FOLDING OF THE GLUCOCORTICOID RECEPTOR BY THE HEAT-SHOCK-PROTEIN (HSP) 90-BASED CHAPERONE MACHINERY - THE ROLE OF P23 IS TO STABILIZE RECEPTOR-HSP90 HETEROCOMPLEXES FORMED BY HSP90-P60-HSP70

In cytosols from animal and plant cells, the abundant heat shock prote in hsp90 is associated with several proteins that act together to asse mble steroid receptors into receptor-hsp90 heterocomplexes. We have re constituted a minimal receptor hsp90 assembly system containing four r equired components, hsp90, hsp70, p60, and p23 (Dittmar, K, D,, Hutchi son, K, A., Owens-Grille, J, IG,, and Pratt, W, B, (1996) J, Biol. Che m, 271, 12833-12839), We have shown that hsp90, p60, and hsp70 are suf ficient for carrying out the folding change that converts the glucocor ticoid receptor (GR) hormone binding domain (HBD) from a non-steroid b inding to a steroid binding conformation, but to form stable GR hsp90 heterocomplexes, p23 must also be present in the incubation mix (Dittm ar, K, D,, and Pratt, W, B. (1997) J, Biol, Chem. 272, 13047-13054), I n this work, we show that addition of p23 to native GR.hsp90 heterocom plexes immunoadsorbed from L cell cytosol or to GR.hsp90 heterocomplex es prepared with the minimal (hsp90.p60.hsp70) assembly system inhibit s both receptor heterocomplex disassembly and loss of steroid binding activity, p23 stabilizes the GR.hsp90 heterocomplex in a dynamic and A TP-independent manner. In contrast to hsp90 that is bound to the GR, f ree hsp90 binds p23 in an ATP-dependent manner, and hsp90 in the hsp90 p60 hsp70 heterocomplex is in a conformation that does not bind p23 a t all, The effect of p23 in the minimal GR heterocomplex assembly syst em is to stabilize GR.hsp90 heterocomplexes once they are formed and i t does not appear to affect the rate of heterocomplex assembly, Molybd ate has the same ability as p23 to stabilize GR heterocomplexes with m ammalian hsp90, but GR heterocomplexes with plant hsp90 are stabilized by p23 and not by molybdate, We propose that incubation of the GR.wit h hsp90 p60]hsp70 forms a GR hsp90 heterocomplex in which hsp90 is in an ATP-dependent conformation, The ATP dependent conformation of hsp90 is required for the hormone binding domain to have a steroid binding site, and binding of p23 to that state of hsp90 stabilizes the GR.hsp9 0 heterocomplex to inactivation and disassembly.