Characterization of Melanocortin Receptors

Aaron S. Goetz1, Diane M. Ignar1

1 GlaxoSmithKline, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 1.28
DOI:  10.1002/0471141755.ph0128s22
Online Posting Date:  November, 2003
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This unit describes a Scintillation Proximity Assay (SPA) for the measurement of ligand binding to melanocortin receptors (MCRs) using membranes prepared from cell lines stably expressing recombinant MCRs. It provides a facile method for determining the affinity of compounds at MC1R, MC3R, MC4R, or MC5R.

Keywords: melanocortins; melanocortin receptors; SPA; MC1R; MC3R; MC4R; MC5R; NDP‐α‐MSH; MSH

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • CHO‐K1 cells expressing a recombinant melanocortin receptor (MC3R, MC4R and MC5R membranes are available from Perkin Elmer Life Sciences)
  • Culture medium (see recipe)
  • Dulbecco's phosphate‐buffered saline (D‐PBS; e.g., Life Technologies)
  • Nonenzymatic cell dissociation solution (Sigma)
  • Membrane preparation buffer (see recipe), ice cold
  • 5 mM HEPES, pH 7.5
  • Assay buffer (see reciperecipe)
  • Wheat‐germ agglutinin SPA beads (Amersham Biosciences)
  • [125I]NDP‐α‐MSH (Amersham Biosciences)
  • Unlabeled NDP‐α‐MSH (Sigma)
  • Dimethyl sulfoxide (DMSO)
  • Test compound: e.g., SHU9119 (Sigma; also see Table 1.28.1)
  • 225‐cm2 cell culture flask
  • Refrigerated centrifuge and 50‐ or 250‐ml centrifuge tubes
  • Polytron homogenizer (Brinkmann)
  • Polypropylene 96‐well microtiter plates
  • White 96‐well OptiPlates (Perkin‐Elmer)
  • Automatic pipettors and siliconized pipet tips
  • Scintillation plate counter
  • Graphing software capable of nonlinear regression (e.g., Prism, GraphPad)
  • Additional reagents and equipment for cell culture (unit 12.1) and protein assay ( appendix 3A)
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Literature Cited

Literature Cited
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   Chen, W., Kelly, M.A., Opitz‐Araya, X., Thomas, R.E., Low, M.J., and Cone, R.D. 1997. Exocrine gland dysfunction in MC5R‐deficient mice: Evidence for coordinated regulation of exocrine gland function by melanocortin peptides. Cell 91:789‐798.
   Chen, A.S., Marsh, D.J., Trumbauer, M.E., Frazier, E.G., Guan, X.M., Yu, H., Rosenbaum, C.I., Vongs, A., Feng, Y., Cao, L., Metzger, J.M., Strack, A.M., Camacho, E., Mellin, T.N., Nunes, C.N., Min, W., Fisher, J., Gopal‐Truter, S., MacIntyre, D.E., Chen, H.J.M., and Van der Ploeg, L.H.T. 2000. Inactivation of the mouse melanocortin‐3 receptor results in increased fat mass and reduced lean body mass. Nature Genet. 26:97‐102.
   Cheng, Y.C. and Prusoff, W.H. 1973. Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50% inhibition of an enzymatic reaction. Biochem. Pharmacol. 22:3099‐3108.
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   Mountjoy, K.G., Robbins, L.S., Mortrud, M.T., and Cone, R.D. 1992. The cloning of a family of genes that encode the melanocortin receptors. Science 257:1248‐1251.
   Schioth, H.B., Muceniece, R. Wikberg, J.E.S., and Chhajlani, V. 1994. Characterization of melanocortin receptor subtypes by radioligand binding analysis. Eur. J. Pharmacol. 288:311‐317.
   Schioth, H.B., Chhajlani, V., Muceniece, R., Klusa, V., and Wikberg, J.E.S. 1996. Major pharmacological distinction of the ACTH receptor from other melanocortin receptors. Life Sci. 59:797‐801.
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