Compartmentalization of the Type I Fcε receptor and MAFA on mast cell membranes

Abstract

The Mast cell Function-associated Antigen (MAFA) is a membrane glycoprotein on rat mast cells (RBL-2H3) expressed at a ratio of ∼ 1:30 with respect to the Type I Fcε receptor (FcεRI). Despite this stoichiometry, clustering MAFA by its specific mAb G63 substantially inhibits secretion of both granular and de novo synthesized mediators induced upon FcεRI aggregation. Since the FcεRIs apparently signal from within raft micro-environments, we investigated possible co-localization of MAFA within these membrane compartments containing aggregated FcεRI. We used cholera toxin B subunit (CTB) to cluster the raft component ganglioside GM1 and studied the effects of this perturbation on rotation of FcεRI 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 ∞) of unperturbed FcεRI or MAFA. However, on cells where FcεRI-IgE has previously been clustered by antigen (DNP 14-BSA), CTB treatment increases the FcεRI-IgE's r ∞ 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 ∞ by 0.010 but leaves FcεRI's unaffected. Evaluation of raft localization of FcεRI 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 FcεRI is clustered by antigen or when MAFA itself is clustered by mAb G63. The large excess of FcεRI over MAFA explains why clustering MAFA does not substantively affect FcεRI dynamics. Moreover, in single-particle tracking studies of individual FcεRI-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 FcεRI 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 FcεRI which themselves signal from within the raft environment.