FOLDING OF THE GLUCOCORTICOID RECEPTOR BY THE RECONSTITUTED HSP90-BASED CHAPERONE MACHINERY - THE INITIAL HSP90-P60-HSP70-DEPENDENT STEP ISSUFFICIENT FOR CREATING THE STEROID-BINDING CONFORMATION

Rabbit reticulocyte lysate contains a multiprotein chaperone system th at assembles steroid receptors into a complex with hsp90, The glucocor ticoid receptor (GR) is bound to hsp90 via its hormone binding domain (HBD), which must be associated with hsp90 to have a steroid binding c onformation, Recently, we have reconstituted a receptor-hsp90 heteroco mplex assembly system with purified rabbit hsp90 and hsp70 and bacteri ally expressed human p23 and p60 (Dittmar, K.D., Hutchison, K.A., Owen s-Grille, J.K., and Pratt, W.B. (1996) J. Biol. Chem. 271, 12833-12839 ), In this work we show that when the GR is incubated with hsp90, hsp7 0, and p60, steroid binding sites are generated despite the absence of p23, In this minimal reconstituted system, the GR is incubated with t he chaperones in the presence of [H-3]triamcinolone acetonide ([H-3]TA ), which binds to the receptor as GR.hsp90 complexes are formed. When molybdate or p23 is also present during the incubation with chaperones at 30 degrees C, the formation of steroid binding sites can be assaye d by incubating the washed GR with [H-3]TA after heterocomplex assembl y at 30 degrees C, However, in the absence of p23 or molybdate, rapid disassembly of GR.hsp90 complexes apparently occurs simultaneously wit h assembly, such that [H-3]TA must be present during the assembly proc ess to trap evidence of conversion of the GR HBD from a non-steroid bi nding to a steroid binding conformation, Mixture of purified rabbit hs p90 and hsp70 with bacterial lysate containing human p60 results in sp ontaneous formation of an hsp90.p60.hsp70 complex that can be adsorbed with anti-p60 antibody, and the resulting immune complex converts the GR HBD to a steroid binding state in an ATP-dependent and K+-dependen t manner. When the GR is incubated with hsp90, hsp70, and p60 in the p resence of the hsp90-binding antibiotic geldanamycin, GR.hsp90.p60.hsp 70 complexes are formed, but they have no steroid binding activity, Ou r data suggest that hsp90, hsp70, and p60 work together as a chaperone complex that possesses all of the folding/unfolding activity necessar y to generate the high affinity steroid binding conformation of the re ceptor.