Nuclear Receptor (PXR), Ligand and Co-Activator Interactions Measured by Total Internal Reflection Fluorescence Microscopy

Abstract

The pregnane X receptor (PXR) is a member of the nuclear receptor family. PXR acts in a ligand-dependent manner, in concert with its dimerization partner, the retinoid X receptor, and co-regulators, to modulate the expression of proteins that metabolize endogenous and exogenous compounds. Here, we report the use of total internal reflection fluorescence microscopy (TIRFM) for examining interactions between the ligand-binding domain of PXR (PXR-LBD), its ligands, and a peptide derived from a co-activator. TIRFM is a surface-specific technique that enables us to study the behavior of fluorescent species close to or at interfaces. Our experimental system consists of biotinylated PXR-LBD immobilized on a fused silica substrate coated with NeutrAvidin and ovalbumin, a ligand (e.g., the antibiotic rifampicin) and a fluorescently labeled, 25 amino acid fragment of the steroid receptor co-activator-1 (F-SRC-1). Using TIRFM, we measured the surface-associated fluorescence as a function of the F-SRC-1 concentration at fixed ligand concentrations. These curves were fit to a model of single-site binding to obtain apparent equilibrium constants. The apparent equilibrium constants as a function of ligand concentration were fit to an appropriate model to obtain binding constants for the ligand/PXR-LBD interaction, F-SRC-1 binding to apo and ligand-bound PXR-LBD and ligand binding to F-SRC-1-bound PXR-LBD. The approach yielded four, previously unmeasured, binding constants. These values indicate that the increase in PXR's affinity for its co-activator peptide upon binding an activating ligand is modest. This observation implies that for gene expression to be significantly upregulated, the signal arising from the activation of PXR must be amplified downstream - possibly when co-activators like SRC-1 start recruiting the cell's transcription machinery. Kinetic data obtained by combining TIR illumination with fluorescence recovery after photobleaching and/or fluorescence correlation spectroscopy may also be discussed.