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Characterization of Melanocortin Receptors

关键词： melanocortin receptors来源：互联网

Abstract
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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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Reagents and Solutions
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Literature Cited
Figures
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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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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).

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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).

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Literature Cited

Literature Cited
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