The Seven Amino Acids (547–553) of Rat Glucocorticoid Receptor Required for Steroid and Hsp90 Binding Contain a Functionally Independent LXXLL Motif That Is Critical for Steroid Binding

Hsp90 cooperates with its co-chaperone Cdc37 to provide obligatory support to numerous protein kinases involved in the regulation of cellular signal transduction pathways. In this report, the crystal structure of the Src family tyrosine kinase Lck was used to guide the creation of kinase constructs to determine features recognized by Hsp90 and its "kinase-specific" co-chaperone Cdc37. Two parameters were assayed: the ability and extent to which the constructs bound to Hsp90 and Cdc37, and the ability of the constructs to trigger saltresistant high affinity complexes with Hsp90 and Cdc37 independent of the presence of molybdate. Although Hsp90 interacted with both the N-terminal and C-terminal lobes (NL and CL, respectively) of the catalytic domains of the kinases, the lobes themselves were not sufficient to trigger the high affinity binding of Hsp90. Only constructs containing a complete N-or C-terminal lobe and part of the adjacent lobe bound to Hsp90 and Cdc37 in salt-stable complexes independent of molybdate. The two minimum constructs that bound Hsp90 and Cdc37 contained the ␣-C-helix and the ␤4-and ␤5-strands of the NL through to end of the CL and the NL through to the ␣-E-helix and the amino acids that cap the helix. Cdc37 interacted with only the NL and minimally required the ␣-C-helix and ␤4-and ␤5-strands of this lobe of Lck. The results indicate that the high affinity binding activity of Hsp90 is triggered through its interaction with adjacent subdomain structures of kinase catalytic domains. Furthermore, the ␣-C-helix and part of its adjoining loop connection to the ␤4-strand appear to be the primary determinants recognized by Cdc37.

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Definition of Protein Kinase Sequence Motifs That Trigger High Affinity Binding of Hsp90 and Cdc37

Prince

Matts

2004

“…This segment lies at the rim of the ligand binding cleft of the receptor (25), and mutations within this segment alter both steroid binding activity and transcriptional activity of the GR (25,26). Although we know that GR LBD mutants lacking this segment do not form heterocomplexes with hsp90, we do not know whether it is the first hsp70-dependent step or the second hsp90-dependent step of assembly that is affected.…”

mentioning

confidence: 91%

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

Morishima

Chen

et al. 2003

Self Cite

A minimal system of five proteins, hsp90, hsp70, Hop, hsp40, and p23, assembles glucocorticoid receptor (GR)⅐hsp90 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 GR⅐hsp70 complex that primes the receptor for subsequent ATPdependent 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 GR⅐hsp70 complexes by atomic force microscopy, and we find the most common stoichiometry to be 1:1, with some complexes of a size~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 GR⅐hsp90 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.Glucocorticoid receptors (GR) 1 are recovered from hormonefree cells as large multiprotein complexes containing a dimer of hsp90 (for review see Refs. 1 and 2). Hsp90 binds to the ligand binding domain (LBD) of the GR, and the steroid binding activity of the GR is absolutely hsp90-dependent (3, 4). When the GR is stripped of its associated hsp90, it immediately loses its ability to bind steroid, and steroid binding activity is regenerated when GR⅐hsp90 heterocomplexes are reformed by the hsp90/hsp70-based chaperone machinery (5, 6). The steroids bind deep in a hydrophobic cleft that appears to be collapsed in the absence of ligand, such that the LBD must change its conformation to allow entry of ligand (7). The hsp90/hsp70-dependent chaperone machinery carries out the ATP-dependent opening of the binding cleft in the GR LBD such that it can be accessed by steroid. In addition to opening the steroid binding cleft, formation of a complex with hsp90 is accompanied by conformational changes that increase the sensitivity of the GR LBD to attack by thiol-derivatizing agents and trypsin (8 -10).The multiprotein chaperone machinery that forms receptor⅐hsp90 heterocomplexes was originally identified in reticulocyte lysate (11,12). This machinery has been reconstituted (13), and a mixture of five purified proteins (hsp90, hsp70, 2 Hop, hsp40, and p23) is now used to achieve efficient receptor⅐hsp90 heterocomplex assembly (14, 15). The chaperones hsp90 and hsp70 are both essential for opening the steroid binding cleft in the GR LBD, and hsp40, Hop (hsp70/hsp90 organizing protein), and p23 act as co-chaperones to increase the rate or extent of GR⅐hsp90 heterocomplex assembly (16). Hop b...

“…Interaction domains critical for HSP90 binding are located within helix 1 of the GR LBD (16), and steroid binding also requires contacts between an LXXLL motif in helix 1 and The resulting fusion protein includes the last six amino acids of the RAR-LBD helix 1 (19,20), as well as the complete GR LBD helix 1 (inferred from the LBD structure of the progesterone receptor (22)). GR domains also included in the fusion are the heptapeptide element essential for HSP90 binding (16) and the putative LXXLL interaction motif (17). N-term, Nterminal; DBD, DNA binding domain.…”

Section: Resultsmentioning

confidence: 99%

A Glucocorticoid/Retinoic Acid Receptor Chimera That Displays Cytoplasmic/Nuclear Translocation in Response to Retinoic Acid

Mackem¹,

Baumann²,

Hager³

2001

Members of the nuclear receptor superfamily play key roles in a host of physiologic and pathologic processes from embryogenesis to cancer. Some members, including the retinoic acid receptor (RAR), are activated by ligand binding but are unaffected in their subcellular distribution, which is predominantly nuclear. In contrast, several members of the steroid receptor family, including the glucocorticoid receptor, are cytoplasmic and only translocate to the nucleus after ligand binding. We have constructed chimeras between RAR and glucocorticoid receptor that selectively respond to RAR agonists but display cytoplasmic localization in the absence of ligand. These chimeric receptors manifest both nuclear translocation and gene activation functions in response to physiological concentrations of RAR ligands. The ability to achieve regulated subcellular trafficking with a heterologous ligand binding domain has implications both for current models of receptor translocation and for structural-functional conservation of ligand binding domains broadly across the receptor superfamily. When coupled to the green fluorescent protein, chimeric receptors offer a powerful new tool to 1) study mechanisms of steroid receptor translocation, 2) detect dynamic and graded distributions of ligands in complex microenvironments such as embryos, and 3) screen for novel ligands of "orphan" receptors in vivo.The glucocorticoid receptor (GR) 1 is a member of the translocating class of steroid receptors. Unliganded GR is found in the cytoplasm and moves rapidly into the nucleus in response to hormone stimulation. Other members of the nuclear receptor superfamily are either complex in their intracellular distributions (e.g. mineralocorticoid receptor (1), progesterone receptor (2, 3), androgen receptor (4), and dioxin receptor (5)) or are found primarily in the nucleus (e.g. estrogen receptor (6), thyroid hormone receptor (see Refs. 7 and 8), and retinoic acid receptor (RAR; see Refs. 7 and 8)). The recent characterization of functional GFP fusions with the steroid/nuclear receptors allows study of real time movement for these molecules within living cells (9). The unliganded GFP-GR is found completely in the cytoplasm, whereas the hormone-activated receptor is located almost exclusively in the nucleus shortly after hormone activation (9 -11). Because nuclear translocation by this intrinsically fluorescent receptor is quite dramatic and easily monitored in real time in vivo, it can provide a straightforward methodology to detect physiological concentrations of receptor ligand in the cellular environment.The availability of chimeric proteins with the nuclear/cytoplasmic translocation properties of GR, and the ligand responsiveness of the nuclear receptors, would provide a powerful translocation assay for nuclear receptor ligands and potentially for novel analogues, as well. Many chimeric receptors have been described (e.g. see Ref. 12), but these fusions have been characterized exclusively in terms of their gene activation potential. We describ...