Co-operative regulation of ligand binding to melanocortin receptor subtypes: Evidence for interacting binding sites

doi:10.1016/j.ejphar.2005.02.021

European Journal of Pharmacology

Volume 512, Issues 2–3, 11 April 2005, Pages 85-95

https://doi.org/10.1016/j.ejphar.2005.02.021 Get rights and content

Abstract

This study evaluates the binding the melanocyte stimulating hormone peptide analogue [¹²⁵I]NDP-MSH to melanocortin receptors MC₁, MC₃, MC₄ and MC₅ in insect cell membranes produced by baculovirus expression systems. The presence of Ca²⁺ was found to be mandatory to achieve specific [¹²⁵I]NDP-MSH binding to the melanocortin receptors. Although association kinetics of [¹²⁵I]NDP-MSH followed the regularities of simple bimolecular reactions, the dissociation of [¹²⁵I]NDP-MSH from the melanocortin receptors was heterogeneous. Eleven linear and cyclic MSH peptides studied displaced the [¹²⁵I]NDP-MSH binding to the studied melanocortin receptors, with the shapes of their competition curves varying from biphasic or shallow to super-steep (Hill coefficients ranging from 0.4 to 1.5). Notably the same peptide often gave highly different patterns on different melanocortin receptor subtypes; e.g. the MC₄ receptor selective antagonist HS131 gave a Hill coefficient of 1.5 on the MC₁ receptor but 0.5–0.7 on the MC_3–5 receptors. Adding a mask of one of the peptides to block its high affinity binding did not prevent other competing peptides to yield biphasic competition curves. The data indicate that the binding of MSH peptides to melanocortin receptors are governed by a complex dynamic homotropic co-operative regulations.

Introduction

The melanocortin receptors belong to the G-protein coupled receptors, and contain five known subtypes, MC_1–5. These subtypes of melanocortin receptors are specific for melanocyte stimulating hormones (MSH) and the MC₂ receptors for adrenocorticotropic hormone (ACTH). The melanocortin receptors show distinct distributions to various parts of the body and evidence suggest that the MC₁ receptors could serve as a novel target for anti-inflammatory therapies, the MC₃ and MC₄ receptors for treatment of sexual dysfunctions, the MC₄ receptors for treatment of eating disorders and the MC₅ receptors for treatment of dysfunctions of exocrine glands (Wikberg et al., 2000). Accordingly, the melanocortin receptors have become important targets for drug developments (Andersson et al., 2001, Wikberg, 2001).

The melanocortin receptors were cloned almost a decade ago and several types of assays were developed for evaluation of their interactions with drugs. Early studies utilised radioligand binding in cells expressing the recombinant receptors. For the MC₁ and MC_3–5 receptors the stable high affinity MSH peptide analogue [¹²⁵I][Nle⁴, d-Phe⁷]α-MSH ([¹²⁵I]NDP-MSH) was used as radioligand (Schiöth et al., 1995, Schiöth et al., 1996b). For the MC₂ receptors [¹²⁵I]-adrenocorticotropic hormone ([¹²⁵I]ACTH) was used (Schiöth et al., 1996a). The melanocortin receptors couple in a positive fashion to adenylate cyclase and assays of cAMP have been used for their characterization in cells expressing the recombinant melanocortin receptors (Chen et al., 1995).

In the course of our studies aiming to develop subtype selective organic agonists and antagonists for melanocortin receptors (Mutulis et al., 2002a, Mutulis et al., 2002b) we were interested in improving the accuracy of our radioligand binding assays. We reasoned that using a simpler system, where the receptors were confined in a membrane fraction rather than in cells, should be beneficial as less confounding effects should become introduced by ligand diffusion into the cells, cell metabolism and alike. During these studies we found that the melanocortin receptors show complex ligand binding patterns, indicating the presence of both positive and negative co-operative interactions. We here report our new findings, which indicate that the melanocortin receptors are subject to much more complex molecular interactions in the cell membrane than was previously thought.

Section snippets

Materials

[¹²⁵I][Nle⁴, d-Phe⁷]α-MSH ([¹²⁵I]NDP-MSH) was prepared radiochemically pure (2190 Ci/mmol) by iodination using chloramine T followed by High Performance Liquid Chromatography (HPLC). [Nle⁴, d-Phe⁷]α-MSH (NDP-MSH), α-MSH, β-MSH, γ¹-MSH, γ²-MSH, Lys-γ¹-MSH, [Nle⁴]-γ²-MSH, Ser-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂ (MS05) (Szardenings et al., 2000), cyclic [Ac-Cys¹¹, d-Nal¹⁴, Cys¹⁸, Asp-NH₂²²]-β-MSH(11–22) (HS014) (Schiöth et al., 1998), cyclic [Ac-Cys³, Nle⁴, Arg⁵, d-Nal⁷, Cys-NH₂¹¹

Effects of Ca²⁺

Some quite early work had suggested that binding of [¹²⁵I]NDP-MSH to native melanocortin receptors in melanoma cells (i.e. the MC₁ receptors) requires the presence of Ca²⁺ (Gerst et al., 1987, Salomon, 1990). In the present study it was shown that Ca²⁺ is mandatory for specific binding of [¹²⁵I]NDP-MSH to all the four studied melanocortin receptors, when these are confined in membranes prepared from insect cells expressing the recombinant human melanocortin receptor variants. Varying the

Discussion

[¹²⁵I]NDP-MSH has been widely used for the characterization of melanocortin receptors (Schiöth et al., 1995, Schiöth et al., 1996b), but little attention has been given on its mechanisms of binding. The data of our present study show that binding of peptides to the different melanocortin receptors are governed by complex regulations, which cannot be described by simple reversible bimolecular reactions. The heterogeneity of binding was revealed already in a detailed analysis on the binding of [

Acknowledgements

Financial support was obtained from the Swedish MRC (04X-05957), the Estonian Science Foundation (6492) and Estonian Ministry of Education and Science (2592).

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Co-operative regulation of ligand binding to melanocortin receptor subtypes: Evidence for interacting binding sites

Abstract

Introduction

Section snippets

Materials

Effects of Ca2+

Discussion

Acknowledgements

Anal. Biochem.

Anal. Biochem.

FEBS Lett.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Protein Expr. Purif.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem. Lett.

Pharmacol. Ther.

Mol. Cell. Endocrinol.

Eur. J. Pharmacol.

Life Sci.

Mol. Cell. Endocrinol.

Neuropeptides

Peptides

Biochem. Pharmacol.

Effects of Ca²⁺