As indicated in Fig. 2, the primed GR-hsp70 complex binds hsp90 and there is more binding if Hop is present (Morishima et al. 2000b). The binding of hsp90 is rapid, and it is the subsequent ATP-dependent opening of the steroid binding cleft that is rate-limiting in the overall assembly process (Kanelakis et al. 2002). In this cleft opening step, the GR-bound hsp90 is converted to its ATP-dependent conformation. To have an open steroid binding cleft, the receptor-bound hsp90 must assume its ATP-dependent conformation (Grenert et al. 1999) and it is only the ATP-dependent conformation of hsp90 that binds p23 (Sullivan et al. 1997). Like the priming step with hsp70, this second, cleft opening step is also K+-dependent, suggesting that the GR-bound hsp70 must be converted from its ATP-dependent to its ADP-dependent conformation while it cooperates with hsp90 to activate steroid binding activity (Morishima et al. 2001).
The cleft-opening reaction is not understood in greater detail, but in reticulocyte lysate, it seems that a series of events are occurring. For example, much of the hsp70 and all of the Hop dissociate during this second step (Smith 1993). Reticulocyte lysate contains the TPR domain immunophilins and PP5; thus dissociation of Hop from the GR-bound hsp90 yields an open TPR acceptor site, permitting binding of FKBP51, FKBP52, CyP-40, or PP5.
It is important to emphasize that the purified five-protein system is a minimal system for efficient assembly of stable GR-hsp90 heterocomplexes. Retic-ulocyte lysate contains other components that likely play important roles in a more complex assembly system. For example, Aha proteins (activator of hsp90 ATPase) are hsp90 co-chaperones present in cell lysates (Panaretou et al. 2002), and addition of purified Aha protein to the five-protein system accelerates the rate of generation of steroid-binding activity. This would be consistent with an iterative process in which GR-bound hsp90 switches between its ATP-bound and ADP-bound conformations during the ATP-dependent cleft opening process.
Small amounts of both Hip (hsc70-interacting protein) and BAG-1 (Bcl-2-associated gene product-1) are present in the hsp90-Hop-hsp70-hsp40 complexes immunoadsorbed from reticulocyte lysate with antibody against Hop (Kanelakis et al. 1999). Hip and BAG-1 are co-chaperones that compete with each other for binding to the ATPase domain of hsp70; thus, they must exist in separate chaperone machinery complexes. Although Hip was regarded as essential for receptor-hsp90 heterocomplex assembly in reticulocyte lysate (Prapapanich et al. 1998), it is not present in the five-protein assembly system and it does not affect GR-hsp90 assembly by that system (Kanelakis et al. 2000). Nelson et al. (2004) have reported that introduction of Hip into yeast enhances hormone-dependent activation of a reporter gene by the GR, but how it works is unclear. At physiological levels, BAG-1 does not affect the rate of GR-hsp90 assembly by the five-protein system, but at high levels, it is inhibitory (Kanelakis et al. 1999). BAG-1(RAP46) is associated with the transformed GR and travels to the nucleus with it (Zeiner and Gehring 1995; Schneikert et al. 2000). BAG-1 has a nonspecific DNA-binding domain that is required for its inhibition of GR-dependent transactivation (Schmidt et al. 2003), but again, the mechanism of the inhibition is not clear.
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