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/.

Structural Complexity and Plasticity of Signaling Regulation at the Melanocortin-4 Receptor

“…The substitution of D7.49N and an additional four other amino acid substitutions and C- and N-terminal truncations in the MC4R construct used for crystallization leads to a significant increase in melting temperature [ 21 ], suggesting that D7.49N might have some structural effect. Of note, a double aspartate constellation (D2.50/D7.49), as observed in MCRs and, e.g., in the P2Y12 [ 155 ], was suggested to potentially bind divalent cations [ 136 ], which is an interesting note with respect to previous reports on the impact of calcium [ 156 , 157 ] and a recent publication on zinc and copper binding in the MC4R [ 96 ]. Both zinc and copper ions in the presence of calcium are presumed to be negative allosteric modulators of ligand binding; however, calcium is required for high-affinity ligand binding.…”

“…Basal activity of GPCRs can be particularly modified by several factors, such as the pH, receptor variants, the level of receptor cell surface expression, or interaction with specific ions [ 94 , 95 ]. As described above, for the MC4R, increased basal activity has been suggested by the action of an intramolecular agonist [ 65 , 79 ] but recently, an alternative mechanism of basal activity regulation in the MC4R by interaction with divalent ions was published [ 96 ]. In particular, zinc ions cause activation of the cAMP pathway in the MC4R, whereby copper reduces MC4R activity to below normal basal signaling levels [ 96 ].…”

“…As described above, for the MC4R, increased basal activity has been suggested by the action of an intramolecular agonist [ 65 , 79 ] but recently, an alternative mechanism of basal activity regulation in the MC4R by interaction with divalent ions was published [ 96 ]. In particular, zinc ions cause activation of the cAMP pathway in the MC4R, whereby copper reduces MC4R activity to below normal basal signaling levels [ 96 ]. Hence, the authors concluded that zinc causes a certain level of permanent MC4R signaling activity as an external factor, which would be a prerequisite for an inverse agonistic effect of AgRP.…”

Alanine Scanning Mutagenesis of the DRYxxI Motif and Intracellular Loop 2 of Human Melanocortin-4 Receptor

“…For example, Na + can allosterically modulate MC4R activity [ 66 ] and D90 2.50 residue, as revealed by NMR structure, participates in maintaining the functional conformation of MC4R by Na + interaction [ 67 ]. Zn 2+ and Cu 2+ are also shown to allosterically modulate constitutive activity of MC4R in the cAMP pathway [ 68 ]. In the crystal structure of MC4R, Ca 2+ was identified to be a cofactor for ligand binding at orthosteric sites and regulate the affinity and potency of α-MSH, but not AgRP, in a selective manner [ 25 ].…”

Structural mechanism of calcium-mediated hormone recognition and Gβ interaction by the human melanocortin-1 receptor

“…Extensive evidence reveal that the divalent ion is of crucial importance for melanocortin signaling. Calcium ion assists melanocortins in binding to their cognate receptors with a better effect than magnesium ion [40][41][42] . Zinc ion activates MC1R and MC4R by acting as an agonist or allosteric modulator 43,44 .…”