Homogeneous fluorescence anisotropy-based assay for characterization of ligand binding dynamics to GPCRs in budded baculoviruses: The case of Cy3B-NDP-α-MSH binding to MC4 receptors

Abstract

Despite the availability of numerous conceptually different approaches for the characterization of ligand- receptor interactions, there remains a great requirement for complementary methods that are suitable for kinetic studies, especially for the characterization of membrane protein systems and G proteincoupled receptors (GPCRs) in particular. One of the potential approaches that inherently fits well for this purpose is fluorescence anisotropy (FA), a method that allows continuous monitoring of ligand binding processes and characterization of ligand binding dynamics. However, significant changes in FA signal of fluorescently labeled ligands can be detected only if the ratio of bound to free fluorescent ligand portions is altered, which means that receptor and ligand concentrations have to be comparable. As most of the GPCRs are normally present at relatively low concentrations in native tissues and conventional receptor preparations from overexpressed systems often generate high background levels due to significant autofluorescence, receptor preparations with sufficiently high receptor concentrations have become a critical requirement for successful FA assay performance. We propose that budded baculoviruses that display GPCRs on their surfaces can be used as a receptor source in FA assays. Here, we describe the experimental setup of this homogeneous budded baculovirus/FA-based assay system for investigation of receptorligand interactions and a novel strategy for FA kinetic data analysis that is taking into account the effect of nonspecific interactions and the depletion of the fluorescent ligand during the binding reaction. The developed budded baculovirus/FA-based assay system brings the experimental data to a level that could solve complex models of ligand-receptor interactions and become a valuable tool for the screening of pharmacologically active compounds. Melanocortin 4 (MC4) receptors and the fluorescent ligand Cy3B-NDP- α-MSH were used as the model system.