Visualization and Mechanism of Assembly of a Glucocorticoid Receptor·Hsp70 Complex That Is Primed for Subsequent Hsp90-dependent Opening of the Steroid Binding Cleft

Murphy, Patrick J.; Morishima, Yosuke; Chen, Haifeng; Galigniana, Mario D.; Mansfield, John F.; Simons, S. Stoney; Pratt, William B.

doi:10.1074/jbc.m304469200

Cited by 49 publications

(43 citation statements)

References 39 publications

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“…As we have previously reported, GR⅐hsp90⅐immunophilin⅐dynein complexes can be assembled by incubating stripped GR immune pellets with rabbit reticulocyte lysate (13). The complexes can be released from the immune pellet by competition with epitope peptide (26), and Fig. 5A shows an immunoblot of three states of the GR released from the immunopellets: the stripped GR (Str) and the stripped GR that was incubated with rabbit reticulocyte lysate in the absence (Ϫ) or presence (ϩ) of the PPIase domain fragment to compete for immunophilin association with the dynein⅐dynactin complex.…”

Section: Fig 2 Dynamitin Is Present In Gr Heterocomplexes a Immunmentioning

confidence: 98%

“…Visualization of GR⅐hsp90⅐Immunophilin⅐Dynein Complexes by Atomic Force Microscopy-We have previously imaged both the GR and GR⅐hsp70 complexes by atomic force microscopy (26). In the event that at least some of the very large receptor complexes with the movement machinery remain intact through the sample preparation procedure, we wanted to use this technique to visualize GR associated with the dynein motor protein complex.…”

Section: Fig 2 Dynamitin Is Present In Gr Heterocomplexes a Immunmentioning

confidence: 99%

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Evidence for Glucocorticoid Receptor Transport on Microtubules by Dynein

Harrell

Murphy

Morishima

et al. 2004

Journal of Biological Chemistry

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114

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Rapid, ligand-dependent movement of glucocorticoid receptors (GR) from cytoplasm to the nucleus is hsp90-dependent, and much of the movement system has been defined. GR⅐hsp90 heterocomplexes isolated from cells contain one of several hsp90-binding immunophilins that link the complex to cytoplasmic dynein, a molecular motor that processes along microtubular tracks to the nucleus. Although it is known that rapid, hsp90-dependent GR movement requires intact microtubules, it has not been shown that the movement is dynein-dependent. Here, we show that overexpression of dynamitin, which blocks movement by dissociating the dynein motor from its cargo, inhibits ligand-dependent movement of the GR to the nucleus. We show that native GR⅐hsp90⅐immnunophilin complexes contain dynamitin as well as dynein and that GR heterocomplexes isolated from cytosol containing paclitaxel and GTP to stabilize microtubules also contain tubulin. The complete movement system, including the dynein motor complex and tubulin, can be assembled under cell-free conditions by incubating GR immune pellets with paclitaxel/GTP-stabilized cytosol prepared from GR ؊ L cells. This is the first evidence that the movement of a steroid receptor is dynein-dependent, and it is the first isolation of a steroid receptor bound to the entire system that determines its retrograde movement.As the initial step in their action, transcription factors, such as steroid receptors, p53, and HSF1, must move in a targeted manner through the cytoplasm to the nucleus. Until recently, there has been little mechanistic understanding of how protein solutes (i.e. non-vesicle-associated proteins) undergo such retrograde trafficking. Because the glucocorticoid receptor (GR)

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Section: Fig 2 Dynamitin Is Present In Gr Heterocomplexes a Immunmentioning

confidence: 98%

Section: Fig 2 Dynamitin Is Present In Gr Heterocomplexes a Immunmentioning

confidence: 99%

Evidence for Glucocorticoid Receptor Transport on Microtubules by Dynein

Harrell

Murphy

Morishima

et al. 2004

Journal of Biological Chemistry

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114

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“…Imaging individual macromolecular particles with AFM can be useful for determining particle homogeneity and for identifying the physical arrangement of constituent components of the imaged particles. While the present method was developed for visualization of GRchaperone protein complexes 1,2 and DNA strands to which the GR can bind, it can be applied broadly to imaging DNA and protein samples from a variety of sources.…”

Section: +mentioning

confidence: 99%

“…Preparing DNA and protein samples to be imaged free of contaminants 1. Isolate biomolecules to be imaged and place in a suitable aqueous buffer.…”

Section: Video Linkmentioning

confidence: 99%

“…Isolate biomolecules to be imaged and place in a suitable aqueous buffer. Proteins to be imaged may be purified using liquid chromatography 6 , immunoadsorption followed by removal of antibody and pellet 1,2 , or Profinity eXact purification and tag removal 7 (Figure 2A). Isolate DNA molecules to be imaged by miniprep purification ( Figure 2B).…”

Section: Video Linkmentioning

confidence: 99%

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Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

Murphy

Shannon

Goertz

2011

JoVE

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Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1) 1,2. Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged.The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation 3 ,such as Ni 2+ or Mg 2+, which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase 4 and a repair enzyme 5 , bound to individual DNA strands. These experiments provide significant insight into the protein-protein and DNA-protein biophysical interactions taking place at the molecular level. Imaging individual macromolecular particles with AFM can be useful for determining particle homogeneity and for identifying the physical arrangement of constituent components of the imaged particles. While the present method was developed for visualization of GRchaperone protein complexes 1,2 and DNA strands to which the GR can bind, it can be applied broadly to imaging DNA and protein samples from a variety of sources.

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