Measurement of Lymphokine Receptors

John W. Lowenthal1, Thomas R. Malek2, Horacio Saragovi3

1 CSIRO Division of Animal Health, Parkville,Victoria, Australia, 2 University of Miami School of Medicine, Miami, Florida, 3 University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.1
DOI:  10.1002/0471142735.im0601s35
Online Posting Date:  May, 2001
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Abstract

One of the most widely studied lymphokine systems is the T lymphocyte growth factor interleukin 2 (IL‐2). This unit describes two basic methods for the quantitation and biochemical characterization of IL‐2 receptors. The first method employs the radioreceptor assay. Support protocols to this technique describe quantification of data via calculations of association and dissociation rates and the Scatchard plot analysis. The second approach detects cell‐surface lymphokine receptors by covalently cross‐linking IL‐2 to its receptor. This method employs the chemical disuccinimidyl suberate (DSS) to achieve irreversible cross‐linking of IL‐2 to IL‐2R.

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of Lymphokine Receptor Number and Affinity Using Radioreceptor Assays
  • Support Protocol 1: Calculation of Association and Dissociation Rates
  • Support Protocol 2: Calculation of IL‐2 Binding Affinity and Receptor Number by Scatchard Plot Analysis
  • Basic Protocol 2: Irreversible Cross‐Linking of Lymphokine to Its Receptor Using Disuccinimidyl Suberate (DSS)
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Lymphokine Receptor Number and Affinity Using Radioreceptor Assays

  Materials
  • Target cells (e.g., cell population to be assayed for receptor)
  • Complete RPMI‐10 medium ( appendix 2A), at 4°C and 37°C
  • recipeComplete RPMI‐10 medium, pH 3.0 (see recipe)
  • recipeBinding oil (see recipe)
  • 600 Ci/mmol 125I‐labeled recombinant human IL‐2 (30 to 40 µCi/µg, DuPont NEN; see Critical Parameters Table 6.1.2)
  • ≥0.5 mg/ml recombinant IL‐2, purified
  • 400‐µl microcentrifuge tubes (Sarstedt) or 96‐well flat‐bottom microtiter plates (Costar)
  • 5‐ to 15‐ml tissue culture tube
  • Tabletop centrifuge, 4°C
  • Additional reagents and equipment for counting cells ( appendix 3A)

Support Protocol 1: Calculation of Association and Dissociation Rates

  Materials
  • Target cells (e.g., cell population to be studied)
  • Hanks balanced salt solution (HBSS; appendix 2A) without phenol red, 4°C
  • recipeComplete RPMI‐10 medium ( appendix 2A ) with 1 mg/ml bovine serum albumin (BSA)
  • 600 Ci/mmol 125I‐labeled recombinant human IL‐2 (30 to 40 µCi/µg, DuPont NEN; see Critical Paramters Table 6.1.2)
  • recipe40 mM disuccinimidyl suberate (DSS) solution (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A), pH 7.4
  • 1 M ammonium acetate
  • recipeExtraction buffer (see recipe)
  • 100 mM phenylmethylsulfonyl fluoride (PMSF) in methanol
  • 1 M iodoacetamide, prepared daily
  • 1% (v/v) Nonidet P‐40 (NP‐40) in recipeextraction buffer (see recipe)
  • 20% (w/v) sodium dodecyl sulfate (SDS)
  • 2× SDS sample buffer (unit 8.4)
  • Anti‐pα5 (CD25) monoclonal antibody (7D4, ATCC #CRL1698)
  • Coomassie brilliant blue (Bio‐Rad)
  • Molecular weight markers (Pharmacia LKB)
  • 5‐ to 15‐ml tissue culture tube (Falcon)
  • Tabletop centrifuge, 4°C
  • 14°C water bath
  • Additional reagents and equipment for counting cells ( appendix 3A), immunoprecipitation (unit 8.3), SDS–polyacrylamide gel electrophoresis (unit 8.4), gel staining (unit 8.9), and autoradiography ( appendix 3J)
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Figures

Videos

Literature Cited

Literature Cited
   Dower, S.K., Kronheim, S.R., March, C.J., Conlon, P.J., Hopp, T.P., Gillis, S., and Urdal, D.L. 1985. Detection and characterization of high‐affinity plasma membrane receptors for human interleukin 1. J. Exp. Med. 162:501‐515.
   Kimura, Y., Takeshita, T., Kondo, M., Ishii, N., Nakamura, M., Van Snick, J., and Sugamura, K. 1995. Sharing of the IL‐2 receptor γ chain with the functional IL‐9 receptor. Int. Immunol . 7:115‐120.
   Kondo, M., Takeshita, T., Higuchi, M., Nakamura, M., Sudo, T., Nishikawa, S.‐I., and Sugamura. K. 1994. Functional participation of the IL‐2 receptor γ chain in IL‐7 receptor complexes. Science 263:1453‐1454.
   Lowenthal, J.W. and Greene, W.C. 1987. Contrasting interleukin 2 binding properties of the α (p55) and β (p70) protein subunits of the human high affinity interleukin 2 receptor. J. Exp. Med. 166:1156‐1161.
   Lowenthal, J.W. and MacDonald, H.R. 1986. Binding and internalization of interleukin 1 by T cells. Direct evidence for high and low affinity classes of interleukin 1 receptor. J. Exp. Med. 164:1060‐1074.
   Lowenthal, J.W., Coethesy, P., Tougne, C., Lees, R., MacDonald, H.R., and Nabholz, M. 1985. High and low affinity IL‐2 receptors: Analysis by IL‐2 dissociation rate and reactivity with a monoclonal anti‐receptor antibody PC61. J. Immunol. 135:3988‐3994.
   Lowenthal, J.W., Castle, B.E., Christiansen, J., Schreurs, J., Rennick, D., Arai, N., Hoy, P., Takabe, Y., and Howard, M. 1988. Expression of high affinity receptors for murine interleukin 4 (BSF‐1) on hemopoetic and nonhemopoetic cells. J. Immunol. 140:456‐464.
   Malek, T.R., Furse, R.K., Fleming, M.L., Fadell, A.J., and He, Y.‐W. 1995. Biochemical identity and characterization of the mouse interleukin 2 receptor β and γ chains J. Interferon Cytokine Res. 15:447‐454.
   Munson, P.J. 1983. LIGAND: A computerized analysis of ligand binding data. Methods Enzymol. 92:543‐577.
   Munson, P.J. and Rodbard, D. 1980. LIGAND: A versatile computerized approach for characterization of ligand‐binding systems. Anal. Biochem. 107:220‐239.
   Ohara, J. and Paul, W.E. 1987. Receptors for B cell stimulatory factor expressed on cells of haematopoietic lineage. Nature 325:537‐540.
   Robb, R.J., Munck, A., and Smith, K.A. 1981. T cell growth factor receptors.Quantitation, specificity and biological relevance. J. Exp. Med. 154:1455‐1474.
   Robb, R.J., Mayer, P.C., and Garlick, R. 1985. Retention of biological activity following radioiodination of human interleukin 2: Comparison with biosynthetically labeled growth factor in receptor binding assays. J. Immunol. Methods 81:15‐30.
   Saragovi, H. and Malek, T.R. 1987. The murine interleukin 2 receptor: Irreversible cross‐linking of radiolabeled interleukin 2 to high affinity interleukin 2 receptors reveals a noncovalently associated subunit. J. Immunol. 139:1918‐1926.
   Scatchard, G. 1949. The attractions of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51:660‐672.
   Sharon, M., Klausner, R.D., Cullens, B.R., Chizzanite, R., and Leonard, W.J. 1986. Novel interleukin‐2 receptor subunit detected by cross‐linking under high affinity conditions. Science 234:859‐862.
Key References
   Munson, 1983. See above.
  Review of computerized Scatchard plot analysis.
   Robb et al., 1981. See above.
  Detailed description of first radiolabeled IL‐2 binding assay.
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