The lectin-dependent function of mutant CALR is required for cytokine-independent growth, whereas its chaperone and polypeptide binding functionalities are dispensable. (A) Schema depicting mutations introduced into the lectin (dark red), polypeptide binding (blue), and chaperone (light green) domains of mutant CALR. (B) Immunoblotting of FLAG immunoprecipitated proteins from 293T cells cotransfected with wild-type CALR, mutant CALR, or mutant CALR lectin-, chaperone-, and polypeptide binding-deficient variants demonstrates that binding between mutant CALR and MPL is lost when residues required for lectin binding are mutated, but binding is retained when residues critical for CALR chaperone and polypeptide binding functionality are mutated. (C) Growth curves in Ba/F3-MPL cells expressing wild-type CALR, mutant CALR, or mutant CALR lectin- (left), chaperone- (center), and polypeptide binding-deficient (right) variants demonstrates that mutant CALR loses its ability to drive cytokine-independent growth when residues required for lectin binding are mutated but retains its ability to drive cytokine-independent growth in when residues critical for CALR chaperone and polypeptide-binding functionality are mutated. (D) In vitro binding assay between purified recombinant CALR^WT or CALR^MUT and purified recombinant ERp57 demonstrates that CALR^WT binds directly to ERp57 but CALR^MUT does not. (E) In vitro binding assay between purified recombinant CALR^WT, CALR^MUT, CALR^MUT-D135L, or CALR^MUT-D317A demonstrates that CALR^MUT binds directly to MPL but not CALR^WT or altered CALR^MUT variants. (F) FLAG pulldown in 293T cells coexpressing CALR^MUT and MPL glycosylation mutants (2xNQ = N117/178Q; 4xNQ = N117/178/298/358Q), shows CALR^MUT binding to MPL-2xNQ but not to MPL-4xNQ.