Budded Baculoviruses as a Tool for a Homogeneous Fluorescence Anisotropy-Based Assay of Ligand Binding to G Protein-Coupled Receptors

Santa Veiksina1, Sergei Kopanchuk1,2, Ago Rinken1,2. 1Institute of Chemistry, University of Tartu, Tartu, Estonia, 2Competence Centre on Reproductive Medicine & Biology, Tartu, Estonia

Implementation of fluorescence methods in studies of ligand binding to their receptors opens new possibilities to characterise these processes. One of the potential approaches is the detection of changes of the fluorescence anisotropy (FA) signal upon binding reaction. However, in order to significantly change the anisotropy of the population of fluorescent ligand molecules some requirements needs to be meet. The concentration of receptor binding sites as well as the dissociation constant of the interaction should be at the same order as the fluorescent ligand’s concentration. It may be difficult to achieve required conditions with widespread receptor preparations and often leads to the high level of background due to the significant autofluorescence. The temporal stability of assays is also of paramount significance when interactions with slow kinetic properties are studied. For solving those obstacles we proposed implementation of budded baculoviruses that display G protein-coupled receptors on their surfaces for the investigation of ligand-receptor interactions using FA technique. We also set up a new strategy for the global analysis of FA kinetic data with mechanistic models that are taking into account the effect of nonspecific interactions and the depletion of the fluorescent ligand during the binding reaction. This allows for quantitative estimation of the total concentration of binding sites in a preparation in absolute terms as well as binding parameters for labelled and unlabelled ligands (1).

Melanocortin 4 (MC4) receptors and the fluorescent ligand Cy3B-NDP-α-MSH were used as the model system. It was revealed that at low Cy3B-NDP-α-MSH concentrations (< 1 nM) interactions between the fluorescent ligand and the MC4 receptor could be described as a simple one-site binding process, whereas at higher Cy3B-NDP-α-MSH concentrations the appearance of additional low affinity receptor binding site, that was inaccessible at lower fluorescent ligand concentrations, becomes evident. These observations are consistent with previously obtained results from MC4 receptor studies with radiolabelled [125I]-NDP-α-MSH (2). The emergence of an additional lower affinity MC4 receptor-ligand interaction site with slow association kinetics observed in present FA-based study supports the previously developed model where ligand binding to MC4 receptor is regulated by tandemly arranged ligand binding sites on the MC4 receptor dimer (2).

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 that was in agreement with previously reported values. Additionally, we demonstrated the applicability of the proposed experimental setup on another GPCR system – it was used for evaluation of a number of functional and pharmacological events of 5-HT1A receptors (3). In summary, the developed budded baculovirus-based led the experimental data to a level that would solve complex models of receptor–ligand interactions and would become as a valuable tool for the screening of pharmacologically active compounds.

(1) Veiksina S et al, Biochimica et Biophysica Acta (BBA)-Biomembranes 1838.1: 372, 2014

(2) Kopanchuk S et al, Neurochemisrty International 49.5: 533, 2006

(3) Tõntson L et al, , Neurochemisrty International, 2014 (in press)