Abstract
GRP94 is an endoplasmic reticulum (ER) localized glycoprotein with Ca2+ binding and protein chaperoning properties. Using a ribozyme driven by a stress-inducible promoter and targeted against grp94 mRNA, we have generated a cell line deficient in its ability to induce GRP94. The effect of the ribozyme is mediated by the cleavage of the grp94 message just downstream of the initiation codon, and not by an antisense effect, as determined by the level of intact grp94 mRNA. Unexpectedly, this cell line's ability to induce GRP78 is also impaired. Transient overexpression of recombinant human lysosomal hydrolase alpha-L-iduronidase in the ribozyme expressing cells indicates that the secretion ratio of this enzyme is reduced by about 6-fold. Additionally, the ribozyme expressing cells showed increased sensitivity to Ca2+ depletion from ER caused by either A23187 or thapsigargin, an ER-Ca(2+)-ATPase inhibitor, but not to tunicamycin. These combined results show that the induction of GRP94 may play important roles in ER to nuclear signaling, protein sorting and secretion, and specific protection against Ca2+ depletion stress.
Highlights
From the Department of Biochemistry and Molecular Biology and the Norris Cancer Center, University of Southern California School of Medicine, Los Angeles, California 90033-0800
Under a variety of stress conditions, it is coregulated with GRP78, known as the immunoglobulin heavy chain binding protein, BiP (Pelham, 1986)
We explore the use of the ribozyme technology to modulate GRP94 expression
Summary
The effect of the ribozyme is mediated by the cleavage of the grp message just downstream of the initiation codon, and not by an antisense effect, as determined by the level of intact grp mRNA This cell line's ability to induce GRP78 is impaired. The ribozyme expressing cells showed increased sensitivity to Ca2 + depletion from ER caused by either A23187 or thapsigargin, an ER-Ca2 + -ATPase inhibitor, but not to tunicamycin These combined results show that the induction of GRP94 may play important roles in ER to nuclear signaling, protein sorting and secretion, and specific protection against Ca2 + depletion stress. GRP94, a 94-kDa protein referred to as ERp99 (Mazzarella and Green, 1987) or endoplasmin (Koch et al, 1986), is an abundant ER glycoprotein It shares 50% amino acid identity to HSP90, the 90-kDa member of the heat shock protein family (Mazzarella and Green, 1987). As low affinity but high capacity Ca 2 + binding proteins, GRPs may play significant roles in protein trafficking (Gething and Sambrook, 1992) and intracellular Ca 2 + homeostasis (Brostrom et al, 1990)
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