Co-operative regulation of ligand binding to melanocortin receptor subtypes: Evidence for interacting binding sites
Introduction
The melanocortin receptors belong to the G-protein coupled receptors, and contain five known subtypes, MC1–5. 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 MC3–5 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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