Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and EGF Receptor

Alexander Sorkin1, Jason E. Duex1

1 Department of Pharmacology, University of Colorado Denver Medical School, Aurora, Colorado
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 15.14
DOI:  10.1002/0471143030.cb1514s46
Online Posting Date:  March, 2010
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Abstract

Binding of epidermal growth factor (EGF) to the EGF receptor (EGFR) initiates signal transduction, ultimately leading to altered gene expression. Ligand‐activated EGFR is also rapidly internalized and then targeted to lysosomes for degradation or recycled back to the plasma membrane. Endocytosis is a major regulator of EGFR signaling. Therefore, elucidation of the mechanisms of EGFR endocytosis is essential for a better understanding of EGFR biology. In order to achieve a comprehensive analysis of these mechanisms, reliable methods for measuring the rates of EGFR protein turnover and the rate parameters for individual steps of EGFR endocytic trafficking must be employed. The protocols in this unit describe methodologies to measure the rates of EGFR synthesis and degradation, to monitor EGF‐induced down‐regulation of surface EGFR, to measure the kinetic rate parameters of internalization, recycling, and degradation of radiolabeled EGF, and to perform radioiodination of EGF by the chloramine T method. Curr. Protoc. Cell Biol. 46:15.14.1‐15.14.20. © 2010 by John Wiley & Sons, Inc.

Keywords: EGF receptor; endocytosis; recycling; degradation; synthesis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measuring EGFR Protein Synthesis
  • Alternate Protocol 1: Measuring EGFR Protein Degradation
  • Basic Protocol 2: Measuring EGFR Down‐Regulation
  • Basic Protocol 3: Measuring Internalization of [125I]EGF
  • Basic Protocol 4: Measuring [125I]EGF Recycling and Degradation
  • Alternate Protocol 2: Measuring [125I]EGF Recycling/Degradation in the Absence of Excess Unlabeled EGF
  • Support Protocol 1: Preparation of [125I]EGF
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Measuring EGFR Protein Synthesis

  Materials
  • EGFR‐expressing cells (most cultured cells of epithelial and fibroblast origin express EGFR at levels from 5,000 to 2,000,000 per cell)
  • Complete DMEM (unit 1.1; use 5% to 10% FBS) or other appropriate cell culture medium
  • DMEM or other appropriate culture medium free of L‐methionine, L‐cysteine, and L‐glutamine (Invitrogen)
  • EasyTag 35S‐labeled amino acids (PerkinElmer)
  • Phosphate‐buffered saline (DPBS; Invitrogen
  • TGH lysis buffer (see recipe) containing 150 mM NaCl
  • EGFR antibody mAb528 (mouse hybridoma, ATCC #HB8509)
  • 20% (v/v) protein A–Sepharose resin (Sigma) in 50 mM HEPES
  • TGH lysis buffer (see recipe) containing no NaCl and 500 mM NaCl
  • 2× sample buffer for SDS‐PAGE (see recipe for 1×)
  • 7.5% acrylamide gel (unit 6.1)
  • EGFR antibody 1005 (Santa Cruz Biotechnology)
  • 60‐mm plastic culture dishes
  • Rubber policemen
  • Platform rotator (e.g., Nutator from Clay Adams)
  • Image analysis software: e.g., ImageJ (NIH freeware)
  • Additional reagents and equipment for cell culture (unit 1.1), determining protein concentration ( appendix 3H), SDS‐PAGE (unit 6.1), immunoblotting and immunodetection (unit 6.2), and phosphor imaging (unit 6.3)

Alternate Protocol 1: Measuring EGFR Protein Degradation

  • Experimental compounds of interest (e.g., EGF)

Basic Protocol 2: Measuring EGFR Down‐Regulation

  Materials
  • EGFR‐expressing cells (most cultured cells of epithelial and fibroblast origin express EGFR at levels from 5,000 to 2,000,000 per cell)
  • Complete DMEM (unit 1.1; use 5% to 10% FBS) or other appropriate cell culture medium
  • Recombinant human culture‐grade EGF (BD Biosciences, cat. no. 354052)
  • Binding medium; DMEM or other medium containing 0.1% (w/v) BSA
  • Sodium acetate buffer, pH 4.5: 0.2 M sodium acetate, pH 4.5 ( appendix 2A)/0.5 M NaCl
  • 1 to 200 ng/ml [125I]EGF (sp. act. ∼200,000 cpm/ng; protocol 7)
  • 1 N NaOH
  • 12‐well plastic culture plates
  • Additional reagents and equipment for cell culture (unit 1.1)

Basic Protocol 3: Measuring Internalization of [125I]EGF

  Materials
  • EGFR‐expressing cells (most cultured cells of epithelial and fibroblast origin express EGFR at levels from 5,000 to 2,000,000 per cell)
  • Complete DMEM (unit 1.1; use 5% to 10% FBS) or other appropriate cell culture medium
  • Binding medium; DMEM or other medium containing 0.1% (w/v) BSA
  • 0.1 to 200 ng/ml [125I]EGF (sp. act., ∼200,000 cpm/ng; protocol 7)
  • Recombinant human culture‐grade EGF (BD Biosciences, cat. no. 354052)
  • Sodium acetate buffer, pH 2.8: 0.2 M acetic acid/0.5 M NaCl, pH 2.5 to 2.8
  • 1 N NaOH
  • 12‐well plastic cell culture plates
  • γ‐counter vials
  • γ counter
  • Additional reagents and equipment for cell culture (unit 1.1)

Basic Protocol 4: Measuring [125I]EGF Recycling and Degradation

  Materials
  • EGFR‐expressing cells (most cultured cells of epithelial and fibroblast origin express EGFR at levels from 5,000 to 2,000,000 per cell)
  • Complete DMEM (unit 1.1; use 5% to 10% FBS) or other appropriate cell culture medium
  • Binding medium: DMEM or other medium containing 0.1% (w/v) BSA
  • 1 to 200 ng/ml [125I]EGF (sp. act., ∼200,000 cpm/ng; protocol 7)
  • Recombinant human culture‐grade EGF (BD Biosciences, cat. no. 354052)
  • Sodium acetate buffer, pH 4.5: 0.2 M sodium acetate buffer, pH 4.3 to 4.5 ( appendix 2A)/0.5 M NaCl
  • Sodium acetate buffer, pH 2.8: 0.2 M acetic acid/0.5 M NaCl, pH 2.5 to 2.8
  • 1 N NaOH
  • 10% (w/v) trichloroacetic acid (TCA)/2% (w/v) phosphotungstic acid (PTA) in H 2O
  • 35‐mm plastic cell culture dishes
  • γ‐counter vials
  • γ counter
  • Refrigerated centrifuge
  • Additional reagents and equipment for cell culture (unit 1.1)

Alternate Protocol 2: Measuring [125I]EGF Recycling/Degradation in the Absence of Excess Unlabeled EGF

  Materials
  • 0.05 M sodium phosphate buffer, pH 7.5 ( appendix 2A) containing 0.075 M NaCl
  • 20 mg/ml BSA in 0.05 M sodium phosphate buffer, pH 7.5 ( appendix 2A) containing 0.075 M NaCl
  • Receptor‐grade mouse EGF (BD Biosciences, cat. no. 354010)
  • 0.5 M sodium phosphate buffer, pH 7.5 ( appendix 2A)
  • 0.1 mCi/µl Na[125I] (PerkinElmer; cat. no. NEZ33A)
  • 2 mg/ml chloramine T (Sigma) in 0.05 M sodium phosphate buffer, pH 7.5 (see appendix 2A for buffer); prepare fresh
  • 4 mg/ml sodium metabisulfite in 0.05 M sodium phosphate buffer, pH 7.5 (see appendix 2A for buffer); prepare fresh
  • PD‐10 (G‐25) Sepharose disposable column (GE Healthcare)
  • γ counter
NOTE: The entire procedure is carried out at room temperature.
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Figures

Videos

Literature Cited

Literature Cited
   Carpenter, G. and Cohen, S. 1976. 125I‐Labeled human epidermal growth factor: Binding internalization, and degradation in human fibroblasts. J. Cell Biol. 71:159‐171.
   Duex, J.E. and Sorkin, A. 2009. RNA interference screen identifies Usp18 as a regulator of EGF receptor synthesis. Mol. Biol. Cell 20:1833‐1844.
   Haigler, H.T., Maxfield, F.R., Willingham, M.C., and Pastan, I. 1980. Dansylcadaverine inhibits internalization of 125I‐epidermal growth factor in BALB 3T3 cells. J. Biol. Chem. 255:1239‐1241.
   Herbst, J.J., Opresko, L.K., Walsh, B.J., Lauffenberger, D.A., and Wiley, H.S. 1994. Regulation of postendocytic trafficking of the epidermal growth factor receptor through endosomal retention. J. Biol. Chem. 269:12865‐12873.
   Resat, H., Ewald, J.A., Dixon, D.A., and Wiley, H.S. 2003. An integrated model of epidermal growth factor receptor trafficking and signal transduction. Biophys. J. 85:730‐743.
   Sorkin, A., Teslenko, L., and Nikolsky, N. 1988. The endocytosis of epidermal growth factor in A431 cells: A pH of microenvironment and the dynamics of receptor complexes dissociation. Exp. Cell Res. 175:192‐205.
   Shtiegman, K., Kochupurakkal, B.S., Zwang, Y., Pines, G., Starr, A., Vexler, A., Citri, A., Katz, M., Lavi, S., Ben‐Basat, Y., Benjamin, S., Corso, S., Gan, J., Yosef, R.B., Giordano, S., and Yarden, Y. Defective ubiquitinylation of EGFR mutants of lung cancer confers prolonged signaling. Oncogene 2007. 26:6968‐6978.
   Todderud, G. and Carpenter, G. 1989. Epidermal growth factor: The receptor and its function. BioFactors 2:11‐15.
   Wiley, H.S. and Cunningham, D.D. 1982. The endocytotic rate constant: A cellular parameter for quantitating receptor‐mediated endocytosis. J. Biol. Chem. 257:4222‐4229.
   Wiley, H.S., Herbst, J.J., Walsh, B.J., Lauffenberger, D.A., Rosenfeld, M.G., and Gill, G.N. 1991. The role of tyrosine kinase activity in endocytosis, compartmentalization and down‐regulation of the epidermal growth factor receptor. J. Biol. Chem. 266:11083‐11094.
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