Molecular and Functional Analysis of Calcium Binding by a Cancer-linked Calreticulin Mutant

Calreticulin (CRT) is an endoplasmic reticulum (ER) chaperone with low affinity calcium binding sites in its C-terminal domain. This region is altered by somatic mutations in the CRT gene (CALR), which drive a subset of myeloproliferative neoplasms (MPN). Perturbations in ER calcium storage and signaling are reported for the MPN type I mutant, CRT_Del52, and are linked to disease pathogenesis. Using recombinant CRT proteins, we found similar low affinity calcium binding characteristics for wild-type CRT and CRT_Del52, as determined by isothermal titration calorimetry (ITC). Residues 340-349, conserved in both wild-type CRT and CRT_Del52, contribute to binding. Furthermore, ER and cytosolic calcium levels and store-operated calcium entry (SOCE) were comparable in CRT knockout (CRT-KO) cells reconstituted with wild-type CRT or CRT_Del52. Notably, the CRT-KO induces expression of multiple ER proteins known to contain low affinity calcium binding sites or regulate ER calcium levels. Overall, these findings indicate that CRT_Del52 retains at least some low affinity calcium binding capacity and that CRT-deficiency induces compensatory cellular changes that maintain ER calcium homeostasis.