Evidence that the endogenous heat-stable glucocorticoid receptor-activating factor is thioredoxin

Extraction of rat liver cytosol with 10% charcoal at 4 degrees C inactivates specific glucocorticoid-binding capacity. The steroid-binding capacity of extracted cytosol can be restored by adding dithiothreitol or by incubating with boiled liver cytosol at 20 degrees C in the presence of 10 mM sodium molybdate. Two components of boiled cytosol are required for receptor activation: NADPH and an endogenous heat-stable protein with an apparent Mr of 12,300 by Sephadex G-50 chromatography. This endogenous receptor-activating protein coelutes on Sephadex G-50 chromatography with endogenous thioredoxin activity, and it can be replaced in the activating system by purified Escherichia coli thioredoxin. These observations suggest that glucocorticoid receptors in cytosol preparations are maintained in a reduced, steroid-binding state by a NADPH-dependent, thioredoxin-mediated reducing system.     

The endogenous heat-stable glucocorticoid receptor stabilizing factor and the H-2 locus

It has recently been suggested that the level of the endogenous glucocorticoid receptor stabilizing factor in mouse liver is regulated by the major hisiocompatibility, H-2. complex (Katsumata et al. [1]). We have developed an asssay for the activity of the endogenous heat-stable factor in mouse liver and have assayed this factor in liver cytosols prepared from two pairs of two H-2 congenic mouse strains. Our results show that the amount of the endogenous factor is the same in all four mouse strains and that it is not regulated by the H-2 locus.     

Evidence that removal of an endogenous metal that stabilizes the untransformed glucocorticoid receptor in cytosol allows ligand-independent receptor transformation

Cytosol preparations contain an endogenous heat-stable factor which stabilizes the glucocorticoid receptor in its untransformed, non DNA-binding form. Elution of a partially purified preparation of this stabilizing factor through a metal chelating resin (Chelex-100) leads to the loss of its ability to inhibit temperature-mediated transformation of the receptor. Sodium molybdate mimicks the ability of this endogenous metal to stabilize the untransformed receptor, and it too is adsorbed by Chelex resin. When an L-cell cytosol preparation containing the glucocorticoid receptor is passed through a column of Chelex-100 resin and then incubated at 15 degrees C, the receptor is rapidly transformed to the DNA-binding state, regardless of whether it is steroid-bound or not. In contrast, whole cytosol containing endogenous metals is transformed to the DNA-binding state only when the receptor is both steroid-bound and exposed to elevated temperature. these data suggest that a metal (or metals) may be involved in conferring the property of ligand-dependency to the transformation process.     

Proof that the endogenous, heat-stable glucocorticoid receptor-activating factor is thioredoxin

Extraction of rat liver cytosol with charcoal inactivates glucocorticoid-binding capacity and receptors can be reactivated to the steroid-binding state by an endogenous reducing system utilizing NADPH and a Mr = 12,000, heat-stable, endogenous, cytosolic protein (Grippo, J. F., Tienrungroj, W., Dahmer, M. K., Housley, P. R., and Pratt, W. B. (1983) J. Biol. Chem. 258, 13658-13664). In this paper we show that NADPH-dependent conversion of the rat liver glucocorticoid receptor from a nonbinding to a steroid-binding form is blocked in an immune-specific manner by antisera raised against purified rat liver thioredoxin reductase or thioredoxin. The inhibition produced by thioredoxin reductase antiserum may be circumvented by dithiothreitol or overcome by addition of purified thioredoxin reductase. These observations prove that the endogenous glucocorticoid receptor-activating factor is thioredoxin and that the enzyme required for generating the steroid-binding conformation of the glucocorticoid receptor by the endogenous receptor-activating system is thioredoxin reductase.     

Purification of the endogenous glucocorticoid receptor stabilizing factor

A ubiquitous, low molecular weight, heat-stable component of cytosol stabilizes the glucocorticoid receptor in its untransformed state in association with hsp90. This heat-stable factor mimics molybdate in its effects on receptor function, and it has the heat stability, charge, and chelation properties of a metal oxyanion [Meshinchi, S., Grippo, J.F., Sanchez, E.R., Bresnick, E.H., & Pratt, W.B. (1988) J. Biol. Chem. 263, 16809-16817]. In this paper, we describe the further purification of the endogenous factor from rat liver cytosol by anion-exchange HPLC (Ion-110) after prepurification by molecular sieving, cation absorption, and charcoal absorption. Elution of the factor with an isocratic gradient of ammonium bicarbonate results in recovery of all of the bioactivity in a single peak which coelutes with inorganic phosphate and contains all of the endogenous molybdenum. The bioactivity can be separated from inorganic phosphate by chromatography of the partially purified endogenous factor on a metal-chelating column of Chelex-100. The chelating procedure results in complete loss of bioactivity with recovery of 98% of the inorganic phosphate in both the column drop-through and a subsequent 1 M NaCl wash. The factor preparation purified through the Ion-110 HPLC step inhibits temperature-mediated dissociation of the immunopurified glucocorticoid receptor-hsp90 complex, but it is considerably more effective at stabilizing the unpurified receptor-hsp90 complex in a Chelex-treated cytosol system that has been depleted of metal components. These observations support the proposal that an endogenous metal can stabilize the binding of hsp90 to the receptor but it is likely that other cytosolic components that are not present in the immunopurified complex must contribute to the stability of the soluble protein-protein complex in cytosol.     

Evidence that the 90-kDa phosphoprotein associated with the untransformed L-cell glucocorticoid receptor is a murine heat shock protein

Two phosphoproteins are adsorbed to protein-A-Sepharose when cytosol from 32P-labeled L-cells is incubated with a monoclonal antibody against the glucocorticoid receptor: one is a 98-100-kDa phosphoprotein that contains the steroid-binding site and the other is a 90-kDa nonsteroid-binding phosphoprotein that is associated with the untransformed, molybdate-stabilized receptor (Housley, P. R., Sanchez, E. R., Westphal, H.M., Beato, M., and Pratt, W.B. (1985) J. Biol. Chem. 260, in press). In this paper we show that the 90-kDa receptor-associated phosphoprotein is an abundant cytosolic protein that reacts with a monoclonal antibody that recognizes the 90-kDa phosphoprotein that binds steroid receptors in the chicken oviduct. The 90-kDa protein immunoadsorbed from L-cell cytosol with this antibody reacts on Western blots with rabbit antiserum prepared against the 89-kDa chicken heat shock protein. Immunoadsorption of molybdate-stabilized cytosol by antibodies against the glucocorticoid receptor results in the presence of a 90-kDa protein that interacts on Western blots with the antiserum against the chicken heat shock protein. The association between the 90-kDa protein and the receptor is only seen by this technique when molybdate is present to stabilize the complex; and when steroid-bound receptors are incubated at 25 degrees C to transform them to the DNA-binding state, the 90-kDa protein dissociates. These observations are consistent with the proposal that the untransformed glucocorticoid receptor in L-cells exists in a complex with the murine 90-kDa heat shock protein.     

The heat-stable cytosolic factor that promotes glucocorticoid receptor binding to DNA is neither thioredoxin nor ribonuclease

Treatment of rat liver cytosol containing temperature-transformed [3H]dexamethasone-bound receptors at 0 degree C with the sulfhydryl modifying reagent methyl methanethiosulfonate (MMTS) inhibits the DNA-binding activity of the receptor, and DNA-binding activity is restored after addition of dithiothreitol (DTT). However, transformed receptors that are treated with MMTS and then separated from low Mr components of cytosol by passage through a column of Sephadex G-50 have very little DNA-binding activity when DTT is added to regenerate sulfhydryl moities. The receptors will bind to DNA if whole liver cytosol or boiled liver cytosol is added in addition to DTT. The effect of boiled cytosol is mimicked by purified rat thioredoxin or bovine RNase A in a manner that does not reflect the reducing activity of the former or the catalytic activity of the latter. This suggests that the reported ability of each of these heat-stable peptides to stimulate DNA binding by glucocorticoid receptors is not a biologically relevant action. We suggest that stimulation of DNA binding of partially purified receptors by boiled cytosol does not constitute a reconstitution of a complete cytosolic system in which the dissociated receptor must associate with a specific heat-stable accessory protein required for DNA binding, as has been suggested in the "two-step" model of receptor transformation recently proposed by Schmidt et al. (Schmidt T.J., Miller-Diener, A., Webb M.L. and Litwack G. (1985) J. biol. Chem. 260, 16255-16262).     

Direct stoichiometric evidence that the untransformed Mr 300,000, 9S, glucocorticoid receptor is a core unit derived from a larger heteromeric complex

We have used three methods to measure the stoichiometry of the glucocorticoid receptor and the 90-kDa heat shock protein (hsp90) in L-cell glucocorticoid receptor complexes that were purified by immunoadsorption to protein A-Sepharose with an anti-receptor monoclonal antibody, followed by a minimal washing procedure that permits retention of receptor-associated protein. In two of the methods, receptor was quantitated by radioligand binding, and receptor-specific hsp90 was quantitated against a standard curve of purified hsp90, either on Coomassie blue stained SDS gels by laser densitometry or on Western blots by quantitative immunoblotting with 125I-labeled counterantibody. The stoichiometry values obtained by densitometry and immunoblotting are 7 and 6 mol of hsp90/mol of receptor, respectively. In a third method, which detects total receptor protein rather than just steroid-bound receptor, the ratio of hsp90 to receptor was determined by immunopurifying receptor complexes from [35S]methionine-labeled L cells, and the amount of 35S incorporated into receptor and hsp90 was corrected for the established methionine content of the respective proteins. In complexes from L cells which are labeled to steady state (48 h), the ratio of hsp90 to GR is 4:1. When immunoadsorbed receptor complexes are washed extensively with 0.5 M NaCl and 0.4% Triton X-100 in the presence of molybdate, the ratio of hsp90 to GR is 2:1. In addition to hsp90, preparations of [35S]methionine-labeled untransformed receptor complex also contain a 55-kDa protein that the conclusion that the untransformed L-cell glucocorticoid receptor exists in cytosol in a much larger heteromeric complex than considered to date.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nanomolar concentrations of untransformed glucocorticoid receptor in nuclei of intact cells

The subcellular distribution of untransformed glucocorticoid-receptor complex in vivo has been studied by chemical crosslinking of intact cells, and using a procedure adequate for correction of experimental errors due to redistribution of components between cytosolic and nuclear fractions. We found that in HeLa S₃ cells 85.4% of total glucocorticoid-receptor complexes are located in nuclei, and 14.6% are cytosolic. If measurements were performed with MCF-7 cells, we determined that the nuclear pool of glucocorticoid-receptor complexes accounts for 75.2% of the total cellular content, whereas the remaining 24.8% are cytosolic. When the subcellular distribution of estrogen-receptor complexes was determined, instead, we found that they are almost exclusively located in nuclei of MCF-7 cells, which contain 88.9% of the total. In order to estimate the molar concentration of receptors in cytosol and nuclei of intact cells, we determined the free water content of the two compartments. The volume of solvent was found to vary in the three cell lines we have studied, and our data showed that these variations are due to the cytosolic fractions, as the free water content of nuclei is essentially the same in those cells. When the free water content and the levels of glucocorticoid-receptor complexes we have measured were used to estimate the molar ncentrations of receptors, we found that these range between 0.4 and 18.9 nM in cytosols, and between 3.9 and 6.3 nM in nuclei of the three cell lines we have studied. We then concluded that the relative distribution of untransformed glucocorticoid-receptor complexes between cytosol and nuclei is cell-specific but their molar concentration in the nuclear compartment does not greatly vary among different cells.     

Absence of detectable ribonucleic acid in the purified, untransformed mouse glucocorticoid receptor

The glucocorticoid receptor (GC-R) isolated from the mouse AtT-20 pituitary tumor cell line exists in three forms. The untransformed (non-DNA-binding), 9.1S species (319K) can be converted into two transformed (DNA-binding) species. One of these (5.2 S, Mr 132K) appears to be composed of one molecule of the hormone-binding, monomeric protein (96K) plus a small RNA, while the second transformed species is the monomeric, hormone-binding subunit (3.8 S, 96K) itself. We wished to determine whether the untransformed GC-R contains RNA or if the monomer binds to RNA subsequent to subunit dissociation (which occurs during receptor transformation). Kinetic studies using both the crude and purified untransformed GC-R show that the untransformed, 9.1S GC-R dissociates into 3.8S monomeric subunits, without forming a transient 5.2S complex. The untransformed receptor was then purified with affinity chromatography, gel filtration, and DEAE-cellulose chromatography. One major protein band, corresponding in size to the GC-R monomer (94K-96K), was observed on sodium dodecyl sulfate-polyacrylamide gels upon silver staining or fluorography of [3H]dexamethasone mesylate covalently labeled receptor. In vivo 32P-labeling of AtT-20 cells, followed by purification of the untransformed GC-R, yielded two major 32P-labeled components (94K-96K and 24K). Both of these bands were protease-sensitive, contained phosphoserine, and were unaffected by ribonuclease treatment. We conclude that the untransformed mouse GC-R is wholly proteinaceous and contains no RNA. Thus, RNA binding occurs subsequent to dissociation of the oligomeric, untransformed GC-R complex into monomers.     

The HIV-1 Vpr and glucocorticoid receptor complex is a gain-of-function interaction that prevents the nuclear localization of PARP-1

The Vpr protein of HIV-1 functions as a vital accessory gene by regulating various cellular functions, including cell differentiation, apoptosis, nuclear factor of kappaB (NF-kappaB) suppression and cell-cycle arrest of the host cell. Several reports have indicated that Vpr complexes with the glucocorticoid receptor (GR), but it remains unclear whether the GR pathway is required for Vpr to function. Here, we report that Vpr uses the GR pathway as a recruitment vehicle for the NF-kappaB co-activating protein, poly(ADP-ribose) polymerase-1 (PARP-1). The GR interaction with Vpr is both necessary and sufficient to facilitate this interaction by potentiating the formation of a Vpr-GR-PARP-1 complex. The recruitment of PARP-1 by the Vpr-GR complex prevents its nuclear localization, which is necessary for Vpr to suppress NF-kappaB. The association of GR with PARP-1 is not observed with steroid (glucocorticoid) treatment, indicating that the GR association with PARP-1 is a gain of function that is solely attributed to HIV-1 Vpr. These data provide important insights into Vpr biology and its role in HIV pathogenesis.     

Evidence that the mitochondrial activator of phosphorylated branched-chain 2-oxoacid dehydrogenase complex is the dissociated E1 component of the complex

The ability of glucagon (10 nM) to increase hepatocyte intracellular cyclic AMP concentrations was reduced markedly by the tumour-promoting phorbol ester TPA (12-O-tetradecanoyl phorbol-13-acetate). The half-maximal inhibitory effect occurred at 0.14 ng/ml TPA. This action occurred in the presence of the cyclic AMP phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) indicating that TPA inhibited glucagon-stimulated adenylate cyclase activity. TPA did not affect either the binding of glucagon to its receptor or ATP concentrations within the cell. TPA did inhibit the increase in intracellular cyclic AMP initiated by the action of cholera toxin (1 microgram/ml) under conditions where phosphodiesterase activity was blocked. TPA did not inhibit glucagon-stimulated adenylate cyclase activity in a broken plasma membrane preparation unless Ca2+, phosphatidylserine and ATP were also present. It is suggested that TPA exerts its inhibitory effect on adenylate cyclase through the action of protein kinase C. This action is presumed to be exerted at the point of regulation of adenylate cyclase by guanine nucleotides.     

Guanylyl cyclase is a heat-stable enterotoxin receptor.

Plasma membrane forms of guanylyl cyclase have been shown to function as natriuretic peptide receptors. We describe a new clone (GC-C) encoding a guanylyl cyclase receptor for heat-stable enterotoxin. GC-C encodes a protein containing an extracellular amino acid sequence divergent from that of previously cloned guanylyl cyclases; however, the protein retains the intracellular protein kinase-like and cyclase catalytic domains. Expression of GC-C in COS-7 cells results in high guanylyl cyclase activity. In addition, heat-stable enterotoxin from E. coli, but not natriuretic peptides, causes marked elevations of cyclic GMP and is specifically bound by cells transfected with GC-C. The enterotoxin fails to elevate cyclic GMP in nontransfected cells or in cells transfected with the natriuretic peptide/guanylyl cyclase receptors. These results show that a heat-stable enterotoxin receptor responsible for acute diarrhea is a plasma membrane form of guanylyl cyclase.     

Purification and identification of the heat-stable factor required for pregnenolone-binding protein activity. Evidence that the factor is adenosine 3',5'-diphosphate.

This paper presents data identifying adenosine 3',5'-diphosphate (3',5'-ADP) as the small heat-stable factor essential for the active steroid binding complex of the adrenocortical pregnenolone-binding protein (PBP). Factor activity obtained from the boiled supernatant of partially purified PBP was isolated by high performance liquid chromatography using weak anion-exchange and hydrophobic (C18) chromatography sequentially. The purified material retained characteristic factor activity and presented a UV spectrum identical to that for authentic 3',5'-ADP. Mass spectroscopic analysis of the isolated factor revealed an M-H ion of appropriate mass (m/z = 426) and a decomposition pattern for the M-H ion that was consistent with the structure of 3',5'-ADP. The studies presented here demonstrate that authentic 3',5'-ADP can categorically substitute for factor prepared from the soluble fraction of the guinea pig adrenal. Specifically, 3',5'-ADP potentiated ligand binding of partially purified native PBP and restored binding capacity to alkaline phosphatase-inactivated PBP in a dose-dependent manner. As is the case for adrenocortical factor activity, these effects were negated by pretreating the 3',5'-ADP with calf intestinal alkaline phosphatase. Other nucleotides similarly tested, including ADP isomers, were ineffective as factor substitutes. The sulfated form of 3',5'-ADP (i.e. 3'-phosphoadenosine 5'-phosphosulfate) demonstrated some potential for restoring binding capacity to phosphatase-inactivated PBP; however, this compound was clearly inhibitory rather than stimulatory for native PBP activity. Taken collectively, the data overwhelmingly demonstrate that 3',5'-ADP is in fact the molecule required by the PBP for high affinity steroid binding complex formation. It is not yet known whether 3',5'-ADP acts allosterically or contributes directly to the structure of the steroid binding site.     

Ribonucleic acid is a component of the oligomeric, transformed mouse AtT-20 cell glucocorticoid receptor

The glucocorticoid receptor from mouse AtT-20 pituitary tumor cells exists in three forms. The largest form is an untransformed (non-DNA-binding), oligomeric species (9.1 S, 8.3 nm, Mr 319 000). Two transformed (DNA-binding) forms can be generated. One is an oligomeric protein (5.2 S, 6-8.3 nm, Mr 132 000-182 000), while the other is the monomeric, hormone-binding subunit (3.8 S, 6 nm, Mr 96 000). The composition of the oligomeric, transformed receptor and its relationship to the monomeric protein were examined. The 3.8S monomer can be isolated from DEAE-cellulose (0.12 M step elution) in a form that continues to sediment at about 3.8 S on molybdate-containing sucrose gradients and at about 4.2 S on molybdate-free gradients. Addition of a non-hormone-binding component isolated from the same DEAE-cellulose column (0.5 M KCl step) can apparently interact with the 3.8-4.2 S monomer, increasing its sedimentation coefficient to 5.2 S (on molybdate-containing gradients) or 6.6 S (on low-salt, molybdate-free gradients). This factor is a macromolecule (nondialyzable) and is heat-stable (100 degrees C, 20 min). A dose-dependent shift to the higher sedimentation coefficient is observed when increasing quantities of the 0.5 M step material are added to the receptor monomer. This activity is abolished when the 0.5 M step material is treated with ribonuclease A. Further, when RNA is purified from the 0.5 M step by phenol/chloroform extraction, its ability to increase the S value of the monomer is retained. Ribonuclease treatment of the untransformed, 9.1S, oligomeric complex does not cause a significant decrease in sedimentation rate, while the same treatment of the 5.2S, oligomeric, transformed receptor (obtained after Sephadex G-25 transformation) causes a decrease in sedimentation rate to about 3.8 S. The addition of bovine liver mRNA and rRNA does not cause a shift in sedimentation rate of the receptor monomer to a discrete, higher sedimenting receptor form. However, the addition of total rabbit liver tRNA or three distinct tRNA species causes a shift in sedimentation to a similar, but not identical, form as that with the 0.5 M step material. We propose that the 5.2S, oligomeric transformed glucocorticoid receptor is composed of one monomeric hormone-binding, protein subunit (Mr 96 000) and a low molecular weight RNA (Mr 36 000). This interaction may be important for the role of the receptor in regulating gene expression.     

Subunit composition of the untransformed glucocorticoid receptor in the cytosol and in the cell.

We have used bifunctional reagents to examine the subunit composition of the non-DNA-binding form of the rat and human glucocorticoid receptor. Treatment of intact cells and cell extracts with a reversible cross-linker, followed by electrophoretic analysis of immunoadsorbed receptor revealed that three proteins of apparent approximate molecular masses, 90, 53 and 14 kDa are associated with the receptor. The first of these was identified immunochemically as a 90-kDa heat-shock protein (hsp90). The complex isolated from HeLa cells contained 2.2 mol hsp90/mol steroid-binding subunit. Cross-linking of the receptor complex in the cytosol completely prevented salt-induced dissociation of the subunits. The cross-linked receptor was electrophoretically resolved into two oligomeric complexes of apparent molecular mass 288 kDa and 347 kDa, reflecting the association of the 53-kDa protein with a fraction of the receptor. Since no higher oligomeric complexes could be generated by cross-linking cell extracts under different conditions, we conclude that most of the untransformed cytosolic receptor is devoid of additional components.     

Purification of splicing factor SF1, a heat-stable protein that functions in the assembly of a presplicing complex.

Splicing factor SF1 represents one of the proteins that function early in the splicing of nuclear pre-mRNA in the formation of a presplicing complex. SF1 was purified to homogeneity from HeLa cell nuclear extracts by column chromatography. It consists of a single polypeptide of 75 kDa and is distinct from other protein factors that function early in spliceosome assembly. SF1 activity is completely resistant to temperatures of up to 100 degrees C. The purified protein does not appear to be associated with RNA-binding, RNA-annealing, or ATPase activity.     

Evidence from the anti-idiotypic network that the acetylcholine receptor is a rabies virus receptor.

We have developed idiotype-anti-idiotype monoclonal antibodies that provide evidence for rabies virus binding to the acetylcholine receptor (AChR). Hybridoma cell lines 7.12 and 7.25 resulted after fusion of NS-1 myeloma cells with spleen cells from a BALB/c mouse immunized with rabies virus strain CVS. Antibody 7.12 reacted with viral glycoprotein and neutralized virus infectivity in vivo. It also neutralized infectivity in vitro when PC12 cells, which express neuronal AChR, but not CER cells or neuroblastoma cells (clone N18), which have no AChR, were used. Antibody 7.25 reacted with nucleocapsid protein. Anti-idiotypic monoclonal antibody B9 was produced from fusion of NS-1 cells with spleen cells from a mouse immunized with 7.12 Fab. In an enzyme-linked immunosorbent assay and immunoprecipitation, B9 reacted with 7.12, polyclonal rabies virus immune dog serum, and purified AChR. The binding of B9 to 7.12 and immune dog serum was inhibited by AChR. B9 also inhibited the binding of 7.12 to rabies virus both in vitro and in vivo. Indirect immunofluorescence revealed that B9 reacted at neuromuscular junctions of mouse tissue. B9 also reacted in indirect immunofluorescence with distinct neurons in mouse and monkey brain tissue as well as with PC12 cells. B9 staining of neuronal elements in brain tissue of rabies virus-infected mice was greatly reduced. Rabies virus inhibited the binding of B9 to PC12 cells. Mice immunized with B9 developed low-titer rabies virus-neutralizing antibody. These mice were protected from lethal intramuscular rabies virus challenge. In contrast, anti-idiotypic antibody raised against nucleocapsid antibody 7.25 did not react with AChR.     

Evidence that the antiestrogen binding site is a histamine or histamine-like receptor

N,N-diethyl-2-[(4 phenylmethyl)-phenoxy]-ethanamine X HCl (DPPE), a compound selective for the antiestrogen binding site, is structurally similar to the aminoethyl ether group of antihistamines. Our studies now reveal that H1-, but not H2-antagonists, also compete for this site in the order: DPPE = hydroxyzine = perchlorperazine greater than phenyltoloxamine greater than pyrilamine greater than diphenhydramine. The affinity of these compounds for the antiestrogen binding site correlates with their in vitro cytotoxicity against MCF-7 and EVSA-T human breast cancer cells. Tamoxifen, DPPE and hydroxyzine also bind to H1 receptors present in digitonin-solubilized rat liver microsomes, but with less affinity than pyrilamine, which is selective for this site; the ratio of H1 to antiestrogen binding sites in this preparation is 4:1. The data suggest that the antiestrogen binding site may be, in whole or in part, a receptor for histamine different from H1 and H2.