Receptors and Hormone Action. Volume III
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The lower portion of the blotting membrane was also stained with monoclonal antibody JJ3 directed against the component p23 However, no immunosignal was observed with either of the highly purified receptor preparations data not shown. Components of the glucocorticoid receptor complex in geldanamycin treated cells. Extracts were submitted to chemical cross-linking with EGS. Portions of the membrane were stained separately for receptor polypeptide with mab49 , hsp90, p59, and p23 since JJ3 did not produce any signal, this part of the blot is not shown.
The above experiments suggest that geldanamycin does not interfere with the intracellular formation of receptor complexes of normal size and composition. Nevertheless, we also checked the size of in vitro synthesized glucocorticoid receptors by gel permeation chromatography Fig. With geldanamycin present during receptor translation and assembly, the major portion of receptors was again in high molecular weight form lower part , and 6-nm receptor material was also apparent.
Interestingly, the high molecular weight peak was shifted from an 8. We also wanted to find out whether the effect of geldanamycin on hormone binding is reversible. This is indeed the case as shown in Fig. After washout of the drug, it takes several hours in culture before glucocorticoid binding activity is regenerated in WEHI-7 cells. Within 4 h we had attained roughly half of the original activity, and after 1 day binding activity had recovered completely. Indeed, treatment of WEHI-7 cells with lactacystin significantly diminished the loss of immunodetectable receptor brought about by geldanamycin lane 3 versus lane 2.
Such a regain of intermediate binding activity with new protein synthesis inhibited was reproducibly seen. Unfortunately, however, prolonged exposure of WEHI-7 cells to cycloheximide affected cellular integrity so that we could not carry these experiments for much longer incubation times. In another set of experiments we used E36ts20 hamster cells that harbor a defect in protein degradation by the ubiquitin-proteasome pathway These cells contain glucocorticoid receptors at 5—fold lower levels than WEHI-7 cells, which nevertheless are detectable by immunoblotting and can be used for binding studies, albeit with somewhat lower accuracy.
We also observed that E36ts20 cells can withstand cycloheximide for extended periods of time before deteriorating.
Geldanamycin treatment for 2 h again decreased hormone binding ability Fig. Moreover, receptor degradation was less prevalent in these cells lane 2 versus lane 1. Perhaps not surprisingly, full restoration of hormone binding was only attained if protein synthesis was allowed to proceed Fig. The heat shock protein hsp90 is one of the most abundant cytosolic proteins in eukaryotes, even under normal growth conditions. It reaches up to the percentage level of the total soluble protein pool. Collectively, hsp90 is a mixture of two proteins of similar size that are the products of two related genes for a review, see Ref.
Studies in yeast have shown that only one of these genes can be inactivated without producing deleterious cell effects Although this points to some vital function s , it is not clear at present which cellular event involving hsp90 is essential for cell growth and survival. Nevertheless, it has become evident over the past years that hsp90 interacts with a host of intracellular proteins.
These include receptors for steroid hormones and aryl hydrocarbons, a variety of protein kinases, and some transcription factors for a review, see Ref. Recently, association with the reverse transcriptase of hepatitis B virus has also been described With respect to steroid hormone receptors, hsp90 appears to play a dual role, as pointed out in the Introduction. The present investigation made use of the antibiotic geldanamycin, which specifically interacts with hsp90 18 and interferes with at least some of its functions.
We found that geldanamycin indeed inhibits steroid hormone action in several target systems. Both the expression of an endogenous hormone-responsive gene and of appropriate reporter constructs were affected in qualitatively similar ways cf. Also, a recent paper 39 that appeared in print while this work was under consideration describes inhibition of glucocorticoid inducibility in transfected cells upon exposure to geldanamycin.
We observed that binding of glucocorticoid, progestin, androgen, and estrogen to the respective receptors in cell extracts is only affected if the drug is administered to the respective target cells under cell growth conditions. There is no effect on hormone binding activity if presynthesized receptors are exposed to geldanamycin. In accordance with this, the drug prevents the generation of hormone binding ability if it is present during in vitro receptor synthesis and assembly, while in vitro transcription or translation per se are not impaired.
This conclusion is in support of recent data from others 40 , 41 who observed an effect of geldanamycin on an hspcontaining multichaperone complex in reticulocyte lysate that is essential for the assembly of receptor heterocomplexes. When Whitesell et al. These observations made us wonder whether the drug might similarly interfere with the association of hsp90 with steroid hormone receptor polypeptides.
However, when we carefully analyzed the inactive glucocorticoid receptor of geldanamycin-treated lymphoma cells we found that the receptor complex was perfectly normal in size. Moreover, the receptor complex that was stabilized by chemical cross-linking and extensively purified by immunoaffinity chromatography and SDS-PAGE was found to contain normal amounts of both hsp90 and the FKbinding immunophilin p56, as compared with the functionally intact receptor of untreated cells.
Therefore, the heterotetrameric structure consisting of one receptor polypeptide, two molecules of hsp90, and one p59 subunit that had previously been established 22 appears to be maintained upon drug treatment. When we analyzed the extensively purified glucocorticoid receptor for p23, no specific immunosignal was obtained. This is of interest because p23 had been detected by copurification with the avian progestin receptor 31 and was repeatedly found in association with in vitro reconstituted receptor complexes 4 , 40 , It is perhaps not surprising that standard coprecipitation techniques as often used result in copurification of proteins like p23 31 , 39 , which we do not find as components of cross-linked and extensively purified receptor heteromers.
Our purification procedure would certainly remove such loosely bound proteins. The above observations on the subunit constitution then lead to the question of how hormone binding ability is affected within the heteromeric receptor of at least similar overall structure.
We suspect that geldanamycin produces conformational changes in receptor heteromers that may be rather subtle but nevertheless functionally important. Observations on the effect of geldanamycin on wild-type and mutated forms of p53 43 in fact support the view that this drug is either able to distinguish between protein conformations or perhaps produces altered conformational states, possibly through the action of heat shock proteins. In any event, hsp90 is known to have chaperoning activity 36 , and geldanamycin may well affect its folding properties on receptors.
The fact that geldanamycin inhibits the receptors for glucocorticoid, progestin, androgen, and estrogen cf.
Steroid-hormone rapid actions, membrane receptors and a conformational ensemble model
Table I supports the view that hsp90 plays the very same role for hormone binding throughout the entire class of steroid hormone receptors. The view that the presence of geldanamycin during receptor complex assembly produces conformational alterations that interfere with the recognition of steroidal ligands is indirectly supported by observations by others who have described increased levels of heat shock protein hsp70 in association with receptors. In fact, hsp70 is a major molecular chaperone that participates in protein folding events and is known to preferentially bind to partially unfolded or malfolded proteins for a review, see Ref.
Increased binding of hsp70 then appears quite logical if, for example, the hormone binding domain of receptors, which also contains the areas of contact with hsp90 for a review, see Ref. The glucocorticoid receptor polypeptide either in vitro translated by reticulocyte lysate or presynthesized and subsequently incubated with this system in the presence of ATP is known to associate with hsp90 for a review, see Ref.
Presumably, this material is malfolded and thus bait for hsp70, as discussed above. Interestingly, geldanamycin did not significantly influence the relative amount of monomeric receptor produced in vitro. The shift in elution profile of heteromeric receptors from an 8. In this context, it is of interest that a minimal receptor assembly system has recently been reconstituted from components of reticulocyte lysate 42 in which the p59 component is not required but hormone binding ability is nevertheless generated.
Furthermore, the chicken progestin receptor reconstituted by reticulocyte lysate in the presence of geldanamycin was found to be devoid of p59 and other immunophilins 40 , Taken together, these data show that the in vitro synthesized and assembled receptor in the presence of geldanamycin is not equivalent to that contained in cells although hormone binding is impaired in both cases. Still another difference between glucocorticoid receptors in cells or produced by in vitro synthesis and assembly in the presence of geldanamycin concerns receptor stability.
In contrast to the effects seen in intact cells see below , no destabilization of receptors was observed in the reticulocyte lysate system although this cell extract is well known for containing the ubiquitin-proteasome pathway of protein degradation for reviews, see Refs. When we analyzed geldanamycin-treated mouse lymphoma cells for immunoreactive glucocorticoid receptor polypeptide, we consistently observed a reduction in receptor levels although the cells had only been in contact with the drug for 2 h.
Receptor loss in cells exposed to geldanamycin turned out to be rather variable in our hands. Also, in transfected HeLa cells a decrease in the stability of glucocorticoid receptors was recently described upon exposure to geldanamycin A receptor half-life of approximately 1 h was reported 39 , in contrast to about 4 h in untreated control cells.
Endocrine Press | Endocrine Society
In other experiments, the progestin receptor was transiently transfected into COS-1 cells Upon treatment with geldanamycin, the hormone binding ability decreased, but there was no loss of immunoreactive receptor although the concentration of the drug was at least fold higher than that used in our cell studies. Enhanced glucocorticoid receptor degradation in geldanamycin-treated cells appears to involve the ubiquitin-proteasome pathway of intracellular protein breakdown.
Support for this view comes from two independent lines of evidence.
E36ts20 cells are known to carry a defect in the ubiquitin-activating enzyme E1 Interestingly, tyrosine kinase p erbB2 is in vivo associated with the glucose-regulated protein GRP94, which has homology to hsp90 Like hsp90, this protein binds geldanamycin, resulting in disruption of the complex Such binding of geldanamycin or the analog herbimycin A to hsp90 or GRP94 further leads to degradation of the respective tyrosine kinase molecules in which the ubiquitin-proteasome pathway was again found to be involved 49 , In experiments in which we first exposed cells to geldanamycin and then removed it by wash out, we found reversal to hormone binding ability.
In principle, this restoration of activity may either be due to reactivation of receptor molecules preexposed to the drug or depend on new synthesis of receptor components. To distinguish between these alternatives, we inhibited protein synthesis.
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We still observed significant regain of hormone binding under such conditions Fig. This then leads to the conclusion that receptors are not locked into an inactive complex upon exposure of living cells to geldanamycin. In intact cells, binding-competent receptor complexes can rather be restored at least in part from preexisting components after dissociation of the drug.
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However, steroid hormone receptors within living cells are certainly not static, but rather dynamic. By continuously cycling between the free receptor form and states bound to heat shock proteins, they are thought to maintain the ability to bind hormone 8 , Therefore, it does not matter much, in principle, for the cellular pool of binding-competent and hence biologically active receptors whether new receptor polypeptides enter the cycle at one point or presynthesized molecules join at another. A comparison of the above described and discussed ansamycin effects on steroid hormone receptors and on tyrosine-specific protein kinases brings up some interesting issues.
While the hsp90 heterocomplex with pp60 v- src is being disrupted 18 , association with the receptor polypeptide is maintained cf. Table I. This indicates that different portions of the hsp90 molecule interact with different partner proteins, which belong to separate cellular signaling systems. We are very grateful to Dr. Rinehart University of Illinois for a sample of geldanamycin, to Dr.
We thank Drs. Liao and R. Hiipakka University of Chicago for providing the Rat1A cells expressing the human androgen receptor, Dr. Cato Forschungszentrum Karlsruhe for NIH 3T3 cells stably transfected with a mouse mammary tumor virus-chloramphenicol acetyltransferase gene construct, and Dr. The monoclonal antibody BuGR2 was kindly provided by Dr. Gametchu Medical College of Wisconsin. We also thank Dr.
Zeiner for stimulating discussions. Type 3 signaling, mediated by transcriptionally inactive cytoplasmic TR isoforms, will be addressed later in this review.