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Characterization of Melanocortin Receptors

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1414
  • Abstract
  • Table of Contents
  • Materials
  • Figures
  • Literature Cited

Abstract

 

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???MSH; MSH

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • 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)
  • [125 I]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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Figures

  •   Figure 1.28.1 Specific [125 I]NDP‐α‐MSH equilibrium binding for cloned human melanocortin receptor subtypes with time. Data are expressed as specific binding ( B o – NSB) versus time in hours. Each curve was generated by repetitive plate counting at specific time points. Data presented represent MC1R (solid diamonds), MC3R (open squares), MC4R (solid triangles), and MC5R (open circles).
    View Image
  •   Figure 1.28.2 Displacement of [125 I]NDP‐α‐MSH binding for SHU9119 at cloned human melanocortin receptor subtype. Data are expressed as specific binding ( B o – NSB) versus molar concentration of SHU9119. Each curve was generated by serial diluting SHU9119 in DMSO. Data presented represent MC1R (solid diamonds), MC3R (open squares), MC4R (solid triangles), and MC5R (open circles).
    View Image

Videos

Literature Cited

Literature Cited
   Butler, A.A., Kesterson, R.A., Khong, K., Cullen, M.J., Pelleymounter, M.A., Dekoning, J., Baetscher, M., and Cone, R.D. 2000. A unique metabolic syndrome causes obesity in the melanocortin‐3 receptor‐deficient mouse. Endocrinology 141:3518‐3521.
   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.
   Cone, R.D., Lu, D., Koppula, S., Vage, D.I., Klungland, H., Boston, B., Chen, W., Orth, D.N., Pouton, C., and Kesterson, R.A. 1996. The melanocortin receptors: Agonists, antagonists, and the hormonal control of pigmentation. Recent Progress Hormone Res. 51:287‐317.
   Fathi, Z., Iben, L.G., and Parker, E.M. 1995. Cloning, expression and tissue distribution of a fifth melanocortin receptor subtype. Neurochem. Res. 20:107‐113.
   Gantz, I., Konda, Y., Tashiro, T., Shimoto, Y., Miwa, H., Munzert, G., Watson, S.J., DelValle, J., and Yamada, T. 1993a. Molecular cloning of a novel melanocortin receptor. J. Biol. Chem 268:8246‐8250.
   Gantz, I., Miwa H., Konda, Y., Shimoto, Y., Tashiro, T., Watson, S.J., DelValle, J., and Yamada, T. 1993b. Molecular cloning, expression and gene localization of a fourth melanocortin receptor. J. Biol. Chem. 268:15174‐15179.
   Gantz, I., Shimoto, Y., Konda, Y., Miwa, H., Dickinson, C.J., and Yamada, T. 1994. Molecular cloning, expression, and characterization of a fifth melanocortin receptor. Biochem. Biophys. Res. Commun. 200:1214‐1220.
   Gobel, J., Saussy, Jr., D. L., and Goetz, A. S. 1999. Development of scintillation proximity assays for alpha adrenoceptors. J. Pharm. Toxicol. Methods, 42:237‐244.
   Lipton, J.M. and Catania, A. 1997. Anti‐inflammatory actions of the neuroimmunomodulator α‐MSH. Immunol. Today 18:140‐145.
   MacNeil, D.J., Howard, A.D., Guan, X., Fong, T.M., Nargund, R.P., Bednarek, M.A., Goulet, M.T., Weinberg, D.H., Strack, A.M., Marsh, D.J., Chen, H.Y., Shen, C.‐P., Chen, A.S., Rosenblum, C.I., MacNeil, T., Tota, M., MacIntyre, E.D., and Van der Ploeg, L.H.T. 2002. The role of melanocortins in body weight regulation: Opportunities for the treatment of obesity. Eur. J. Pharmacol. 450:93‐109.
   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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