Proximity relationships between the type I receptor for Fc epsilon (Fc epsilon RI) and the mast cell function-associated antigen (MAFA) studied by donor photobleaching fluorescence resonance energy transfer microscopy.
Clustering of the mast cell function-associated antigen (MAFA) on the surface of rat mucosal type mast cells line 2H3 (RBL-2H3) leads to suppression of the secretory response induced by the type I Fc epsilon receptor (Fc epsilon RI). In order to establish a possible association between MAFA and Fc epsilon RI we measured fluorescence resonance energy transfer (FRET) between the MAFA-specific monoclonal antibody (mAb) G63 and Fc epsilon RI-bound ligands as well as between Fc epsilon RI-bound ligands themselves using the donor photobleaching FRET (pbFRET) technique. Average FRET efficiencies between 6 and 9% were determined after low-temperature incubation with fluorescent dye conjugated mAb G63 bound to MAFA (donor) and IgE bound to Fc epsilon RI (acceptor) on RBL-2H3 cells. Subsequent cross-linking of IgE by a polyvalent antigen caused no change in FRET efficiencies. These results suggest that the MAFA is located in the vicinity of the Fc epsilon RI on resting cells, and that clustering of the Fc epsilon RI leads to no significant change in the proximity of the two molecular species. In view of the sequence motif identified in the cytosolic tail of the MAFA and the observed changes in its phosphorylation upon antigen stimulation (Guthmann et al., Proc. Natl. Acad. Sci. USA 1995, 92: 9397-9401), the present study suggests that the secretory response inhibition by MAFA interferes with the signal transduction cascade initiated via the Fc epsilon RI. An additional finding was that clustering of the Fc epsilon RI by antigen showed a clear increase in the efficiency of FRET between Fc epsilon RI-bound IgE molecules conjugated with fluorescent donor and acceptor.
Jürgens
L
L
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Arndt-Jovin
D
D
Pecht
I
I
Jovin
T M
TM
D000818:Animals; D002460:Cell Line; D007073:Immunoglobulin E / Q000737:chemistry; D037102:Lectins / Q000737:chemistry*; D037181:Lectins, C-Type; D008407:Mast Cells / Q000737:chemistry*; D008562:Membrane Glycoproteins / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D051381:Rats; D017455:Receptors, IgE / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
C094696:Klrg1 protein, rat; D037102:Lectins; D037181:Lectins, C-Type; D008562:Membrane Glycoproteins; D017455:Receptors, IgE; D007073:Immunoglobulin E