Effects of calreticulin deficiency and myeloproliferative neoplasm (MPN)-linked mutation on cellular calcium signaling.

Calreticulin (CRT) is an endoplasmic reticulum (ER) chaperone containing multiple low affinity calcium-binding sites in its acidic C-terminal domain. Somatic mutations of the CRT gene (CALR) that are drivers of a subset of myeloproliferative neoplasms (MPN) alter the C-terminal domain. Perturbations in ER calcium storage and signaling are reported for an MPN type I mutant, CRT(Del52), and have been linked to disease pathogenesis. The effects of the mutations on calcium binding are, however, yet to be quantified. Using recombinant purified proteins, we found that residues 340-349 in the CRT C-terminal domain, conserved in both the wild-type CRT and CRT(Del52), are major contributors to low affinity calcium binding measured using isothermal titration calorimetry (ITC). Quantitative measurements in various engineered cell lines also indicate overall similarities in ER and cytosolic calcium levels and signals in CRT knockout (CRT-KO) cells reconstituted with wild-type CRT or CRT(Del52). Notably, the CRT-KO induces transcriptional changes to various genes in the cellular calcium signaling pathways. Thus, CRT(Del52) retains at least some low affinity calcium binding sites, and any measured effects of ER and cytosolic calcium signal changes in cells with CRT perturbations may not be solely determined by its calcium binding functions.