An Unusual Conformation of γ-Melanocyte-Stimulating Hormone Analogues Leads to a Selective Human Melanocortin 1 Receptor Antagonist for Targeting Melanoma Cells

B16 murine melanoma cells selected in vivo for enhanced liver metastatic ability (B16-LS9) show on the one hand an increased expression and constitutive activation of the proto-oncogene c-met (the receptor for hepatocyte growth factor/scatter factor), and on the other hand a more differentiated phenotype, when compared to the parental cell line, B16-F1. Following this observation, we have tried to establish whether there is a direct relationship between differentiation and c-met expression in B16 melanoma cells. Treatment of these cells with differentiating agents indicated that c-met expression was strongly induced by melanocyte stimulating hormone, while retinoic acid had almost no influence. c-met induction was triggered by engagement of the melanocortin receptor, cAMP elevation and PKA/PKC(α) activation, as respectively shown by the effects of ACTH, cAMP elevating agents and specific PK inhibitors. Regulation of c-met expression via the melanocortin receptor and cAMP raises the intriguing possibility that autocrine and/or paracrine mechanisms acting in vivo on this circuit might influence (through c-met expression and activation) the metastatic behavior of these tumor cells, which we have shown to be dependent on their c-met expression.

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New Melanocortin 1 Receptor Binding Motif Based on the C-Terminal Sequence of ?-Melanocyte-Stimulating Hormone

Basic & Clinical Pharmacology & Toxicology ◽

10.1111/j.1742-7843.2006.pto_459.x ◽

2006 ◽

Vol 99 (4) ◽

pp. 287-293 ◽

Cited By ~ 3

Author(s):

Helgi B. Schiöth ◽

Ruta Muceniece ◽

Ilga Mutule ◽

Jarl E. S. Wikberg

Keyword(s):

Receptor Binding ◽

Binding Motif ◽

Terminal Sequence ◽

Melanocortin 1 Receptor ◽

Melanocyte Stimulating Hormone ◽

Stimulating Hormone

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Isolation and partial purification of a melanocyte-stimulating hormone receptor from B16 murine melanoma cells. A novel approach using a cleavable biotinylated photoactivated ligand and streptavidin-coated magnetic beads

Biochemical Journal ◽

10.1042/bj2860377 ◽

1992 ◽

Vol 286 (2) ◽

pp. 377-382 ◽

Cited By ~ 22

Author(s):

A R H Ahmed ◽

G W J Olivier ◽

G Adams ◽

M E Erskine ◽

R G Kinsman ◽

...

Keyword(s):

Magnetic Beads ◽

Melanoma Cells ◽

Partial Purification ◽

Affinity Column ◽

Ligand Complex ◽

Melanocyte Stimulating Hormone ◽

Receptor Complexes ◽

Novel Approach ◽

B16 Mouse Melanoma ◽

Stimulating Hormone

The alpha-melanocyte-stimulating hormone (alpha-MSH) receptor of B16 mouse melanoma cells was characterized by photoaffinity labelling using radiolabelled photoactive derivatives of alpha-MSH. A doublet band of 43-46 kDa representing a ligand-receptor complex was identified. A novel adaptation of the streptovadin/biotin-based affinity system was used to isolate the alpha-MSH receptor. A probe was synthesized which contained biotin connected to a photolabelled alpha-MSH analogue via a cleavable disulphide linker and which displayed high affinity for the alpha-MSH receptor. Streptavidin-coated magnetic beads were used as a solid support instead of an affinity column. Covalently linked probe-receptor complexes solubilized in Triton X-100 were equilibrated with the beads, and after magnetic separation and washing, specifically bound complexes were treated with dithiothreitol to cleave the disulphide bridge in the biotin-peptide spacer arm and so release the receptor-ligand complex. The identity of the isolated protein was established by SDS/PAGE analysis. Methods to achieve purification to homogeneity and to allow quantitative isolation of the receptor are discussed.

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