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

Abstract

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/. (Figure presented.).