Abstract. Calreticulin is an ubiquitous and highly conserved high capacity Ca2+-binding protein that plays a major role in Ca 2÷ storage within the lumen of the ER. Here, using L fibroblast cell lines expressing different levels of calreticulin, we show that calreticulin plays a role in the control of cell adhesiveness via regulation of expression of vinculin, a cytoskeletal protein essential for cell-substratum and cell-cell attachments. Both vinculin protein and mRNA levels are increased in cells overexpressing calreticulin and are downregulated in cells expressing reduced level of calreticulin. Abundance of actin, talin, cx 5 and 131 integrins, pp125 focal adhesion kinase, and a-catenin is not affected by the differential calreticulin expression. Overexpression of calreticulin increases both cell-substratum and cell-cell adhesiveness of L fibroblasts that, most surprisingly, establish vinculin-rich cell-ceU junctions. Upregulation of calreticulin also affects adhesion-dependent phenomena such as cell motility (which decreases) and cell spreading (which increases). Downregulation of calreticulin brings about inverse effects.Cell adhesiveness is Ca > regulated. The level of calreticulin expression, however, has no effect on either the resting cytoplasmic Ca 2+ concentration or the magnitude of FGF-induced Ca 2÷ transients. Calreticulin, however, participates in Ca 2+ homeostasis as its level of expression affects cell viability at low concentrations of extracellular Ca 2÷. Consequently, we infer that it is not the Ca 2÷ storage function of calreticulin that affects cell adhesiveness. Neither endogenous calreticulin nor overexpressed green fluorescent proteincalreticulin construct can be detected outside of the ER. Since all of the adhesion-related effects of differential calreticulin expression can be explained by its regulation of vinculin expression, we conclude that it is the ER-resident calreticulin that affects cellular adhesiveness.
Calreticulin, a Ca2؉ storage protein and chaperone in the endoplasmic reticulum, also modulates cell adhesiveness. Overexpression of calreticulin correlates with (i) increased cell adhesiveness, (ii) increased expression of N-cadherin and vinculin, and (iii) decreased protein phosphorylation on tyrosine. Among proteins that are dephosphorylated in cells that overexpress calreticulin is -catenin, a structural component of cadherindependent adhesion complexes, a member of the armadillo family of proteins and a part of the Wnt signaling pathway. We postulate that the changes in cell adhesiveness may be due to calreticulin-mediated effects on a signaling pathway from the endoplasmic reticulum, which impinges on the Wnt signaling pathway via the cadherin/catenin protein system and involves changes in the activity of protein-tyrosine kinases and/or phosphatases. The endoplasmic reticulum (ER)1 plays a critical role in the synthesis and chaperoning of membrane-associated proteins and secreted proteins (1). It is also an important site for the storage and release of Ca 2ϩ (2). In fact, the ER is the major signal transducing organelle within the cell, continuously responding to extracellular stimuli by releasing Ca 2ϩ (3,4). The ER is also sensitive to intracellular signaling; in response to a variety of stimuli signals are transduced from the ER to the cytoplasm, the plasma membrane, and the nucleus, and they affect a variety of cellular activities including Ca 2ϩ influx, protein folding, and synthesis, cholesterol synthesis, and transcription (5-8). Signaling from the extracellular space to the cell interior has long been the subject of intense research. In contrast, little is known about intracellular signaling from the ER. The lumen of the ER is a unique environment, which contains a high concentration of Ca 2ϩ -binding chaperones (2). Calreticulin, a major Ca 2ϩ -binding protein resident in the ER, is ubiquitous in eukaryotic cells (9). It functions as a molecular chaperone (10, 11) and also participates in ER-dependent Ca 2ϩ homeostasis (2, 9). Calreticulin also affects processes external to the ER, such as steroid-sensitive gene expression and cell adhesiveness (12). Although the Ca 2ϩ storage and chaperone functions of calreticulin are consistent with its localization to the lumen of the ER and with its structure, it is not obvious how it can affect cell adhesiveness at the level of the plasma membrane. To investigate the mechanisms behind calreticulindependent modulation of cell adhesiveness, we used well characterized mouse L fibroblasts differentially expressing calreticulin (13-15). Here we show that stable overexpression of fulllength, ER-targeted calreticulin correlates with an increased adhesiveness in transformed fibroblasts, such that their cohesion resembles that of epithelial cells in culture. We also show that changes in the expression of calreticulin affect the tyrosine phosphorylation of cellular proteins, including -catenin. -Catenin is a structural component of cadherin-mediated adhesion comple...
Calreticulin is a major Ca2+ binding protein in the endoplasmic reticulum of non-muscle cells. In this report we show that calreticulin protein is strongly induced by heat shock. Activation and attenuation of the heat shock transcriptional response is caused by heat shock factor that binds to 5'-flanking sequences of heat shock responsive genes, the heat shock element. The smallest stretch of DNA that shows detectable binding of heat shock factor in vitro contains a two-sequence unit nGAAnnTTCn which exists in the 5'-flanking region of calreticulin DNA (5'-gGAAccCAGcgTTC-3'). The present data provide direct evidence that calreticulin expression can be modulated by heat shock. Thus, our results strengthen the hypothesis that calreticulin, in addition to its function as a cellular Ca2+ store, is a multifunctional protein which performs at least some of its functions from the lumen of the ER.
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