Allosteric modulation of human melanocortin-4 receptor (MC4R) by zinc and copper ions and characterization of spotted sea bass MC4R

Abstract

Since the metal ions have been shown to play an important role in regulating signal transduction of G protein-coupled receptors (GPCRs), we investigated whether the Zn2+ and Cu2+ regulate the pharmacology of human melanocortin-4 receptor (hMC4R). We showed Zn2+ and Cu2+ were not able to displace 125I-NDP-MSH but exerted allosteric effects on the binding affinities and signaling properties of endogenous agonist (a-melanocyte stimulating hormone, a-MSH) at hMC4R. In addition, Zn2+ and Cu2+ induced biased signaling at hMC4R as they selectively modulated the canonical cAMP signaling but failed to regulate the ERK1/2 signaling. Notably, Cu2+ stimulated a biphasic cAMP accumulation not only at wild-type (WT) but also at obesity-associated gain-of-function mutant MC4Rs, suggesting this biphasic action might constitute as novel therapeutic opportunities for obese patients with these mutations. We also showed the Zn2+ cannot influence RM493 (a second-generation of MC4R agonist)-induced cAMP accumulation at WT and rescued loss-of-function mutant MC4Rs. This is probably due to the fact that the histidine residue of central amino acid motif (His-Phe-Arg-Trp) of RM493 and a-MSH coordinate with hMC4M at different binding pockets. Compared to WT hMC4R, the D122A, a “WT-like” variant, exerted significantly decreased cAMP producing in response to Zn2+, suggesting D122, the binding site of calcium, also serves as a potential binding site of Zn2+ at hMC4R. Our study might provide novel therapeutic applications for repurposing or designing drugs with metal ions combining allosteric and biased properties.

The MC4R plays important roles in regulation of multiple physiological processes including energy homeostasis, reproduction, sexual function and other functions in mammals. Recent studies suggested that teleost MC4Rs have different physiological functions and pharmacological characteristics when compared to mammalian MC4Rs. In this study, we investigated spotted sea bass (Lateolabrax maculatus) MC4R (LmMC4R) physiology and pharmacology. Spotted sea bass mc4r consisted of a 984 bp open reading frame encoding a protein of 327 amino acids. LmMC4R was homologous to those of several teleost MC4Rs and hMC4R. qRT-PCR and in situ hybridization revealed that mc4r transcripts were highly expressed in the brain, followed by pituitary and liver. Brain mc4r transcripts were down-regulated in long-term and short-term fasting challenges. LmMC4R was a functional receptor with lower maximal binding and higher basal activity than hMC4R. AgRP could displace 125I-NDP-MSH and serve as the inverse agonist. THIQ, Ipsen 5i and ML00253764 were not able to displace 125I-NDP-MSH but could affect intracellular cAMP accumulation, suggesting that they were allosteric ligands for LmMC4R. In summary, we cloned spotted sea bass MC4R, and showed that it had different pharmacological properties compared to hMC4R, and potentially different functions.

In summary, we showed Zn2+ and Cu2+ served as biased allosteric modulators at hMC4R. We also investigated the physiology and pharmacology of spotted sea bass MC4R.