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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.

Melanocortin‐4 receptors (MC4R) are unique among G‐protein‐coupled receptors (GPCRs) as they have endogenous ligands that can exhibit inverse agonistic properties in the case of elevated basal activity. It is known that the constitutive activity of GPCRs strongly affects the ligand‐dependent physiological responses, but little is known about these regulatory mechanisms. Since several metal ions have been shown to be important modulators of the signal transduction of GPCRs, we hypothesized that metal ions regulate the basal activity of MC4Rs. Implementation of a fluorescence anisotropy assay and novel redshifted fluorescent peptides enabled kinetic characterization of ligand binding to MC4R expressed on budded baculoviruses. We show that Ca2+ is required for high‐affinity ligand binding, but Zn2+ and Cu2+ in the presence of Ca2+ behave as negative allosteric modulators of ligand binding to MC4R. FRET‐based cAMP biosensor was used to measure the activation of MC4R stably expressed in CHO‐K1 cells. At low micromolar concentrations, Zn2+ caused MC4R‐dependent activation of the cAMP pathway, whereas Cu2+ reduced the activity of MC4R even below the basal level. These findings indicate that at physiologically relevant concentrations can Zn2+ and Cu2+ function as MC4R agonists or inverse agonists, respectively. This means that depending on the level of constitutive activity induced by Zn2+ ions, the pharmacological effect of orthosteric ligands of MC4R can be switched from a partial to an inverse agonist. Open Science Badges This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. More information about the Open Science badges can be found at https://cos.io/our-services/open-science-badges/.

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