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

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Calreticulin (CRT) is an endoplasmic reticulum (ER) chaperone with crucial calcium-binding roles.
• Somatic mutations in the CALR gene drive myeloproliferative neoplasms (MPNs), altering CRT's C-terminal domain.
• MPN-associated CRT mutations, like CRT_Del52, are linked to ER calcium dysregulation and disease pathogenesis.

Purpose of the Study:

• Quantify the impact of CRT mutations on calcium binding affinity.
• Investigate ER and cytosolic calcium signaling in cells with wild-type versus mutant CRT.
• Determine the contribution of calcium binding to CRT's role in cellular calcium homeostasis.

Main Methods:

• Isothermal titration calorimetry (ITC) to measure calcium binding affinity of purified recombinant CRT proteins.
• Engineering cell lines to express wild-type CRT, CRT_Del52 mutant, or CRT knockout (CRT-KO).
• Quantitative measurements of ER and cytosolic calcium levels and signaling dynamics.

Main Results:

• Specific C-terminal residues (340-349) are identified as major contributors to CRT's low-affinity calcium binding.
• CRT_Del52 retains significant low-affinity calcium binding capacity.
• CRT knockout cells reconstituted with wild-type or CRT_Del52 showed similar ER/cytosolic calcium levels, but CRT-KO induced transcriptional changes in calcium signaling pathways.

Conclusions:

• The C-terminal domain of calreticulin is critical for its low-affinity calcium binding.
• CRT_Del52, an MPN-associated mutant, retains substantial calcium-binding function.
• Observed cellular calcium signaling alterations in CRT-perturbed cells may involve mechanisms beyond direct calcium binding.