GRP78, also known as BiP, is one of the better-characterized molecular chaperones. It has been implicated in protein folding and also calcium sequestration in the endoplasmic reticulum. When the cells are subjected to endoplasmic reticulum stress, in particular the depletion of stored calcium and/or the accumulation of abnormal proteins, the rate of transcription of grp78 is enhanced. Previous studies have shown that the core region of the rat grp78 promoter (-170 to -135), which is 95% conserved with the human grp78 core (-133 to -98), is one of the key regulatory elements. Using ligation-mediated PCR, we have found that there are specific changes in factor occupancy after stress induction and the major changes occur within a cluster of bases located in the 3' half of the gip core, whereas other regulatory elements are constitutively occupied. This inducible binding to the 3' half of the human grp78 core region is observed under diverse stress signals, suggesting a common mechanism for the gip stress response. Nonetheless, the lack of constitutive in vivo protection at this region is not due to the absence of a binding factor in nuclear extracts. Using in vitro gel mobility shift assays, we detected a constitutive binding activity which exhibits specificity and affinity to the stress-inducible region. Through sodium dodecyl sulfate-polyacrylamide gel electrophoresis size fractionation and renaturation analysis, the activity is found in polypeptides with molecular sizes of 65 to 75 kDa. After a three-step purification scheme including core affinity column chromatography, we purified p7OCORE, which is about 70 kDa in its monomeric form. The purified p7OCORE is sufficient to form a complex specific to the stress-inducible region.The 78-kDa glucose-regulated protein (GRP78), also known as the immunoglobulin heavy-chain-binding protein (BiP), is a major cellular protein localized in the lumen of the endoplasmic reticulum (ER). It is a member of the 70-kDa heat shock protein (HSP70) protein family (46) and, in an ATP-dependent manner, assists in the translocation, folding, and assembly of oligomeric proteins (10, 17). In the capacity of a molecular chaperone protein, GRP78 binds to a wide repertoire of proteins traversing through the ER. Another function of GRP78 may involve its calcium-binding capabilities. It is postulated that GRP78, as well as other ER-localized GRPs, can bind and regulate the mobilization of sequestered calcium in the ER compartment, thus playing a key role in the calcium-dependent protein folding and transport processes (36,41).The grp78 gene system provides an interesting model for studying the transcriptional activation of ER protein genes (16,17). As a gene encoding a ubiquitous ER protein performing normal physiological functions, grp78 is transcribed at a basal level constitutively in many different tissues and cell types (32). Its rate of transcription, however, is induced when the ER compartment is subjected to stress. Inducers for grp78 include tunicamycin, which blocks glycosylation, ...

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GRP78, also known as BiP, is one of the better-characterized molecular chaperones. It has been implicated in protein folding and also calcium sequestration in the endoplasmic reticulum. When the cells are subjected to endoplasmic reticulum stress, in particular the depletion of stored calcium and/or the accumulation of abnormal proteins, the rate of transcription of grp78 is enhanced. Previous studies have shown that the core region of the rat grp78 promoter (-170 to -135), which is 95% conserved with the human grp78 core (-133 to -98), is one of the key regulatory elements. Using ligation-mediated PCR, we have found that there are specific changes in factor occupancy after stress induction and the major changes occur within a cluster of bases located in the 3' half of the grp core, whereas other regulatory elements are constitutively occupied. This inducible binding to the 3' half of the human grp78 core region is observed under diverse stress signals, suggesting a common mechanism for the grp stress response. Nonetheless, the lack of constitutive in vivo protection at this region is not due to the absence of a binding factor in nuclear extracts. Using in vitro gel mobility shift assays, we detected a constitutive binding activity which exhibits specificity and affinity to the stress-inducible region. Through sodium dodecyl sulfate-polyacrylamide gel electrophoresis size fractionation and renaturation analysis, the activity is found in polypeptides with molecular sizes of 65 to 75 kDa. After a three-step purification scheme including core affinity column chromatography, we purified p70CORE, which is about 70 kDa in its monomeric form. The purified p70CORE is sufficient to form a complex specific to the stress-inducible region.