Abstract
A minimal system of five proteins, hsp90, hsp70, Hop, hsp40, and p23, assembles glucocorticoid receptor (GR).hsp90 heterocomplexes and causes the simultaneous opening of the steroid binding cleft to access by steroid. The first step in assembly is the ATP-dependent and hsp40 (YDJ-1)-dependent formation of a GR.hsp70 complex that primes the receptor for subsequent ATP-dependent activation by hsp90, Hop, and p23. This study focuses on three aspects of the GR priming reaction with hsp70. First, we have visualized the primed GR.hsp70 complexes by atomic force microscopy, and we find the most common stoichiometry to be 1:1, with some complexes of a size approximately 1:2 and a few complexes of larger size. Second, in a recent study of progesterone receptor priming, it was shown that hsp40 binds first, leading to the notion that it targets hsp70 to the receptor. We show here that hsp40 does not perform such a targeting function in priming the GR. Third, we focus on a short amino-terminal segment of the ligand binding domain that is required for GR.hsp90 heterocomplex assembly. By using two glutathione S-transferase (GST)/ligand binding domain fusions with (GST/520C) and without (GST/554C) hsp90 binding and steroid binding activity, we show that the priming step with hsp70 occurs with GST/554C, and it is the subsequent assembly step with hsp90 that is defective.
Highlights
Hsp40, and p23, assembles glucocorticoid receptor bind deep in a hydrophobic cleft that appears to be collapsed in (GR)1⁄7hsp90 heterocomplexes and causes the simultane- the absence of ligand, such that the ligand binding domain (LBD) must change its ous opening of the steroid binding cleft to access by conformation to allow entry of ligand [7]
By using two glutathione S-transferase (GST)/ligand bindpendent chaperone machinery carries out the ATP-dependent opening of the binding cleft in the Glucocorticoid receptors (GR) LBD such that it can be accessed by steroid
We have used L cell cytosol as a source of GR in all of the stoichiometry experiments (Figs. 1–5), because ϳ100% of the L cell GR is bound to hsp90, and nearly complete GR1⁄7hsp90 reassembly and activation of steroid binding activity is achieved when the stripped GR is incubated with the purified assembly system [19]
Summary
Vol 278, No 37, Issue of September 12, pp. 34764 –34773, 2003 Printed in U.S.A. Visualization and Mechanism of Assembly of a Glucocorticoid Receptor1⁄7Hsp Complex That Is Primed for Subsequent Hsp90-dependent Opening of the Steroid Binding Cleft*. By using the two-step assembly protocol, we show that a fusion protein lacking the requisite segment for hsp binding (GST/ 554C) forms a primed 554C1⁄7hsp complex that can bind hsp90/Hop, but hsp dissociates during the second assembly step, and no steroid binding activity is regenerated. This suggests that it is the second assembly step with hsp that is defective when the segment at the rim of the ligand binding cleft is not present
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