PROOF THAT HSP70 IS REQUIRED FOR ASSEMBLY OF THE GLUCOCORTICOID RECEPTOR INTO A HETEROCOMPLEX WITH HSP90

Incubation of immunopurified glucocorticoid receptor with rabbit retic ulocyte lysate forms a complex between the receptor and hsp90, with si multaneous conversion of the receptor from a non-steroid binding but D NA binding state typical of the transformed receptor back to the stero id binding, non-DNA binding state typical of the untransformed recepto r (Scherrer, L. C., Dalman, F. C., Masse, E., Meshinchi, S., and Pratt , W. B. (1990) J. Biol. Chem. 265, 21397-21400). The receptor heteroco mplex 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, 14047-14 053). In this work, we selectively depleted reticulocyte lysate of hsp 70 by passing it through a column of ATP-agarose. The hsp70-depleted l ysate contains hsp90, but it cannot form a receptor-hsp90 heterocomple x. hsp70 purified from mouse L cells binds to immunopurified glucocort icoid receptor but does not convert it to the steroid binding state. A ddition of purified hsp70 to the hsp70-depleted lysate reactivates the heterocomplex assembly system, permitting formation of a receptor-hsp 90-hsp70 complex, with the receptor being returned to the high affinit y steroid-binding conformation. These data are consistent with a model in which the protein-unfolding activity of hsp70 is required for hsp9 0 binding to the hormone binding domain of the glucocorticoid receptor . The hsp56 immunophilin component of the native receptor heterocomple x is also present in the reconstituted receptor heterocomplex in an hs p70-dependent manner. In addition to hsp70, other as yet unidentified factors in reticulocyte lysate are required for receptor heterocomplex assembly.