title: 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 mutagenesis to map amino acid residues that are critical in calreticulin function. We have focused on two cysteine residues (Cys(88) and Cys(120)), which form a disulfide bridge in the N-terminal domain of calreticulin, on a tryptophan residue located in the carbohydrate binding site (Trp(302)), and on certain residues located at the tip of the "hairpin-like" P-domain of the protein (Glu(238), Glu(239), Asp(241), Glu(243), and Trp(244)). 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 Glu(238), Glu(239), Asp(241), and Glu(243) mutants. The Cys(88) and Cys(120) mutants rescued the calreticulin-deficient phenotype only partially ( approximately 40%), and the Trp(244) and Trp(302) mutants did not rescue it at all. We identified four amino acid residues (Glu(239), Asp(241), Glu(243), 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), Asp(241), and Glu(243) 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.
authors.affiliation: Membrane Protein Research Group and the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Virginie
authors.initials: V
authors.lastname: Martin
authors.forename: Jody
authors.initials: J
authors.lastname: Groenendyk
authors.forename: Simone S
authors.initials: SS
authors.lastname: Steiner
authors.forename: Lei
authors.initials: L
authors.lastname: Guo
authors.forename: Monika
authors.initials: M
authors.lastname: Dabrowska
authors.forename: J M Robert
authors.initials: JM
authors.lastname: Parker
authors.forename: Werner
authors.initials: W
authors.lastname: Müller-Esterl
authors.forename: Michal
authors.initials: M
authors.lastname: Opas
authors.forename: Marek
authors.initials: M
authors.lastname: Michalak
mesh_terms: D000818:Animals; D015153:Blotting, Western; D001920:Bradykinin / Q000737:chemistry; D002118:Calcium / Q000737:chemistry; D037282:Calreticulin / Q000737:chemistry* / Q000235:genetics; D002460:Cell Line; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D006360:Heat-Shock Proteins / Q000737:chemistry; D051379:Mice; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000378:metabolism*; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D011485:Protein Binding; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D020349:Surface Plasmon Resonance; D014357:Trypsin / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry
chemical_list: D037282:Calreticulin; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D014364:Tryptophan; D014357:Trypsin; C513195:Pdia3 protein, mouse; D019704:Protein Disulfide-Isomerases; D003545:Cysteine; D001920:Bradykinin; D002118:Calcium