Steroid hormone receptors and heat shock proteins.
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Animals↗
Biomedical subjects
U Gehring
Publications and source records attributed to U Gehring.
Interference between proteins Hap46 and Hop/p60, which bind to different domains of the molecular chaperone hsp70/hsc70.
Several structurally divergent proteins associate with molecular chaperones of the 70-kDa heat shock protein (hsp70) family and modulate their activities. We investigated the cofactors Hap46 and Hop/p60 and the effects of their binding to mammalian hsp70 and the cognate form hsc70. Hap46 associates with the amino-terminal ATP binding domain and stimulates ATP binding two- to threefold but inhibits binding of misfolded protein substrate to hsc70 and reactivation of thermally denatured luciferase in an hsc70-dependent refolding system. By contrast, Hop/p60 interacts with a portion of the carboxy-terminal domain of hsp70s, which is distinct from that involved in the binding of misfolded proteins. Thus, Hop/p60 and substrate proteins can form ternary complexes with hsc70. Hop/p60 exerts no effect on ATP and substrate binding but nevertheless interferes with protein refolding. Even though there is no direct interaction between these accessory proteins, Hap46 inhibits the binding of Hop/p60 to hsc70 but Hop/p60 does not inhibit the binding of Hap46 to hsc70. As judged from respective deletions, the amino-terminal portions of Hap46 and Hop/p60 are involved in this interference. These data suggest steric hindrance between Hap46 and Hop/p60 during interaction with distantly located binding sites on hsp70s. Thus, not only do the major domains of hsp70 chaperones communicate with each other, but cofactors interacting with these domains affect each other as well.
Adenosine Triphosphate↗
Proteins interacting with the molecular chaperone hsp70/hsc70: physical associations and effects on refolding activity.
We investigated several hsp70/hsc70 interacting proteins and established by two independent techniques that hsp40 and Hop/p60 specifically interact with the 257 residue carboxy-terminal domain of hsp70 while Hap-46 and Hip/p48 bind the 383 residue amino-terminal ATP binding domain. Hap-46 and Hip/p48 competed for binding to hsc70, while Hap-46 had no effect on the binding of either Hop/p60 or hsp40 to hsc70. Hap-46 inhibited the refolding of thermally denatured firefly luciferase in an hsc70 and hsp40 dependent assay, and this effect was largely compensated by Hop/p60. These interacting proteins thus appear to cooperate in affecting the chaperoning activity of hsp70/hsc70.
Carrier Proteins↗
Mammalian protein RAP46: an interaction partner and modulator of 70 kDa heat shock proteins.
A ubiquitously expressed nuclear receptor-associating protein of approximately 46 kDa (RAP46) was identified recently. Interaction experiments with in vitro-translated proteins and proteins contained in cell extracts revealed that a great variety of cellular regulators associate with RAP46. However, in direct interaction tests by the far-Western technique, only 70 kDa proteins showed up and were identified as members of the 70 kDa heat shock protein (hsp70) family. Interaction is specific since not all members of the hsp70 family bind to RAP46; interaction occurs through their ATP-binding domain. RAP46 forms complexes with hsp70 in mammalian cells and interacts with hsp70 in the yeast two-hybrid system. Consistent with the fact that hsp70 can bind a multitude of proteins, we identified heteromeric complexes of RAP46-hsp70 with some selected proteins, most notably c-Jun. Complex formation is increased significantly by pre-treatment with alkaline phosphatase, thus suggesting modulation of interactions by protein phosphorylation. We observed that RAP46 interferes with efficient refolding of thermally denatured luciferase. Moreover, ATP-dependent binding of misfolded proteins to hsp70 was greatly inhibited by RAP46. These data suggest that RAP46 functions as a regulator of hsp70 in higher eukaryotes.
Alkaline Phosphatase↗
The function of steroid hormone receptors is inhibited by the hsp90-specific compound geldanamycin.
The ansamycin antibiotic geldanamycin, which specifically interacts with the heat shock protein hsp90, was used to study the function of hsp90 in steroid hormone receptors. We observed inhibition of glucocorticoid-specific gene induction in several responsive cell systems. Hormone binding abilities of receptors for glucocorticoid, progestin, androgen, and estrogen were inhibited upon exposing intact cells to geldanamycin. Inhibition was only seen when geldanamycin was applied to cell cultures under growth conditions or was present during in vitro synthesis; presynthesized receptors in cell extracts were not affected. Upon withdrawal of geldanamycin, glucocorticoid binding ability was regained; this was partially independent of de novo protein synthesis. Geldanamycin caused decreased levels of immunoreactive glucocorticoid receptors in wild-type cells with enhanced degradation occurring through the ubiquitin-proteasome pathway. Analysis of receptors from treated cells revealed a heteromeric structure of normal size in which the receptor polypeptide is complexed with normal amounts of hsp90 and the immunophilin p59. These data support the view that hsp90 actively participates in steroid-induced signal transduction, and they suggest that geldanamycin affects receptor action without disrupting hsp90-containing heterocomplexes per se. Nevertheless, complexes synthesized and assembled in vitro in the presence of geldanamycin differ from receptors of cellular origin.
3T3 Cells↗
Phospholipase A2 in human placental serum.
Intervillous blood was collected from term placentae at delivery, and sera were tested for phospholipase A2 under various experimental conditions. Enzyme activity was found to develop upon extended storage in the cold or at 37 degrees C. The enzyme is reversibly inhibited by dithiothreitol, requires Ca++ ions for activity, and tolerates various detergents. The apparent molecular weight is 42 kDa. In all these parameters the serum enzyme behaves similar to the 42 kDa phospholipase A2 which we recently purified to homogeneity from thoroughly washed placental tissue. Serum phospholipase A2 appears to be generated by proteolytic processing from a slightly larger inactive precursor which was detected immunochemically. Most likely this protein originates from fetal cells and may be released by membrane damage. We conclude that both placental serum and tissue harbour a novel type of phospholipase A2 which is distinct from cytosolic and secretory phospholipases A2. Preference for arachidonate containing substrate suggests a role in eicosanoid production within gestational tissues.
Blood↗
Molecular chaperoning of steroid hormone receptors.
The study of the large, unactivated form of steroid receptors has led to the discovery of an hsp90/hsp70-based multicomponent protein folding system(s). For steroid receptors, the hsp90 chaperone system determines both repression of transcriptional activity in the absence of hormone and the proper folding of the hormone binding domain to produce the steroid binding conformation. Like steroid receptors, a number of other regulators of transcription and some protein kinases are now known to be associated with hsp90. Given the abundance of the proteins comprising the hsp90 chaperone system and the apparent ubiquity of the system in the animal and plant kingdoms, this system is thought to serve a fundamental role for protein folding, function and possibly trafficking within the cytoplasm and nucleus. In this chapter, we discuss the work on steroid receptor heterocomplex composition that has led to the discovery of new chaperone proteins and we summarize the mechanistic information developed in cell-free studies of receptor heterocomplex assembly.
Carrier Proteins↗
A protein that interacts with members of the nuclear hormone receptor family: identification and cDNA cloning.
In search of proteins which interact with activated steroid hormone receptors, we screened a human liver lambda gt11 expression library with the glucocorticoid receptor. We identified and cloned a cDNA sequence of 1322 bp that encodes a protein of 274 aa. This protein consists predominantly of hydrophilic amino acids and contains a putative bipartite nuclear localization signal. The in vitro translated receptor-associating protein runs in SDS/polyacrylamide gels with an apparent molecular mass of 46 kDa. By use of the bacterially expressed fusion protein with glutathione S-transferase we have found that interaction is not limited to the glucocorticoid receptor but included other nuclear receptors--most notably, the estrogen and thyroid receptors. Binding also occurs with the glucocorticoid receptor complexed with the antiglucocorticoid RU 38486, with the estrogen receptor complexed with the antiestrogen 4-hydroxytamoxifen or ICI 164,384, and even with receptors not complexed with ligand. Association with steroid hormone receptors depends on prior receptor activation--i.e., release from heat shock proteins. The sequence identified here appears to be a general partner protein for nuclear hormone receptors, with the gene being expressed in a variety of mammalian tissues.
Amino Acid Sequence↗
A novel phospholipase A2 from human placenta.
A major soluble phospholipase A2 of human term placenta was characterized and purified about 15,000-fold to homogeneity. The apparent molecular mass as determined in SDS/polyacrylamide gels is 42 kDa. The enzyme is inhibited by dithiothreitol indicating the presence of disulphide bridges which are essential for activity. Studies with known phospholipase A2 inhibitors revealed no immediate relationship to either secretory or cytosolic phospholipases A2. The placental enzyme prefers liposomes of phosphatidylcholine and has a distinct preference for arachidonic acid in the sn-2 position. It tolerates various detergents. Roughly 10 microM Ca2+ is required for activity, but it cannot be replaced by Mg2+ or Mn2+; Zn2+, Cu2+ and Fe3+ are inhibitory. In immunoblots, the placental enzyme was not detected by two separate antisera specific for type-II phospholipases A2 but reacted very weakly with antisera directed against cytosolic phospholipase A2. From these data we suggest that this enzyme is a novel form of phospholipase A2 which may be involved in arachidonic acid mobilization both during the course of pregnancy and at parturition.
Amino Acid Sequence↗
Subunit structure of the nonactivated human estrogen receptor.
The nonactivated estrogen receptor of human MCF-7 mammary carcinoma cells was investigated with respect to stoichiometry of protein subunits. The native receptor complex stabilized by molybdate had a molecular mass of approximately 300 kDa. Chemical cross-linking with several bifunctional reagents resulted in complete stabilization of the same receptor form of approximately 300 kDa and was achieved both in cell extracts and in intact cells. Incubation of the cross-linked receptor with a receptor-specific monoclonal IgG1 antibody increased the molecular mass by approximately 135 kDa--i.e., no more than one immunoglobulin molecule bound to the complex. Partial and progressive cross-linking of affinity-labeled receptors revealed patterns of labeled bands upon denaturing gel electrophoresis indicative of a heteromeric structure. The completely cross-linked receptor was purified to homogeneity and analyzed for protein components. In addition to the receptor polypeptide of approximately 65 kDa, we detected the heat shock proteins hsp90 and p59; the hsp90 band was roughly twice as intense as the p59 band. The heat shock protein hsp70 and the 40-kDa cyclophilin were not detected as components of the highly purified cross-linked receptor of approximately 300 kDa. We suggest a heterotetrameric structure consisting of one receptor polypeptide, two hsp90 molecules, and one p59 subunit, for which the molecular mass adds up to approximately 300 kDa. Thus, the nonactivated estrogen receptor has a molecular architecture homologous to those of glucocorticoid and progesterone receptors, even though phylogenetically the estrogen receptor gene forms a distinct subgroup within the gene family of nuclear hormone receptors.
Antibodies, Monoclonal↗
Signal transduction pathways leading to arachidonic acid release from neutrophilic HL-60 cells. The involvement of G protein, protein kinase C and phospholipase A2.
Arachidonic acid release from undifferentiated and neutrophilic HL-60 cells was studied. In neutrophilic cells it was stimulated by N-formyl-Met-Leu-Phe and mastoparan by a mechanism involving Gi protein and phospholipase C and was largely dependent on diacyglycerol lipase. Maximum release from both cell types was achieved with fluoride and required cellular energy. Inhibitor studies suggest that arachidonic acid release by fluoride stimulation leads to phospholipase A2 activation with signal transduction involving phospholipase C and protein kinase C. Only neutrophilic cells responded to phorbol ester if Ca(2+)-ionophore was simultaneously present but this effect was abolished by extended treatment with phorbol ester. Thus, protein kinase C plays a major role in highly stimulated neutrophilic cells. These cells are differently equipped with protein kinase C isoenzymes compared with undifferentiated cells. In contrast, both cell types contain similar levels of type II and cytosolic phospholipases A2, the former being by far the more prevalent.
Arachidonic Acid↗
Expression screening for interacting proteins using immunochemical detection.
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Animals↗
Cloning of 5' cDNA regions by inverse PCR.
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Cloning, Molecular↗
Glucocorticoid receptor expression during differentiation of human promyeloic leukemia cells.
The human promyeloic leukemia cell line HL-60 can be triggered in culture to differentiate into several cell types of the myeloid lineage in response to a variety of chemical stimuli. We used this cell system in order to investigate the changes in glucocorticoid receptors which occur concomitantly with such cellular differentiations. Neutrophilic granulocytes obtained by the addition of dimethyl sulfoxide or retinoic acid to the culture medium showed only slight changes in cellular glucocorticoid receptor levels and receptor-specific mRNA as compared to undifferentiated control cells. Monocytic cells induced by incubation with dihydroxy-vitamin D3 had a moderate increase in receptor hormone-binding activity. However, differentiation toward macrophages by exposure to phorbol ester resulted in a 5- to 6- fold increase in both cellular hormone-binding capacity and immunochemically cross-reacting receptor protein. An even greater increase in glucocorticoid receptor-specific mRNA was observed. These data suggest that the receptor is regulated at the mRNA level and that de novo receptor synthesis occurs during macrophage differentiation, thus making these cells potentially more susceptible to glucocorticoid-induced effects.
Cell Differentiation↗
The structure of glucocorticoid receptors.
The glucocorticoid receptor of mouse thymic lymphoma cells was investigated. The receptor-hormone complex in cytosolic extracts has a Stokes' radius of 82 A and Mw approximately 330 kDa. In the presence of salt at high concentrations, however, the receptor-complex has a Stokes' radius of 60 A and Mw approximately 120 kDa. This receptor form is able to interact with DNA. Chemical cross-linking was used to stabilize the high molecular weight receptor structure against subunit dissociation and this was found to prevent receptor activation to DNA binding. The affinity labeled receptor was submitted to progressive cross-linking and the intermediate cross-linked forms were analyzed. This led to the conclusion that the high molecular weight receptor structure is a hetero-tetramer consisting of one receptor polypeptide of approximately 100 kDa, two molecules of the 90 kDa heat shock protein hsp90 and an additional protein subunit. The latter was unequivocally identified by immunochemical techniques as the 59 kDa protein p59. The 70 kDa heat shock protein was found not to be a bona fide receptor component but was a contaminant of our immunopurification procedure. Cross-linking studies also showed that the receptor exists in the high molecular weight form in intact cells and in the absence of hormone.
Animals↗
Heterotetrameric structure of the human progesterone receptor.
Nonactivated progesterone receptors in extracts of human T47D mammary carcinoma cells were investigated. Chemical cross-linking with dimethyl suberimidate resulted in complete stabilization of the A and B receptors with an average molecular mass of 340 kDa. For analyzing the subunit structure, we concentrated on the larger B receptor, which was separated from the A form by immunoaffinity chromatography. Progressive cross-linking of the photoaffinity-labeled receptor resulted in patterns of labeled bands in SDS gels, which are indicative of a heterotetrameric structure. It consists of one receptor polypeptide in association with two 90-kDa subunits and one polypeptide of approximately 60 kDa. The completely cross-linked B receptor has a molecular mass of approximately 390 kDa. To identify the subunits, the oligomeric B receptor was cross-linked with a cleavable bisimidate, highly purified by immunoaffinity chromatography, and analyzed by gel electrophoresis and immunoblotting. The receptor polypeptide has a mass of 116.5 kDa. The 90-kDa band was identified as the heat shock protein hsp90 and was roughly twice as intense as the receptor polypeptide. By use of specific antibodies, we identified the fourth receptor subunit as a 59-kDa protein (p59); we did not obtain any evidence for the heat shock protein hsp70 being a receptor component. We suggest an analogous heterotetrameric structure for the nonactivated A receptor.
Chromatography, Affinity↗
Structure of the glucocorticoid receptor in intact cells in the absence of hormone.
The nonactivated glucocorticoid receptor (Mr approximately 330,000, Strokes radius = 82 A) contained in cell extracts and complexed with a steroidal ligand was previously investigated by chemical cross-linking. It was identified as a heterotetramer composed of one receptor polypeptide, two molecules of the 90-kDa heat shock protein hsp90, and one 59-kDa protein subunit (Rexin, M., Busch, W., and Gehring, U. (1991) J. Biol. Chem. 266, 24601-24605). We now have used the cross-linking technique to investigate the receptor structure in intact WEHI-7 mouse lymphoma cells at 37 degrees C and under steroid-free conditions. Using immunochemical methods we show that the receptor present in whole cells likewise exists as a high molecular weight structure of Strokes radius 82 A. It has a subunit composition identical to that of the nonactivated receptor-steroid complex in cell extracts. This is the first account of a steroid hormone receptor in its native state as it is contained in target cells under physiological conditions and before a hormonal signal is received.
Animals↗
Protein components of the nonactivated glucocorticoid receptor.
The nonactivated glucocorticoid receptor (Mr approximately 350,000) of WEHI-7 mouse lymphoma cells was investigated with respect to the stoichiometry of protein subunits. Cross-linking patterns obtained by affinity labeling and denaturing gel electrophoresis revealed a heterotetramer consisting of one receptor polypeptide in association with two 90- and one approximately 50-kDa subunits. The receptor stabilized by molybdate, disulfide bond formation, or chemical cross-linking was purified roughly 6000-fold by immunoaffinity chromatography and analyzed by gel electrophoresis and immunoblotting. The 90-kDa component was consistently detected in a 2:1 ratio with respect to the receptor polypeptide and was identified as the 90-kDa heat shock protein, hsp90. A 70-kDa heat shock protein was found in both stabilized and nonstabilized receptors and bound to the immunomatrix independent of receptor. The additional receptor subunit was unequivocally identified as the 59-kDa protein previously described (Tai, P.-K. K., Maeda, Y., Nakao, K., Wakim, N. G., Duhring, J. L., and Faber, L. E. (1986) Biochemistry 25, 5269-5275). This component was found only in complexes cross-linked via amino groups. It was removed from the molybdate-stabilized receptor under our purification conditions, thus leaving behind a trimer composed of the receptor polypeptide and two molecules of hsp90. In the absence of hormone, the receptor had the same subunit composition as in its presence.
Animals↗