Identification by mutational analysis of amino acid residues essential in the chaperone function of calreticulin

Abstract

Calreticulin is a Ca2+-binding chaperone that resides in the lumen of the endoplasmic reticulum and is involved in the regulation of intracellular Ca2+ homeostasis and in the folding of newly synthesized glycoproteins. In this study, we have used site-specific mutagenes is to map amino acid residues that are critical in calreticulin function. We have focused on two cysteine residues (Cys88 and Cys120), which form a disulfide bridge in the N-terminal domain of calreticulin, on a tryptophan residue located in the carbohydrate binding site (Trp302), and on certain residues located at the tip of the "hairpin-like" P-domain of the protein (Glu238, Glu239, Asp 241, Glu243, and Trp244). Calreticulin mutants were expressed in crt-/- fibroblasts, and bradykinin-dependent Ca2+ release was measured as a marker of calreticulin function. Bradykinin-dependent Ca2+ release from the endoplasmic reticulum was rescued by wild-type calreticulin and by the Glu238, Glu 239, Asp241, and Glu243 mutants. The Cys 88 and Cys120 mutants rescued the calreticulin-deficient phenotype only partially (∼40%), and the Trp244 and Trp 302 mutants did not rescue it at all. We identified four amino acid residues (Glu239, Asp241, Glu243, and Trp 244) at the hairpin tip of the P-domain that are critical in the formation of a complex between ERp57 and calreticulin. Although the Glu 239, Asp241, and Glu243 mutants did not bind ERp57 efficiently, they fully restored bradykinin-dependent Ca2+ release in crt-/- cells. This indicates that binding of ERp57 to calreticulin may not be critical for the chaperone function of calreticulin with respect to the bradykinin receptor. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.