Merry R. Sherman

Structural similarity of molybdate-stabilized steroid receptors in human breast tumors, uteri and leukocytes

Abstract Hydrodynamic parameters of receptors for three classes of steroids from several benign and malignant human tissues have been evaluated in buffers of similar conductivity containing Na 2 MoO 4 and/or KCl. Estrogen receptors in breast tumor cytosols were labeled with [ 3 H]-estradiol, progcstin receptors in cytosols from breast tumors and benign or carcinomatous uterine endometrium with [ 3 H]-promegestone (R5020) and glucocorticoid receptors in normal lymphocytes and cells from patients with acute lymphoblastic and nonlymphocytic leukemias with [ 3 H]-triamcinolone acetonide. Receptor-bound steroid was resolved from free or loosely bound steroid by charcoal-dextran treatment or chromatography on 9-ml columns of Sephadex LH-20 (Pharmacia) prior to analysis by glycerol gradient centrifugation or filtration on 120-ml columns of Agarose A-1.5 m (BioRad). Ultracentrifugal and Chromatographic patterns obtained in control buffers were heterodisperse, and the recovery and predominant receptor form(s) detected depended on numerous experimental variables: time and temperature of incubation, protein concentration of the cytosol and concentration of salt in the fractionation buffers (30, 50 or 150 mM KCl). In contrast, analyses in hypotonic buffers containing 20 mM Na 2 MoO 4 revealed highly consistent results for both the sedimentation coefficient (s 20 ,w) and Stokes radius ( R s ) of all of the receptors studied. In view of this similarity among the corresponding parameters, the data from 47 gradients and 26 chromatograms were pooled to obtain the following values for the “average” molybdate-stabilized receptor in human tissues: s 20 w = 9.6 ± 0.3 S and R s = 77 ± 4 A (mean ± SD). In a protein of normal density and solvalion, these parameters indicate a molecular weight of ~310,000 and an axial ratio of 11, for a prolate ellipsoid. The observation of similarly large, asymmetric forms of these receptors in buffers containing both 20 mM Na 2 MoO 4 and 120 mM KCl implies that they are not artifactual aggregates formed during extraction and analysis in hypotonic buffers. The remarkable conservation of this structure among receptors for three classes of steroids in both benign and malignant specimens of human breast, uterus and leukocytes suggests that most of this structure is essential to receptor function. Purification and detailed characterization of the molybdate-stabilized complexes should facilitate the elucidation of the common pathways of receptor action in benign and steroid-responsive malignant tissues and the detection of any structural defects in receptors in steroid-resistant cancers.

Binding of glucocorticoid 21-OIC acids and esters to molybdate-stabilized hepatic receptors

A new class of antiinflammatory steroids, the glucocorticoid acid esters, have been synthesized by modifying the 17β-ketol side chain of cortisol and prednisolone. The competititive binding of several steroid 21-oic acids and their methyl esters to receptors in rat liver cytosol was studied at 4°C in the presence of 20 mM Na2MoO4, which was previously shown to stabilize these and other receptors. Charcoal-dextran assays of the inhibition of [3H]-dexamethasone binding indicated the following relative affinities: dexamethasone > prednisolone > cortisol > methyl prednisolonate (methyl 11β,17-dihydroxy-3,20-dioxo-pregna-1,4-dien-21-oate) ≅: methyl cortisolate > methyl 20-dihydroprednisolonate (methyl 11β,17,20ξ-trihydroxy-3-oxo-pregna-1,4-dien-21-oate) ≅ methyl 20-dihydrocortisolate > 20-dihydroprednisolonic acid (11β,17,20ξ-trihydroxy-3-oxo-pregna-1,4-dien-21-oic acid) ≅ 20-dihydrocortisolic acid. There was no detectable competition by methyl 20α- or methyl 20β-cortolate (methyl 3α,11β,17,20-tetrahydroxy-5β-pregnan-21-oate). Although the affinity of methyl prednisolonate for hepatic cytosol receptors was

Glucocorticoid Receptor Cleavage by Leupeptin-Sensitive Enzymes in Rat Kidney Cytosol

75-fold lower than that of prednisolone under these conditions, the local antiinflammatory potency of the ester was similar to that of the parent compound, as determined in separate studies. There was no detectable metabolism of [3H]-labeled methyl prednisolonate during incubation with liver cytosol for 5 h at 4°C. n nThe receptors, labeled with [3H]-triamcinolone acetonide, were characterized by glycerol gradient centrifugation and filtration on Agarose A-1.5 m in buffers containing 30 or 50 mM KCl (controls) or 20mM Na2MoO4. The heterodisperse patterns of labeled complexes in the control gradients and columns were replaced in the presence of molybdate by a single peak with a sedimentation coefficient of 9.3 ± 0.1 S (SD, n = 17) and a Stokes radius of 75–82 A. In a protein of normal density, these parameters indicate a molecular weight of 2.9–3.2 × 105. Methyl prednisolonate specifically inhibited the binding of [3H]triamcinolone acetonide to this receptor form.

Discussion Paper: Mero‐Receptor Formation From a Larger Subcomponent of the Oviduct Progesterone Receptor*

The proteolytic origin of small fragments of both the glucocorticoid and mineralocorticoid receptors in rat kidney cytosol was inferred from the effects of leupeptin, a bacterial tripeptide that inhibits many proteases [Sherman, M.R. et al., (1978). Federation Proc. 37:167--173]. In the present study, the smallest fragment of the glucocorticoid receptor containing the steroid-binding site, the mero-receptor, was characterized with respect to the Stokes radius (RS = 23 +/- 3 A) and the isoelectric point (pI = 5.9 at 4 degrees). Chromatography of cytosol labeled with [3H]triamcinolone acetonide on Sephadex LH-20 (Pharmacia) in aqueous buffer resolved the steroid-receptor complex from the unmodified free steroid and from steroid metabolites and contaminants. This technique facilitated analyses of the leupeptin-stabilized receptor form by isoelectric focusing (pU = 5.9 at 4 degrees) and centrifugation in glycerol gradients (s20,w = 9--11 S in 50mM KCl). When this large complex in fresh cytosol was analyzed on Agarose (Bio-Rad) at a high flow rate, it had RS congruent to 60 A in 50 mM KCl and RS congruent to 30 A in 400 mM KCl. These analytical studies with leupeptin indicate the need for inexpensive, irreversible inhibitors of proteolytic enzymes for the purification of intact receptors, holo-receptors, from kidney and other tissues. Specific proteases can then be applied to dissect the holo-receptor into the globular mero-receptor, proximal to the steroid-binding site, and the asymmetric region(s), distal segment(s), that may be involved in the nuclear interactions.

“Defective” Receptors in Steroid-Resistant Conditions may be Proteolytic Artifacts

In the initial characterization of [3H]progesterone-labeled macromolecules in chick oviduct cytosol, Sherman et a / . described two proteins, AI and A2, that ( I ) were stimulated or diminished in parallel on administration or withdrawal of in rivo estrogen treatment; ( 2 ) had similar or identical affinities for progesterone, indicated by linear plots of (bound steroid)- vs (free steroid)- for the mixture; (3) were partially resolved and shown to have large Stokes radii by agarose gel filtration in 0.3 M KCI; (4) sedimented as a single 4s peak in KCI-containing density gradients. indicating highly asymmetric shapes in view of the large Stokes radii; and ( 5 ) . in low salt media, formed large aggregates that were analyzed by gel filtration, electrophoresis, and ultracentrifugation. The next step was to analyze the complexes extracted from nuclei by KCI after oviduct slices and [3Hlprogesterone were warmed to 37°C. Agarose filtration of the nuclear extract revealed equal quantities of the same two components. A I and Az, that were found in oviduct cytosol.2 This result implied that A I and A2 were transferred to the nuclei by a concerted mechanism and that both were involved in the subsequent nuclear events. I n a later study, the two [3H]progesterone-labeled complexes eluted from diethylaminoethyl (DEAE) -cellulose columns by low and high KCI concentrations were identified as the A and B subunits,3 but their relationships to forms AI and AP of Sherman et a / . I were not established. showed the correlations among the complexes detected by gel electrophoresis, chromatography on DEAE columns or filters, protamine precipitation, and gel filtration. They numbered the receptor forms in order of elution from agarose in high-salt buffer, starting with the aggregates at the void volume, 1. followed by the two included peaks I1 and Ill (AI and Az of the earlier publication). With the electrophoretic procedure of Miller et a / . , 5 they demonstrated the apparent identity of form I1 from gel filtration with the high-salt eluate from DEAE (B)3 and the similarity of form I11 from gel filtration with the low-salt eluate from DEAE (A).3 A further step in the analysis of this receptor was the demonstration by Sherman er that all of the determinants of specific, high-affinity steroid binding resided in a portion of the receptor with a molecular weight of about 20,000. This moiety, later termed the mero-receptor by analogy with the fragments of m y o ~ i n . ~ had the following properties: it was obtained in the presence of specific divalent cations, including Ca2+ but not Mg2+; it had the same equilibrium dissociation constants for a wide range of steroids as does unfractionated cytosol: More recently, Sherman et

Fractionation of Diverse Steroid-Binding Proteins: Basic and Clinical Applications

The specific question addressed in this report is whether the resistance to steroid treatment of certain tissues or tumors which appear to contain a normal quantity of steroid-binding sites may be due to structural defects in the receptors. This question may be seen as part of the more general question of whether there are intrinsic variations in the structures of receptors for a given class of steroids in different healthy tissues, in healthy vs. malignant tissues or in different types of tumors. Our experimental approach to these questions has involved the stabilization and precise physicochemical characterization of the receptors. To date, we have studied the estrogen and progestin receptors from human breast cancers and benign and malignant gynecologic specimens and the glucocorticoid receptors from several healthy and malignant rodent tissues and from normal human lymphocytes and various types of leukemic cells. Chromatographic and ultracentrifugal analyses in buffers of low ionic strength, containing 20 mM Na2MoO4 as the stabilizer, have revealed each of these receptors to be a large, oligomeric complex, characterized by remarkably similar values of the Stokes radius, sedimentation coefficient, molecular weight and axial ratio. In the absence of adequate stabilization, however, we found that the receptors for three classes of steroids in extracts of some healthy, steroid-responsive tissues, such as rat kidney and human uterine endometrium, are invariably degraded by endogenous proteinases. The extent of such cleavage is increased considerably by freezing the tissues prior to homogenization. Studies designed to distinguish the intact receptors from the products of proteolysis have included the characterization of receptors in cytosols prepared from mixtures of rat liver and kidney. The results strongly support the interpretation that the smaller size of the receptors detected in kidney cytosol reflects their cleavage by the more active proteinases in that tissue. The sizes and shapes of the receptors in cytosols from various tissues were found to be correlated with the activities of specific endopeptidases, assayed fluorometrically with peptidyl derivatives of 7-amino-4-methylcoumarin (AMC). These studies suggested that the receptors are vulnerable to cleavage by lysine-specific endopeptidases, detected with t-butyloxycarbonyl-L-valyl-L-leucyl-L-lysyl-AMC. An enzyme of this specificity was partially purified from rat kidney cytosol and tested for its ability to digest the glucocorticoid receptors from rat liver cytosol.(ABSTRACT TRUNCATED AT 400 WORDS)

Estrogen Receptor Cleavage and Plasminogen Activation by Enzymes in Human Breast Tumor Cytosol

Two types of diversity in steroid-binding proteins have become apparent: 1) the presence of several intracellular receptors and/or serum steroid-binding components in a tissue; and 2) the existence of multiple forms of individual receptors. Resolution of these components is clearly essential to understanding the mechanisms of action of one or more steroids in a target cell. The techniques that can be used to fractionate such mixtures and characterize the constituents include analytical gel filtration, Polyacrylamide gel electrophoresis, preparative ion exchange filtration and polyamine precipitation, in addition to density gradient centrifu-gation. Applications of these techniques to a clinical problem, the influence of steroids on breast cancer, and a fundamental problem, the subunit structure of chick oviduct progesterone receptors, will be described in this chapter.