Interaction of Hsp90 with the nascent form of the mutant epidermal growth factor receptor EGFRvIII.
EGFRvIII is a mutant epidermal growth factor that promotes aggressive growth of glioblastomas. We made a plasmid that directed the expression of an EGFRvIII with three copies of the Flag epitope at its amino terminus. Flag-tagged EGFRvIII was expressed at the same levels as unmodified EGFRvIII, and showed the same subcellular localization. However, the Flag epitope could only be detected on EGFRvIII present in the endoplasmic reticulum; the epitope was covalently modified during trafficking of the receptor through the Golgi so that it was no longer recognized by anti-Flag antibody. This property was exploited to selectively purify nascent EGFRvIII from glioblastoma cells. Nascent EGFRvIII was found to copurify with a set of other proteins, identified by mass spectrometry as the two endoplasmic reticulum chaperones Grp94 and BiP, and the two cytosolic chaperones Hsc70 and Hsp90. The Hsp90-associated chaperone Cdc37 also co-purified with EGFRvIII, suggesting that Hsp90 binds EGFRvIII as a complex with this protein. Geldanamycin and radicicol, two chemically unrelated inhibitors of Hsp90, decreased the expression of EGFRvIII in glioblastoma cells. These studies show that nascent EGFRvIII in the endoplasmic reticulum associates with Hsp90 and Cdc37, and that the Hsp90 association is necessary to maintain expression of EGFRvIII.
Lavictoire
Sylvie J
SJ
Ottawa Regional Cancer Centre, Centre for Cancer Therapeutics, Ottawa, Ontario K1H 1C4, Canada.
Parolin
Doris A E
DA
Klimowicz
Alex C
AC
Kelly
John F
JF
Lorimer
Ian A J
IA
D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D018833:Chaperonins; D029721:Drosophila Proteins; D004721:Endoplasmic Reticulum / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D018832:Molecular Chaperones / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D015398:Signal Transduction
C498362:CDC37 protein, human; D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; D018841:HSP90 Heat-Shock Proteins; D018832:Molecular Chaperones; C410723:epidermal growth factor receptor VIII; D066246:ErbB Receptors; D018833:Chaperonins