Characterization of ligand binding to melanocortin 4 receptors using fluorescent peptides with improved kinetic properties

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

“…We have previously successfully applied the FA‐based assay for the characterization of ligand binding to MC 4 R using different fluorescent ligands (Veiksina et al, , ; Link et al, ). In this study, we discovered that even low concentrations of EDTA in the presence of 1 mM Ca 2+ substantially improved the level of the FA signal, which corresponds to fluorescent ligand binding to MC 4 R in BBV particles.…”

“…In this study, we discovered that even low concentrations of EDTA in the presence of 1 mM Ca 2+ substantially improved the level of the FA signal, which corresponds to fluorescent ligand binding to MC 4 R in BBV particles. This FA signal increase was studied using two fluorescent ligands, one of which has a higher affinity (UTBC101) and the other faster kinetics (UTBC102) (Link et al, ). Determination of the binding properties of these fluorescent ligands in the presence of EDTA revealed apparent K d values of 0.23 ± 0.07 nM and 3.3 ± 0.9 nM for UTBC101 and UTBC102, respectively (Figure ).…”

“…The Cy3B‐labeled UTBC101 (Cy3B‐Nle‐c[Asp‐His‐DNal(2’)‐Arg‐Trp‐Lys]‐NH 2 ), UTBC102 (Ac‐Lys(Cy3B)‐c[Asp‐His‐DNal(2’)‐Arg‐Trp‐Lys]‐NH 2 ) (CEPEP EESTI, Estonia), and TAMRA‐labeled NDP‐α‐MSH (TAMRA‐Ser‐Tyr‐Ser‐Nle‐Glu‐His‐DPhe‐Arg‐Trp‐Gly‐Lys‐Pro‐Val‐NH 2 ) (AnaSpec, Cat# AS‐60564–1) were characterized as described previously (Veiksina et al, ; Link et al, ). Aliquots of those ligands were stored at −90°C in DMSO.…”

“…The kinetic rate constants of ligand binding have been determined using the fluorescently labeled peptide NDP‐α‐MSH (Veiksina et al, ), but the reaction equilibrium is not reached because of slow dissociation of the MC 4 R‐fluorescent ligand complex. Therefore, two novel redshifted peptides with faster kinetics have been implemented to resolve a broader range of modulatory effects (Link, Veiksina, Rinken, and Kopanchuk, ). The FRET‐based biosensors have opened up novel possibilities also for monitoring the activation of signal transduction pathways in real‐time (Unen et al, ).…”

Baculovirus as Versatile Vectors for Protein Display and Biotechnological Applications

“…The β 2 -adrenergic receptor was expressed on the surface of recombinant baculovirus with homogenous receptor function and post-translational modification in contrast to display on the surface of the infected insect cell (Loisel et al, 1997). This baculovirus virion display technique was subsequently applied to the study of target binding by the human leukotriene B 4 receptor (BLT1) (Masuda et al, 2003), serotonin 1A receptors (5-HT 1A ) (Tõntson et al, 2014), melanocortin 4 receptors (MC4) (Veiksina et al, 2014;Link et al, 2017), and dopamine D 1 receptors (Sakihama et al, 2008a;Allikalt and Rinken, 2017). The baculovirus particles were found in all of these studies to be a homogenous and far more stable system for membrane-bound GPCRs, allowing for their use in different experimental procedures and maintaining signal for long periods of time (Veiksina et al, 2014;Allikalt and Rinken, 2017).…”