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 2MoO 4 and/or KCl. Estrogen receptors in breast tumor cytosols were labeled with [ 3H]-estradiol, progcstin receptors in cytosols from breast tumors and benign or carcinomatous uterine endometrium with [ 3H]-promegestone (R5020) and glucocorticoid receptors in normal lymphocytes and cells from patients with acute lymphoblastic and nonlymphocytic leukemias with [ 3H]-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 2MoO 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 20w = 9.6 ± 0.3 S and R s = 77 ± 4 Å (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 2MoO 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.