Compartmentalization of the Type I Fc epsilon 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 Fc epsilon receptor (Fc epsilon RI). Despite this stoichiometry, clustering MAFA by its specific mAb G63 substantially inhibits secretion of both granular and de novo synthesized mediators induced upon Fc epsilon RI aggregation. Since the Fc epsilon RIs apparently signal from within raft micro-environments, we investigated possible co-localization of MAFA within these membrane compartments containing aggregated Fc epsilon RI. We used cholera toxin B subunit (CTB) to cluster the raft component ganglioside GM1 and studied the effects of this perturbation on rotation of Fc epsilon RI 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 Fc epsilon RI or MAFA. However, on cells where Fc epsilon RI-IgE has previously been clustered by antigen (DNP(14)-BSA), CTB treatment increases the Fc epsilon RI-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 Fc epsilon RI's unaffected. Evaluation of raft localization of Fc epsilon RI 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 Fc epsilon RI is clustered by antigen or when MAFA itself is clustered by mAb G63. The large excess of Fc epsilon RI over MAFA explains why clustering MAFA does not substantively affect Fc epsilon RI dynamics. Moreover, in single-particle tracking studies of individual Fc epsilon RI-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 Fc epsilon RI 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 Fc epsilon RI which themselves signal from within the raft environment.
Barisas
B George
BG
Department of Chemistry, Colorado State University, Fort Collins, CO 80523, USA. barisas@lamar.colostate.edu
Smith
Steven M
SM
Liu
Jingjing
J
Song
Jinming
J
Hagen
Guy M
GM
Pecht
Israel
I
Roess
Deborah A
DA
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects; D002772:Cholera Toxin / Q000494:pharmacology; D037181:Lectins, C-Type / Q000032:analysis* / Q000378:metabolism*; D008407:Mast Cells / Q000737:chemistry / Q000187:drug effects / Q000276:immunology*; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000378:metabolism*; D021962:Membrane Microdomains / Q000737:chemistry* / Q000187:drug effects; D051381:Rats; D017455:Receptors, IgE / Q000032:analysis* / Q000378:metabolism*
C094696:Klrg1 protein, rat; D037181:Lectins, C-Type; D008562:Membrane Glycoproteins; D017455:Receptors, IgE; D002772:Cholera Toxin