Abstract
Previous studies showed that 78-kDa glucose-regulated protein (GRP78) is known to form complexes with heterologous proteins that are processed through the ER, as well as HSP70, a member of the HSP70 family like GRP78, have been reported that form large multimeric structures thought to be important for chaperone activity. In order to delineate the possibility that GRP78 form oligomers, we used purified recombinant GRP78 from Escherihia coli, which indicated a single band in SDS-polyacrylamide gel electrophoresis, showed the large mobility shift of recombinant GRP78 after glutaraldehyde crosslinking and multiple forms in HPLC gel filtration. These results indicate that the protein may exist in multiple species. The relative amount of multiple species was dependent upon ATP: when recombinant GRP78 was incubated with ATP, there was a conversion of the higher molecular weight species to the monomer and dimer forms. Exposure of H460 Non-Small Cell Lung Cancer cells to 5 nM thapsigargin (TG), a sarcoendoplasmic Ca2+-ATPases inhibitor, leads to an immediate suppression of general protein synthesis followed by an enhanced synthesis of the 78-kDa glucose-regulated protein, GRP78. By using coomassie brilliant blue stain and autoradiographic analysis with [35S]methionine-labeled of the two dimensional Blue native / SDS polyacrylamide gel electrophoresis (2D BN-/ SDS-PAGE) revealed that the total GRP78 and the newly synthesized GRP78 existed in different multi-protein complex, respectively. Thus, although we have no evidence that the role of various oligomeric species in multi-protein complex, these observations raised the possibility that the different multiple species of GRP78 might be involved in regulating its activity in the ER and that the role of ATP in dissociating GRP78-protein complexes may be to disaggregate GRP78 higher molecular weight oligomers.