Budded baculoviruses as a tool for a homogeneous fluorescence anisotropy-based assay of ligand binding to G protein-coupled receptors: The case of melanocortin 4 receptors

Abstract

We present here the implementation of budded baculoviruses that display G protein-coupled receptors on their surfaces for the investigation of ligand-receptor interactions using fluorescence anisotropy (FA). Melanocortin 4 (MC4) receptors and the fluorescent ligand Cy3B-NDP-α-MSH were used as the model system. The real-time monitoring of reactions and the high assay quality allow the application of global data analysis with kinetic mechanistic models that take into account the effect of nonspecific interactions and the depletion of the fluorescent ligand during the reaction. The receptor concentration, affinity and kinetic parameters of fluorescent ligand binding as well as state anisotropies for different fluorescent ligand populations were determined. At low Cy3B-NDP-α-MSH concentrations, a one-site receptor-ligand binding model described the processes, whereas divergence from this model was observed at higher ligand concentrations, which indicated a more complex mechanism of interactions similar to those mechanisms that have been found in experiments with radioactive ligands. The information obtained from our kinetic experiments and the inherent flexibility of FA assays also allowed the estimation of binding parameters for several MC4 receptor-specific unlabelled compounds. In summary, the FA assay that was developed with budded baculoviruses led the experimental data to a level that would solve complex models of receptor-ligand interactions also for other receptor systems and would become as a valuable tool for the screening of pharmacologically active compounds.

Probing the pharmacology of G protein-coupled receptors with fluorescent ligands

“…Using a fluorescent antagonist that emits at green wavelengths, the assay can be adapted to determine the affinity of fluorescent antagonists emitting at red wavelengths and vice versa (Vernall et al, 2013). Although the throughput of this system is not as high as for FRET or fluorescence polarisation assays (Allen et al, 2000;Veiksina et al, 2014;Zwier et al, 2010), one advantage of this imaging-based technique is the ability to simultaneously determine the affinity and efficacy of an unknown ligand and potentially to correlate this to what is happening at the single cell level.…”

Ligand Residence Time at G-protein–Coupled Receptors—Why We Should Take Our Time To Study It

“…Such a high level of receptor binding sites, to allow reliable measurements, is usually difficult to achieve, and one also has to be aware of substantial background autofluorescence. One possible solution, other than the use of purified proteins, has been shown by the use of budding baculoviruses, which display GPCRs on their surfaces at such high density that these assays became suitable (Veiksina et al, 2014).…”

Characterization of 5-HT1A receptors and their complexes with G-proteins in budded baculovirus particles using fluorescence anisotropy of Bodipy-FL-NAN-190

“…Fluorescence anisotropy data were blank corrected for each experiment before data were pooled and fitted for determination of K d and receptor concentration (taking ligand depletion and nonspecific binding into account) as described in (Veiksina et al, 2014). Briefly, two binding surfaces (in the presence or absence of competing ligand) with variable baculovirus amounts and variable Bodipy-FL-NAN-190 concentrations were globally fitted to the set of equations for equilibrium binding experiments, which were described (including the syntax used for GraphPad Prism™) in the Supplementary material of Veiksina et al (2014). All parameters were globally shared and some additional constrains were used (K d , concentrations of specific and nonspecific binding sites P0 and ligands' anisotropy values P0 and 60.4 (limitation for one-photon excitation).…”

“…High receptor expression levels and low background fluorescence may be required to increase the signal to noise ratio of such assays in order to achieve an easily detectable signal and GPCR expression in baculovirus particles addresses both of these issues (Veiksina et al, 2014). Bodipy-FL-NAN-190 was recently developed by CellAura as an addition to their line of fluorescent serotonergic ligands and is especially interesting for fluorescence anisotropy based applications as the fluorophore in conjugated to the ligand using a restrained linker, which should result in a large change in anisotropy upon binding to its target.…”