Compartmentalization of the Type I Fcepsilon receptor and MAFA on mast cell membranes.
The Mast cell Function- associated Antigen (MAFA) is a membrane glycoprotein on rat mast cells (RBL-2H3) expressed at a ratio of approximately 1:30 with respect to the Type I Fcepsilon receptor (FcepsilonRI). Despite this stoichiometry, clustering MAFA by its specific mAb G63 substantially inhibits secretion of both granular and de novo synthesized mediators induced upon FcepsilonRI aggregation. Since the FcepsilonRIs apparently signal from within raft micro-environments, we investigated possible co-localization of MAFA within these membrane compartments containing aggregated FcepsilonRI. We used cholera toxin B subunit ( CTB) to cluster the raft component ganglioside GM1 and studied the effects of this perturbation on rotation of FcepsilonRI and MAFA by time-resolved phosphorescence anisotropy of erythrosin-conjugated probes. CTB treatment would be expected to substantially inhibit rotation of raft-associated molecules. Experimentally, CTB has no effect on rotational parameters such as the long-time anisotropy (r(infinity)) of unperturbed FcepsilonRI or MAFA. However, on cells where FcepsilonRI-IgE has previously been clustered by antigen (DNP(14)-BSA), CTB treatment increases the FcepsilonRI-IgE's r(infinity) by 0.010 and MAFA's by 0.014. Similarly, CTB treatment of cells where MAFA had been clustered by mAb G63 increases MAFA's r(infinity) by 0.010 but leaves FcepsilonRI's unaffected. Evaluation of raft localization of FcepsilonRI and MAFA using sucrose gradient ultracentrifugation of Triton X-100 treated membrane fragments demonstrates that a significant fraction of MAFA molecules sediments with rafts when FcepsilonRI is clustered by antigen or when MAFA itself is clustered by mAb G63. The large excess of FcepsilonRI over MAFA explains why clustering MAFA does not substantively affect FcepsilonRI dynamics. Moreover, in single-particle tracking studies of individual FcepsilonRI-IgE or MAFA molecules, these proteins, upon clustering by antigen, move into small membrane compartments of reduced, but similar, dimensions. This provides additional indication of constitutive interactions between FcepsilonRI and MAFA. Taken together, these results of distinct methodologies suggest that MAFA functions within raft microdomains of the RBL-2H3 cell membrane and thus in close proximity to the FcepsilonRI which themselves signal from within the raft environment.[1]References
- Compartmentalization of the Type I Fcepsilon receptor and MAFA on mast cell membranes. Barisas, B.G., Smith, S.M., Liu, J., Song, J., Hagen, G.M., Pecht, I., Roess, D.A. Biophys. Chem. (2007) [Pubmed]
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