Abstract
Protein 4.1R, a member of the 4.1 family, functions as a bridge between cytoskeletal and plasma membrane proteins. It is expressed in T cells, where it binds to a linker for activation of T cell (LAT) family member 1 and inhibits its phosphorylation and downstream signaling events after T cell receptor triggering. The role of the 4.1R protein in cell activation through other immunoreceptors is not known. In this study, we used 4.1R-deficient (4.1R-KO) and 4.1R wild-type (WT) mice and explored the role of the 4.1R protein in the high-affinity IgE receptor (FcεRI) signaling in mast cells. We found that bone marrow mast cells (BMMCs) derived from 4.1R-KO mice showed normal growth in vitro and expressed FcεRI and c-KIT at levels comparable to WT cells. However, 4.1R-KO cells exhibited reduced antigen-induced degranulation, calcium response, and secretion of tumor necrosis factor-α. Chemotaxis toward antigen and stem cell factor (SCF) and spreading on fibronectin were also reduced in 4.1R-KO BMMCs, whereas prostaglandin E2-mediated chemotaxis was not affected. Antibody-induced aggregation of tetraspanin CD9 inhibited chemotaxis toward antigen in WT but not 4.1R-KO BMMCs, implying a CD9-4.1R protein cross-talk. Further studies documented that in the absence of 4.1R, antigen-mediated phosphorylation of FcεRI β and γ subunits was not affected, but phosphorylation of SYK and subsequent signaling events such as phosphorylation of LAT1, phospholipase Cγ1, phosphatases (SHP1 and SHIP), MAP family kinases (p38, ERK, JNK), STAT5, CBL, and mTOR were reduced. Immunoprecipitation studies showed the presence of both LAT1 and LAT2 (LAT, family member 2) in 4.1R immunocomplexes. The positive regulatory role of 4.1R protein in FcεRI-triggered activation was supported by in vivo experiments in which 4.1R-KO mice showed the normal presence of mast cells in the ears and peritoneum, but exhibited impaired passive cutaneous anaphylaxis. The combined data indicate that the 4.1R protein functions as a positive regulator in the early activation events after FcεRI triggering in mast cells.
Highlights
Cells of the immune system, to other cell types, communicate with their environment via a plethora of surface receptors recognizing various soluble or membrane-bound ligands
The extent of degranulation was evaluated by measurement of enzymatic activity of β-glucuronidase released into the extracellular space
Lower degranulation was observed in 4.1R-KO cells activated with various concentrations of thapsigargin (0.25– 1.0 μM; Figure 1G), which induces the release of Ca2+ from intracellular stores by inhibiting the endoplasmic reticulum (ER) Ca2+-ATPase [49]
Summary
Cells of the immune system, to other cell types, communicate with their environment via a plethora of surface receptors recognizing various soluble or membrane-bound ligands. Recognition of such ligands initiates intracellular signaling events resulting, depending on the overall cellular context, in the activation or inhibition of various immune effector mechanisms, cell differentiation, and/or chemotaxis. It is known that the transmembrane adaptor protein, phosphoprotein associated with glycosphingolipid-enriched membrane microdomains (PAG), is an anchor of C-terminal Src kinase, a potent inhibitor of Src family kinases, our studies with bone marrow mast cells (BMMCs) derived from mice with PAG knockout (KO) showed that PAG enhances FcεRI-induced degranulation and chemotaxis responses, but inhibits c-KIT-induced degranulation [6, 7]. Other studies indicated an essential role of actin and tubulin in mast cell signaling [19,20,21,22,23,24,25], but the connection between the plasma membrane located adaptor proteins, other signal transduction molecules, and cytoskeletal components is not fully understood
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