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

  • Basic Protocol 1: Screening in Melanophores using Transiently Expressed GPCRs
  • Alternate Protocol 1: 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 1: Screening in Melanophores using Transiently Expressed GPCRs

  Materials
  • Xenopus laevis melanophores (Arena Pharmaceuticals; http://www.arenapharm.com)
  • Conditioned fibroblast medium (CFM; see protocol 3)
  • 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 H 2O, 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 protocol 3recipe for CFM)
  • 50/50 Percoll: 50% (v/v) Percoll (Sigma) 50% (v/v) CFM (see protocol 3recipe for CFM)
  • cDNA encoding GPCR (Chen et al., , )
  • 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)
  • α‐Melanin stimulating hormone (α‐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.

Alternate Protocol 1: GPCR Constitutive Screening in Melanophores

  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 1: Production of Conditioned Fibroblast Medium

  • pcDNA3.1 (Invitrogen)
  • CFM (see protocol 3) 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

Videos

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 β‐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 α‐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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