Calreticulin is an ER (endoplasmic reticulum) luminal Ca²⁺-buffering chaperone. The protein is involved in regulation of intracellular Ca²⁺ homoeostasis and ER Ca²⁺ capacity. The protein impacts on store-operated Ca²⁺ influx and influences Ca²⁺-dependent transcriptional pathways during embryonic development. Calreticulin is also involved in the folding of newly synthesized proteins and glycoproteins and, together with calnexin (an integral ER membrane chaperone similar to calreticulin) and ERp57 [ER protein of 57 kDa; a PDI (protein disulfide-isomerase)-like ER-resident protein], constitutes the ‘calreticulin/calnexin cycle’ that is responsible for folding and quality control of newly synthesized glycoproteins. In recent years, calreticulin has been implicated to play a role in many biological systems, including functions inside and outside the ER, indicating that the protein is a multi-process molecule. Regulation of Ca²⁺ homoeostasis and ER Ca²⁺ buffering by calreticulin might be the key to explain its multi-process property.

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.

To test the role of ER luminal environment in apoptosis, we generated HeLa cell lines inducible with respect to calreticulin and calnexin and investigated their sensitivity to drug-dependent apoptosis. Overexpression of calreticulin, an ER luminal protein, resulted in an increased sensitivity of the cells to both thapsigargin- and staurosporine-induced apoptosis. This correlated with an increased release of cytochrome c from the mitochondria. Overexpression of calnexin, an integral ER membrane protein, had no significant effect on drug-induced apoptosis. In contrast, calreticulin-deficient cells were significantly resistant to apoptosis and this resistance correlated with a decreased release of cytochrome c from mitochondria and low levels of caspase 3 activity. This work indicates that changes in the lumen of the ER amplify the release of cytochrome c from mitochondria, and increase caspase activity, during drug-induced apoptosis. There may be communication between the ER and mitochondria, which may involve Ca²⁺ and play an important role in conferring cell sensitivity to apoptosis. Apoptosis may depend on both the presence of external apoptosis-activating signals, and, as shown in this study, on an internal factor represented by the ER.