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. 1995 Apr 1;129(1):47–54. doi: 10.1083/jcb.129.1.47

Recruitment of epidermal growth factor receptors into coated pits requires their activated tyrosine kinase

PMCID: PMC2120379  PMID: 7698994

Abstract

EGF-receptor (EGF-R) tyrosine kinase is required for the down- regulation of activated EGF-R. However, controversy exists as to whether ligand-induced activation of the EGF-R tyrosine kinase is required for internalization or for lysosomal targeting. We have addressed this issue using a cell-free assay that selectively measures the recruitment of EGF-R into coated pits. Here we show that EGF bound to wild-type receptors is efficiently sequestered in coated pits. In contrast, sequestration of kinase-deficient receptors occurs inefficiently and at the same basal rate of endocytosis of unoccupied receptors or receptors lacking any cytoplasmic domain. Sequestration of deletion mutants of the EGF-R that lack autophosphorylation sites also requires an active tyrosine kinase. This suggests that a tyrosine kinase substrate(s) other than the EGF-R itself, is required for its efficient ligand-induced recruitment into coated pits. Addition of a soluble EGF-R tyrosine kinase fully and specifically restores the recruitment of kinase-deficient EGF-R into coated pits providing a powerful functional assay for identification of these substrate(s).

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Selected References

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  1. Bjorge J. D., Chan T. O., Antczak M., Kung H. J., Fujita D. J. Activated type I phosphatidylinositol kinase is associated with the epidermal growth factor (EGF) receptor following EGF stimulation. Proc Natl Acad Sci U S A. 1990 May;87(10):3816–3820. doi: 10.1073/pnas.87.10.3816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cantley L. C., Auger K. R., Carpenter C., Duckworth B., Graziani A., Kapeller R., Soltoff S. Oncogenes and signal transduction. Cell. 1991 Jan 25;64(2):281–302. doi: 10.1016/0092-8674(91)90639-g. [DOI] [PubMed] [Google Scholar]
  3. Carpenter G. Receptor tyrosine kinase substrates: src homology domains and signal transduction. FASEB J. 1992 Nov;6(14):3283–3289. doi: 10.1096/fasebj.6.14.1385243. [DOI] [PubMed] [Google Scholar]
  4. Carter L. L., Redelmeier T. E., Woollenweber L. A., Schmid S. L. Multiple GTP-binding proteins participate in clathrin-coated vesicle-mediated endocytosis. J Cell Biol. 1993 Jan;120(1):37–45. doi: 10.1083/jcb.120.1.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chang C. P., Kao J. P., Lazar C. S., Walsh B. J., Wells A., Wiley H. S., Gill G. N., Rosenfeld M. G. Ligand-induced internalization and increased cell calcium are mediated via distinct structural elements in the carboxyl terminus of the epidermal growth factor receptor. J Biol Chem. 1991 Dec 5;266(34):23467–23470. [PubMed] [Google Scholar]
  6. Chang C. P., Lazar C. S., Walsh B. J., Komuro M., Collawn J. F., Kuhn L. A., Tainer J. A., Trowbridge I. S., Farquhar M. G., Rosenfeld M. G. Ligand-induced internalization of the epidermal growth factor receptor is mediated by multiple endocytic codes analogous to the tyrosine motif found in constitutively internalized receptors. J Biol Chem. 1993 Sep 15;268(26):19312–19320. [PubMed] [Google Scholar]
  7. Chen W. S., Lazar C. S., Lund K. A., Welsh J. B., Chang C. P., Walton G. M., Der C. J., Wiley H. S., Gill G. N., Rosenfeld M. G. Functional independence of the epidermal growth factor receptor from a domain required for ligand-induced internalization and calcium regulation. Cell. 1989 Oct 6;59(1):33–43. doi: 10.1016/0092-8674(89)90867-2. [DOI] [PubMed] [Google Scholar]
  8. Chen W. S., Lazar C. S., Poenie M., Tsien R. Y., Gill G. N., Rosenfeld M. G. Requirement for intrinsic protein tyrosine kinase in the immediate and late actions of the EGF receptor. 1987 Aug 27-Sep 2Nature. 328(6133):820–823. doi: 10.1038/328820a0. [DOI] [PubMed] [Google Scholar]
  9. Damke H., Baba T., Warnock D. E., Schmid S. L. Induction of mutant dynamin specifically blocks endocytic coated vesicle formation. J Cell Biol. 1994 Nov;127(4):915–934. doi: 10.1083/jcb.127.4.915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Felder S., LaVin J., Ullrich A., Schlessinger J. Kinetics of binding, endocytosis, and recycling of EGF receptor mutants. J Cell Biol. 1992 Apr;117(1):203–212. doi: 10.1083/jcb.117.1.203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Felder S., Miller K., Moehren G., Ullrich A., Schlessinger J., Hopkins C. R. Kinase activity controls the sorting of the epidermal growth factor receptor within the multivesicular body. Cell. 1990 May 18;61(4):623–634. doi: 10.1016/0092-8674(90)90474-s. [DOI] [PubMed] [Google Scholar]
  12. Gill G. N., Kawamoto T., Cochet C., Le A., Sato J. D., Masui H., McLeod C., Mendelsohn J. Monoclonal anti-epidermal growth factor receptor antibodies which are inhibitors of epidermal growth factor binding and antagonists of epidermal growth factor binding and antagonists of epidermal growth factor-stimulated tyrosine protein kinase activity. J Biol Chem. 1984 Jun 25;259(12):7755–7760. [PubMed] [Google Scholar]
  13. Glenney J. R., Jr, Chen W. S., Lazar C. S., Walton G. M., Zokas L. M., Rosenfeld M. G., Gill G. N. Ligand-induced endocytosis of the EGF receptor is blocked by mutational inactivation and by microinjection of anti-phosphotyrosine antibodies. Cell. 1988 Mar 11;52(5):675–684. doi: 10.1016/0092-8674(88)90405-9. [DOI] [PubMed] [Google Scholar]
  14. Gout I., Dhand R., Hiles I. D., Fry M. J., Panayotou G., Das P., Truong O., Totty N. F., Hsuan J., Booker G. W. The GTPase dynamin binds to and is activated by a subset of SH3 domains. Cell. 1993 Oct 8;75(1):25–36. [PubMed] [Google Scholar]
  15. Haigler H. T., McKanna J. A., Cohen S. Direct visualization of the binding and internalization of a ferritin conjugate of epidermal growth factor in human carcinoma cells A-431. J Cell Biol. 1979 May;81(2):382–395. doi: 10.1083/jcb.81.2.382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Helin K., Beguinot L. Internalization and down-regulation of the human epidermal growth factor receptor are regulated by the carboxyl-terminal tyrosines. J Biol Chem. 1991 May 5;266(13):8363–8368. [PubMed] [Google Scholar]
  17. Herman P. K., Stack J. H., Emr S. D. An essential role for a protein and lipid kinase complex in secretory protein sorting. Trends Cell Biol. 1992 Dec;2(12):363–368. doi: 10.1016/0962-8924(92)90048-r. [DOI] [PubMed] [Google Scholar]
  18. Herskovits J. S., Shpetner H. S., Burgess C. C., Vallee R. B. Microtubules and Src homology 3 domains stimulate the dynamin GTPase via its C-terminal domain. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11468–11472. doi: 10.1073/pnas.90.24.11468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Honegger A. M., Dull T. J., Felder S., Van Obberghen E., Bellot F., Szapary D., Schmidt A., Ullrich A., Schlessinger J. Point mutation at the ATP binding site of EGF receptor abolishes protein-tyrosine kinase activity and alters cellular routing. Cell. 1987 Oct 23;51(2):199–209. doi: 10.1016/0092-8674(87)90147-4. [DOI] [PubMed] [Google Scholar]
  20. Honegger A. M., Schmidt A., Ullrich A., Schlessinger J. Separate endocytic pathways of kinase-defective and -active EGF receptor mutants expressed in same cells. J Cell Biol. 1990 May;110(5):1541–1548. doi: 10.1083/jcb.110.5.1541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Joly M., Kazlauskas A., Fay F. S., Corvera S. Disruption of PDGF receptor trafficking by mutation of its PI-3 kinase binding sites. Science. 1994 Feb 4;263(5147):684–687. doi: 10.1126/science.8303278. [DOI] [PubMed] [Google Scholar]
  22. Lamaze C., Baba T., Redelmeier T. E., Schmid S. L. Recruitment of epidermal growth factor and transferrin receptors into coated pits in vitro: differing biochemical requirements. Mol Biol Cell. 1993 Jul;4(7):715–727. doi: 10.1091/mbc.4.7.715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Murphy L. C., Dotzlaw H., Wong M. S., Miller T., Mrockowski B., Gong Y., Murphy L. J. Epidermal growth factor: receptor and ligand expression in human breast cancer. Semin Cancer Biol. 1990 Oct;1(5):305–315. [PubMed] [Google Scholar]
  24. Scaife R., Gout I., Waterfield M. D., Margolis R. L. Growth factor-induced binding of dynamin to signal transduction proteins involves sorting to distinct and separate proline-rich dynamin sequences. EMBO J. 1994 Jun 1;13(11):2574–2582. doi: 10.1002/j.1460-2075.1994.tb06547.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schmid S. L., Smythe E. Stage-specific assays for coated pit formation and coated vesicle budding in vitro. J Cell Biol. 1991 Sep;114(5):869–880. doi: 10.1083/jcb.114.5.869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Smythe E., Redelmeier T. E., Schmid S. L. Receptor-mediated endocytosis in semiintact cells. Methods Enzymol. 1992;219:223–234. doi: 10.1016/0076-6879(92)19024-z. [DOI] [PubMed] [Google Scholar]
  27. Sorkin A., Carpenter G. Interaction of activated EGF receptors with coated pit adaptins. Science. 1993 Jul 30;261(5121):612–615. doi: 10.1126/science.8342026. [DOI] [PubMed] [Google Scholar]
  28. Sorkin A., Helin K., Waters C. M., Carpenter G., Beguinot L. Multiple autophosphorylation sites of the epidermal growth factor receptor are essential for receptor kinase activity and internalization. Contrasting significance of tyrosine 992 in the native and truncated receptors. J Biol Chem. 1992 Apr 25;267(12):8672–8678. [PubMed] [Google Scholar]
  29. Sorkin A., Waters C. M. Endocytosis of growth factor receptors. Bioessays. 1993 Jun;15(6):375–382. doi: 10.1002/bies.950150603. [DOI] [PubMed] [Google Scholar]
  30. Starbuck C., Lauffenburger D. A. Mathematical model for the effects of epidermal growth factor receptor trafficking dynamics on fibroblast proliferation responses. Biotechnol Prog. 1992 Mar-Apr;8(2):132–143. doi: 10.1021/bp00014a007. [DOI] [PubMed] [Google Scholar]
  31. Walton G. M., Chen W. S., Rosenfeld M. G., Gill G. N. Analysis of deletions of the carboxyl terminus of the epidermal growth factor receptor reveals self-phosphorylation at tyrosine 992 and enhanced in vivo tyrosine phosphorylation of cell substrates. J Biol Chem. 1990 Jan 25;265(3):1750–1754. [PubMed] [Google Scholar]
  32. Wedegaertner P. B., Gill G. N. Activation of the purified protein tyrosine kinase domain of the epidermal growth factor receptor. J Biol Chem. 1989 Jul 5;264(19):11346–11353. [PubMed] [Google Scholar]
  33. Wells A., Welsh J. B., Lazar C. S., Wiley H. S., Gill G. N., Rosenfeld M. G. Ligand-induced transformation by a noninternalizing epidermal growth factor receptor. Science. 1990 Feb 23;247(4945):962–964. doi: 10.1126/science.2305263. [DOI] [PubMed] [Google Scholar]
  34. Wiley H. S. Anomalous binding of epidermal growth factor to A431 cells is due to the effect of high receptor densities and a saturable endocytic system. J Cell Biol. 1988 Aug;107(2):801–810. doi: 10.1083/jcb.107.2.801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wiley H. S., Herbst J. J., Walsh B. J., Lauffenburger D. A., Rosenfeld M. G., Gill G. N. The role of tyrosine kinase activity in endocytosis, compartmentation, and down-regulation of the epidermal growth factor receptor. J Biol Chem. 1991 Jun 15;266(17):11083–11094. [PubMed] [Google Scholar]
  36. Yano H., Nakanishi S., Kimura K., Hanai N., Saitoh Y., Fukui Y., Nonomura Y., Matsuda Y. Inhibition of histamine secretion by wortmannin through the blockade of phosphatidylinositol 3-kinase in RBL-2H3 cells. J Biol Chem. 1993 Dec 5;268(34):25846–25856. [PubMed] [Google Scholar]

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