Characterization of Chemokine Receptors

Alexander Scheer1, Amanda E.I. Proudfoot1

1 Serono Pharmaceutical Research Institute, Plan‐les‐Ouates
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 1.24
DOI:  10.1002/0471141755.ph0124s14
Online Posting Date:  November, 2001
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Abstract

This unit describes the procedures for measuring binding of a radiolabeled chemokine to chemokine receptors in cells or cell membranes. The whole‐cell binding assay can be used for both purified leukocytes as well as transfected cell lines expressing chemokine receptors. Two basic protocols are described. The first is applicable to all cells expressing chemokine receptors, both primary cultures as well as recombinantly transfected cell lines expressing a single chemokine receptor. The second is used principally for transfected cell lines and measures chemokine binding to cell membranes using scintillation proximity assay (SPA) methodology, but does not require washing steps and thus is applicable to automated high‐throughput screening strategies. An alternative procedure is also described which also uses SPA methodology to measure GTP‐gamma‐S binding to chemokine receptors in cell membranes. The two basic protocols can be used to determine the binding of compounds to chemokine receptors, while the alternate protocol determines the effects of compounds on the chemokine‐stimulated activation of the receptor.

     
 
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Table of Contents

  • Basic Protocol 1: Binding of Radiolabeled Chemokines to Cells Expressing Chemokine Receptors
  • Basic Protocol 2: Competition Equilibrium Assay Using Scintillation Proximity Assay (SPA) Technology on Membranes Expressing a Chemokine Receptor
  • Alternate Protocol 1: SPA‐Based [35S]GTPγS Binding to Cell Membranes Expressing Chemokine Receptors
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Binding of Radiolabeled Chemokines to Cells Expressing Chemokine Receptors

  Materials
  • Unlabeled recombinant chemokine: prepare in‐house or purchase (e.g., PeproTech, R&D Systems, Dictagene, Gryphon Sciences)
  • HEPES binding buffer (see recipe)
  • Radiolabeled (i.e., 125I) chemokine ligand (Amersham)
  • Transfected cells expressing a single chemokine receptor (ATCC: http://www.attc.org) or purified leukocytes (unit 12.4)
  • Phosphate buffered saline (PBS; unit 12.4) containing recipe1 mM EDTA ( appendix 2A)
  • 0.4% (w/v) trypan blue solution (Life Technologies): store in a dark bottle and filter before use or after prolonged storage
  • Wash buffer, ice cold: HEPES binding buffer containing 0.5 M NaCl
  • Optiphase Supermix scintillation liquid (Wallac)
  • Hemacytometer
  • 96‐well opaque MultiScreen plates fitted with a 0.65‐µm hydrophilic low‐protein‐binding membrane (Millipore)
  • Multichannel pipettor (optional)
  • Plate shaker
  • MultiScreen manifold filtration system (Millipore)
  • Vacuum pump
  • Wallac Microbeta Trilux counter
  • PRISM software (GraphPad Software)
NOTE: The radiolabeled ligand should be prepared in a laboratory equipped for protection from γ‐irradiation.

Basic Protocol 2: Competition Equilibrium Assay Using Scintillation Proximity Assay (SPA) Technology on Membranes Expressing a Chemokine Receptor

  Materials
  • Confluent cells expressing a recombinant chemokine receptor (e.g., CHO, HEK) grown in 75‐ml flasks in appropriate growth medium
  • PBS (unit 12.4) with and without 1 mM EDTA
  • Breakage buffer (see recipe)
  • HEPES binding buffer (see recipe)
  • Wheat‐germ agglutinin (WGA)‐coated SPA beads (Amersham)
  • 125I‐labeled chemokine (Amersham or NEN Life Sciences)
  • 1 to 5 µM unlabeled chemokine (ReproTech)
  • 5‐mm‐diameter Polytron PT1200 homogenizer
  • Ultracentrifuge and appropriate tubes
  • 96‐deep‐well microtiter plates (i.e., Corning low‐binding plate)
  • Wallac Microbeta Trilux counter
  • Graphing software (e.g., GraphPad PRISM)
  • Additional reagents and equipment for Bradford protein assay ( appendix 3A)

Alternate Protocol 1: SPA‐Based [35S]GTPγS Binding to Cell Membranes Expressing Chemokine Receptors

  • Cell membranes expressing a given chemokine receptor (see protocol 2, steps to )
  • GTPγS assay buffer (see recipe)
  • GDP (Roche)
  • Individual or multiple chemokines
  • Individual or multiple test compounds
  • 1200 Ci/mmol guanosine 5′‐(γ‐[35S]thio)triphosphate ([35S]GTPγS; NEN Life Sciences)
  • 1 mM unlabeled guanosine 5′‐O‐3‐thiotriphosphate (GTPγS; Roche)
  • Shaker
  • Centrifuge with microplate carrier
CAUTION: Radioactive materials require special handling; all supernatants must be considered radioactive waste and disposed of appropriately.
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Figures

Videos

Literature Cited

Literature Cited
   Elsner, J., Mack, M., Brühl, H., Dulkys, Y., Kimmig, D., Simmons, G., Clapham, P.R., Schlöndorff, D., Kapp, A., Wells, T.N.C., and Proudfoot, A.E.I. 2000. Differential activation of CC chemokine receptors by AOP‐RANTES. J. Biol. Chem. 275:7787‐7794.
   Gerard, C. 1999. Chemokine receptors and ligand specificity: Understanding the enigma. In Chemokines and Cancer (B.J. Rollins, ed.) pp. 21‐31. Humana, Totowa, NJ.
   Hoogewerf, A.J., Kuschert, G.S., Proudfoot, A.E.I., Borlat, F., Clark, L.I., Power, C.A., and Wells, T.N.C. 1997. Glycosaminoglycans mediate cell surface oligomerization of chemokines. Biochemistry 36:13570‐13578.
   Imai, T., Baba, M., Nishimura, M., Kakizaki, M., Takagi, S., and Yoshie, O. 1997. The T cell‐directed CC chemokine TARC is a highly specific biological ligand for CC chemokine receptor 4. J. Biol. Chem. 272:15036‐15042.
   Kledal, T.N., Rosenkilde, M.M., and Schwartz, T.W. 1998. Selective recognition of the membrane‐bound CX3C chemokine, fractalkine, by the human cytomegalovirus‐encoded broad‐spectrum receptor US28. FEBS Lett. 441:209‐214.
   Luster, A.D. 1998. Chemokines: Chemotactic cytokines that mediate inflammation. N. Engl. J. Med. 338:436‐445.
   Neote, K., DiGregorio, D., Mak, J.Y., Horuk, R., and Schall, T.J. 1993. Molecular cloning, functional expression, and signaling characteristics of a C‐C chemokine receptor. Cell. 72:415‐425.
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   Ponath, P.D. 1998. Chemokine receptor antagonists: Novel therapeutics for inflammation and AIDS. Exp. Opin. Invest. Drugs 7:1‐18.
   Power, C.A., Meyer, A., Nemeth, K., Bacon, K.B., Hoogewerf, A.K., Proudfoot, A.E.I., and Wells, T.N.C. 1995. Molecular cloning and functional expression of a novel CC chemokine receptor cDNA from a human basophilic cell line. J. Biol. Chem. 270:19495‐19500.
   Proudfoot, A.E.I. and Power, C.A. 2001. The chemokine system: novel broad‐spectrum therapeutics targets. Curr. Opin. Pharm. 1:417‐424.
   Proudfoot, A.E.I., Power, C.A., Hoogewerf, A.J., Montjovent, M.O., Borlat, F., Offord, R.E., and Wells, T.N.C. 1996. Extension of recombinant human RANTES by the retention of the initiating methionine produces a potent antagonist. J. Biol. Chem. 271:2599‐2603.
   Proudfoot, A.E., Wells, T.N., and Clapham, P.R. 1999. Chemokine receptors: Future therapeutic targets for HIV? Biochem. Pharmacol. 57:451‐463.
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   Schwarz, M.K. and Wells, T.N.C. 1999. Recent developments in modulating chemokine networks. Exp. Opin. Ther. Patents 9:1471‐1490.
   Simmons, G., Clapham, P.R., Picard, L., Offord, R.E., Rosenkilde, M.M., Schwartz, T.W., Buser, R., Wells, T.N.C. and Proudfoot, A.E. 1997. Potent inhibition of HIV‐1 infectivity in macrophages and lymphocytes by a novel CCR5 antagonist. Science 276:276‐279.
   Trkola, A., Gordon, C., Matthews, J., Maxwell, E., Ketas, T., Czaplewski, L., Proudfoot, A.E., and Moore, J.P. 1999. The CC‐chemokine RANTES increases the attachment of human immunodeficiency virus type 1 to target cells via glycosaminoglycans and also activates a signal transduction pathway that enhances viral infectivity. J. Virol. 73:6370‐6379.
   Wells, T.N.C., Power, C.A. and Proudfoot, A.E.I. 1998. Definition, function and pathophysiological significance of chemokine receptors. Trends Pharmacol. Sci. 19:376‐380.
   Weng, Y.M., Siciliano, S.J., Waldburger, K.E., Sirotinameisher, A., Staruch, M.J.D., Gould, S.L., Daugherty, B.L., Springer, M.S., and DeMartino, J.A. 1998. Binding and functional properties of recombinant and endogenous CXCR3 chemokine receptors. J. Biol. Chem. 273:18288‐18291.
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