Functional Screening in the Melanophore Bioassay

Channa Jayawickreme1, Howard Sauls1, Chris Watson1, David Moncol1, Thomas Rimele1, Terry Kenakin1

1 GlaxoSmithKline, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 12.9
DOI:  10.1002/0471141755.ph1209s29
Online Posting Date:  July, 2005
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Abstract

The melanophore bioassay is a robust, sensitive, and versatile procedure for screening G protein–coupled receptors in a variety of formats. Because melanophores contain a wide variety of G proteins, they can be employed as a sensitive, real-time response system for studying transfected receptors and for defining equilibria for drug effects. This assay can be run in 96-well microtiter plates or in open-lawn 1536 format, and can yield conventional agonist-antagonist as well as constitutive assays.

Keywords: melanophore; G-protein coupled receptors; high throughput screening; constitutive screening

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol: Screening in Melanophores using Transiently Expressed GPCRs
  • Alternate Protocol: GPCR Constitutive Screening in Melanophores
  • Support Protocol 1: Production of Conditioned Fibroblast Medium
  • Support Protocol 2: Generation of a Stable Melanophore Cell Line
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol: Screening in Melanophores using Transiently Expressed GPCRs

 Materials
  • Xenopus laevis melanophores (Arena Pharmaceuticals; http://www.arenapharm.com)
  • Conditioned fibroblast medium (CFM; see Support Protocol 1)
  • 0.7× PBS, pH 7.4 (for trypsinization; see recipe)
  • 0.7× trypsin solution: mix 100 ml 1× trypsin-EDTA (Invitrogen) with 42 ml sterile H2O, filter using 0.22-µm filter, and store up to 1 month at 4°C
  • 20/80 Percoll: 20% (v/v) Percoll (Sigma)/80% (v/v) CFM (see recipe for CFM)
  • 50/50 Percoll: 50% (v/v) Percoll (Sigma) 50% (v/v) CFM (see recipe for CFM)
  • cDNA encoding GPCR (Chen et al., 1998, 2000)
  • 0.7× PBS, pH 7.0 (for electroporation; see recipe), cold
  • 5 mM test compounds dissolved in 100% DMSO
  • Melanophore assay buffer (MAB; see recipe) containing 10 nM melatonin (Sigma)
  • -Melanin stimulating hormone (-MSH; Sigma)
  • Melanophore assay buffer (MAB; see recipe) containing 10 nM melatonin (Sigma) and 1% (v/v) DMSO
  • 27°C incubator
  • 225-cm2 tissue culture flasks
  • 50- and 15-ml conical centrifuge tubes
  • Tabletop centrifuge
  • 400-µl electroporation cuvettes (available from BTX)
  • Electroporator (e.g., Electro Cell Manipulator, Model BT 600 from BTX)
  • Hemacytometer
  • 75-cm2 tissue culture flasks (optional)
  • 96-well flat bottom tissue culture plates (24-well plates or 60-mm dishes may also be used)
  • 340 ATTC microtiter plate reader (SLT LabInstruments)

NOTE: All reagents should be used at room temperature unless otherwise noted.

Support Protocol 1: Production of Conditioned Fibroblast Medium

 Materials
  • Xenopus laevis melanophore fibroblasts (Arena Pharmaceuticals; http://www.arenapharm.com)
  • Regular Frog Medium (RFM; see recipe)
  • 27°C incubator
  • 225-cm2 tissue culture flasks or cell factories
  • 1-liter, 0.22-µm bottle-top filters

Support Protocol 2: Generation of a Stable Melanophore Cell Line

 Additional Materials (also see Basic Protocol)
  • pcDNA3.1 (Invitrogen)
  • CFM (see Support Protocol 1) containing 512 µg/ml G418: add 10.24 ml of 50 mg/ml geneticin (G418, Invitrogen) to 1 liter CFM; filter sterilize using a 0.22-µm filter and store at 4°C
  • Natural ligand for transfected receptor
  • Soldering iron
  • Cloning cylinders (Bellco 2090-01010) autoclaved in a glass petri dish
  • Sterile forceps
  • Vacuum grease autoclaved in a glass petri dish
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Figures

  •  FigureFigure 12.9.1 Melanophore pigment dispersion. (A) Melanophores responding to activation of a Gi-coupled receptor: melanosome aggregation. (B) Melanophores responding to activation of a Gs- or Gq-coupled receptor: melanosome dispersion.
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  •  FigureFigure 12.9.2 Temporal effects of agonists on melanophores transfected with 8 µg of human calcitonin receptor type 2 cDNA. (A) Effects of 0.1 nM human calcitonin (hCAL; Sigma; filled circles), 1 nM hCAL (open circles), and 10 nM hCAL (open triangles) with time. (B) Effects of the inverse agonist AC66 at 1 nM (filled circles), 10 nM (open circles), and 100 nM (open triangles) with time. (C) Dose-response curves for hCAL and AC66.
    View Image
  •  FigureFigure 12.9.3 Reversal of calcitonin receptor constitutive activity with inverse agonist AC512 in XM-B2-1 melanophore cells. Cells were transfected with 32 µg cDNA for human calcitonin receptor type 2. Responses shown to seven concentrations of the inverse agonist AC512 (synthesized as described in Chen et al., 1998).
    View Image
  •  FigureFigure 12.9.4 Effect of transfection with human calcitonin receptor type 2 (hCTR2) cDNA on basal activity and response to human calcitonin (hCAL; Sigma; filled circles) and the inverse agonist AC512 (open circles). Cells transfected with 16 µg (A) and 32 µg (B) of hCTR2 cDNA. Redrawn from Chen et al., 2000.
    View Image
  •  FigureFigure 12.9.5 SDF-1–mediated receptor activity of CXCR-4 transiently transfected into melanophores. Effect of hSDF-1 (human stromal cell derived factor 1, Peprotech) obtained following 30 min, 60 min, 90 min, 120 min, or 210 min of incubation. The lack of response to IL-8 (a CXCR1 agonist) in transfected cells is also shown.
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  •  FigureFigure 12.9.6 Effects of different levels of CXCR4 transfection on responses to (A) hSDF-1 and (B) basal and maximal agonist response. Panel A shows dose-response curves to hSDF-1 in melanophores transfected with CXCR4 cDNA at 10 µg (filled circles), 20 µg (open circles), 40 µg (filled squares), and 80 µg (open triangles). Either 20 µg or 40 µg would be acceptable for future experiments, as they both give approximately the same window of expression. Panel B illustrates dependence of basal constitutive receptor activity (open circles) and maximal response to hSDF-1 (filled circles) on the concentration of CXCR4 cDNA used for transfection.
    View Image
  •  FigureFigure 12.9.7 Effects of pertussis toxin (PTX) treatment on constitutive and agonist-induced responses of melanophores transfected with the human CXC chemokine receptor 4 (CXCR4). Responses to hSDF-1 in melanophores not treated (filled circles) and treated with PTX (open circles). Melanophores transfected with 20 µg (A), 40 µg (B), 80 µg (C), and 100 µg (D) of CXCR4 cDNA.
    View Image

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Literature Cited

Literature Cited
    Chen, W.-J., Jayawickreme, C., Watson, C., Wolfe, L., Holmes, W., Ferris, R., Armour, S., Dallas, W., Chen, G., Boone, L., Luther, M., and Kenakin, T.P. 1998. Recombinant human CXC-Chemokine Receptor-4 in melanophores are linked to Gi protein: Seven transmembrane coreceptors for human immunodificiency virus entry into cells. Mol. Pharmacol. 53:177-181.
    Chen, G., Way, S., Armour, S., Watson, C., Queen, K., Jayawickreme, C.K., Chen, W., and Kenakin, T. 2000. Use of constitutive G protein-coupled receptor activity for drug discovery. Mol. Pharmacol. 57:125-134.
    Gross, S.P., Tuma, M.C., Deacon, S.W., Serpinskaya, A.S., Reilein, A.R., and Gelfand, V.I. 2002. Interactions and regulation of molecular motors in Xenopus melanophores. J. Cell Biol. 156:855-865.
    Graminski, G.F. and Lerner, M.R. 1994. A rapid bioassay for platelet-derived growth factor -receptor tyrosine kinase function. Bio/Technology 12:1008-1011.
    Graminski, G.F., Jayawickreme, C.K., Potenza, M.N., and Lerner, M.R. 1993. Pigment dispersion in frog melanophores can be induced by a phorbol ester or stimulation of a recombinant receptor that activates phospholipase C. J. Biol. Chem. 268:5957-5964.
    Jayawickreme, C.K., Graminski, G.F., Quillan, J.M., and Lerner, M.R. 1994a. Creation and functional screening of a multi-use peptide library. Proc. Natl. Acad. Sci. U.S.A. 91:1614-1618.
    Jayawickreme, C.K., Graminski, G.F., Quillan, J.M., and Lerner, M.R. 1994b. Discovery and structure-function analysis of -melanocyte-stimulating hormone antagonists. J. Biol. Chem. 269:29846-29854.
    Jayawickreme, C.K., Sauls, H., Bolio, N., Ruan, J., Moyer, M., Burkhart, W., Marron, B., Rimele, T., and Shaffer, J. 1999. Use of a cell-based lawn format assay to rapidly screen a 442,368 bead-based library. J. Pharmacol. Toxicol. Methods 42:189-198.
    Karne, S., Jayawickreme, C.K., and Lerner, M.R. 1993. Cloning and characterization of an endothelin-3 specific receptor (ETC receptor) from Xenopus laevis dermal melanophores. J. Biol. Chem. 26:19126-19133.
    Lerner, M.R. 1994. Tools for investigating functional interactions between ligands and G-protein-coupled receptors. Trends Neurosci. 17:142-146.
    McClintock, T.S., Graminski, G.F., Potenza, M.N., Jayawickreme, C.K., Roby-Shemkovitz, A., and Lerner, M.R. 1993. Functional expression of recombinant G-protein-coupled receptors monitored by video imaging of pigment movement in melanophores. Anal. Biochem. 209:298-305.
    Potenza, M.N., Graminski, G.F., and Lerner, M.R. 1992. A method for evaluating the effects of ligands upon G protein-coupled receptors using a recombinant melanophore-based bioassay. Anal. Biochem. 206:315-322.
    Potenza, M.N., Graminski, G.F., Schmauss, C., and Lerner, M.R. 1994. Functional expression and characterization of human D2 and D3 dopamine receptors. J. Neurosci. 14:1463-1476.
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