Lipid-based, protein-based, and steric interactions synergize to facilitate transmembrane signaling stimulated by antigen-clustering of IgE receptors

Abstract

Antigen (Ag) crosslinking of immunoglobulin E-receptor (IgE-Fc{varepsilon}RI) complexes in mast cells stimulates transmembrane (TM) signaling, requiring phosphorylation of the clustered Fc{varepsilon}RI by lipid-anchored Lyn tyrosine kinase. Previous studies showed that this stimulated coupling between Lyn and Fc{varepsilon}RI occurs in liquid ordered (Lo)-like nanodomains of the plasma membrane and that Lyn binds directly to cytosolic segments of Fc{varepsilon}RI that it initially phosphorylates for amplified activity. Net phosphorylation above a non-functional threshold is achieved in the stimulated state, but not in the resting state, and current evidence supports the hypothesis that this relies on disruption by Ag-crosslinking of a balance between Lyn and tyrosine phosphatase activities. However, the structural interactions that underlie the stimulation process remain poorly defined. This study evaluates the relative contributions and functional importance of different types of interactions leading to supra-threshold phosphorylation of Ag-crosslinked IgE-Fc{varepsilon}RI in live rat basophilic leukemia (RBL) mast cells. Our high-precision diffusion measurements by Imaging Fluorescence Correlation Spectroscopy (ImFCS) on multiple structural variants of Lyn and other lipid-anchored probes confirm subtle, stimulated stabilization of the Lo-like nanodomains and concomitant sharpening of segregation from liquid-disordered (Ld)-like regions. With other structural variants we determine that lipid-based interactions are essential for access by Lyn leading to phosphorylation of and protein-based binding to clustered Fc{varepsilon}RI. By contrast, TM tyrosine phosphatase, PTP, is excluded from these regions by steric repulsion of TM segments and preference for Ld-like regions. Overall, we establish a synergy of lipid-based, protein-based, and steric interactions underlying functional TM signaling in mast cells. SIGNIFICANCE STATEMENTLipid organization of the plasma membrane is known to be important for facilitating protein interactions in transmembrane signaling. However, the orchestration of these interactions in live cells has been elusive. We employed ImFCS to systemically investigate the interplay of lipids and proteins during signaling in mast cells, initiated as phosphorylation of Ag-crosslinked IgE-Fc{varepsilon}RI by lipid-anchored Lyn kinase. We find lipid-based interactions are first required for protein-based phosphorylation of the clustered Fc{varepsilon}RI within Lo-like nanodomains. Transmembrane phosphatases must be excluded from these regions, and we find this is mediated by their preference for Ld-like regions and by steric exclusion from the clustered Fc{varepsilon}RI proteins. ImFCS provides quantitative characterization of the functional link between features of plasma membrane organization and transmembrane signaling.