Independent and Cooperative Roles of Adaptor Molecules in Proximal Signaling during FcεRI-Mediated Mast Cell Activation

Mast cell activation during allergic inflammation is mediated by the high-affinity immunoglobulin E (IgE)-binding receptor FcɛRI. Cross-linking of FcɛRI on mast cells by IgE/cognate antigen complexes results in the rapid release of a wide array of inflammatory mediators, including vasoactive amines and cytokines/chemokines that give rise to allergic symptoms, ranging in severity from simple urticaria to anaphylactic shock and death (14). As allergy affects ∼30% of the population in developed countries (13), much attention has been placed on studying the signal transduction mechanisms involved in mast cell activation downstream of FcɛRI in hopes of finding novel targets for therapeutic intervention. Signal transduction downstream of FcɛRI is initiated by the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) contained in the signaling components (β and γ chains) of the FcɛRI complex (30, 37). Once phosphorylated, these chains serve as docking sites for several protein tyrosine kinases (PTKs), including Lyn and spleen tyrosine kinase (Syk) (9, 19, 34). Recruitment of Syk to the membrane by FcɛRI results in the phosphorylation of scaffold proteins known as adaptor molecules. Adaptor proteins lack enzymatic activity but instead contain protein-binding domains that are critical for the formation of a multimolecular complex, which orchestrates downstream signaling in a temporal and spatial manner. The adaptor molecules Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) and linker of activated T cells 1 (LAT1) organize the assembly of a proximal signaling complex downstream of FcɛRI. Failure to form this complex is detrimental to FcɛRI-mediated mast cell function, as demonstrated by the finding that both SLP-76-deficient (22, 29, 41) and LAT1-deficient (25, 31, 32) mast cells display severely diminished degranulation and cytokine/chemokine production following FcɛRI ligation. Similar proximal signaling complexes are formed downstream of several different ITAM-containing receptors. Much of our understanding of the role of adaptor molecules in signal transduction has come from identification of phosphoproteins during T-cell receptor (TCR)-mediated activation of the human Jurkat T-cell line (1, 33). These studies eventually led to a paradigm describing the sequence of events in the formation of the SLP-76/LAT1 signaling complex. According to this model, SLP-76 is found constitutively bound to Grb2-related adaptor downstream of Shc (GADS) (24) and resides in the cytosol. Upon TCR activation, the tyrosines of membrane-resident LAT1 are phosphorylated and become attachment sites for proteins such as phospholipase Cγ (PLCγ) and GADS (43, 45). SLP-76 is drawn to the membrane through a GADS/LAT1 interaction, which then permits Syk family PTKs to maximally phosphorylate the N-terminal tyrosines of SLP-76 (5, 10). Several lines of evidence support this model whereby a LAT1/SLP-76 module organizes TCR signaling. First, both SLP-76- and LAT1-deficient Jurkat T cells display similar biochemical defects, such as diminished PLCγ and extracellular signal-regulated kinase (ERK) activation (10, 42). Second, T cells in SLP-76−/− and LAT1−/− mice are blocked at the same stage of development (7, 44). Third, SLP-76 can be coimmunoprecipitated with LAT1 but not with LAT1 harboring tyrosine-to-phenylalanine mutations (45). Finally, expression of a fusion protein comprised of the membrane-localizing domain of LAT1 and SLP-76 that forces localization of SLP-76 to the plasma membrane rescues the TCR-induced functional defects of both SLP-76- and LAT1-deficient Jurkat T cells (3). This model implies a mutually dependent relationship between SLP-76 and LAT1, where SLP-76 and LAT1 rely on each other to carry out their roles. One might suspect that this model for LAT1/SLP-76 function would operate in all other cells that utilize these adaptor molecules for ITAM-containing receptor-mediated signaling. However, the published defects of LAT1-deficient mast cells in FcɛRI-mediated signaling appeared milder than those of SLP-76-deficient mast cells, although a direct comparison has never been reported. In the present study, we show that LAT1-deficient mast cells display distinct functional and biochemical defects compared to SLP-76-deficient mast cells, implying that unlike in T cells, SLP-76 may not depend entirely on LAT1 for its function in mast cells. Surprisingly, the membrane recruitment and phosphorylation of SLP-76 were also preserved in LAT1−/− mast cells. We show that LAT2 (also known as non-T-cell activation linker [NTAL] or linker for activation of B cells [LAB]), which is not expressed in naive T cells but is expressed in mast cells (15), is responsible for phosphorylation and plasma membrane recruitment of SLP-76 in the absence of LAT1. However, LAT2 cannot support all LAT1/SLP-76-associated functions, such as sustained Ca2+ flux, likely due to decreased stability of the LAT2/SLP-76/PLCγ complex. Comparison of SLP-76−/− LAT1−/− and SLP-76−/− mast cells also revealed that some functions of LAT1 could occur independently of SLP-76. We propose that although SLP-76 and LAT1 are interdependent for many of their functions, LAT2/SLP-76 interactions and SLP-76-independent LAT1 functions mediate positive signaling downstream of FcɛRI in mast cells.