Abstract
Calreticulin is a ubiquitously expressed Ca2+-binding protein of the endoplasmic reticulum (ER), which inhibits DNA binding in vitro and transcriptional activation in vivo by steroid hormone receptors. Transient transfection assays were carried out to investigate the effects of different intracellular targeting of calreticulin on transactivation mediated by glucocorticoid receptor. BSC40 cells were transfected with either calreticulin expression vector (ER form of calreticulin) or calreticulin expression vector encoding calreticulin minus leader peptide, resulting in cytoplasmic localization of the recombinant protein. Transfection of BSC40 cells with calreticulin expression vector encoding the ER form of the protein led to 40-50% inhibition of the dexamethasone-sensitive stimulation of luciferase expression. However, in a similar experiment, but using the calreticulin expression vector encoding cytoplasmic calreticulin, dexamethasone-stimulated activation of the luciferase reporter gene was inhibited by only 10%. We conclude that the ER, but not cytosolic, form of calreticulin is responsible for inhibition of glucocorticoid receptor-mediated gene expression. These effects are specific to calreticulin, since overexpression of the ER lumenal proteins (BiP, ERp72, or calsequestrin) has no effect on glucocorticoid-sensitive gene expression. The N domain of calreticulin binds to the DNA binding domain of the glucocorticoid receptor in vitro; however, we show that the N+P domain of calreticulin, when synthesized without the ER signal sequence, does not inhibit glucocorticoid receptor function in vivo. Furthermore, expression of the N domain of calreticulin and the DNA binding domain of glucocorticoid receptor as fusion proteins with GAL4 in the yeast two-hybrid system revealed that calreticulin does not interact with glucocorticoid receptor under these conditions. We conclude that calreticulin and glucocorticoid receptor may not interact in vivo and that the calreticulin-dependent modulation of the glucocorticoid receptor function may therefore be due to a calreticulin-dependent signaling from the ER.
Highlights
§ Recipient of a studentship from the Alberta Heritage Foundation for Medical Research and the Heart and Stroke Foundation of Canada
In order to determine the level of calreticulin expression in BSC40 cells transiently transfected with calreticulin expression vector (10 g), we performed quantitative immunological analysis of calreticulin-transfected and control cells using antibodies raised against calreticulin (Fig. 3A)
In order to examine a role of calreticulin in regulation of the steroid-sensitive gene expression, the protein was targeted either to the lumen of the endoplasmic reticulum (ER) or to the cytoplasm followed by analysis of the effect of calreticulin on the glucocorticoid receptordependent reporter gene expression
Summary
Materials—Pipes, luciferin, glycine, coenzyme A, dithiothreitol, ATP, Nonidet P-40, Triton X-100, dexamethasone, FITC-ConA, and o-nitrophenyl--D-galactopyranoside were from Sigma. First the XhoI/SstI DNA restriction fragment, containing cDNA encoding full-length calreticulin, was excised from the pSCR vector and inserted into pBluescript to form pBCR. The synthetic oligodeoxynucleotides were phosphorylated with DNA kinase, annealed, and inserted into the NotI and StyI sites of the pBCR vector to generate the pBCR-DT plasmid This ligation reaction deleted nucleotides 1290 –1299 of the cDNA encoding calreticulin resulting in the loss of three amino acid residues, 394 –396 (Fliegel et al, 1989), and the addition of 12 amino acids encoding the DT. The XhoI/SacI fragment from pBCR-DT was cloned into XhoI/ SacI restriction sites of pSVL plasmid to generate pSCR-DT calreticulin expression vector.
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