Compartmentalized Activation of the High Affinity Immunoglobulin E Receptor within Membrane Domains

Abstract

The earliest known step in the activation of the high affinity IgE receptor, Fc⑀RI, is the tyrosine phosphorylation of its ␤ and ␥ subunits by the Src family tyrosine kinase, Lyn. We report here that aggregation-dependent association of Fc⑀RI with specialized regions of the plasma membrane precedes its tyrosine phosphorylation and appears necessary for this event. Tyrosine phosphorylation of ␤ and ␥ occurs in intact cells only for Fc⑀RI that associate with these detergent-resistant membrane domains, which are enriched in active Lyn. Furthermore, efficient in vitro tyrosine phosphorylation of Fc⑀RI subunits occurs only for those associated with isolated domains. This association and in vitro phosphorylation are highly sensitive to low concentrations of detergent, suggesting that lipid-mediated interactions with Lyn are important in Fc⑀RI activation. Participation of membrane domains accounts for previously unexplained aspects of Fc⑀RI-mediated signaling and may be relevant to signaling by other multichain immune receptors.The plasma membrane contains specialized regions that have distinct compositions and can serve unique functions in the regulation of cell surface receptor activation. For example, caveolae have been shown to associate with certain signaling proteins (1, 2) and have been implicated in receptor activation (3-6), vesicular transport (7,8), and the uptake of small molecules (9). Compositionally related membrane domains, which lack the invaginated morphology of caveolae as well as the membrane protein caveolin, have also been identified and biochemically separated from caveolae (10). These membrane domains, like caveolae, are resistant to solubilization in nonionic detergents such as Triton X-100, are enriched in sphingolipids and glycosylphosphatidylinositol-linked proteins, and are associated with palmitoyl-anchored signaling molecules including Src family tyrosine kinases (10 -14). Detergent-resistant membrane domains isolated from rat basophilic leukemia (RBL) 1 cells, a mast cell line, contain at least 30% of the cellular Lyn, a Src family tyrosine kinase, and no detectable caveolin (15).Aggregation of Fc⑀RI on mast cells and basophils by multivalent antigens leads to phosphorylation of immunoreceptor tyrosine-based activation motifs within the ␤ and ␥ receptor subunits by . This initiates a signaling cascade culminating in secretion of inflammatory mediators and cytokines that play an important role in the allergic response (20, 21). The molecular mechanism by which aggregation of Fc⑀RI initiates its phosphorylation by Lyn is incompletely understood. Selective binding of Lyn directly to unphosphorylated Fc⑀RI ␤ (22) has been proposed to mediate an initial transphosphorylation of aggregated Fc⑀RI (23), but this does not account for the capacity of Fc⑀RI lacking the ␤ subunit (24, 25) or chimeric receptors containing only the ␥ cytoplasmic tail (26 -28) to become tyrosine-phosphorylated upon aggregation. The involvement of detergent-resistant membrane domains in Fc⑀RI signaling was recently sugge...

Signaling assemblies formed in mast cells activated via Fcϵ receptor I dimers

“…Such high-density complexes were also observed in cells solubilized with 0.5% Triton X-100, i.e. after treatment that prevented the association of aggregated Fc 4 RI with DRM (not shown), suggesting that they represent complexes of Fc 4 RI with the cytoskeleton [5,10]. Pretreatment of the IgE-sensitized cells for 40 min with 10 mM methyl-g -cyclodextrin (MBCD) before adding anti-IgE Ab or anti-Fc 4 RI mAb P Fig.…”

“…To test how the extent of aggregation of Fc 4 RI correlates with association of the receptors with DRM, cells were exposed to 125 I-labeled 5.14 or H10 mAb for 5 min and solubilized in ice-cold 0.06% Triton X-100; this solubilization procedure preserves the association of aggregated Fc 4 RI with DRM [5,6]. In control cells exposed to 125 I-IgE instead of anti-Fc 4 RI Ab, most of the 125 I-IgEFc 4 RI complexes (94±8%; mean ± SD, n=4) were found in the high-density fractions of the sucrose gradient (fractions [11][12][13][14][15][16][17][18][19][20][21][22], and only a small portion (5.8±2.2%) was found in the low-density fractions (fractions 1-10; Fig.…”

“…Further analysis indicated that Fc 4 RI was only weakly associated with DRM in resting cells and that this association increased upon Fc 4 RI aggregation [5]. Confocal microscopy with green fluorescence-labeled cytoplasmic probes proved that aggregated Fc 4 RI colocalized with Lyn and Syk [6,7].…”

Topography of plasma membrane microdomains and its consequences for mast cell signaling

“…Biochemical studies with detergent-solubilized cells implied that FceRI-mediated activation is initiated by coalescence of aggregated FceRI with DRM [10,12] and that F-actin is involved in this process [19,20]. However, EM observations failed to discern any significant increase in association between Thy-1 and aggregated FceRI [12,15].…”

“…Biochemical studies of isolated detergent-resistant membranes (DRM) showed that they are rich not only in GPI-anchored proteins, but also in palmitoylated Src family protein tyrosine kinases [6,7] and palmitoylated transmembrane adaptor proteins, including linker for activation of T cells (LAT) [8] and non-T cell activation linker (NTAL) [9]. Importantly, in nonactivated mast cells, the FceRI is excluded from DRM, whereas in FceRI-activated cells most of the receptor is associated with DRM in a cholesterol-dependent manner [10][11][12]. These findings suggested that FceRI-mediated activation is initiated through association of the aggregated FceRI with membrane microdomains possessing signal transduction machinery.…”