Development of a Novel Ligand Binding Assay for Estrogen Receptor

Nuclear receptors undergo conformational changes when they bind their cognate ligands. It should be possible to monitor these changes in vivo using resonance energy transfer between flurophores. The existence of inherently fluorescent proteins such as the variants of jellyfish green fluorescent protein GFP suggests that this problem may be approached by making fusions of these proteins to nuclear receptors. We set out to study this problem using the estrogen receptor ER, a nuclear receptor known to undergo a conformational change upon ligand binding. We have proposed to generate a novel intrinsic ligand binding assay for the estrogen receptor based on ligand dependent conformational changes detected by fluorescence resonance energy transfer FRET between complimentary fluorescent proteins. We are in the process of cloning double and single chimeras of the estrogen receptor and the various fluorescent proteins into mammalian CMV expression vectors. We have extended the number of chimeras that we are generating because of the advent of new fluorescent proteins now available from Clontech, which include cyan, yellow and red fluorescent protein vectors. These new fluorescent proteins are more optimal for FRET than the original blue and green variants.