The goal of this research is to develop an experimental evolution system in which selection pressure is used to evolve human estrogen receptors (ERs) with affinity for novel ligands. This system will allow the structure function relationships that determine interactions between receptors and their ligands to be evaluated using the exponential efficiency of natural selection, a major advance over currently used methods of mutagenesis and screening to evaluate mutant receptors for novel functions, as well as provide a tool for understanding the coevolution of receptor-ligand interactions. Initial experiments will seek to evolve a human ERalpha that has developed increased affinity for, and is fully transactivated by, two major breast cancer drugs--the selective estrogen receptor modulator, tamoxifen, and the pure antiestrogen, ICI182, 780. This novel experimental approach addresses fundamental questions about the dynamics and mechanism of breast cancer disease progression seen clinically by recapitulating, in the controlled laboratory environment, the process by which Eralpha proteins evolve altered affinity for and resistance to first-line breast cancer drugs. This experimental system is comprised of a yeast strain, engineered to constitutively express human ERalpha, whose growth rate is dependent upon the expression of nutrient genes regulated by estrogen response elements. Expression of these growth regulating nutrient genes is driven by ERalpha binding of selected ligands (tamoxifen or ICI182, 780). Thus, variant ERs having increased affinity for these ligands will arise by random mutations and increase in frequency in the experimental yeast population due to natural selection. Over many generations of selection, ERs that use the ligands tamoxifen and ICI182, 780 as high-affinity agonists will evolve. Variant ERs will be sequenced, functionally characterized using reporter gene assays, and evaluated by homology modeling for three-dimensional structural alterations. Such an experimental system will allow the rigorous testing of hypotheses about ER-ligand structure function relationships and the dynamics of receptor-ligand coevolution.
