdel52 variant in all cells in mice (loss of calreticulin Ca 2+ pool) results in embryonic lethality 110 thus validating the original finding that the loss of calreticulin Ca 2+ pool in cardiomyocytes is responsible for embryonic lethality. 93,235ken together, these findings firmly establish that calreticulin Ca 2+ pool is well integrated into Ca 2+ -dependent transcriptional events.
Importantly, the loss of high-capacity Ca 2+ binding to calreticulin and consequently the loss of the calreticulin ER Ca 2+ pool makes the protein functionally ineffective and leads to pathology.

| Increased calreticulin Ca 2+ pool in the heart
Although the abundance of calreticulin and the size of the calreticulin Ca 2+ pool is initially is highly in the embryonic heart, the abundance of calreticulin and concomitantly the size of the calreticulin Ca 2+ pool, sharply decreases in postnatal and adult cardiomyocytes. 93is turns out to be important for the development of cardiac conductive system because maintaining a high level of calreticulin Ca 2+ pool in the postnatal heart results in impaired systolic function, sinus bradycardia and prolonged atrioventricular (AV) node dysfunction with progressive prolongation of the P-R interval that result in complete heart block and sudden death in mice. 249This is reminiscent of a complete heart block seen in children. 250 The increased of the calreticulin Ca 2+ pool in the adult heart enhances mechanical work potential of cardiomyocytes and activates the IRE1α branch of the UPR all leading to cardiac fibrosis and heart failure. 251,252Increased mechanical work of cardiomyocytes triggers activation of UPR in cardiac fibroblasts leading to fibrosis and cardiac remodelling. 252Remarkably, blocking the activation of the UPR pathway by tauroursodeoxycholic acid (TUDCA) prevents cardiac fibrosis. 246The mechanisms of action of TUDCA on UPR signalling are not well understood but likely involve TUDCA-dependent alteration of cellular and/or ER Ca 2+ (managed by calreticulin). 253 and transcriptional signalling, protein quality control, lipid metabolism and ER stress responses, to name a few. 2 Thus, it is entirely predictable that loss of the ER-associated calreticulin Ca 2+ pool in cardiomyocytes explains impairment of heart development and embryonic lethality. 93,231In contrast mice lacking cardiac calsequestrin (loss of the SR calsequestrin-associated Ca 2+ pool) are viable and fertile but only manifest dysfunction in RyR2 channel and conduction abnormalities. 256,257This further illustrates that cardiomyocytes not only have two functionally distinct membrane systems (ER and SR) 2 but most importantly they have two functionally different Ca 2+ pools; one highly specialized calsequestrin Ca 2+ pool (driving excitation-contraction coupling) and an ER calreticulin Ca 2+ pool that is responsible for handling many Ca 2+ -dependent cellular processes (transcription, stress responses, protein quality control and turnover, lipid metabolism).
In summary, one lesson from the heart is that cardiac cells are unique because there is functional compartmentalization of Ca 2+ in cardiomyocytes with the SR Ca 2+ being specialized for muscle mechanical function and the ER Ca 2+ , handled by calreticulin for general cellular functions.
Calsequestrin Ca 2+ pool evolved as Ca 2+ pool dedicated to supply Ca 2+ for excitation-contraction coupling.Another lesson from the heart is that the Ca 2+ pool under the control of calreticulin is not only essential for cardiogenesis and development of muscle conductive system, but most importantly, it impacts on cells Ca 2+ and transcriptional signalling, protein quality control, energy metabolism and ER stress responses.
The calreticulin Ca 2+ pool must be tightly controlled as any increase or decrease in calreticulin Ca 2+ pool results in pathologies.

| CON CLUDING THOUG HTS
Ca 2+ has emerged through evolution as a ubiquitous signalling molecule and integral part of the physiology and biology of the organism.Eukaryotic cells developed Ca 2+ stores system of the ER, which serves as an internally controlled source of Ca 2+ for organellar and cellular communications, essential for survival.The story of calreticulin begun over 40 years ago with a question: how is Ca 2+ handled in the lumen of the ER?This led to the discovery of calreticulin, a protein that today is universally accepted as the major Ca 2+ handling protein in all cells that have ER.Over three decades of studying calreticulin demonstrates that calreticulin plays the role of the ER Ca 2+ gatekeeper (Figure 8).Both the role of calreticulin as the ER Ca 2+ gatekeeper and the interdependence of calreticulin and Ca 2+ form the foundation of ER Ca 2+ signalling and, therefore, influences many cellular processes (Figure 8).The protein maintains ER Ca 2+ supply under different physiological conditions, manages access to Ca 2+ , how Ca 2+ is used depending on the environmental events, and makes sure Ca 2+ is not misused.

F I G U R E 8
Calreticulin as the ER Ca 2+ gatekeeper.As the Ca 2+ gatekeeper calreticulin maintains ER Ca 2+ supply, manages access to Ca 2+ and how Ca 2+ is used.Ca 2+ is supplied to the ER from the extracellular space and cytoplasm by combined action of SOCE and SERCA.Depending on the environmental conditions/cellular needs Ca 2+ is released from the Ca 2+ gatekeeper calreticulin to manage ER 'local' events or other needed 'services' including stress responses, energy metabolism and cellular and organellar communication.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The author declares no conflict of interest.

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linking ER luminal redox environment to Ca 2+ handling.Being a member of the F I G U R E 2 Three-dimensional structure of calreticulin.The crystal structure of the human calreticulin CALR-1.The location of peptide binding, the high-affinity Ca 2+ binding site and Zn 2+ binding site is indicated.The PDIA3 and PDIA9 binding site is located at the tip of the P-domain.A stretch of acidic amino acid residues in the C-domain is involved in a high-capacity Ca 2+ binding is shown.The broken line represents unstructured regions of the protein.The model is based on X-ray, Cryo-EM and NMR data: PDB ID:1HHN, PDB ID:3POW. 38,62-oxidoreductases family of folding enzymes it is likely that PDIA1 also docks on the tip of the P-domain.Most importantly, under physiological conditions interactions between the P-domain of calreticulin and oxidoreductases are sensitive to ER luminal Ca 2+ provided by calreticulin.
or release of calreticulin F I G U R E 3 Model of calreticulin variants identified in myeloproliferative neoplasm.The frameshift within the exon 9 of the calreticulin gene generates a novel C-terminus with most of negatively charges amino acid residues of the native C-domain replaced with positively charged amino acid residue.The two most common calreticulin mutations are shown in the Figure: a 52 base pair deletion (CALRdel52) and a 5 base pair insertion (CALRins5).These mutations generate novel amino acid sequence of the C-domain (shown in red).The ER retrieval KDEL sequence is lost in both mutants.Amino acid sequence of the native C-domain (wild-type) is also shown.
These important processes are Ca 2+ sensitive and they depend on the presence of ER luminal Ca 2+ supplied by calreticulin.Not surprisingly, F I G U R E 4 Model of calreticulin depicting protein variants identified in sudden death patients.Shown is the location of calreticulin mutations identified in sudden unexpected death patients.A direct link between mutations of the calreticulin gene and sudden death has not been established.Information on possible related mutations was received from Drs. D.J. Tester and M.J. Ackerman at the Windland Smith Rice Sudden Death Genomics Laboratory at the Mayo Clinic in Rochester, Minnesota.MPN calreticulin C-domain deletion mutants are functionally impaired in facilitating MHC-I Class II or Class I assembly

F I G U R E 5
Calreticulin and calnexin.Calreticulin (A) and calnexin (B) share amino acid sequence similarities in their N-terminal globular N-domain and proline-rich P-domain.Calreticulin is an ER-resident soluble protein (A) whereas calnexin is a type 1 integral ER membrane protein (B).The C-terminal regions of the proteins are unique for each protein.Calreticulin has a high-capacity Ca 2+ binding C-domain whereas C-terminal domain of calnexin is extended into cytosol.Calreticulin model is based on PDB ID:1HHN, PDB ID:3POW.Calnexin model based on PDB ID:1JHN.TM, transmembrane.
Furthermore, in the embryonic stem (ES) cell model of cardiogenesis the increasing calreticulin Ca 2+ pool or knocking-down the InsP 3 R/Ca 2+ channel (Ca 2+ export protein) prevents proper development of ES-derived pacemaker cells. 236Thus, the role of calreticulin in delivering Ca 2+ to the F I G U R E 7 Calreticulin and calsequestrin.Calreticulin (A) and calsequestrin (Casq2) (B) are major Ca 2+ binding proteins in the lumen of the ER and the junctional SR, respectively.The proteins share amino acid sequence similarities of their unstructured and flexible C-terminal domains responsible for high-capacity Ca 2+ binding.Three thioredoxin fold domains of calsequestrin are indicated in the Figure (domains I, II, III).The N-terminal regions of calreticulin (A) and calsequestrin (B) are unique for each protein.Calreticulin model is based on PDB ID:1HHN, PDB ID:3POW.Calsequestrin model based on PDB ID:2VAF.InsP 3 R is essential for proper development of pacemaker activity during early cardiogenesis and foetal life.

| 13 of 19 MICHALAK
The ER is an extensive network of membranes that occupies a major proportion of the CRN, and calreticulin, as the ER Ca 2+ gatekeeper, partitions Ca 2+ to different regions of the CRN for many cellular functions and needs.This explains why calreticulin turns up in numerous studies involving cell function.AUTHO R CO NTR I B UTI O N S Marek Michalak: Conceptualization (lead); data curation (lead); formal analysis (lead); funding acquisition (lead); investigation (lead); project administration (lead); visualization (lead); writing -original draft (lead); writing -review and editing (lead).ACK N O WLE D G E M ENTS I am grateful to past and present members of our laboratory with whom I have had the pleasure to enjoy the calreticulin journey.They have greatly contributed to the advancement of our knowledge of calreticulin biology and beyond.Thanks to all calreticulinologists around the world for your support of the biennial International Calreticulin Workshops running strong since 1994.Thanks to David J. Tester and Michael J. Ackerman (Mayo Clinic, Rochester, Minnesota) for sharing information on calreticulin mutants identified in sudden unexpected death patients.Thanks to Alexej Verkhratsky for thoughtful and stimulating comments.I thank the anonymous reviewers for their insightful comments and suggestions.I am extremely grateful to Luis B. Agellon, a dear friend and an individual with a brilliant mind.This review would have not been possible without his unconditional support, encouragement and thoughtprovoking endless Zoom sessions.FU N D I N G I N FO R M ATI O N Research in our laboratory is supported by a generous donation from the Kenneth and Sheelagh McCourt family; University Hospital Foundation; Canadian Institutes of Health Research; Natural Sciences & Engineering Research Council of Canada; Canada Foundation for Innovation; Multiple Sclerosis Society; Women and Children's Health Research Institute; SynAD.