ANALYSIS OF C-KIT RECEPTOR DIMERIZATION BY FLUORESCENCE RESONANCE ENERGY-TRANSFER

Stem cell factor (SCF) binding to the c-kit receptor triggers homodime rization and intermolecular tyrosine phosphorylation of the c-kit rece ptor, thus initiating signal transduction, Receptor dimerization is a critical early step in this process. Prior biochemical studies of c-ki f receptor dimerization have mainly used affinity cross-linking techni ques, which are beset with problems including low efficiency of cross- linking and the usual requirement for radiolabeled SCF to detect the c ross-linked complex. We used the fluorescence resonance energy transfe r (FRET) technique to examine the effects of SCF and other hematopoiet ic cytokines on c-kit receptor dimerization. The nonneutralizing anti- c-kit receptor monoclonal antibody 104D2 was directly conjugated to fl uorescein isothiocyanate (FITC) or to the carbocyanine dye Cy3 and use d to label cytokine-responsive human hematopoietic cell lines. The abi lity of SCF to induce c-kit receptor dimerization was assessed by flow cytometric analysis of FRET between the donor fluorochrome FITC and t he acceptor fluorochrome Cy3. SCF induced a dose-dependent increase in c-kit receptor dimerization that correlated well with the concentrati ons of SCF required to stimulate cell proliferation, Receptor dimeriza tion was detectable within 3 minutes after the addition of SCF and was maximal 30 minutes after the addition of SCF. Confocal microscopy sho wed redistribution of the c-hit receptor (from a diffuse distribution on the cell surface to ''caps'' at one end of the cell) within 3 minut es after SCF addition, followed by receptor internalization. Reappeara nce of the c-kit receptor on the cell surface required new protein syn thesis, 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 (Tp o), stimulated c-kit receptor dimerization detectable by FRET, and tyr osine phosphorylation of the c-kif receptor, These results suggest tha t exposure to Epo can activate the c-kit receptor and provide further evidence for cross-talk between the Epo and c-kif receptors in human h ematopoietic cell lines, Studies with progeny of burst-forming unit-er ythroid (BFU-E) suggest that the FRET technique is sufficiently sensit ive to detect c-kit receptor dimerization on normal human hematopoieti c cells. (C) 1998 by The American Society of Hematology.