Folding of the glucocorticoid receptor by the reconstituted Hsp90-based chaperone machinery. The initial hsp90.p60.hsp70-dependent step is sufficient for creating the steroid binding conformation.
Rabbit reticulocyte lysate contains a multiprotein chaperone system that assembles steroid receptors into a complex with hsp90. The glucocorticoid receptor (GR) is bound to hsp90 via its hormone binding domain (HBD), which must be associated with hsp90 to have a steroid binding conformation. Recently, we have reconstituted a receptor.hsp90 heterocomplex assembly system with purified rabbit hsp90 and hsp70 and bacterially expressed human p23 and p60 (Dittmar, K. D., Hutchison, K. A., Owens-Grillo, 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, hsp70, and p60, steroid binding sites are generated despite the absence of p23. In this minimal reconstituted system, the GR is incubated with the chaperones in the presence of [3H]triamcinolone acetonide ([3H]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 assayed by incubating the washed GR with [3H]TA after heterocomplex assembly at 30 degrees C. However, in the absence of p23 or molybdate, rapid disassembly of GR.hsp90 complexes apparently occurs simultaneously with assembly, such that [3H]TA must be present during the assembly process to trap evidence of conversion of the GR HBD from a non-steroid binding to a steroid binding conformation. Mixture of purified rabbit hsp90 and hsp70 with bacterial lysate containing human p60 results in spontaneous 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+-dependent manner. When the GR is incubated with hsp90, hsp70, and p60 in the presence of the hsp90-binding antibiotic geldanamycin, GR.hsp90.p60. hsp70 complexes are formed, but they have no steroid binding activity. Our data suggest that hsp90, hsp70, and p60 work together as a chaperone complex that possesses all of the folding/unfolding activity necessary to generate the high affinity steroid binding conformation of the receptor.
Dittmar
K D
KD
Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
Pratt
W B
WB
D000818:Animals; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D017510:Protein Folding; D011817:Rabbits; D011965:Receptors, Glucocorticoid / Q000737:chemistry* / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism
D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D011965:Receptors, Glucocorticoid