Critical role for cholesterol in Lyn-mediated tyrosine phosphorylation of Fc epsilon RI and their association with detergent-resistant membranes

Tyrosine phosphorylation of the high affinity immunoglobulin (Ig)E receptor (Fc epsilon RI) by the Src family kinase Lyn is the first known biochemica l step that occurs during activation of mast cells and basophils after cros s-linking of Fc epsilon RI by antigen. The hypothesis that specialized regi ons in the plasma membrane, enriched in sphingolipids and cholesterol, faci litate the coupling of Lyn and Fc epsilon RI was tested by investigating fu nctional and structural effects of cholesterol depletion on Lyn/Fc epsilon RI interactions. We find that cholesterol depletion with methyl-beta-cyclod extrin substantially reduces stimulated tyrosine phosphorylation of Fc epsi lon RI and other proteins while enhancing more downstream events that lead to stimulated exocytosis, In parallel to its inhibition of tyrosine phospho rylation, cholesterol depletion disrupts the interactions of aggregated Fc epsilon RI and Lyn on intact cells and also disrupts those interactions wit h detergent-resistant membranes that are isolated by sucrose gradient ultra centrifugation of lysed cells. Importantly, cholesterol repletion restores receptor phosphorylation together with the structural interactions. These r esults provide strong evidence that membrane structure, maintained by chole sterol, plays a critical role in the initiation of Fc epsilon RI signaling.