Proof that hsp70 is required for assembly of the glucocorticoid receptor into a heterocomplex with hsp90.
Incubation of immunopurified glucocorticoid receptor with rabbit reticulocyte lysate forms a complex between the receptor and hsp90, with simultaneous conversion of the receptor from a non-steroid binding but DNA binding state typical of the transformed receptor back to the steroid binding, non-DNA binding state typical of the untransformed receptor (Scherrer, L. C., Dalman, F. C., Massa, E., Meshinchi, S., and Pratt, W. B. (1990) J. Biol. Chem. 265, 21397-21400). The receptor heterocomplex formed by the lysate also contains hsp70 and is formed in an ATP-dependent and cation-selective manner (Hutchison, K. A., Czar, M.J., Scherrer, L. C., and Pratt, W.B. (1992) J. Biol. Chem. 267, 14047-14053). In this work, we selectively depleted reticulocyte lysate of hsp70 by passing it through a column of ATP-agarose. The hsp70-depleted lysate contains hsp90, but it cannot form a receptor-hsp90 heterocomplex. hsp70 purified from mouse L cells binds to immunopurified glucocorticoid receptor but does not convert it to the steroid binding state. Addition of purified hsp70 to the hsp70-depleted lysate reactivates the heterocomplex assembly system, permitting formation of a receptor-hsp90-hsp70 complex, with the receptor being returned to the high affinity steroid-binding conformation. These data are consistent with a model in which the protein-unfolding activity of hsp70 is required for hsp90 binding to the hormone binding domain of the glucocorticoid receptor. The hsp56 immunophilin component of the native receptor heterocomplex is also present in the reconstituted receptor heterocomplex in an hsp70-dependent manner. In addition to hsp70, other as yet unidentified factors in reticulocyte lysate are required for receptor heterocomplex assembly.
Hutchison
K A
KA
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109.
Dittmar
K D
KD
Czar
M J
MJ
Pratt
W B
WB
D000818:Animals; D002474:Cell-Free System; D002848:Chromatography, DEAE-Cellulose; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006360:Heat-Shock Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007739:L Cells; D046911:Macromolecular Substances; D051379:Mice; D008954:Models, Biological; D011485:Protein Binding; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D011965:Receptors, Glucocorticoid / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D012156:Reticulocytes / Q000378:metabolism
D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; D011965:Receptors, Glucocorticoid