Abstract
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.
Highlights
Commercial preparations of rabbit reticulocyte lysate which are used for cell-free protein translation contain a system that assembles steroid receptors into multiprotein heterocomplexes that contain the 90-kDa heat shock protein, hsp901 [1, 2]
In experiments using wheat germ extract instead of reticulocyte lysate to reconstitute GR1⁄7hsp90 heterocomplexes, we found that GR1⁄7plant hsp90 complexes were very unstable, but formation of steroid binding sites could be detected by having [3H]triamcinolone acetonide (TA) present while the glucocorticoid receptor (GR) was incubated with the wheat germ extract [3]
We use this technique of heterocomplex assembly in the presence of ligand to show that a mixture of hsp90, hsp70, and p60 is sufficient to generate the steroid binding conformation of the GR hormone binding domain (HBD), but a stable heterocomplex is formed only when p23 or molybdate is present during assembly
Summary
Commercial preparations of rabbit reticulocyte lysate which are used for cell-free protein translation contain a system that assembles steroid receptors into multiprotein heterocomplexes that contain the 90-kDa heat shock protein, hsp901 [1, 2]. Because the hormone binding domain (HBD) of the glucocorticoid receptor (GR) must be bound to hsp for it to bind steroid [4], the conversion of receptors from a non-steroid binding state to a steroid binding state by the hsp90-based chaperone system can be used as a rapid folding assay to detect the formation of GR1⁄7hsp complexes in which the HBD is in the high affinity steroid binding conformation [2, 5] Using both formation of steroid binding sites and direct measurement of the formation of receptor1⁄7hsp complexes, a number of details of heterocomplex assembly by reticulocyte lysate have been established. Smith et al [15] predicted that the combining factor was a 60-kDa protein (p60) that coimmunoadsorbed with hsp and hsp70 They had observed this p60 in progesterone receptor (PR) complexes formed in reticulocyte lysate when ATP was limiting [6] or at early times of heterocomplex assembly [16]. P60 is required for GR1⁄7hsp heterocomplex assembly by reticulocyte lysate [20]
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