Single plasmids expressing human steroid hormone receptors and a reporter gene for use in yeast signaling assays

Abstract

Single plasmids designed to express the six human type I steroid hormone receptors and detect signaling activity are described in this report. These stably replicating plasmids reported ligand-induced transcriptional activation via lacZ assays in Baker's yeast (Saccharomyces cerevisiae). The ligand concentrations needed to activate signaling in yeast expressing these plasmids spanned five orders of magnitude as based on comparisons of EC(50) values. Radicicol, a direct inhibitor of heat shock protein 90 (Hsp90) and an indirect inhibitor of steroid hormone receptor signaling, was used to determine the functional utility of this yeast reporter system. The inhibitory effect of radicicol was similar on the signaling of all six steroid hormone receptors and was distinguishable from cytotoxic effects that occurred with higher concentrations. These yeast plasmids provide a high throughput system for comparative assessment of steroid hormone receptor signaling and may be useful in screening for pharmacological or xenobiotic activities.

Figure 1. General Structure of pRR Plasmids

The pRR series of plasmids is designed to express the individual type I human steroid hormone receptors, as depicted by the receptor cDNA sequence (labeled RECEPTOR cDNA). The different response elements (5-RE) upstream of a minimal cytochrome C (CYC) promoter that direct β-galactosidase reporter gene (LACZ) regulation are described in Table 2. Selective plasmid maintenance in yeast is conferred by the TRP1 gene. The autonomously replicating sequence (ARS) and centromere sequence (CEN) provide replication and segregation functions, respectively, in yeast. Replication and selective propagation in E. coli are conferred by the Col E1 or f1 origins and the β-lactamase gene (AMP), respectively.

Figure 2. Dose Response Curves for Yeast with pRR Plasmids

Yeast containing pRR plasmids were treated with ligands for 18 hr and then assessed for signaling using lacZ assays. The scale on the x-axis is logarithmic, and data were normalized to a maximal 100% ligand response to facilitate graphical comparisons. Symbols representing each receptor signaling profile are noted in the panel, and the error bars indicate the standard error of the mean. The ligands used to activate receptors were testosterone (AR), 17β-estradiol (ERfα and β), dexamethasone (GR), aldosterone (MR), and progesterone (PR).

Figure 3. Cytotoxicity of Radicicol

Yeast cultures were treated with a range of radicicol concentrations and assessed after 18 h in culture. The curve reflects radicicol cytotoxicity as measured by relative culture density, with error bars indicating the standard error of the means. The scale of the x-axis is logarithmic, and asterisks indicate points that are significantly different from the control (100%).