Fc epsilon RI-mediated recruitment of p53/56lyn to detergent-resistant membrane domains accompanies cellular signaling.
Detergent-resistant plasma membrane structures, such as caveolae, have been implicated in signalling, transport, and vesicle trafficking functions. Using sucrose gradient ultracentrifugation, we have isolated low-density, Triton X-100-insoluble membrane domains from RBL-2H3 mucosal mast cells that contain several markers common to caveolae, including a src-family tyrosine kinase, p53/56lyn. Aggregation of Fc epsilon RI, the high-affinity IgE receptor, causes a significant increase in the amount of p53/56lyn associated with these low-density membrane domains. Under our standard conditions for lysis, IgE-Fc epsilon RI fractionates with the majority of the solubilized proteins, whereas aggregated receptor complexes are found at a higher density in the gradient. Stimulated translocation of p53/56lyn is accompanied by increased tyrosine phosphorylation of several proteins in the low-density membrane domains as well as enhanced in vitro tyrosine kinase activity toward these proteins and an exogenous substrate. With a lower detergent-to-cell ratio during lysis, significant Fc epsilon RI remains associated with these membrane domains, consistent with the ability to coimmunoprecipitate tyrosine kinase activity with Fc epsilon RI under similar lysis conditions [Pribluda, V. S., Pribluda, C. & Metzger, H. (1994) Proc. Natl. Acad. Sci. USA 91, 11246-11250]. These results indicate that specialized membrane domains may be directly involved in the coupling of receptor aggregation to the activation of signaling events.
Field
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Department of Chemistry, Cornell University, Ithaca, NY 14853, USA.
Holowka
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Baird
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D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000276:immunology; D002499:Centrifugation, Density Gradient; D003902:Detergents / Q000494:pharmacology*; D004351:Drug Resistance; D015151:Immunoblotting; D015471:Leukemia, Basophilic, Acute; D019000:Phosphotyrosine / Q000032:analysis; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000378:metabolism*; D051381:Rats; D017455:Receptors, IgE / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D015398:Signal Transduction / Q000276:immunology*; D014407:Tumor Cells, Cultured; D019061:src-Family Kinases / Q000378:metabolism*
D003902:Detergents; D017455:Receptors, IgE; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases