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. 2002 Mar 1;362(Pt 2):499–505. doi: 10.1042/bj3620499

Evidence for the direct interaction between calmodulin and the human epidermal growth factor receptor.

Hongbing Li 1, Antonio Villalobo 1
PMCID: PMC1222412  PMID: 11853560

Abstract

Previous work from our laboratory has demonstrated that the Ca(2+)-calmodulin complex inhibits the intrinsic tyrosine kinase activity of the epidermal growth factor receptor (EGFR), and that the receptor can be isolated by Ca(2+)-dependent calmodulin-affinity chromatography [San José, Bengurija, Geller and Villalobo (1992) J. Biol. Chem. 267, 15237-15245]. Moreover, we have demonstrated that the cytosolic juxtamembrane region of the human receptor (residues 645-660) binds calmodulin in a Ca(2+)-dependent manner when this segment forms part of a recombinant fusion protein [Martijn-Nieto and Villalobo (1998) Biochemistry 37, 227-236]. However, demonstration of the direct interaction between calmodulin and the whole receptor has remained elusive. In this work, we show that calmodulin, in the presence of Ca(2+), forms part of a high-molecular-mass complex built upon covalent cross-linkage of the human EGFR immunoprecipitated from two cell lines overexpressing this receptor. Although several calmodulin-binding proteins co-immunoprecipitated with the EGFR, suggesting that they interact with the receptor, we demonstrated using overlay techniques that biotinylated calmodulin binds directly to the receptor in a Ca(2+)-dependent manner without the mediation of any adaptor calmodulin-binding protein. Calmodulin binds to the EGFR with an apparent dissociation constant (K'(d)) of approx. 0.2-0.3 microM. Treatment of cells with epidermal growth factor, or with inhibitors of protein kinase C and calmodulin-dependent protein kinase II, or treatment of the immunoprecipitated receptor with alkaline phosphatase, does not significantly affect the binding of biotinylated calmodulin to the receptor.

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Selected References

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  1. Benguría A., Martín-Nieto J., Benaim G., Villalobo A. Regulatory interaction between calmodulin and the epidermal growth factor receptor. Ann N Y Acad Sci. 1995 Sep 7;766:472–476. doi: 10.1111/j.1749-6632.1995.tb26698.x. [DOI] [PubMed] [Google Scholar]
  2. Billingsley M. L., Pennypacker K. R., Hoover C. G., Brigati D. J., Kincaid R. L. A rapid and sensitive method for detection and quantification of calcineurin and calmodulin-binding proteins using biotinylated calmodulin. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7585–7589. doi: 10.1073/pnas.82.22.7585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Countaway J. L., McQuilkin P., Gironès N., Davis R. J. Multisite phosphorylation of the epidermal growth factor receptor. Use of site-directed mutagenesis to examine the role of serine/threonine phosphorylation. J Biol Chem. 1990 Feb 25;265(6):3407–3416. [PubMed] [Google Scholar]
  4. Countaway J. L., Nairn A. C., Davis R. J. Mechanism of desensitization of the epidermal growth factor receptor protein-tyrosine kinase. J Biol Chem. 1992 Jan 15;267(2):1129–1140. [PubMed] [Google Scholar]
  5. Davis R. J. Independent mechanisms account for the regulation by protein kinase C of the epidermal growth factor receptor affinity and tyrosine-protein kinase activity. J Biol Chem. 1988 Jul 5;263(19):9462–9469. [PubMed] [Google Scholar]
  6. Feinmesser R. L., Wicks S. J., Taverner C. J., Chantry A. Ca2+/calmodulin-dependent kinase II phosphorylates the epidermal growth factor receptor on multiple sites in the cytoplasmic tail and serine 744 within the kinase domain to regulate signal generation. J Biol Chem. 1999 Jun 4;274(23):16168–16173. doi: 10.1074/jbc.274.23.16168. [DOI] [PubMed] [Google Scholar]
  7. Hayashi N., Matsubara M., Takasaki A., Titani K., Taniguchi H. An expression system of rat calmodulin using T7 phage promoter in Escherichia coli. Protein Expr Purif. 1998 Feb;12(1):25–28. doi: 10.1006/prep.1997.0807. [DOI] [PubMed] [Google Scholar]
  8. Holbrook M. R., Slakey L. L., Gross D. J. Thermodynamic mixing of molecular states of the epidermal growth factor receptor modulates macroscopic ligand binding affinity. Biochem J. 2000 Nov 15;352(Pt 1):99–108. [PMC free article] [PubMed] [Google Scholar]
  9. Hunter T., Ling N., Cooper J. A. Protein kinase C phosphorylation of the EGF receptor at a threonine residue close to the cytoplasmic face of the plasma membrane. Nature. 1984 Oct 4;311(5985):480–483. doi: 10.1038/311480a0. [DOI] [PubMed] [Google Scholar]
  10. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  11. Livneh E., Dull T. J., Berent E., Prywes R., Ullrich A., Schlessinger J. Release of a phorbol ester-induced mitogenic block by mutation at Thr-654 of the epidermal growth factor receptor. Mol Cell Biol. 1988 Jun;8(6):2302–2308. doi: 10.1128/mcb.8.6.2302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lund K. A., Lazar C. S., Chen W. S., Walsh B. J., Welsh J. B., Herbst J. J., Walton G. M., Rosenfeld M. G., Gill G. N., Wiley H. S. Phosphorylation of the epidermal growth factor receptor at threonine 654 inhibits ligand-induced internalization and down-regulation. J Biol Chem. 1990 Nov 25;265(33):20517–20523. [PubMed] [Google Scholar]
  13. Martín-Nieto J., Villalobo A. The human epidermal growth factor receptor contains a juxtamembrane calmodulin-binding site. Biochemistry. 1998 Jan 6;37(1):227–236. doi: 10.1021/bi971765v. [DOI] [PubMed] [Google Scholar]
  14. Sako Y., Minoghchi S., Yanagida T. Single-molecule imaging of EGFR signalling on the surface of living cells. Nat Cell Biol. 2000 Mar;2(3):168–172. doi: 10.1038/35004044. [DOI] [PubMed] [Google Scholar]
  15. San José E., Benguría A., Geller P., Villalobo A. Calmodulin inhibits the epidermal growth factor receptor tyrosine kinase. J Biol Chem. 1992 Jul 25;267(21):15237–15245. [PubMed] [Google Scholar]
  16. Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell. 2000 Oct 13;103(2):211–225. doi: 10.1016/s0092-8674(00)00114-8. [DOI] [PubMed] [Google Scholar]
  17. Theroux S. J., Latour D. A., Stanley K., Raden D. L., Davis R. J. Signal transduction by the epidermal growth factor receptor is attenuated by a COOH-terminal domain serine phosphorylation site. J Biol Chem. 1992 Aug 15;267(23):16620–16626. [PubMed] [Google Scholar]
  18. Ullrich A., Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell. 1990 Apr 20;61(2):203–212. doi: 10.1016/0092-8674(90)90801-k. [DOI] [PubMed] [Google Scholar]
  19. Weiss A., Schlessinger J. Switching signals on or off by receptor dimerization. Cell. 1998 Aug 7;94(3):277–280. doi: 10.1016/s0092-8674(00)81469-5. [DOI] [PubMed] [Google Scholar]
  20. Zwick E., Hackel P. O., Prenzel N., Ullrich A. The EGF receptor as central transducer of heterologous signalling systems. Trends Pharmacol Sci. 1999 Oct;20(10):408–412. doi: 10.1016/s0165-6147(99)01373-5. [DOI] [PubMed] [Google Scholar]

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