Calreticulin And Its Functional Diversity

Structure and Molecular Features of Calreticulin

Two types of CBPs are associated with the endoplasmic reticulum (ER), namely reticulocalbin (an EF-hand CBP whose localisation and function are discussed in Chapter 12 and calreticulin). Calreticulin is a non-EF-hand CBP that is ubiquitously associated with the ER of a variety of tissues. It is regarded as the nonmuscle equivalent of calsequestrin, which is associated with the sarcoplasmic reticulum (SR). Calreticulin, which was identified as a CBP and isolated from SR (Ostwald and MacLennan, 1974), is believed to be a homologue of calnexin, which is a 64-kDa transmembrane protein (Hammond and Helenius, 1995). It is a highly conserved protein. Calreticulin obtained from rat liver has a molecular size of 60 kDa. In some cell-free systems a 62-kDa protein has been detected, which is probably processed into 60-kDa calreticulin (Denning et al. 1997). Hershberger and Tuan (1998) have cloned a full-length cDNA for calreticulin from mouse trophoblast coding for a 57-kDa protein showing a high degree of sequence homology to calreticulin. Yamamoto and Nakamura (1996) isolated and sequenced the ER-associated calnexin from Rana rugosa, which shared 77% sequence homology with calreticulin. Two distinct iso-forms of calretinin have been isolated from spinach leaves and from the pollen of Liriodendron tulipifera L. and Ginkgo biloba L. (Navazio et al. 1995, 1998; Nardi et al. 1998). These are reported to show very low sequence homology with animal calreticulin. However, maize calreticulin is said to be a 48-kDa protein (as deduced from its cDNA sequence), highly acidic in nature, sharing 77 to 92% sequence homology with other plant calreticulins, and have approximately 50% homology with animal calreticulin (Dresselhaus et al. 1996).

Calreticulin consists of 400 amino acid residues. It has three distinct domains: an N-terminal domain containing 180 amino acid residues, a C-terminal domain with acidic residues and lysines, and a middle domain (P-domain) that is composed of three repeats of a 17 amino acid motif. The C-domain binds calcium with high capacity but low affinity. In contrast, the P-domain binds calcium with low capacity but high affinity. Overall, calreticulin may be seen as a high-capacity but low-affinity CBP.

The mouse calreticulin gene spans 4.2 kbp of genomic DNA and contains nine exons and eight introns (Waser et al. 1997). In the mouse the gene is located on chromosome 8 (Rooke et al. 1997). It should be pointed out here that P. Lin et al. (1998) state that nucleobindin, a mammalian protein showing a high degree of homology to calreticulin, is an EF-hand CBP. This protein is found in the cytosol and is associated with membranes, in the latter case mainly with the luminal surface of Golgi membranes.

Regulation of Calreticulin Expression

The expression of calreticulin appears to be regulated by intracellular calcium levels. The promoter region of the calreticulin gene contains elements responsive to the calcium ionophore A23187 and to agents such as thapsigargin, which can raise intracellular calcium levels by releasing calcium from intracellular stores. Both A23187 and thapsi-gargin have been shown to increase transcription of the calreticulin gene. It is not affected by changes in extracellular or cytoplasmic levels of calcium. This has led to the suggestion that loss of calcium levels in the intracellular stores could induce gene transcription (Waser et al. 1997). Calreticulin gene expression is also up-regulated by other stimuli such as heat shock and heavy metals like zinc and cadmium (Nguyen et al. 1996; Szewczenko-Pawlikowski et al. 1997). A number of heavy metal ions, e.g., Ni2+, Zn2+, Cu2+, and La3+, stimulate the release of calcium from intracellular stores (McNulty and Taylor, 1999). Hyperthermia also raises intracellular calcium levels by calcium mobilisation from intracellular stores as well as by simulating its influx into the cell (Itagaki et al. 1998). Therefore, the up-regulation of calreticulin gene expression might, again, be mediated by the stress response of depletion of calcium held in the intracellular stores. This is in sharp contrast with the effects of hyperthermia and thapsigargin on the expression of S100A4. Both treatments have been shown to down-regulate S100A4 expression (Parker and Sherbet, 1992; Albertazzi et al. 1998a). This might suggest a different mode of regulation from that of calreticulin.

The thesis that the calreticulin gene is regulated by androgen has been advocated vigorously by N. Zhu et al. (1998). This is based on the finding that androgen ablation down-regulates and its restoration up-regulates both calreticulin mRNA and protein expression in the prostate. Calreticulin is expressed at higher levels in the prostate than in the seminal vesicle and other organs and muscle. The regulation by androgen occurs only in the prostate and seminal vesicles. In vitro, the induction of calreticulin by androgen is not inhibited by inhibitors of protein synthesis, hence the suggestion that the calreticulin gene might be regulated by androgen. Furthermore, in androgen-sensitive LNCaP prostate cancer cell lines, androgen is able to block apoptosis induced by the calcium ionophore A23187. This effect of androgen can be negated by antisense calreticulin oligonucleotides (N. Zhu et al. 1999).

Phosphorylation of Calreticulin

Calreticulin phosphorylation occurs under different physiological conditions. However, it is unclear at present whether this is a physiological mechanism by which calreticulin activity is regulated. A phosphorylated form of calreticulin is detectable in cells infected with rubella virus (RV). It has been reported that the binding of phosphorylated calreticulin to the 3'-end of RV genomic RNA is necessary for initiating RNA replication (Singh et al. 1994). Phosphorylation is a requirement also in the binding of calreticulin to Leischmania donovani RNA (Joshi et al. 1996). Calreticulin from spinach leaves is phosphorylated by protein kinase (casein kinase) CK2, but those of animal origin are not substrates for this kinase. It appears that this is due to structural differences between the two types of calreticulin (Baldan et al. 1996). CK2 was identified as an ER-associated kinase involved in the phosphorylation of calreticulin (Ou et al. 1992). CK2 might be localised in the lumen of the ER (N.G. Chen et al. 1996). The in vitro phosphorylation of calreticulin and other proteins in plasma membrane-enriched fractions has been attributed also to other protein kinases susceptible to inhibition by the PKC inhibitor staurosporine (Droil-lard et al. 1997). CK2 also phosphorylates calnexin in vitro as well as in vivo. Serine residues that occur in the C-terminal half of the cytosolic domain of calnexin were exclusively phosphorylated (Wong et al. 1998). Sphingosine-dependent kinases

(SDKs) are another kinase variety that have been shown recently to phosphorylate calreticulin. SDK1 has been found specifically to phosphorylate calreticulin and protein disulphide isomerase (PDI) (Megidish et al. 1999). Heat shock proteins are also substrates of SDKs. Calreticulin, heat shock proteins, and PDI have a common function as molecular chaperones. Furthermore, calreticulin and PDI both occur in the ER. Therefore, these observations might be the closest researchers have come to suggesting a relationship between calreticulin function and phosphorylation. In spite of these various findings, how phosphorylation of these molecules regulates their participation in their apparently diverse physiological functions is yet to be elucidated. At present, no changes in the state of calreticulin phosphorylation have been found to correlate with a specific function. Furthermore, it has been suggested that calreticulin could occur in a constitutively phosphorylated form (Cala, 1999). This would deny the process of phosphorylation any regulatory control over calreticulin function. Nevertheless, the induction of apoptosis of HL-60 cells by geranylge-raniol has been shown to be accompanied by a decrease in calreticulin as well as a decrease in the phosphorylation of another protein of 36 kDa molecular size. These reductions occur prior to DNA fragmentation (Nakajo et al. 1996). The occurrence of these events in parallel suggests that calreticulin may influence the phosphorylation of the 36-kDa protein.

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