Identification by mutational analysis of amino acid residues essential in the chaperone function of calreticulin
- 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.
Verfasserangaben: | Virginie Martin, Jody Groenendyk, Simone S. Steiner, Lei Guo, Monika Dabrowska, J.M. Robert Parker, Werner Müller-EsterlORCiDGND, Michal Opas, Marek MichalakORCiD |
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URN: | urn:nbn:de:hebis:30:3-762456 |
DOI: | https://doi.org/10.1074/jbc.M508302200 |
ISSN: | 0021-9258 |
Pubmed-Id: | https://pubmed.ncbi.nlm.nih.gov/16291754 |
Titel des übergeordneten Werkes (Englisch): | Journal of biological chemistry |
Verlag: | American Society for Biochemistry and Molecular Biology Publications |
Verlagsort: | Bethesda, Md |
Dokumentart: | Wissenschaftlicher Artikel |
Sprache: | Englisch |
Datum der Veröffentlichung (online): | 04.01.2021 |
Jahr der Erstveröffentlichung: | 2005 |
Veröffentlichende Institution: | Universitätsbibliothek Johann Christian Senckenberg |
Datum der Freischaltung: | 29.04.2024 |
Jahrgang: | 281.2006 |
Ausgabe / Heft: | 4 |
Seitenzahl: | 9 |
Erste Seite: | 2338 |
Letzte Seite: | 2346 |
Institute: | Medizin |
Biochemie, Chemie und Pharmazie / Biochemie und Chemie | |
DDC-Klassifikation: | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit | |
Sammlungen: | Universitätspublikationen |
Lizenz (Deutsch): | Creative Commons - CC BY - Namensnennung 4.0 International |