Interaction of aggregated native and mutant IgE receptors with the cellular skeleton.

Abstract

When aggregated, cell surface proteins become resistant to solubilization by detergents, presumably because of aggregation-induced or -stabilized interactions between the membrane protein and the cytoskeleton or plasma membrane skeleton. We genetically engineered variants of the tetrameric high-affinity receptor for IgE (Fc6RI) to identify a site on its a, (3, or y chains that mediates such putative interactions. Using flow cytofluorometry, we studied rat basophilic leukemia cells, transiently transfected COS cells, and stably transfected P815 cells bearing wild-type and mutated receptors. We observed that (i) solubilization was markedly dependent on the degree of aggregation, the extent varying somewhat with the cell type and, particularly at lower levels of aggregation, with the time after addition of detergent; (iu) truncation of no single cytoplasmic domain of the a, 13, or y chains ablated the insolubilization effect; and (ii) incomplete receptors were also efficiently insolubilized by aggregation. Thus receptors consisting only of a and y chains, a "receptor" consisting of only the ectodomain of the a chain attached to the plasma membrane by a glycosyl-phosphatidyl inositol anchor, and "receptors" consisting only of minimally modified Y chains were resistant to solubilization after aggregation. We conclude that no unique subunit or domain of FcRI mediates the insolubilization phenomenon. Our results support a model in which the bridging of membrane proteins leads to their becoming nonspecifically enmeshed in a network of membrane skeletal proteins on either the outside and/or the inside of the membrane so that dissolution of the lipid bilayer becomes irrelevant.Aggregation of a variety of membrane proteins leads to changes in their topological properties: their translational and rotational mobility is markedly decreased, they become segregated into clusters ("patching" and "capping"), they may be internalized by coated pits or by an alternative mechanism, and they become resistant to solubilization by detergents that dissolve the lipid bilayer in which the proteins are embedded. Most workers have plausibly postulated that these phenomena are related to interactions between the aggregated protein and other cellular components such as those that constitute the cytoskeleton or plasma membrane skeleton.The receptor that binds IgE with high affinity (Fc6RI) has been widely studied with respect to these phenomena because of the relative ease with which its aggregation can be systematically altered and the changes in its properties can be monitored (1-3). The receptor is known to consist of three transmembrane peptides (one a chain and two y chains) and a single 83 chain with four transmembrane domains and substantial N-terminal and C-terminal cytoplasmic domains (4). However, as yet no specific regions of this tetrameric (a, 13, y2) receptor have been implicated as mediating the interactions that lead to the changes in the receptor's topological characteristics, nor have the cellular components with whic...

Divalency of the monoclonal antibody 5-1-6 is required for induction of proteinuria in rats

“…element sueh as microfilamcnls may be responsible for movcmenl of receptors inlo patches ami caps 12O.2I|. However, such movement has recently been reported lo be associated wilh lhe interaction of reeeptors, and one of the components of the membrane skeleton rather than with the cytoskcleton [6,22]. It is possible that in our model, the critical signalling event initiating the increase of glomerular pcrmcabilily occurs during cross-linking and aggregation ofthe epitopes.…”

Transmembrane signaling by the high-affinity IgE receptor on membrane preparations.

“…The final concentrations were 10 mM CHAPS, 2 mM lipids, 0.5 mM Na3VO4, 5 mM Na4P207, 5 mM EDTA, and the same protein inhibitors as noted above. The samples were left on ice for a minimum of 30 min (10). The cell specimens and the first sonicate were centrifuged for 1 min at 14,000 x g at 40C and 0.5 ml of supernatant was withdrawn for analysis; for the centrifuged sonicate and subsequent membrane fractions, this centrifugation step was omitted.…”

“…The principal reagents used in these studies have been described (10 (GIBCO/BRL) and containing 32p (200-500 ,uCi/ml; 1 Ci = 37 GBq) before trypsin treatment.…”

“…Just before sonication, protease inhibitors were added to yield final concentrations of aprotinin (200 kallikrein inhibitor units/ml), leupeptin (10 ,ug/mI), and pepstatin A (10 ug/ml). The cell disrupter (model W-225R; Heat Systems/Ultrasonic) was used at maximal output, approximately three times for 30 sec each.…”

Inhibition of Antigen-Induced Mediator Release from IgE-Sensitized Cells by a Monoclonal Anti-FcεRI α-Chain Receptor Antibody: Implications for the Involvement of the Membrane-Proximal α-Chain Region in FcεRI-Mediated Cell Activation

“…It is known that receptor aggregation results in an increased association of Fc⑀RI with membrane skeletal proteins (32), and it has been suggested that lipid-mediated interactions between Fc⑀RI and the plasma membrane occur before tyrosine phosphorylation (33). Evidence is accumulating that initiation of Fc⑀RI signaling depends on how Fc⑀RI and tyrosine kinase Lyn interact with cholesteroland sphingolipid-rich regions within the plasma membrane (34).…”