Generation of a mammalian cell line deficient in glucose-regulated protein stress induction through targeted ribozyme driven by a stress-inducible promoter.
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.
Little
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Department of Biochemistry and Molecular Biology, University of Southern California School of Medicine, Los Angeles 90033-0800, USA.
Lee
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D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis* / Q000172:deficiency / Q000235:genetics; D006360:Heat-Shock Proteins / Q000096:biosynthesis*; D007068:Iduronidase / Q000096:biosynthesis; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000172:deficiency / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D016337:RNA, Catalytic / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D014162:Transfection
D000091342:Endoplasmic Reticulum Chaperone BiP; D018840:HSP70 Heat-Shock Proteins; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger; C018551:glucose-regulated proteins; D007068:Iduronidase; D002118:Calcium