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Analysis of c-kit Receptor Dimerization by Fluorescence
Resonance Energy Transfer
Virginia C. Broudy,
Nancy L. Lin,
Hans-Jörg Bühring,
Norio Komatsu, and
Terrance J. Kavanagh
From the Departments of Medicine and Environmental Health, University
of Washington, Seattle; the Department of Medicine, University of
Tübingen, Tübingen, Germany; and the Department of
Medicine, Jichi Medical School, Tochigi, Japan.
Stem cell factor (SCF) binding to the c-kit receptor
triggers homodimerization and intermolecular tyrosine phosphorylation of the c-kit receptor, thus initiating signal transduction.
Receptor dimerization is a critical early step in this process. Prior
biochemical studies of c-kit receptor dimerization have mainly
used affinity cross-linking techniques, which are beset with problems
including low efficiency of cross-linking and the usual requirement for radiolabeled SCF to detect the cross-linked complex. We used the fluorescence resonance energy transfer (FRET) technique to examine the
effects of SCF and other hematopoietic cytokines on c-kit receptor dimerization. The nonneutralizing anti-c-kit receptor monoclonal antibody 104D2 was directly conjugated to fluorescein isothiocyanate (FITC) or to the carbocyanine dye Cy3 and used to label
cytokine-responsive human hematopoietic cell lines. The ability of SCF
to induce c-kit receptor dimerization was assessed by flow
cytometric analysis of FRET between the donor fluorochrome FITC and the
acceptor fluorochrome Cy3. SCF induced a dose-dependent increase in
c-kit receptor dimerization that correlated well with the
concentrations of SCF required to stimulate cell proliferation. Receptor dimerization was detectable within 3 minutes after the addition of SCF and was maximal 30 minutes after the addition of SCF.
Confocal microscopy showed redistribution of the c-kit receptor
(from a diffuse distribution on the cell surface to "caps" at one
end of the cell) within 3 minutes after SCF addition, followed by
receptor internalization. Reappearance of the c-kit receptor on
the cell surface required new protein synthesis, suggesting that the
c-kit receptor is not recycled to the cell surface after internalization. Finally, erythropoietin (Epo), but not the
structurally and functionally related cytokine thrombopoietin (Tpo),
stimulated c-kit receptor dimerization detectable by FRET, and
tyrosine phosphorylation of the c-kit receptor. These results
suggest that exposure to Epo can activate the c-kit receptor
and provide further evidence for cross-talk between the Epo and
c-kit receptors in human hematopoietic cell lines. Studies with
progeny of burst-forming unit-erythroid (BFU-E) suggest
that the FRET technique is sufficiently sensitive to detect
c-kit receptor dimerization on normal human hematopoietic cells.
Blood, Vol. 91 No. 3 (February 1), 1998:
pp. 898-906
© 1998 by The American Society of Hematology.
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