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

Abstract

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, CRTDel52, and are linked to disease pathogenesis. Using recombinant CRT proteins, we found similar low affinity calcium binding characteristics for wild-type CRT and CRTDel52, as determined by isothermal titration calorimetry (ITC). Residues 340-349, conserved in both wild-type CRT and CRTDel52, 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 CRTDel52. 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 CRTDel52 retains at least some low affinity calcium binding capacity and that CRT-deficiency induces compensatory cellular changes that maintain ER calcium homeostasis. SummaryPathogenic CRTDel52 retains at least partial low-affinity calcium-binding capacity. Additionally, live-cell imaging and flow cytometry with ratiometric calcium probes indicate that ER and cytosolic calcium levels, as well as store-operated calcium entry (SOCE), are comparable in CRT-deficient cells rescued with either wild-type CRT or CRTDel52.