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PubMed2000

The conformation of calreticulin is influenced by the endoplasmic reticulum luminal environment.

In order to understand the dynamics of the endoplasmic reticulum (ER) luminal environment, we investigated the role of Ca(2+), Zn(2+), and ATP on conformational changes of calreticulin. Purified calreticulin was digested with trypsin in the presence or absence of Ca(2+), Zn(2+), and ATP. At low Ca(2+) concentration (<100 micrometer), calreticulin is rapidly and fully degraded by trypsin, indicating that under these conditions the protein is in a highly trypsin-susceptible conformation. Increasing Ca(2+) concentration up to 500 micrometer or 1 mm resulted in protection of the full-length calreticulin and in generation of the 27-kDa fragment highly resistant to trypsin digestion. The 27-kDa protease-resistant core of the protein represented the NH(2)-terminal half of calreticulin and was identified by its reactivity with specific antibodies and by NH(2)-terminal amino acid sequence analysis. Ca(2+)-dependent changes in calreticulin's sensitivity to proteolysis indicate that agonist-induced fluctuation in the free ER luminal Ca(2+) concentration may affect the protein conformation and function. Trypsin digestion of calreticulin in the presence of Zn(2+) resulted in the formation of a 17-kDa central protease-resistant core in the protein corresponding to the central region of the protein, indicating that under these conditions the N- and C-domains of the protein are in an extended conformation. Here we also show that calreticulin is an ATP-binding protein but that it does not contain detectable ATPase activity. Digestion of the protein with trypsin in the presence of Mg(2+)-ATP protects the full-length protein. These results indicate that calreticulin may undergo frequent, ion-induced conformation changes, which may affect its function and its ability to interact with other proteins in the lumen of the ER.

Adenosine Triphosphate↗
PubMed2000

Thrombospondin mediates focal adhesion disassembly through interactions with cell surface calreticulin.

Thrombospondin induces reorganization of the actin cytoskeleton and restructuring of focal adhesions. This activity is localized to amino acids 17-35 in the N-terminal heparin-binding domain of thrombospondin and can be replicated by a peptide (hep I) with this sequence. Thrombospondin/hep I stimulate focal adhesion disassembly through a mechanism involving phosphoinositide 3-kinase activation. However, the receptor for this thrombospondin sequence is unknown. We now report that calreticulin on the cell surface mediates focal adhesion disassembly by thrombospondin/hep I. A 60-kDa protein from endothelial cell detergent extracts has homology and immunoreactivity to calreticulin, binds a hep I affinity column, and neutralizes thrombospondin/hep I-mediated focal adhesion disassembly. Calreticulin on the cell surface was confirmed by biotinylation, confocal microscopy, and by fluorescence-activated cell sorting analyses. Thrombospondin and calreticulin potentially bind through the hep I sequence, since thrombospondin-calreticulin complex formation can be blocked specifically by hep I peptide. Antibodies to calreticulin and preincubation of thrombospondin/hep I with glutathione S-transferase-calreticulin block thrombospondin/hep I-mediated focal adhesion disassembly and phosphoinositide 3-kinase activation, suggesting that calreticulin is a component of the thrombospondin-induced signaling cascade that regulates cytoskeletal organization. These data identify both a novel receptor for the N terminus of thrombospondin and a distinct role for cell surface calreticulin in cell adhesion.

Amino Acid Sequence↗
PubMed2003

Identification of an N-domain histidine essential for chaperone function in calreticulin.

Calreticulin is an endoplasmic reticulum (ER) luminal Ca(2+)-binding chaperone involved in folding of newly synthesized glycoproteins via the "calreticulin-calnexin cycle." We reconstituted ER of calreticulin-deficient cells with N-terminal histidine (His25, His82, His128, and His153) calreticulin mutants and carried out a functional analysis. In crt(-/-) cells bradykinin-dependent Ca2+ release is altered, and the reestablishment of bradykinin-dependent Ca2+ release was used as a marker for calreticulin function. Bradykinin-dependent Ca2+ release from the ER was rescued by wild type calreticulin and by the His25, His82, or His128 mutant but not by the His153 mutant. Wild type calreticulin and the His25, His82, and His128 mutants all prevented in vitro thermal aggregation of malate dehydrogenase and IgY, whereas the His153 mutant did not, indicating that His153 chaperone function was impaired. Biophysical analysis of His153 mutant revealed that conformation changes in calreticulin mutant may be responsible for the loss of its chaperone activity. We conclude that mutation of a single amino acid residue in calreticulin has devastating consequences for its chaperone function, indicating that mutations in chaperones may play a significant role in protein folding disorders.

Animals↗
PubMed1997

Regulation of calreticulin gene expression by calcium.

We have isolated and characterized a 12-kb mouse genomic DNA fragment containing the entire calreticulin gene and 2.14 kb of the promoter region. The mouse calreticulin gene consists of nine exons and eight introns, and it spans 4.2 kb of genomic DNA. A 1.8-kb fragment of the calreticulin promoter was subcloned into a reporter gene plasmid containing chloramphenicol acetyltransferase. This construct was then used in transient and stable transfection of NIH/ 3T3 cells. Treatment of transfected cells either with the Ca2+ ionophore A23187, or with the ER Ca2+-ATPase inhibitor thapsigargin, resulted in a five- to sevenfold increase of the expression of chloramphenicol acetyltransferase protein. Transactivation of the calreticulin promoter was also increased by fourfold in NIH/3T3 cells treated with bradykinin, a hormone that induces Ca2+ release from the intracellular Ca2+ stores. Analysis of the promoter deletion constructs revealed that A23187- and thapsigargin-responsive regions are confined to two regions (-115 to -260 and -685 to -1,763) in the calreticulin promoter that contain the CCAAT nucleotide sequences. Northern blot analysis of cells treated with A23187, or with thapsigargin, revealed a fivefold increase in calreticulin mRNA levels. Thapsigargin also induced a fourfold increase in calreticulun protein levels. Importantly, we show by nuclear run-on transcription analysis that calreticulin gene transcription is increased in NIH/3T3 cells treated with A23187 and thapsigargin in vivo. This increase in gene expression required over 4 h of continuous incubation with the drugs and was also sensitive to treatment with cycloheximide, suggesting that it is dependent on protein synthesis. Changes in the concentration of extracellular and cytoplasmic Ca2+ did not affect the increased expression of the calreticulin gene. These studies suggest that stress response to the depletion of intracellular Ca2+ stores induces expression of the calreticulin gene in vitro and in vivo.

3T3 Cells↗
PubMed1991

High-level bacterial expression, purification and characterization of human calreticulin.

To investigate its cellular function and role in autoimmune disease pathogenesis, we have bacterially expressed human calreticulin, a major calcium-binding protein in the endoplasmic reticulum and a human autoantigen. This is the first report describing the heterologous expression of calreticulin from any source. The recombinant calreticulin constituted approximately 32% of the soluble Escherichia coli proteins, and was purified to apparent homogeneity by ion exchange and hydrophobic liquid chromatography. As does the bona fide protein, the recombinant calreticulin binds calcium and undergoes changes in its conformation upon Zn2+ binding. We take this as a strong indication that the folding of the E.coli-expressed calreticulin is very similar, if not identical, to that of the authentic protein. Moreover, the bacterially expressed calreticulin readily reacted with anti-human and anti-rabbit antibodies, and the anti-recombinant calreticulin antibodies immunoreacted with HeLa calreticulin. The availability of this expression system will allow us to carry out site-specific and deletion mutagenesis analysis in structure--function studies of calreticulin.

Amino Acid Sequence↗
PubMed2004

Interaction of C1q with the receptor calreticulin requires a conformational change in C1q.

The interaction between C1q and the chaperone calreticulin was studied under various conditions. When both proteins were present in equal amounts in solution, no interaction could be demonstrated. However, C1q immobilized on a hydrophobic surface, exposed to heat-treatment or bound to immunoglobulins (Igs) showed a strong, rapid and specific binding of calreticulin. The interaction appeared to be a two-step process, and the initial phase of interaction was sensitive to high concentrations of salt but not to a physiological salt concentration. The following strong binding was insensitive to salt and extremes of pH but sensitive to strongly denaturing agents (urea and guanidine). The sensitivity to salt during the initial phase of interaction was practically identical to that observed when calreticulin was bound to type V collagen. Binding between C1q and calreticulin could be inhibited by serum amyloid P component and by proteinase K-digested ovalbumin, and the binding of calreticulin to proteinase K-digested ovalbumin was shown to be inhibited by C1q. The data indicate that C1q binds stably to the peptide-binding site of calreticulin and that the initial binding of calreticulin to C1q involves the collagen-like domain of the C1q molecule. In conclusion, our results suggest calreticulin as a potential receptor for an altered conformation of C1q as occurs during binding to Igs. Thus, the chaperone and protein-scavenging function of calreticulin may extend from the endoplasmic reticulum to the topologically equivalent cell surface, where it may contribute to the elimination of immune complexes and apoptotic cells.

Animals↗
PubMed2006

Ultrastructural analysis of development of myocardium in calreticulin-deficient mice.

BACKGROUND: Calreticulin is a Ca2+ binding chaperone of the endoplasmic reticulum which influences gene expression and cell adhesion. The levels of both vinculin and N-cadherin are induced by calreticulin expression, which play important roles in cell adhesiveness. Cardiac development is strictly dependent upon the ability of cells to adhere to their substratum and to communicate with their neighbours. RESULTS: We show here that the levels of N-cadherin are downregulated in calreticulin-deficient mouse embryonic hearts, which may lead to the disarray and wavy appearance of myofibrils in these mice, which we detected at all investigated stages of cardiac development. Calreticulin wild type mice exhibited straight, thick and abundant myofibrils, which were in stark contrast to the thin, less numerous, disorganized myofibrils of the calreticulin-deficient hearts. Interestingly, these major differences were only detected in the developing ventricles while the atria of both calreticulin phenotypes were similar in appearance at all developmental stages. Glycogen also accumulated in the ventricles of calreticulin-deficient mice, indicating an abnormality in cardiomyocyte metabolism. CONCLUSION: Calreticulin is temporarily expressed during heart development where it is required for proper myofibrillogenesis. We postulate that calreticulin be considered as a novel cardiac fetal gene.

Animals↗
PubMed1998

BiP and calreticulin form an abundant complex that is independent of endoplasmic reticulum stress

BiP is found in association with calreticulin, both in the presence and absence of endoplasmic reticulum stress. Although the BiP-calreticulin complex can be disrupted by ATP, several properties suggest that the calreticulin associated with BiP is neither unfolded nor partially or improperly folded. (1) The complex is stable in vivo and does not dissociate during 8 hr of chase. (2) When present in the complex, calreticulin masks epitopes at the C terminus of BiP that are not masked when BiP is bound to an assembly-defective protein. And (3) overproduction of calreticulin does not lead to the recruitment of more BiP into complexes with calreticulin. The BiP-calreticulin complex can be disrupted by low pH but not by divalent cation chelators. When the endoplasmic reticulum retention signal of BiP is removed, complex formation with calreticulin still occurs, and this explains the poor secretion of the truncated molecule. Gel filtration experiments showed that BiP and calreticulin are present in distinct high molecular weight complexes in which both molecules interact with each other. The possible functions of this complex are discussed.

Journal Article↗
PubMed1999

Determination of a putative phosphate-containing peptide in calreticulin.

Calreticulin is an abundant endo/sarcoplasmic reticulum (ER/SR) protein that may carry out multiple functions inside cells. Except for calreticulin, all of the major ER/SR Ca2+-binding proteins are substrates for protein kinase CK2 in vitro, which led us to hypothesize that native calreticulin might exist in the phosphorylated form. To investigate this possibility, we purified calreticulin from cardiac microsomes and verified its identity by immunoblot analysis and sequencing of tryptic peptides. Purified calreticulin, like cardiac calsequestrin, contained endogenous phosphate as determined by a Malachite green assay for phosphate. Previous analyses of cardiac calsequestrin have localized phosphate to a single tryptic peptide containing serine phosphate on sites phosphorylated by protein kinase CK2. Using a similar procedure, we analyzed calreticulin tryptic peptides with Malachite green, localizing phosphate binding to a single calreticulin peptide 367LKEEEEDKK. As this peptide contains no phosphorylatable residues, our results suggest that calreticulin may tightly bind phosphate or a phosphate-containing molecule at this site.

Animals↗
PubMed1994

Calreticulin is released from activated neutrophils and binds to C1q and mannan-binding protein.

The Ca2+ storage protein calreticulin is associated with the endoplasmic reticulum and shares a high degree of amino acid homology with the surface receptor C1q-R. In this study, flow cytometric analysis detected calreticulin on the neutrophil surface, which decreased during stimulation probably as a consequence of shedding, as calreticulin was found by ELISA in the cell supernatants of stimulated cells. Antibodies raised against C1q-R and calreticulin demonstrated a high degree of immunological cross-reactivity for purified calreticulin as determined by dot blot analysis. Western blots of neutrophil subcellular fractions located calreticulin in both the cytosol and cell membrane fractions; C1q-R was largely confined to the cell membrane. Calreticulin and C1q-R both bind to C1q and mannan-binding protein. Therefore, calreticulin may be shed on cell activation and may be associated with the cell membrane, where it can potentially interact with C1q and serum lectins. The implications of this are discussed.

Calcium-Binding Proteins↗
PubMed1997

Modulation of the retinoic acid and retinoid X receptor signaling pathways in P19 embryonal carcinoma cells by calreticulin.

Calreticulin is a widely expressed calcium binding protein that can bind to an amino acid sequence motif, KXGFFKR, which is present in the cytoplasmic domain of all integrin alpha-subunits. Closely related sequences, KXFFKR and KXFFRR, are encoded in the DNA-binding domain of all members of the steroid/thyroid/retinoid receptor superfamily and it has recently been demonstrated that calreticulin inhibits their activity both in vitro and in vivo. Here we present novel evidence that calreticulin can interfere directly with the retinoic acid (RARs) and retinoid X (RXRs) receptor pathways. Calreticulin exhibits the ability to inhibit DNA-binding activity of both heterodimeric RAR/RXR and homodimeric RXR complexes in vitro. Inhibition of RXR binding to DNA is achieved with a concentration of calreticulin that is approximately fourfold lower than that required for inhibition of RAR/RXR binding to a cognate binding site. Coprecipitation experiments suggest a direct protein:protein interaction between calreticulin and retinoid receptors. Stable overexpression of calreticulin in P19 embryonal carcinoma cells significantly decreases the rapid activation of the endogenous RA-responsive RARbeta gene, abrogates the ability of endogenous RAR/RXR complexes to bind to DNA, and inhibits the emergence of the RA-induced differentiated phenotype. These data demonstrate that calreticulin can interfere with the two distinct retinoid signaling pathways through a mechanism likely involving direct protein:protein interactions and that disruption of the retinoid signal alters biological processes in vivo.

Amino Acid Sequence↗
PubMed1993

Comparison of cDNAs from bovine brain coding for two isoforms of calreticulin.

Calreticulin is a major calcium-binding protein of the endoplasmic reticulum of non-muscle cells. In addition to a 1.9-kb calreticulin mRNA, some evidence has suggested the existence of another transcript of 3.75 kb, which is very similar to calreticulin. We report here the isolation and sequencing of cDNA clones from a bovine brain lambda gt11 cDNA library, two of which appear to code for calreticulin and a third for a novel isoform of calreticulin. The deduced amino-acid sequence of the novel clone shares high similarity with mouse calreticulin in the C-terminal 318 amino acids. However, its N-terminal sequence is completely divergent. Northern blot analysis of bovine cerebral cortex RNA indicates that the conserved region of the clone hybridizes to two messages of 1.9 kb and 3.75 kb. The divergent region of this clone hybridizes to the 3.75-kb message, but not to the 1.9-kb message. We believe that this novel clone corresponds to an alternate form of calreticulin which is identical to calreticulin toward the C-terminus, but completely different at the N-terminal region, and that this isoform is encoded by a much larger message.

Amino Acid Sequence↗
PubMed2006

Identification of calreticulin as a marker for phagocytosis of apoptotic cells in Drosophila.

Apoptotic cell phagocytosis is initiated through the specific interaction between markers for phagocytosis present at the surface of targets and their receptors of phagocytes. Although many molecules have been proposed to be phagocytosis markers and receptors in mammals, information as to the identity of those molecules is limited for invertebrate animals. Calreticulin, a molecular chaperone that functions in the lumen of the endoplasmic reticulum, was recently reported to be the second general marker, the membrane phospholipid phosphatidylserine being the first, for mammalian apoptotic cells to be recognized by phagocytes. We here asked whether or not calreticulin serves as a marker for phagocytosis in Drosophila. Phagocytosis of apoptotic S2 cells by Drosophila hemocyte-derived l(2)mbn cells, which we previously showed to occur independent of phosphatidylserine, was inhibited by the addition of anti-calreticulin antibody. This inhibition was observed when the target cells, but not phagocytes, were pre-incubated with the antibody. In addition, RNA interference-mediated reduction of calreticulin expression in apoptotic S2 cells, but not in l(2)mbn cells, reduced the level of phagocytosis. An immunocytochemical analysis revealed that calreticulin is widely distributed at the surface of viable S2 cells. After the induction of apoptosis, cell surface calreticulin seemed to form aggregates, with no change in its amount. Furthermore, in embryos of a mutant Drosophila strain that expresses calreticulin at a reduced level, the level of phagocytosis of apoptotic cells was about a half of that observed in embryos of a wild-type strain. These results collectively indicate that calreticulin is the first molecule to be identified as a marker for phagocytosis of apoptotic cells by Drosophila phagocytes.

Animals↗
PubMed1998

Role of calreticulin in regulating intracellular Ca2+ storage and capacitative Ca2+ entry in HeLa cells.

Calreticulin is a Ca2+ binding protein located primarily in the endoplasmic reticulum (ER) lumen of non-excitable cells, where it is considered to be involved mainly in Ca2+ storage and buffering. However, there is increasing evidence to implicate the protein in other facets of Ca2+ signalling. In this study, we sought to establish more clearly the role of the protein in the regulation of intracellular Ca2+ signalling. Generating HeLa cells stably transfected with GFP-tagged calreticulin (GFPCRT) allowed to us to select cells by FACS in which calreticulin was expressed at ten times its endogenous levels. Using transiently expressed aequorin as a Ca2+ indicator in these cells, we investigated the role of calreticulin in intracellular Ca2+ storage, IP3-mediated Ca2+ release, and capacitative Ca2+ entry. The data showed that the capacity of the ionomycin-sensitive Ca2+ store was doubled in over-expressing cells, indicating that although calreticulin has a role in Ca2+ storage within the lumen, other lumenal proteins are also likely to be involved. No difference was observed in the release of Ca2+ from the IP3-sensitive store in response to prolonged single stimulation with histamine in the absence of extracellular Ca2+, but use of short, sequential pulses of histamine and ATP revealed that calreticulin may exert an effect upon IP3-mediated Ca2+ release. Two different experimental approaches indicated that calreticulin participates in the regulation of capacitative Ca2+ entry. In the presence of extracellular Ca2+, the histamine-generated cytosolic Ca2+ signal was significantly lower in GFPCRT cells than those in control cells. Induction of capacitative Ca2+ entry by complete emptying of the store using the SERCA pump inhibitor, cyclopiazonic acid also showed that the influx component was significantly reduced in the GFPCRT cells. Use of ER-targeted apoaequorin acting as a luciferase demonstrated that the resting ER free [Ca2+] in the GFPCRT cells was lower than that in control cells. These data implicate calreticulin in the control of IP3-mediated Ca2+ release and capacitative Ca2+ entry, which may involve direct interaction with Ca2+ signalling components or control of ER free [Ca2+].

Adenosine Triphosphate↗
PubMed2000

Evidence of [Ca(2+)]i elevation by anti-calreticulin immunoreactive protein in neurons.

Calreticulin is a multifunctional Ca(2+)-binding protein. The effect of anti-calreticulin antibody on intracellular free calcium concentration [Ca(2+)]i was studied in cultured neurons using fura-2 based microfluorometry. Anti-calreticulin increased [Ca(2+)]i in a dose dependent manner. The anti-calreticulin antibody produced a rapid transient [Ca(2+)]i peak followed by a long slowly decaying plateau. Anti-calreticulin induced extracellular Ca(2+) influx in cultured neuron cells was blocked partially by N-methyl-D-aspartate receptor (NMDAR) antagonist 2-amino-5-phosphonovaleric acid (AP5) and spider polyamine toxin JSTX-3, which is recognized as a blocker of glutamatergic nervous system. Furthermore, anti-calreticulin induced intracellular Ca(2+) desensitized NMDAR. Dual immunofluorescent staining studies revealed that NMDAR co-localized with calreticulin in the cultured neurons. Thus, the signal transduction system of NMDA might be closely concerned with the extracellular calreticulin like protein.

Amphibians↗
PubMed1998

Oligosaccharide binding characteristics of the molecular chaperones calnexin and calreticulin.

Calnexin and calreticulin are homologous molecular chaperones of the endoplasmic reticulum. Their binding to newly synthesized glycoproteins is mediated, at least in part, by a lectin site that recognizes the early N-linked oligosaccharide processing intermediate, Glc1Man9GlcNAc2. We compared the oligosaccharide binding specificities of calnexin and calreticulin in an effort to determine the basis for reported differences in their association with various glycoproteins. Using mono-, di-, and oligosaccharides to inhibit the binding of Glc1Man9GlcNAc2 to calreticulin and to a truncated, soluble form of calnexin, we show that the entire Glc alpha 1-3Man alpha 1-2Man alpha 1-2Man structure, extending from the alpha 1-3 branch point of the oligosaccharide core, is recognized by both proteins. Furthermore, analysis of the binding of monoglucosylated oligosaccharides containing progressively fewer mannose residues suggests that for both proteins the alpha 1-6 mannose branch point of the oligosaccharide core is also essential for recognition. Consistent with their essentially identical substrate specificities, calnexin and calreticulin exhibited the same relative affinities when competing for binding to the Glc1Man9GlcNAc2 oligosaccharide. Thus, differential glycoprotein binding cannot be attributed to differences in the lectin specificities or binding affinities of calnexin and calreticulin. We also examined the effects of ATP, calcium, and disulfide reduction on the lectin properties of calnexin and calreticulin. Whereas oligosaccharide binding was only slightly enhanced for both proteins in the presence of high concentrations of a number of adenosine nucleotides, removal of bound calcium abrogated oligosaccharide binding, an effect that was largely reversible upon readdition of calcium. Disulfide reduction had no effect on oligosaccharide binding by calnexin, but binding by calreticulin was inhibited by 70%. Finally, deletion mutagenesis of calnexin and calreticulin identified a central proline-rich region characterized by two tandem repeat motifs as a segment capable of binding oligosaccharide. This segment bears no sequence homology to the carbohydrate recognition domains of other lectins.

Adenosine Triphosphate↗
PubMed2001

Calreticulin is an interleukin-3-sensitive calcium-binding protein in human basophil leukocytes.

BACKGROUND: IL-3 enhances basophil histamine release upon stimulation with any known secretagogue. The molecular mechanism behind this regulation is not known, although some observations suggest that IL-3 modulates the calcium part of the signal transduction mechanism. The inhibitory action of glucocorticoids on basophils can be reversed by stimulation with IL-3. METHODS: Calcium-binding proteins in the basophil cell line KU812 were identified by two-dimensional gel electrophoresis, Calcium-overlay assay, N-terminal sequence analysis, and mass spectometry. The presence of the same proteins in purified human basophil leukocytes was established by comigration of KU812 and human basophil proteins on the two-dimensional gels. The expression of the calcium-binding proteins in the absence and presence of IL-3 and/or anti-IgE was determined by densitometric measurement of the spots on the two-dimensional gels. RESULTS: Calreticulin was identified on the two-dimensional gel of KU812 proteins. A protein with exactly the same migration pattern was found on the gels of proteins from purified human basophils. Immunoblotting with a specific antihuman calreticulin antibody confirmed that this protein was calreticulin. Subsequent analysis showed that the expression of calreticulin in the basophils is upregulated twofold upon stimulation with rhIL-3, even in doses below those needed for enhancement of histamine release. CONCLUSIONS: The expression of calreticulin in human basophil leukocytes is regulated by IL-3. Calreticulin is known to modulate IP3-dependent Ca2+ influx in different cell systems, and calreticulin overexpression inhibits steroid-induced transcriptional activation. Therefore, modulation of calreticulin expression may be one mechanism by which IL-3 exerts its effects on human basophils.

Basophils↗
PubMed1998

Induction of calreticulin expression in response to amino acid deprivation in Chinese hamster ovary cells.

The role of calreticulin as a stress-induced molecular chaperone protein of the endoplasmic reticulum is becoming more apparent. We characterize here the induction of calreticulin in response to complete amino acid deprivation in Chinese hamster ovary cells. Amino acid deprivation caused a 4-fold increase in calreticulin protein levels over a period of 4-10 h. In addition to an overall increase in protein levels, the glycosylation of calreticulin was increased. This glycosylation event was blocked by tunicamycin and was not required for the increase in calreticulin protein levels. Immunofluorescence studies localized calreticulin to the ER of CHO cells, and no significant change was observed after amino acid deprivation. Northern-blot analysis showed that calreticulin mRNA levels were increased approx. 10-fold in response to complete amino acid deprivation. The response was sensitive to actinomycin D and alpha-amanitin, implying that regulation is primarily at the level of transcription. These results are similar to the large increases in asparagine synthetase mRNA observed in response to amino acid deprivation, but the amino acid-deprivation-response element identified to be involved in asparagine synthetase induction is absent from the calreticulin promoter.

Amino Acids↗