Loss of proliferative potential during terminal differentiation coincides with the decreased abundance of a subset of heterogeneous ribonuclear proteins.

The decrease in abundance of a subset of highly conserved basic nuclear proteins is established to correlate with the loss of proliferative potential in association with the process of terminal differentiation in murine mesenchymal stem cells and human keratinocytes. These proteins, designated P2Ps for proliferation potential proteins, have apparent molecular masses of 30-40 kD, are associated with the 30-40S substructures of nuclear hnRNP complexes, and are recognized by antibodies made against core proteins of hnRNP particles. They also share an epitope in common with heat shock protein-90 (hsp90) and are recognized by two mAbs against hsp90. Two-dimensional electrophoretic Western blots furthermore show that P2Ps make up a subset of hnRNP proteins. Cells that possess these proteins express the potential to proliferate whether or not they are traversing the cell cycle. These include rapidly growing cells, reversibly growth-arrested cells, and nonterminally differentiated cells. In contrast, cells that have irreversibly lost their proliferative potential, such as terminally differentiated cells, show a marked reduction in the abundance of P2Ps as determined by immunodetection on Western blots. A correlation, therefore, exists between the presence of this subset of nuclear proteins and the proliferative potential in two cell types. These results raise the possibility that as a subset of hnRNP proteins, P2Ps may mediate posttranscriptional control of the processing of specific RNAs required for cell proliferation.

Molecular chaperone machines: chaperone activities of the cyclophilin Cyp-40 and the steroid aporeceptor-associated protein p23.

Molecular chaperones are essential proteins that participate in the regulation of steroid receptors in eukaryotes. The steroid aporeceptor complex contains the molecular chaperones Hsp90 and Hsp70, p48, the cyclophilin Cyp-40, and the associated proteins p23 and p60. In vitro folding assays showed that Cyp-40 and p23 functioned as molecular chaperones in a manner similar to that of Hsp90 or Hsp70. Although neither Cyp-40 nor p23 could completely refold an unfolded substrate, both proteins interacted with the substrate to maintain a nonnative folding-competent intermediate. Thus, the steroid aporeceptor complexes have multiple chaperone components that maintain substrates in an intermediate folded state.

Molecular cloning of the chicken progesterone receptor.

To define the functional domains of the progesterone receptor required for gene regulation, complementary DNA (cDNA) clones encoding the chicken progesterone receptor have been isolated from a chicken oviduct lambda gt11 cDNA expression library. Positive clones expressed antigenic determinants that cross-reacted with six monospecific antibodies derived from two independent sources. A 36-amino acid peptide sequence obtained by microsequencing of purified progesterone receptor was encoded by nucleotide sequences in the longest cDNA clone. Analysis of the amino acid sequence of the progesterone receptor deduced from the cDNA clones revealed a cysteine-rich region that was homologous to a region found in the estrogen and glucocorticoid receptors and to the avian erythroblastosis virus gag-erb-A fusion protein. Northern blot analysis with chicken progesterone receptor cDNA's indicated the existence of at least three messenger RNA species. These messages were found only in oviduct and could be induced by estrogens.

Steroid receptor interactions with heat shock protein and immunophilin chaperones.

We have provided a historical perspective on a body of steroid receptor research dealing with the structure and physiological significance of the untransformed 9S receptor that has often confused both novice and expert investigators. The frequent controversies and equivocations of earlier studies were due to the fact that the native, hormone-free state of these receptors is a large multiprotein complex that resisted description for many years because of its unstable and dynamic nature. The untransformed 9S state of the steroid and dioxin receptors has provided a unique system for studying the function of the ubiquitous, abundant, and conserved heat shock protein, hsp90. The hormonal control of receptor association with hsp90 provided a method of manipulating the receptor heterocomplex in a manner that was physiologically meaningful. For several steroid receptors, binding to hsp90 was required for the receptor to be in a native hormone-binding state, and for all of the receptors, hormone binding promoted dissociation of the receptor from hsp90 and conversion of the receptor to the DNA-binding state. Although the complexes between tyrosine kinases and hsp90 were discovered earlier, the hormonal regulation or steroid receptor association with hsp90 permitted much more rapid and facile study of hsp90 function. The observations that hsp90 binds to the receptors through their HBDs and that these domains can be fused to structurally different proteins bringing their function under hormonal control provided a powerful linkage between the hormonal regulation of receptor binding to hsp90 and the initial step in steroid hormone action. Because the 9S receptor hsp90 heterocomplexes could be physically stabilized by molybdate, their protein composition could be readily studied, and it became clear that these complexes are multiprotein structures containing a number of unique proteins, such as FKBP51, FKBP52, CyP-40, and p23, that were discovered because of their presence in these structures. Further analysis showed that hsp90 itself exists in a variety of native multiprotein heterocomplexes independent of steroid receptors and other 'substrate' proteins. Cell-free systems can now be used to study the formation of receptor heterocomplexes. As we outlined in the scheme of Fig. 1, the multicomponent receptor-hsp90 heterocomplex assembly system is being reconstituted, and the importance of individual proteins, such as hsp70, p60, and p23, in the assembly process is becoming recognized. It should be noted that our understanding of the mechanism and purpose of steroid receptor heterocomplex assembly is still at an early stage. We can now speculate on the roles of receptor-associated proteins in receptor action, both as individuals and as a group, but their actual functions are still vague or unknown. We can make realistic models about the chaperoning and trafficking of steroid receptors, but we don't yet know how these processes occur, we don't know where chaperoning occurs in the cell (e.g. Is it limited to the cytoplasm? Is it a diffuse process or does chaperoning occur in association with structural elements?), and, with the exception of the requirement for hormone binding, we don't know the extent to which the hsp90-based chaperone system impacts on steroid hormone action. It is not yet clear how far the discovery of this hsp90 heterocomplex assembly system will be extended to the development of a general understanding of protein processing in the cell. Because this assembly system is apparently present in all eukaryotic cells, it probably performs an essential function for many proteins. The bacterial homolog of hsp90 is not an essential protein, but hsp90 is essential in eukaryotes, and recent studies indicate that the development of the cell nucleus from prokaryotic progenitors was accompanied by the duplication of genes for hsp90 and hsp70 (698). (ABSTRACT TRUNCATED)

Characterization of a novel 23-kilodalton protein of unactive progesterone receptor complexes.

Immunoprecipitation of unactivated avian progesterone receptor results in the copurification of hsp90, hsp70, and three additional proteins, p54, p50, and p23. p23 is also present in immunoaffinity-purified hsp90 complexes along with hsp70 and another protein, p60. Antibody and cDNA probes for p23 were prepared in an effort to elucidate the significance and function of this protein. Antibodies to p23 detect similar levels of p23 in all tissues tested and cross-react with a protein of the same size in mice, rabbits, guinea pigs, humans, and Saccharomyces cerevisiae, indicating that p23 is a conserved protein of broad tissue distribution. These antibodies were used to screen a chicken brain cDNA library, resulting in the isolation of a 468-bp partial cDNA clone encoding a sequence containing four sequences corresponding to peptide fragments isolated from chicken p23. This partial clone was subsequently used to isolate a full-length human cDNA clone. The human cDNA encodes a protein of 160 amino acids that does not show homology to previously identified proteins. The chicken and human cDNAs are 88% identical at the DNA level and 96.3% identical at the protein level. p23 is a highly acidic phosphoprotein with an aspartic acid-rich carboxy-terminal domain. Bacterially overexpressed human p23 was used to raise several monoclonal antibodies to p23. These antibodies specifically immunoprecipitate p23 in complex with hsp90 in all tissues tested and can be used to immunoaffinity isolate progesterone receptor complexes from chicken oviduct cytosol.

Binding of heat shock proteins to the avian progesterone receptor.

The protein composition of the avian progesterone receptor was analyzed by immune isolation of receptor complexes and gel electrophoresis of the isolated proteins. Nonactivated cytosol receptor was isolated in association with the 90-kilodalton (kDa) heat shock protein, hsp90, as has been described previously. A 70-kDa protein was also observed and was shown by Western immunoblotting to react with an antibody specific to the 70-kDa heat shock protein. Thus, two progesterone receptor-associated proteins are identical, or closely related, to heat shock proteins. When the two progesterone receptor species, A and B, were isolated separately in the absence of hormone, both were obtained in association with hsp90 and the 70-kDa protein. However, activated receptor isolated from oviduct nuclear extracts was associated with the 70-kDa protein, but not with hsp90. A hormone-dependent dissociation of hsp90 from the cytosolic form of the receptor complex was observed within the first hour of in vivo progesterone treatment, which could explain the lack of hsp90 in nuclear receptor complexes. In a cell-free system, hsp90 binding to receptor was stabilized by molybdate but disrupted by high salt. These treatments, however, did not alter the binding of the 70-kDa protein to receptor. Association of the 70-kDa protein with the receptor could be disrupted by the addition of ATP at elevated temperatures (23 degrees C). The receptor-associated 70-kDa protein is an ATP-binding protein, as demonstrated by its affinity labeling with azido[32P]ATP. These results indicate that the two receptor-associated proteins interact with the progesterone receptor by different mechanisms and that they are likely to affect the structure or function of the receptor in different ways.

Identification of a 60-kilodalton stress-related protein, p60, which interacts with hsp90 and hsp70.

Immunoaffinity purification of hsp90 from chick oviduct cytosol reveals two major proteins, hsp70 and a 60-kDa protein (p60), copurifying with hsp90. A similar result is obtained when hsp90 is immunoaffinity purified from chick liver and brain cytosols, avian fibroblasts, and rabbit reticulocyte lysate. This p60 is the same protein previously identified in certain assembly complexes of chick progesterone receptor generated in a cell-free reconstitution system. Tryptic and cyanogen bromide peptide fragments were generated from gel-purified p60, and partial N-terminal sequences were determined from eight peptides. The sequences show a striking similarity to the sequence of a 63-kDa human protein (IEF SSP 3521) whose abundance is increased in MRC-5 fibroblasts following simian virus 40 transformation. A monoclonal antibody was prepared against avian p60; Western immunoblot analysis showed that p60 was present in each of eight chick tissues examined and in each of the human, rat, rabbit, and Xenopus tissues tested. Immunoaffinity purifications from both chick oviduct cytosol and rabbit reticulocyte lysate using anti-p60 and anti-hsp70 monoclonal antibodies confirm that there is a relatively abundant complex in these extracts containing hsp90, hsp70, and p60. This complex appears to comprise an important functional unit in the assembly of progesterone receptor complexes. However, judging from the abundance and widespread occurrence of this multiprotein complex, hsp90, hsp70, and p60 probably function interactively in other systems as well.

Immunocytochemical study of progesterone receptors in hyperplastic and neoplastic endometrial tissues.

Endometrial carcinoma is the most common genital malignancy in North America. However its pathogenesis, in particular its relationship with hyperplasia is not clear. To understand steroid hormonal interactions in the genesis and growth of human endometrial hyperplasia and carcinoma, we have assayed progesterone receptors in hyperplasia and neoplastic human endometrium by immunocytochemistry. The presence of progesterone receptors in target tissues is known to be a marker of both estrogen and progesterone action. The receptors were identified in fresh-frozen sections using a mouse monoclonal antiprogesterone receptor antibody (alpha PR6). The progesterone receptor content was high in the epithelium of hyperplasia without cytological atypia and low in the epithelium of endometrial intraepithelial neoplasia (hyperplasia with cytological atypia). In carcinomas there was a heterogenous distribution of progesterone receptors in the epithelium but low as compared to hyperplastic endometria without cytological atypia. The stroma contained relatively high progesterone receptor levels irrespective of whether the epithelium was hyperplastic or neoplastic.

Response of human breast cancer cells to heat shock and chemotherapeutic drugs.

Previous studies have shown that certain chemotherapeutic drugs are less effective on tumor cells when cells have been previously exposed to hyperthermia. In the present study, we have evaluated whether specific modifications in heat shock protein (hsp) expression are associated with resistance to anticancer drugs. RNA levels for hsp90, hsp70, and hsp27 were studied by Northern and slot blots, while proteins were studied by two-dimensional gel electrophoresis, in MCF-7/BK and MDA-MB-231 breast cancer cells. The sensitivities of these cells to doxorubicin, colchicine, 5-fluorouracil, cisplatin, actinomycin D, and methotrexate were tested by clonogenic assays. These techniques were applied to both cell lines before (control) and after heat shock. The study revealed that elevated hsp70 and hsp27 levels were associated with doxorubicin resistance. In addition, the presence of phosphorylated hsp27 isoforms was also associated with doxorubicin resistance. The study showed that elevated hsps were not associated with multidrug resistance. Heat shock did not induce P170 glycoprotein mRNA overexpression or resistance to the other drugs tested. We also found that the level of doxorubicin protection conferred by the overexpression of hsp was lower than that obtained in cells expressing a multidrug resistance phenotype (MDA-A1R cells). In these cells, heat shock did not confer additional doxorubicin resistance and hsp27 phosphorylation was deficient. Our studies suggest that specific hsps are associated with doxorubicin resistance in certain human breast cancer cells and that this mechanism seems to be independent of the multidrug resistance system.

Interaction of progesterone receptor with immobilized adenosine triphosphate.

Affinity chromatography has been used to study the binding of ATP to cyto-plasmic progesterone receptors of hen oviduct. A resin which selectively binds the receptor protein was prepared by linking ATP covalently to Sepharose 4B through a 6-carbon bridge of adipic acid dihydrazide. Receptor bound to the affinity resin was recovered in a single peak upon gradient elution with KCl (0.2-1 M) or ATP (0-0.1 M). While affinity chromatography was normally accomplished using the [3H]progesterone receptor complex, the hormone was not necessary for ATP binding under the conditions employed. The chromatography of crude receptor preparations allowed up to 100-fold purification with greater than 80% recovery of the receptor. The semipurified receptor appeared intact when analysed by sucrose gradient centrifugation, polyacrylamide gel electrophoresis, and DEAE-cellulose chromatography. The latter procedure separated the receptor into two components, A and B, both of which were capable of binding ATP. Although a specific biochemical role of ATP in hormone receptor action has not been demonstrated, the present studies support this possibility and, in addition, offer a convenient and reliable step for the purification of progesterone receptors.

Identification of the 90 kDa substrate of rat liver type II casein kinase with the heat shock protein which binds steroid receptors.

It was recently reported that the type II casein kinase of rat liver cytosol co-purified with a major 90 kDa substrate when subjected to gel filtration at low ionic strength. The identity of the 90 kDa substrate was unknown. We have verified this report and have shown that the 90 kDa substrate is recognized by a monoclonal antibody prepared against the 90 kDa heat shock protein. This ubiquitous phosphoprotein is known to increase in abundance in cells subjected to heat stress and has been shown to complex steroid receptors and certain retrovirus tyrosine kinases.

Recent Advances in the Study of hsp90 Structure and Mechanism of Action.

The 90kDa heat shock protein, hsp90, is a major molecular chaperone of the cell that appears to have particular significance to cellular regulatory processes. New tools and approaches have revealed a number of target proteins for hsp90, most of which are protein kinases or transcription factors. While the mechanism of action of hsp90 is not well understood, reasonable models have emerged describing some functional domains of this protein, the importance of conformational transitions for its activity and its role within a multi-component chaperoning pathway of the cell.

Binding of p23 and hsp90 during assembly with the progesterone receptor.

Upon incubation in rabbit reticulocyte lysate, the unactivated progesterone receptor (PR) associates with the heat shock proteins hsp90 and hsp70, the immunophilins FKBP52, FKBP54, and CyP-40, and another protein p23. We have previously described a protein complex between p23, hsp90, and the immunophilins that forms in rabbit reticulocyte lysate in the absence of the PR. Immunodepletion of p23 from lysate prevented the binding of hsp90 and CyP-40 to the PR, suggesting that hsp90, CyP-40, and p23 bind the receptor as a complex. We have further examined the properties of this p23 complex to determine how it is involved in receptor assembly in vitro. Use was made of three chemical probes, sodium molybdate, the nonhydrolyzable ATP analog, 5'-adenylylimidodiphosphate, and the hsp90-binding agent geldanamycin. Molybdate has previously been shown to stabilize the heat-induced dissociation of hsp90 and p23 from the PR. This stabilization is not mediated through the PR, as molybdate stabilizes the heat-induced dissociation of hsp90 from p23 even in the absence of the PR. Molybdate also stabilizes both p23 and PR complexes under conditions of low ATP and magnesium concentration. The ATP analog, 5'-adenylylimidodiphosphate, which does not support the assembly of PR complexes, promotes both the assembly and stabilization of p23 complexes. Geldanamycin disrupts p23 complexes, and when PR complexes are treated with this agent, p23, CyP-40, and some hsp90 are lost from the receptor. Thus, all three of these chemical agents appear to target the p23 complex, which is thought to enter at the last step in the assembly of the PR complex. A model is presented to relate these findings to previous models and another complex between hsp90, hsp70, and p60 that appears to be an intermediate in PR assembly.

Reconstitution of progesterone receptor with heat shock proteins.

Nonactivated chick progesterone receptor from hypotonic tissue extracts exists in a large complex containing the heat shock proteins hsp90 and hsp70 plus additional smaller proteins; activation of receptor to a DNA-binding form involves the dissociation of proteins from the complex. Whereas numerous attempts to reversibly bind components to the activated receptor have been unsuccessful, we now report conditions that promote the reassociation of hsp90 and hsp70 to progesterone receptor. Cytosolic receptor was dissociated from hsp90 and hsp70 by treatment with 0.5 M KCl and 10 mM ATP in the absence of progesterone. It was then purified by binding to immunoaffinity resins. After wash steps, the receptor-resin complex was incubated in rabbit reticulocyte lysate at 30 C, rewashed, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Saturable binding of rabbit hsp90 and hsp70 to chick receptor was found after incubation with reticulocyte lysate; hsp binding was temperature dependent, but not dependent on exogenous ATP. Incubation of dissolved receptor with oviduct cytosol, from which receptor was obtained, or with purified hsp did not result in hsp binding. Furthermore, mixing oviduct cytosol with lysate inhibited hsp reconstitution, suggesting negative factors for hsp binding in oviduct cytosol. The steroid-binding domain of the receptor was required, since no hsp binding was observed in the reconstitution system using a receptor mutant lacking this domain. When the receptor was isolated in the presence of progesterone, reconstitution with hsp90 and hsp70 did not occur. This is consistent with the in vivo effects of progesterone in promoting hsp dissociation.

Interaction of the progesterone receptor with binding proteins for FK506 and cyclosporin A.

T47D human breast carcinoma cells and the chicken oviduct were used to study the structure of the nonactivated progesterone receptor (PR) complex. Immunoprecipitation of PR (B form) from cytosol extracts was performed using monoclonal antibody PR6, a cross-reactive antibody prepared to chicken PR. Analysis of the PR complex by sodium dodecyl sulfate gels and Western immuno-blotting revealed the presence of several specific copurifying proteins. Consistent with previous reports, the two heat shock proteins, hsp90 and hsp70, were shown to be present. A third 59-kilodalton (kDa) protein observed previously was confirmed to be p59 (also called hsp56 or FKBP52), which has been shown to bind the immunosuppressant drug FK506. Two additional PR-associated proteins were observed that had not been previously recognized with human PR. These have molecular masses of 54-kDa and 23-kDa and have been shown by Western blotting to be related to the proteins p54 and p23 that are associated with chicken PR. P23 is a novel protein of unknown function and p54 or FKBP54 has been recently shown to be another FK506-binding protein related to p59. Finally, the cyclosporin A-binding protein, CyP-40, could be detected in isolated chicken PR complexes and in PR complexes that were reconstituted in vitro, but this protein was not detected in human PR complexes, which are less stable than chicken PR complexes in cytosol extracts. The functional significance of FK506 and cyclosporin A-binding proteins to hormone action was tested using a T47D cell line that contained a progestin reporter gene, MMTV-CAT. Treatment with cyclosporin A had no effect on the basal level of CAT expression, but it caused a dramatic increase in the sensitivity and magnitude of the response to the synthetic progestin, R5020. The enhanced response elicited by drug treatment was blocked by the antiprogestin RU486 indicating that this effect was receptor-mediated. While cyclosporin A enhanced progestin action in T47D cells, it inhibited a PR/reporter gene system in L cells. The drugs FK506 and rapamycin had no effect on progestin action in T47D cells, but they stimulated glucocorticoid action in T47D cells. Thus, the effects of these immunosuppressant drugs vary with the cell type and hormonal system that is tested. Whether these drug effects relate directly to the immunophilins bound in receptor complexes remains unknown.

Structure-function properties of the chicken progesterone receptor A synthesized from complementary deoxyribonucleic acid.

The chicken progesterone receptor (PR) cDNA has been cloned and sequenced in our laboratory. Functional receptor A was synthesized from cDNA in two independent systems, by transient transfection of receptor-negative COS M6 cells and by in vitro transcription and translation. These receptors exhibited DNA and hormone binding properties similar to the native PR from oviduct. The ability of receptor to induce target gene transcription was measured by cotransfection of receptor-negative CV-1 cells with expression vectors containing the receptor A cDNA and a progesterone-inducible promotor linked to the chloramphenicol acetyl transferase (CAT) gene. In these assays, receptor A produced hormone-dependent induction of CAT activity. In order to define the functional domains of receptor A, expression constructs coding for C-terminal deletion proteins were prepared. Deletion of the C-terminus resulted in loss of hormone binding activity as well as a loss of CAT induction. However, when 290 amino acids were removed from the C-terminus, this severely truncated receptor protein produced hormone-independent target gene activation. Mutant receptor proteins which retained the highly conserved cysteine-rich (C1) region were able to bind to DNA-cellulose, although removal of 290 amino acids from the C-terminus resulted in reduced affinity for DNA. Deletion of part or all of the C1 region resulted in loss of both DNA-binding and transcriptional activation capacities. These results confirm that C1 functions in DNA binding and transcriptional activation and that hormone binding activity can be localized to the C-terminal half of the protein.

Sequence and expression of a functional chicken progesterone receptor.

We have cloned and sequenced 4.5 kilobases (Kb) of cDNA encoding the chicken progesterone receptor. The complete cDNA contains an open reading frame of 2361 nucleotides in length and encodes a polypeptide of 787 amino acids with a mol wt of 85.9 K. At least four mRNA species have been detected in chick oviduct cells. Direct sequencing of variant cDNAs has suggested that two of the mRNAs (4.5 Kb and 3.6 Kb) differ only in the length of their 3'-untranslated regions. A third mRNA (1.8 Kb) produces a truncated polypeptide which encodes the immunoreactive NH2 terminal sequence of the receptor but lacks the hormone binding regional and half of the DNA-binding domain. The polypeptide expressed from the receptor cDNA in progesterone receptor negative Cos M-6 cells is indistinguishable from oviduct progesterone receptor in terms of hormone binding and antibody reactivity. Furthermore, the cloned receptor is capable of activating transcription of a target gene. This activation is progesterone dependent (with half-maximal stimulation at approximately 3.3 x 10(-10) M) and specific for the target gene.

Interaction of radicicol with members of the heat shock protein 90 family of molecular chaperones.

The Hsp90 family of proteins in mammalian cells consists of Hsp90 alpha and beta, Grp94, and Trap-1 (Hsp75). Radicicol, an antifungal antibiotic that inhibits various signal transduction proteins such as v-src, ras, Raf-1, and mos, was found to bind to Hsp90, thus making it the prototype of a second class of Hsp90 inhibitors, distinct from the chemically unrelated benzoquinone ansamycins. We have used two novel methods to immobilize radicicol, allowing for detailed analyses of drug-protein interactions. Using these two approaches, we have studied binding of the drug to N-terminal Hsp90 point mutants expressed by in vitro translation. The results point to important drug contacts with amino acids inside the N-terminal ATP/ADP-binding pocket region and show subtle differences when compared with geldanamycin binding. Radicicol binds more strongly to Hsp90 than to Grp94, the Hsp90 homolog that resides in the endoplasmic reticulum. In contrast to Hsp90, binding of radicicol to Grp94 requires both the N-terminal ATP/ADP-binding domain as well as the adjacent negatively charged region. Radicicol also specifically binds to yeast Hsp90, Escherichia coli HtpG, and a newly described tumor necrosis factor receptor-interacting protein, Trap-1, with greater homology to bacterial HtpG than to Hsp90. Thus, the radicicol-binding site appears to be specific to and is conserved in all members of the Hsp90 family of molecular chaperones from bacteria to mammals, but is not present in other molecular chaperones with nucleotide-binding domains.

Immunofluorescence colocalization of the 90-kDa heat-shock protein and microtubules in interphase and mitotic mammalian cells.

A mouse monoclonal antibody (AC88) that was raised against the 88-kDa heat-shock protein of the water mold, Achlya ambisexualis, and that cross-reacts with the 90-kDa mammalian heat-shock protein (hsp90), and an antibody against tubulin were used to localize hsp90 and microtubules, respectively, in the same cultured rat endothelial and PtK1 epithelial cells by indirect immunofluorescence. AC88 and tubulin antibodies labeled the same structures in cells at all stages of the cell cycle, regardless of whether cells were permeabilized before or after fixation. Labeling of cell structures by both AC88 and anti-tubulin antibodies was identically affected by treating cells with colcemid. Double labeling with AC88 and anti-tubulin antibodies in interphase and mitotic cells is consistent with the conclusion that all microtubules are labeled and that no subclass of microtubules is preferentially labeled. Fluorescent labeling by AC88 was prevented by preabsorption of the antibody with purified rat hsp90 but was unaffected by preabsorption with purified 6S tubulin dimer. In contrast to AC88, fluorescent labeling by an anti-tubulin antibody was prevented by preabsorption with tubulin dimer but was unaffected by preabsorption with rat hsp90. Western-blot analysis demonstrated no cross-reactivity of AC88 for tubulin and no cross-reactivity of the anti-tubulin antibody for hsp90. A polyclonal antiserum fraction from a rabbit immunized with the 89-kDa heat-shock protein from chicken also labeled the mitotic apparatus in dividing cells and, somewhat less distinctly, fibrous structures in interphase cells. Labeling by hsp89 anti-serum was prevented by absorption with hsp90. AC88 also labeled microtubules in cultured mouse (L929 and 3T3), rat (endothelium and TRST), hamster (CHO) and primate (BSC, COS-1 and HeLa) cell lines. The demonstration of colocalization of hsp90 with microtubules should provide a valuable clue to eventual understanding of the cellular function of this ubiquitous, conserved and abundant stress-response protein.

Estrogen receptors in the chick oviduct.

An estradiol binding component has been identified in the cytoplasmic fraction of the immature chick oviduct. The method used to resolve this receptor differed from the standard sucrose gradient centrifugation approach in that tritiated hormone was present throughout the sucrose gradient. This modification was necessary to preserve the hormone complex during centrifugation. Under these conditions, an similar to 8 S binding component was demonstrated which underwent dissociation to a similar to 5 S component in high ionic strength medium. Binding specificity determinations revealed that this receptor preferentially bound estrogens. Quantitative binding analysis showed that a limited class of binding sites was present with a dissociation constant (K-d) for estradiol of similar to 8.6 times 10-10M. These properties indicate that this binding component may function as a biologic receptor for estrogens in the oviduct.