Effects of Molybdate and Endogenous Inhibitors on Steroid-Receptor Inactivation, Transformation, and Translocation

Abstract

Michigan 48 i d9 INTRODUCTIONTo exert their specific effects on the regulation of gene expression, steroid honnones must first bind to cytoplasmic receptors that are subsequently trans located to specific acceptor sites in the cell nucleus. Prior to this translocation, the steroid-receptor complex is transfonned by a temperature-dependent pro cess to a state that can bind to nuclear components. This general model of binding, followed by receptor transfonnation and translocation to the nucleus, was originally proposed in 1968 on the basis of studies in intact cells (25,37).The initial events in this pathway, honnone binding and receptor transfonna tion, can be readily studied under cell-free conditions. Although clearly valid cell-free models of nuclear translocation have not been developed, binding of transfonned receptors to nuclei or to DNA-cellulose can be assayed in a quantitative manner and is employed as an operational method of assaying translocation of receptors that have been transfonned under cell-free condi tions. Both group VI-A transition-metal oxyanions (42,66,92) and a small, heat-stable endogenous factor (7,9,24,43,77,79,80) have been found to 67 0066-4278/84/0315-0067$02. 00 Annu. Rev. Physiol. 1984.46:67-81. Downloaded from www.annualreviews.org Access provided by University of Pittsburgh on 02/09/15. For personal use only. Quick links to online content Further ANNUAL REVIEWS 68 DAHMER, HOUSLEY & PRA 11'stabilize unoccupied steroid receptors and to inhibit the transfonnation of receptors to the DNA-binding state in cell-free assay systems. Macromolecu lar, heat-labile factors that inhibit translocation oftransfonned receptors have been identified in cytosols prepared from a variety of tissues (12,19,55,87). We review here the actions, under cell-free conditions, of transition metal oxyanions and the two endogenous inhibitors on steroid receptor stability, transfonnation, and translocation. REGULATION OF RECEPTORS BY PHOSPHATE AND SULFHYDRYL MOIETIESIn order to understand the rationale behind some of the experiments described here, it is important to understand the potential role of receptor phosphate and the clear role of receptor sulfhydryl moieties in detennining the ability of steroid receptors to bind hormone. It has recently been demonstrated that the murine glucocorticoid receptor in L929 cells (34) and the avian progesterone receptor in hen oviduct (20) are phosphorylated in vivo. In both cases the major phosphorylated species isolated by affmity chromatography is a protein ofMr == 90,000:-92,000 (90-92K) that is phosphorylated on a serine moiety (or perhaps moieties). In the case of the glucocorticoid receptor, a minor species at lOOK is also phosphorylated. In the case of the progesterone receptor, a second phosphorylated receptor species tha� migrates at about 109K in sodium dodecyl sulfate-polyacrylamide gels has been identified..Several observations in cell-free systems suggest that phosphorylation of steroid receptors may be important for determining their ability to bind the hormon...

A cellular factor stimulates ligand-dependent release of hsp90 from the basic helix-loop-helix dioxin receptor.

“…In cell-free model systems, reassociation between hsp90 and the purified glucocorticoid receptor is, however, promoted by rabbit reticulocyte lysate (43). Moreover, as soon as it is translated in reticulocyte lysate, the glucocorticoid receptor becomes associated with hsp90 (7,13). To demonstrate glucocorticoid receptor-hsp90 interaction, we employed in coimmunoprecipitation experiments a monoclonal IgM antibody capable of recognizing both free hsp90 and hsp90 complexed with other proteins (34).…”

“…The total reactions were then subjected to coimmunoprecipitation with hsp90-specific antibodies, and the products were analyzed by SDS-PAGE and fluorography as described in the legend to Fig. 1. receptor in the presence of sodium molybdate, a compound known to stabilize hsp9O interaction with a number of steroid hormone receptors (7). Finally, the Hepa-1 stimulatory activity cosedimented on sucrose gradients with Arnt immunoreactivity.…”

Evidence that the modulator of the glucocorticoid-receptor complex is the endogenous molybdate factor.

“…Activation is the process by which the steroid-receptor complex is converted to its DNA-binding form (1,2). In 1976 our laboratory reported the existence of modulator (3), and since our initial finding (4,5), several other laboratories have also observed this activation inhibitor in crude or partially purified preparations (6)(7)(8)(9)(10)(11)(12)(13)(14). In addition to inhibiting GRC activation, crude or partially purified preparations of modulator also stabilize the steroid-binding ability of the unoccupied glucocorticoid receptor (UGR; refs.…”

“…Exogenous sodium molybdate (Na2MoO4) has also been observed to inhibit GRC activation and stabilize the UGR (13,14,16). It is not known why Na2MoO4 has these activities, but it is generally thought that molybdate interacts directly with the glucocorticoid receptor (13,14,(17)(18)(19).…”

“…It is not known why Na2MoO4 has these activities, but it is generally thought that molybdate interacts directly with the glucocorticoid receptor (13,14,(17)(18)(19). Furthermore, Na2MoO4 also inhibits the activation and stabilizes the steroid binding of other steroid hormone receptors (13,14).…”