Calreticulin is a Ca2+-binding chaperone in the endoplasmic reticulum (ER), and calreticulin gene knockout is embryonic lethal. Here, we used calreticulin-deficient mouse embryonic fibroblasts to examine the function of calreticulin as a regulator of Ca2+ homeostasis. In cells without calreticulin, the ER has a lower capacity for Ca2+ storage, although the free ER luminal Ca2+ concentration is unchanged. Calreticulin-deficient cells show inhibited Ca2+ release in response to bradykinin, yet they release Ca2+ upon direct activation with the inositol 1,4,5-trisphosphate (InsP3). These cells fail to produce a measurable level of InsP3 upon stimulation with bradykinin, likely because the binding of bradykinin to its cell surface receptor is impaired. Bradykinin binding and bradykinin-induced Ca2+ release are both restored by expression of full-length calreticulin and the N + P domain of the protein. Expression of the P + C domain of calreticulin does not affect bradykinin-induced Ca2+ release but restores the ER Ca2+ storage capacity. Our results indicate that calreticulin may play a role in folding of the bradykinin receptor, which affects its ability to initiate InsP3-dependent Ca2+ release in calreticulin-deficient cells. We concluded that the C domain of calreticulin plays a role in Ca2+ storage and that the N domain may participate in its chaperone functions.

Calreticulin is an endoplasmic reticulum (ER) luminalmutant 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.The endoplasmic reticulum (ER) 1 plays an essential role in a variety of cellular processes, including Ca 2ϩ homeostasis, protein and lipid synthesis, and post-translational modification and folding of membrane-associated and secreted proteins (1). The ER ensures that only correctly folded proteins proceed through the secretory pathway and directs misfolded proteins to ER-associated degradation (2, 3). The lumen of the ER is a dynamic environment that contains numerous molecular chaperones and Ca 2ϩ -binding proteins that are designed for these tasks. Molecular chaperones are proteins that bind to misfolded/unfolded proteins in a transient manner to assist in their folding.Calreticulin is a Ca 2ϩ -binding chaperone that resides in the lumen of the ER and is involved in modulation of Ca 2ϩ homeostasis and in the folding of newly synthesized glycoproteins via the "calreticulin-calnexin cycle" (4 -7). Calreticulin and calnexin are both ER lectins, which bind transiently to virtually all newly synthesized glycoproteins (5-7). Chaperone-assisted protein folding has been studied extensively using Escherichia coli GroEL heat shock proteins, which are cytoplasmic (8). Numerous studies have been carried out on ER-associated chaperones (2-7); yet, the molecular features of calreticulin that confer its chaperone function have not yet been determined (7).Three distinct structural domains have been identified in calreticulin: the amino-terminal, globular N-domain; the central P-domain; and the carboxyl-terminal C-domain (7). NMR (9), modeling (10), and biochemical studies (11) indicate that the globular N-domain and the "extended arm" P-domain of calreticulin may form a functional protein-folding unit (10). This region of calreticulin contains a Zn 2ϩ binding site and one disulfide bond, and it may also bind ATP (12)(13)(14). When calreticulin binds Zn 2ϩ , it undergoes dramatic conformational changes (15). Chemical modification of calreticulin has revealed that four histidines located in the N-domain of the protein (His 25 , His 82 , His 128 , and His 153 ) are involved in the Zn 2ϩ binding (12). The Zn 2ϩ -dependent conformational change in calreticulin affects its ability to bind to unfolded protein/ glycoprotein substrates in vitro (16), suggesting that conformational changes in calreticulin may modify its chaperone function. The role of the Zn 2ϩ binding histidine residues in calreticulin function is not known.Calreticulin deficiency is embryonic lethal, and cells derived from calreticulin knockout embryos have impaired Ca 2ϩ homeostasis and compromised protein folding and quality control (11,17). The availability of calret...

The endoplasmic reticulum is a heterogeneous compartment with respect to the distribution of its Ca2+-handling proteins, namely the Ca2+-binding proteins, the Ca2+ pumps and the Ca2+ release channels. The nonuniform distribution of these proteins may explain the functional heterogeneity of the endoplasmic reticulum, such as the generation of spatially complex Ca2+ signals, Ca2+ homeostasis, and protein folding and quality control.

Calreticulin is an endoplasmic reticulum Ca 2؉ -storage protein, which influences gene expression and cell adhesion. In this study, we show that calreticulin induces fibronectin gene expression and matrix deposition, leading to differences in cell spreading and focal adhesion formation in cells differentially expressing calreticulin. We further show that these effects of calreticulin occur via a c-Src-regulated pathway and that c-Src activity is inversely related to calreticulin abundance. Since c-Src is an important regulator of focal contact turnover, we investigated the effect of c-Src inhibition on cells differentially expressing calreticulin. Inhibition of c-Src rescued the poorly adhesive phenotype of the calreticulin-underexpressing cells in that they became well spread, commenced formation of numerous focal contacts, and deposited a rich fibronectin matrix. Importantly, we show that c-Src activity is dependent on releasable Ca 2؉ from the endoplasmic reticulum, thus implicating Ca 2؉ -sensitive pathways that are affected by calreticulin in cellsubstratum adhesion. We propose that calreticulin affects fibronectin synthesis and matrix assembly via the regulation of fibronectin gene expression. In parallel, calcium-dependent effects of calreticulin on c-Src activity influence the formation and/or stability of focal contacts, which are instrumental in matrix assembly and remodeling.Calreticulin, a Ca 2ϩ -binding protein of the endoplasmic reticulum (ER), 3 has been shown to be involved in a great number of cellular processes (1). It is an important chaperone, working in conjunction with calnexin and protein disulfide isomerase; it affects intracellular Ca 2ϩ homeostasis via its Ca 2ϩ storage capacity and its effects on both the SERCA pumps and inositol 1,4,5-trisphosphate receptors (2, 3). Calreticulin has been shown to affect cell adhesion via the induction of vinculin and N-cadherin expression and its involvement in ␤-catenin-associated pathways (4, 5). Finally, calreticulin overexpression causes a decrease in total cellular tyrosine phosphorylation levels (5, 6). Fibronectin is a large glycoprotein which is secreted by the cell into the extracellular matrix (ECM) as a soluble dimer that, via cellular interactions, is deposited as a fibrillar meshwork that is bound to the surface of cells. Fibronectin affects the formation and the stability of cell-substratum adhesions, and conversely, cell-substratum adhesions may affect fibronectin matrix deposition (7,8). Fibronectin assembly cannot proceed without the presence of cells (9), which implies intracellular inside-out signaling pathways that are crucial for fibronectin fibrillogenesis (10). Fibronectin matrices are essential for embryonic development, wound healing, and tumorigenesis (11) and as such are both spatially and temporally regulated (11). Thus, it is crucial to discern the mechanisms by which a fibronectin matrix is deposited and regulated.Fibronectin is bound to and regulated by cells at specific sites of cell-substratum adhesions that serve to l...

BackgroundCalreticulin, a Ca2+-buffering chaperone of the endoplasmic reticulum, is highly expressed in the embryonic heart and is essential for cardiac development. After birth, the calreticulin gene is sharply down regulated in the heart, and thus, adult hearts have negligible levels of calreticulin. In this study we tested the role of calreticulin in the adult heart.Methodology/Principal FindingsWe generated an inducible transgenic mouse in which calreticulin is targeted to the cardiac tissue using a Cre/loxP system and can be up-regulated in adult hearts. Echocardiography analysis of hearts from transgenic mice expressing calreticulin revealed impaired left ventricular systolic and diastolic function and impaired mitral valve function. There was altered expression of Ca2+ signaling molecules and the gap junction proteins, Connexin 43 and 45. Sarcoplasmic reticulum associated Ca2+-handling proteins (including the cardiac ryanodine receptor, sarco/endoplasmic reticulum Ca2+-ATPase, and cardiac calsequestrin) were down-regulated in the transgenic hearts with increased expression of calreticulin.Conclusions/SignificanceWe show that in adult heart, up-regulated expression of calreticulin induces cardiomyopathy in vivo leading to heart failure. This is due to an alternation in changes in a subset of Ca2+ handling genes, gap junction components and left ventricle remodeling.