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 protein-coupled 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 receptor-ligand 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.

The constitutive activity of melanocortin‐4 receptors in cAMP pathway is allosterically modulated by zinc and copper ions

“…Aliquots of those ligands were stored at −90°C in DMSO. The FA‐based assay with the implementation of the homogenous BBV was set up as described previously (Veiksina et al, ). In brief: experiments were carried out in buffer A with modifications described in the specific assay setups in a final volume of 100 μl using 96‐well half area, flat‐bottom polystyrene NBS microtiter plates (Corning, Cat# 3,993).…”

“…In the current work, we used FA‐based ligand binding (Veiksina et al, ) and FRET‐based functional assays of the cAMP pathway (Mazina, Allikalt, et al, ) to study the effects of several divalent metal ions on the MC 4 R. Studying the metal ion effects from a ligand binding and functional perspective is especially important in the case of an allosteric modulation mechanism, where a negative modulator of the ligand binding may be a positive modulator of the signal transduction or vice versa. We observed this phenomenon for Zn 2+ and Cu 2+ ions, which are both negative allosteric modulators of ligand binding, but Zn 2+ acts as an activator and Cu 2+ as an inhibitor of the basal activity of MC 4 R in cAMP pathway.…”

Image-Based Cell-Size Estimation for Baculovirus Quantification

“…BVs encoding melanocortin 4 receptor (MC 4 R) and dopamine 1 receptor (D 1 R) were prepared as described previously (19,20), and BacMam BVs containing sequences for different cAMP biosensors, which were developed by Kees Jalinks laboratory (21), were prepared as described previously (22). A complete list of the BVs used in this study, along with their symbols, is presented in Table 1.…”

“…The Sf9 cell line from Spodoptera frugiperda pupal ovarian tissue (ThermoFisher Scientific, Waltham, MA) was cultivated for all experiments at 27C in EX-CELL 420 serum-free medium for insect cells (referred hereafter as the medium) (Sigma-Aldrich Chemie GmbH, Darmstadt, Germany), as described previously (19). BVs encoding melanocortin 4 receptor (MC 4 R) and dopamine 1 receptor (D 1 R) were prepared as described previously (19,20), and BacMam BVs containing sequences for different cAMP biosensors, which were developed by Kees Jalinks laboratory (21), were prepared as described previously (22).…”

Dynamics of ligand binding to GPCR: Residence time of melanocortins and its modulation

“…Such a high level of receptor binding sites is usually difficult to achieve with natural tissues and therefore first attempts to implement this method have been performed with reconstituted systems [23]. The membranes of baculovirusinfected Sf9 cells demonstrated very reasonable anisotropy signals connected with ligand binding [24], while budded baculoviruses from these cells displaying GPCR on their surfaces are even more suitable targets for this type of assay [29].…”