Fluorescent-labeled growth factor molecules serve as probes for receptor binding and endocytosis

Kermit L Carraway, Richard A. Cerione

Research output: Contribution to journalArticle

36 Scopus citations

Abstract

In this report, we describe the applicability of fluorescein-labeled EGF (FITC-EGF) in monitoring the interaction between EGF and its cellular receptor in real time. This work takes advantage of previous studies that demonstrated that EGF may be labeled at its amino terminus with FITC without significant deleterious effects on the binding of the growth factor to its receptor or on the ability of the growth factor to activate the intrinsic tyrosine kinase activity of the receptor. When suspended human epidermoid carcinoma (A431) cells were treated with FITC-EGF, a biphasic quenching of the FITC fluorescence was observed. Both phases were blocked when the experiments were performed in the presence of excess unlabeled EGF. The first phase, in which the FITC emission was quenched by 8-10%, was complete within 30 s. This rapid quenching was attributed to changes in the rotational mobility of the EGF molecule that accompany its binding to receptors. The slower phase required 20-30 min for completion and resulted in the further quenching of the FITC fluorescence by 30-40%. This slower phase appeared to reflect the internalization of the receptor and its routing to acidic intracellular compartments. The rapid fluorescence decay phase was used to determine the rate constants (kon and koff) for the interaction of FITC-EGF with receptors on the surface of cells.

Original languageEnglish (US)
Pages (from-to)12039-12045
Number of pages7
JournalBiochemistry
Volume32
Issue number45
StatePublished - 1993
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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