Analysis of c-kit receptor dimerization by fluorescence resonance energy transfer.
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.
Broudy
V C
VC
Department of Medicine, University of Washington, Seattle 98195-7710, USA.
Lin
N L
NL
Bühring
H J
HJ
Komatsu
N
N
Kavanagh
T J
TJ
D002232:Carbocyanines; D002455:Cell Division; D002460:Cell Line; D016207:Cytokines / Q000494:pharmacology; D019281:Dimerization; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006412:Hematopoietic Stem Cells / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D018613:Microscopy, Confocal; D019009:Proto-Oncogene Proteins c-kit / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D019089:Stem Cell Factor / Q000378:metabolism / Q000494:pharmacology
D002232:Carbocyanines; D016207:Cytokines; D005456:Fluorescent Dyes; D019089:Stem Cell Factor; C089187:cyanine dye 3; D019009:Proto-Oncogene Proteins c-kit; D016650:Fluorescein-5-isothiocyanate