Receptor Reporter Systems

David J. Burns1

1 Abbott Laboratories, Abbott Park, Illinois
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 6.2
DOI:  10.1002/0471141755.ph0602s00
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Receptor‐linked reporter systems allow the study of pharmacological properties of known receptors, identification of new receptor subtypes, examination of the specific signal transduction pathways linked to receptor activation, and development of functional assays. Reporter systems provide high‐throughput screening methods that allow researchers to expeditiously identify and characterize compounds that modulate the activities of therapeutically relevant receptors or influence events downstream of receptor activation. This unit provides two representative protocols for coupling cell‐surface receptors to reporter molecules that signal receptor activation. The first is a luciferase‐based assay and the second uses green fluorescent protein (GFP). This unit provides a comparison of several reporter assays as well as reporter systems that rely on chloramphenicol acetyltransferase activity.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Luciferase Reporter Assay
  • Basic Protocol 2: Green Fluorescent Protein (GFP) Reporter Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Luciferase Reporter Assay

  Materials
  • TNF‐α–sensitive cell line (e.g., human embryonic kidney 293 cells)
  • Recombinant plasmid: TNF‐α–inducible promoter and puromycin resistance gene subcloned into pGL3 vector containing enhanced luciferase gene (Promega)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 0.25% (w/v) trypsin/1 mM EDTA in PBS (trypsin/EDTA solution)
  • DMEM‐10 with 1 µg/ml puromycin
  • TNF‐α (Endogen)
  • Reporter lysis buffer (Promega)
  • Luciferase assay reagent (Promega)
  • Recombinant luciferase (Molecular Probes)
  • 175‐cm2 tissue culture flask
  • 50‐ml centrifuge tubes
  • Hemacytometer
  • 96‐well microtiter plate (e.g., VWR Microfluor U‐bottom plates)
  • Luminometer (e.g., Wallac Victor for manual addition of luciferase assay reagent or Labsystems Luminoskan for automatic injection of reagent)

Basic Protocol 2: Green Fluorescent Protein (GFP) Reporter Assay

  Materials
  • TNF‐α‐sensitive cell line (e.g., human embryonic kidney 293 cells)
  • Recombinant plasmid: TNF‐α‐inducible promoter subcloned into pEGFP vector containing enhanced green fluorescent protein (GFP) gene and neomycin resistance gene (Clontech)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 0.25% (w/v) trypsin/1 mM EDTA in recipePBS (trypsin/EDTA solution)
  • DMEM‐10 with 1 mg/ml geneticin (G‐418)
  • TNF‐α (Endogen)
  • 175‐cm2 tissue culture flask
  • 50‐ml centrifuge tubes
  • Hemacytometer
  • 96‐well microtiter plate (e.g., Packard white, clear‐bottom tissue culture–treated plates)
  • Fluorometer (e.g., Wallac Victor or Molecular Dynamics Biolumin 960)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.) 1998. Current Protocols in Molecular Biology. John Wiley & Sons, New York.
   Broach, J.R. and Thorner, J. 1996. High‐throughput screening for drug discovery. Nature 384:14‐16.
   Bronstein, I., Fortin, J., Stanley, P.E., Stewart, G.S.A.B., and Kricka, L.J. 1994. Chemiluminescent and bioluminescent reporter gene assays. Anal. Biochem. 219:169‐181.
   Campbell, A.K. and Sala‐Newby, G. 1993. Bioluminescent and chemiluminescent indicators for molecular signaling and function in living cells. In Fluorescent and Luminescent Probes for Biological Activity (W.T. Mason, ed.) pp. 58‐82. Academic Press, San Diego.
   Castanon, M.J. and Spevak, W. 1994. Functional coupling of human adenosine receptors to a ligand‐dependent reporter gene system. Biochem. Biophys. Res. Commun. 198:626‐631.
   Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., and Prasher, D.C. 1994. Green fluorescent protein as a marker for gene expression. Science 263:802‐805.
   Chen, W., Schields, T.S., Stork, P.J.S., and Cone, R.D. 1995. A colorimetric assay for measuring activation of GS‐ and Gq‐coupled signaling pathways. Anal. Biochem. 226:349‐354.
   Cormack, B.P., Valdivia, R.H., and Falkow, S. 1996. FACS‐optimized mutants of the green fluorescent protein (GFP). Gene 173:33‐38.
   Craig, F.F., Simmonds, A.C., Watmore, D., McCapra, F., and White, M.R.H. 1991. Membrane‐permeable luciferin esters for assay of firefly luciferase in live intact cells. Biochem. J. 276:637‐641.
   Crameri, A., Whitehorn, E.A., Tate, E., and Stemmer, W.P.C. 1996. Improved green fluorescent protein by molecular evolution using DNA shuffling. Nature Biotechnol. 14:315‐319.
   Cubitt, A.B., Heim, R., Adams, S.R., Boyd, A.E., Gross, L.A., and Tsien, R.Y. 1995. Understanding, improving and using green fluorescent proteins. Trends Biochem. Sci. 20:448‐455.
   Delagrave, S., Hawtin, R.E., Silva, C.M., Yang, M.M., and Youvan, D.C. 1995. Red‐shifted excitation mutants of the green fluorescent protein. Bio/Technology 13:151‐154.
   Drews, J. 1996. Genomic sciences and the medicine of tomorrow. Nature Biotechnol. 14:1516‐1518.
   Dhundale, A. and Goddard, C. 1996. Reporter assays in the high throughput screening laboratory: A rapid and robust first look. J. Biomol. Screening 1:115‐118.
   Heim, R., Prasher, D.C., and Tsien, R.Y. 1994. Wavelength mutations and posttranslational oxidation of green fluorescent protein. Proc. Natl. Acad. Sci. U.S.A. 91:12501‐12504.
   Heim, R., Cubitt, A.B., and Tsien, R.Y. 1995. Improved green fluorescence. Nature 373:663‐664.
   Kolb, A.J. and Neumann, K. 1996. Luciferase measurements in high throughput screening. J. Biomol. Screening 1:85‐88.
   Lerner, M.R. 1994. Tools for investigating functional interactions between ligands and G‐protein‐coupled receptors. Trends Neurosci. 17:142‐146.
   Luyten, W.H.M.L. and Leysen, J.E. 1993. Receptor cloning and heterologous expression—Towards a new tool for drug discovery. Trends Biotechnol. 11:248‐253.
   Major, J.S. 1995. Challenges of high throughput screening against cell surface receptors. J. Recept. Signal Transduct. Res. 15:595‐607.
   Martin, C.S. and Bronstein, I. 1994. Imaging of chemiluminescent signals with cooled CCD camera systems. J. Biolumin. Chemilumin. 9:145‐153.
   Martin, C.S., Wight, P.A., Dobretsova, A., and Bronstein, I. 1996. Dual luminescence‐based reporter gene assay for luciferase and β‐galactosidase. BioTechniques 21:520‐524.
   McClintock, T.S., Graminski, G.F., Potenza, M.N., Jayawickreme, C.K., Robey‐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.
   Milligan, G., Marshall, F., and Rees, S. 1996. G16 as a universal G protein adapter: Implications for agonist screening strategies. Trends Pharmacol. Sci. 17:235‐237.
   Montmayeur, J.‐P. and Borrelli, E. 1991. Transcription mediated by a cAMP‐responsive promoter element is reduced upon the activation of dopamine D2 receptors. Proc. Natl. Acad. Sci. U.S.A 88:3135‐3139.
   Owicki, J.C., Parce, J.W., Kercso, K.M., Sigal, G.B., Muir, V.C., Venter, J.C., Fraser, C.M., and McConnel, H.M. 1990. Continuous monitoring of receptor‐mediated changes in the metabolic rates of living cells. Proc. Natl. Acad. Sci. U.S.A. 87:4007‐4011.
   Potenza, M.N. and Lerner, M.R. 1992. A rapid and quantitative bioassay for evaluating the effects of ligands upon receptors that modulate cAMP levels in a melanophore cell line. Pigm. Cell Res. 5:372‐378.
   Roda, A., Pasini, P., Musiani, M., Girotti, S., Baraldini, M., Carrea, G., and Suozzi, A. 1996. Chemiluminescent low‐light imaging of biospecific reactions on macro‐ and microsamples using a videocamera‐based luminograph. Anal. Chem. 68:1073‐1080.
   Samson, M., Labbe, O., Mollereau, C., Vassart, G., and Parmentier, M. 1996. Molecular cloning and functional expression of a new human CC‐chemokine receptor gene. Biochemistry 35:3362‐3367.
   Schroeder, K.S. and Neagle, B.D. 1996. FLIPR: A new instrument for accurate, high throughput optical screening. J. Biomol. Screening 1:75‐80.
   Sista, P., Edmiston, S., Dargess, J.W., Robinson, S., and Burns, D.J. 1994. A cell‐based assay for the identification of protein kinase C activators and inhibitors. Mol. Cell. Biochem. 141:129‐134.
   Strosberg, A.D. and Leysen, J.E. 1991. Receptor‐based assays. Curr. Opin. Biotechnol. 2:30‐36.
   Stratowa, C., Himmler, A., and Czernilofsky, A.P. 1995. Use of a luciferase reporter system for characterizing G‐protein‐linked receptors. Curr. Opin. Biotechnol. 6:574‐581.
   Tate, C.G. and Grisshammer, R. 1996. Heterologous expression of G‐protein‐coupled receptors. Trends Biotechnol. 14:426‐430.
   Wallace, R.W. 1996. Packard and Aurora Bioscience to codevelop ultra‐HTS technologies. Drug Discov. Today 8:313‐314.
   Weyer, U., Schafer, R., Himmler, A., Mayer, S.K., Burger, E., Czernilofsky, A.P., and Stratowa, C. 1993. Establishment of a cellular assay system for G protein–linked receptors: Coupling of human NK2 and 5‐HT2 receptors to phospholipase C activates a luciferase reporter gene. Receptors Channels 1:193‐200.
   Woods, K.V. 1995. Marker proteins for gene expression. Curr. Opin. Biotechnol. 6:50‐58.
   Yang, J. and Thomason, D.B. 1993. An easily synthesized, photolyzable luciferase substrate for in vivo luciferase activity measurement. BioTechniques 15:848‐850.
   Zylka, M.J. and Schnapp, B.J. 1996. Optimized filter set and viewing conditions for the S65T mutant of GFP in living cells. BioTechniques 21:220‐226.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library