High-affinity Binding of Epidermal Growth Factor (EGF) to EGF Receptor Is Disrupted by Overexpression of Mutant Dynamin (K44A)*
- Tove Ringerike‡,
- Espen Stang‡,
- Lene E. Johannessen‡,
- Dagny Sandnes§,
- Finn Olav Levy¶‖ and
- Inger Helene Madshus‡**
- From the ‡Institute of Pathology,¶MSD Cardiovascular Research Center, and ‖Institute for Surgical Research, University of Oslo, The National Hospital, N-0027 Oslo and the §Department of Pharmacology, University of Oslo, P. O. Box 1057, N-0316 Oslo, Norway
Abstract
Activation of the epidermal growth factor receptor (EGFR) kinase was analyzed in cells conditionally defective for clathrin-dependent endocytosis by overexpression of mutant dynamin (K44A). EGF-induced autophosphorylation of the EGFR on ice was strongly reduced in cells overexpressing mutant dynamin, and consistently, binding analyses showed that high-affinity EGFRs were lost. In the absence of mutant dynamin the cells displayed both high- and low-affinity EGFR. At 4 °C EGF-EGFR localized mainly outside coated pits regardless of expression of mutant dynamin. However, also low-affinity EGFR efficiently moved to coated pits upon incubating cells at 37 °C. Thus, expression of mutant dynamin disrupts high-affinity binding of EGF, but not ligand-induced recruitment of EGFR to clathrin-coated pits.
Footnotes
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↵* This work was supported by The Norwegian Cancer Society, The National Council for Science and the Humanities, Medinnova, Nordic Insulin Foundation Committee, The Anders Jahre’s Foundation for the Promotion of Science, Blix Legacy and Bruuns Legacy.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵** To whom correspondence should be addressed: Institute of Pathology, The National Hospital, N-0027 Oslo, Norway. Tel.: 47-22868609; Fax: 47-22112261; E-mail: i.h.madshus{at}labmed.uio.no.
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↵1 The abbreviations used are: EGF, epidermal growth factor; EGFR, EGF receptor; BSA, bovine serum albumin; PLCγ, phospholipase C γ; PKC, protein kinase C; IP3, inositol 1,4,5-trisphosphate.
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- Received April 3, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
