Effect of tyrosine kinase inhibitors on clathrin-coated pit recruitment and internalization of epidermal growth factor receptor.
J Biol Chem
; 277(30): 27433-41, 2002 Jul 26.
Article
em En
| MEDLINE
| ID: mdl-12021271
ABSTRACT
Several inhibitors of epidermal growth factor receptor (EGFR) kinase and Src family kinases (SFK) were employed to study the role of these kinases in EGFR internalization through clathrin-coated pits. The EGFR kinase-specific compound PD158780 substantially diminished EGFR internalization. PP2, an inhibitor of SFK, had a moderate effect on EGFR internalization in several types of cells, including cells lacking SFK, indicating that the inhibition of endocytosis by PP2 is mediated by kinases other than SFK. In contrast, SU6656, a more specific inhibitor of SFK, did not affect EGFR internalization. To examine what stage of internalization requires receptor kinase activity, we established a quantitative assay based on three-dimensional fluorescence microscopy that measures co-localization of an EGF-rhodamine conjugate and a fluorescently tagged clathrin adaptor protein complex, AP-2. Interestingly, recruitment of EGFR into coated pits did not require physiological temperature because the maximal accumulation of EGFR in coated pits was observed at 4 degrees C. Pretreatment of the cells with PD158780 prevented EGFR recruitment into coated pits, whereas the inhibitor did not block the internalization of receptors that had first been allowed to enter the coated pits at 4 degrees C. These data demonstrate that the activation of receptor kinase is essential for the initial, coated pit recruitment step of endocytosis.
Buscar no Google
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas Tirosina Quinases
/
Clatrina
/
Receptores ErbB
Limite:
Animals
Idioma:
En
Revista:
J Biol Chem
Ano de publicação:
2002
Tipo de documento:
Article
País de afiliação:
Estados Unidos