title: Visualization and mechanism of assembly of a glucocorticoid receptor.Hsp70 complex that is primed for subsequent Hsp90-dependent opening of the steroid binding cleft.
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.
authors.affiliation: Department of Pharmacology, the University of Michigan Medical School, Ann Arbor, Michigan 48109, USA. pjmurphy@umich.edu
authors.forename: Patrick J M
authors.initials: PJ
authors.lastname: Murphy
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Morishima
authors.forename: Haifeng
authors.initials: H
authors.lastname: Chen
authors.forename: Mario D
authors.initials: MD
authors.lastname: Galigniana
authors.forename: John F
authors.initials: JF
authors.lastname: Mansfield
authors.forename: S Stoney
authors.initials: SS
authors.lastname: Simons
authors.forename: William B
authors.initials: WB
authors.lastname: Pratt
mesh_terms: D000818:Animals; D001665:Binding Sites; D018840:HSP70 Heat-Shock Proteins / Q000097:blood* / Q000302:isolation & purification / Q000648:ultrastructure; D018841:HSP90 Heat-Shock Proteins / Q000097:blood*; D007700:Kinetics; D018625:Microscopy, Atomic Force; D011817:Rabbits; D011965:Receptors, Glucocorticoid / Q000097:blood* / Q000302:isolation & purification / Q000648:ultrastructure; D012156:Reticulocytes / Q000502:physiology
chemical_list: D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D011965:Receptors, Glucocorticoid