Fluorescence anisotropy assay for pharmacological characterization of ligand binding dynamics to melanocortin 4 receptors

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

“…In this case one does not have to physically separate bound ligand from unbound, and one can monitor the binding as process in real time. This method has been used to characterize ligand binding to receptors of hormones like endothelin (Junge et al, 2010) and melanocortin (Veiksina et al, 2010). However, it has also been demonstrated for GPCRs of small molecules like acetylcholine for mACh receptors (Huwiler et al, 2010) and serotonin (Tõntson et al, 2014).…”

BacMam System for FRET-Based cAMP Sensor Expression in Studies of Melanocortin MC1 Receptor Activation

“…After revealing allosteric interactions between binding sites of MCR, 3 models of tandemly arranged ligandbinding sites 4 and ago-allosteric modulation 5 were proposed. Implementation of a fluorescence anisotropy/intensity assay with a dye-labeled NDP-α-MSH for MC 4 R allowed monitoring of ligand binding in real time 6 and revealed complexities in the binding processes, which have a big impact on the apparent affinities. How these processes influence the biological activity of MCR ligands remains open.…”

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

“…This change, detected as fluorescence anisotropy, can be used as a measure of ligand binding and be monitored in real time. The FA method has been successfully 6 used for characterization of ligand binding to peptide receptors, such as chemokine CXC4 [23] and melanocortin MC4 [24], receptors of small molecules, such as muscarinic M1 [25] and serotoninergic 5-HT1A [26], and binding of nucleotides to G proteins [27]. It is important to mention here that the FA assay by nature is ratiometric, and signal can be detected only if the ratio of bound to free fluorescent ligand has significantly changed [28].…”

“…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].…”