Marek Michalak

Ca2+ signaling and calcium binding chaperones of the endoplasmic reticulum.

The endoplasmic reticulum is a centrally located organelle which affects virtually every cellular function. Its unique luminal environment consists of Ca(2+) binding chaperones, which are involved in protein folding, post-translational modification, Ca(2+) storage and release, and lipid synthesis and metabolism. The environment within the lumen of the endoplasmic reticulum has profound effects on endoplasmic reticulum function and signaling, including apoptosis, stress responses, organogenesis, and transcriptional activity. Calreticulin, a major Ca(2+) binding (storage) chaperone in the endoplasmic reticulum, is a key component of the calreticulin/calnexin cycle which is responsible for the folding of newly synthesized proteins and glycoproteins and for quality control pathways in the endoplasmic reticulum. The function of calreticulin, calnexin and other endoplasmic reticulum proteins is affected by continuous fluctuations in the concentration of Ca(2+) in the endoplasmic reticulum. Thus, changes in Ca(2+) concentration may play a signaling role in the lumen of the endoplasmic reticulum as well as in the cytosol. Recent studies on calreticulin-deficient and transgenic mice have revealed that calreticulin and the endoplasmic reticulum may be upstream regulators in the Ca(2+)-dependent pathways that control cellular differentiation and/or organ development.

The Anti-adhesive Activity of Thrombospondin Is Mediated by the N-terminal Domain of Cell Surface Calreticulin

Thrombospondin (TSP) induces reorganization of the actin cytoskeleton and restructuring of focal adhesions through binding of amino acids (aa) 17–35 (hep I peptide) of thrombospondin to a cell surface form of calreticulin (CRT). In this report we provide further evidence for the involvement of calreticulin in thrombospondin signaling and characterize thrombospondin-calreticulin interactions. Wild type but notcrt −/− cells respond to hep I/TSP. Responsiveness can be restored by incubation of cells with exogenous calreticulin or by transfection with calreticulin. Thrombospondin forms complexes with the CRT-N-domain that are enhanced by physiologic levels of calcium and zinc. Consistent with thrombospondin/CRT-N-domain binding, only the CRT-N-domain blocks hep I- and thrombospondin-stimulated focal adhesion disassembly. A series of glutathione S-transferase-N-domain mutants were used to map the sequence within the N-domain that interacts with TSP/hep I. A construct containing aa 1–43 but not a construct of aa 1–31 supported thrombospondin binding and focal adhesion disassembly. A series of overlapping peptides were used to further map the thrombospondin-binding site. Peptides spanning aa 19–36 (RWIESKHKSDFGKFVLSS) blocked hep I-stimulated focal adhesion disassembly, indicating that the TSP/hep I-binding site is located to this sequence in calreticulin. A mutant fusion protein lacking aa 19–36 (glutathione S-transferase-CRTΔhep I) failed to restore responsiveness to hep I in crt −/−cells, bind thrombospondin, or competitively block focal adhesion disassembly, providing evidence for the role of this calreticulin sequence in mediating thrombospondin signaling.

Is all of the endoplasmic reticulum created equal? The effects of the heterogeneous distribution of endoplasmic reticulum Ca2+-handling proteins

The endoplasmic reticulum is a heterogeneous compartment with respect to the distribution of its Ca2+-handling proteins, namely the Ca2+-binding proteins, the Ca2+ pumps and the Ca2+ release channels. The nonuniform distribution of these proteins may explain the functional heterogeneity of the endoplasmic reticulum, such as the generation of spatially complex Ca2+ signals, Ca2+ homeostasis, and protein folding and quality control.

Identification of an N-domain Histidine Essential for Chaperone Function in Calreticulin

Calreticulin is an endoplasmic reticulum (ER) luminal Ca2+-binding chaperone involved in folding of newly synthesized glycoproteins via the “calreticulin-calnexin cycle.” We reconstituted ER of calreticulin-deficient cells with N-terminal histidine (His25, His82, His128, and His153) calreticulin mutants and carried out a functional analysis. In crt-/- cells bradykinin-dependent Ca2+ release is altered, and the reestablishment of bradykinin-dependent Ca2+ release was used as a marker for calreticulin function. Bradykinin-dependent Ca2+ release from the ER was rescued by wild type calreticulin and by the His25, His82, or His128 mutant but not by the His153 mutant. Wild type calreticulin and the His25, His82, and His128 mutants all prevented in vitro thermal aggregation of malate dehydrogenase and IgY, whereas the His153 mutant did not, indicating that His153 chaperone function was impaired. Biophysical analysis of His153 mutant revealed that conformation changes in calreticulin mutant may be responsible for the loss of its chaperone activity. We conclude that mutation of a single amino acid residue in calreticulin has devastating consequences for its chaperone function, indicating that mutations in chaperones may play a significant role in protein folding disorders.

Calreticulin in cardiac development and pathology

Calreticulin is a Ca(2+) binding/storage chaperone resident in the lumen of endoplasmic reticulum (ER). The protein is an important component of the calreticulin/calnexin cycle and the quality control pathways in the ER. In mice, calreticulin deficiency is lethal due to impaired cardiac development. This is not surprising because the protein is expressed at high level at early stages of cardiac development. Overexpression of the protein in developing and postnatal heart leads to bradycardia, complete heart block and sudden death. Recent studies on calreticulin-deficient and transgenic mice revealed that the protein is a key upstream regulator of calcineurin-dependent pathways during cardiac development. Calreticulin and ER may play important role in cardiac development and postnatal pathologies.

Compromised calnexin function in calreticulin-deficient cells.

Calnexin and calreticulin are molecular chaperones, which are involved in the protein folding, assembly, and retention/retrieval. We know that calreticulin-deficiency is lethal in utero, but do not understand the contribution of chaperone function to this phenotype. Here we studied protein folding and chaperone function of calnexin in the absence of calreticulin. We show that protein folding is accelerated and quality control is compromised in calreticulin-deficient cells. Calnexin-substrate association is severely reduced, leading to accumulation of unfolded proteins and a triggering of the unfolded protein response (UPR). PERK and Ire1alpha and eIF2alpha are also activated in calreticulin-deficient cells. We show that the absence of calreticulin can have devastating effects on the function of the others, compromising overall quality control of the secretory pathway and activating UPR-dependent pathways.

Ultrastructural analysis of development of myocardium in calreticulin-deficient mice

BackgroundCalreticulin is a Ca2+ binding chaperone of the endoplasmic reticulum which influences gene expression and cell adhesion. The levels of both vinculin and N-cadherin are induced by calreticulin expression, which play important roles in cell adhesiveness. Cardiac development is strictly dependent upon the ability of cells to adhere to their substratum and to communicate with their neighbours.ResultsWe show here that the levels of N-cadherin are downregulated in calreticulin-deficient mouse embryonic hearts, which may lead to the disarray and wavy appearance of myofibrils in these mice, which we detected at all investigated stages of cardiac development. Calreticulin wild type mice exhibited straight, thick and abundant myofibrils, which were in stark contrast to the thin, less numerous, disorganized myofibrils of the calreticulin-deficient hearts. Interestingly, these major differences were only detected in the developing ventricles while the atria of both calreticulin phenotypes were similar in appearance at all developmental stages. Glycogen also accumulated in the ventricles of calreticulin-deficient mice, indicating an abnormality in cardiomyocyte metabolism.ConclusionCalreticulin is temporarily expressed during heart development where it is required for proper myofibrillogenesis. We postulate that calreticulin be considered as a novel cardiac fetal gene.

Calreticulin in the heart

Calreticulin is a Ca2+ binding/storage chaperone resident protein of the endoplasmic reticulum. This protein plays a key role in the calreticulin/calnexin cycle and the quality control pathways in the endoplasmic reticulum. Calreticulin deficiency is lethal due to impaired cardiac development. However, over-expression of the protein in developing and postnatal heart leads to bradycardia, complete heart block and sudden death. Ultrastructural evidence indicates that the deficiency associated with the absence of calreticulin in the heart may be due to a defect in the development of the contractile apparatus and/or a defect in development of the conductive system as well as a metabolic abnormality. Collectively, we postulate that calreticulin and endoplasmic reticulum plays an important role in cardiac development and postnatal pathologies. (Mol Cell Biochem 263: 137–142, 2004)

Endoplasmic reticulum in the heart, a forgotten organelle?

Our hypothesis is that sarcoplasmic and endoplasmic reticulum Ca2+ stores may be functionally distinct compartments in cardiomyocytes. Sarcoplasmic reticulum Ca2+ store is responsible for control of excitation-contraction coupling whereas endoplasmic reticulum compartment may provide Ca2+ for housekeeping and transcriptional functions.

Introduction to Calreticulin

Over 30 years ago calreticulin, then known as the high affinity calcium binding protein (HACBP), was identified and purified from isolated skeletal muscle sarcoplasmic reticulum vesicles.1,2 Surprisingly, it took almost 20 years to realize that the protein is a major component of the endoplasmic reticulum (ER) in non-muscle cells3. However, today, calreticulin is considered one of the best markers for the ER. In 1989 isolation of cDNA encoding calreticulin was reported3,4 and provided a useful tool to carry out biochemical, molecular biological and cell biological studies of the protein. This led to a number of advances on the structure and function of calreticulin. The recent application of calreticulin gene deletion in mice,5,6 C. elegans 7,8 and in Dictyostelium 9 have led to exciting discoveries of the role of calreticulin in organogenesis and several pathologies. Moreover, long awaited structural studies on calreticulin10 and calnexin11 provided the first insights into 3D structure of ER luminal proteins and their domains. This will have a tremendous impact on the future studies on these and other ER chaperones.