gp49B1 Inhibits IgE-initiated Mast Cell Activation through Both Immunoreceptor Tyrosine-based Inhibitory Motifs, Recruitment ofsrc Homology 2 Domain-containing Phosphatase-1, and Suppression of Early and Late Calcium Mobilization

Abstract

We define by molecular, pharmacologic, and physiologic approaches the proximal mechanism by which the immunoglobulin superfamily member gp49B1 inhibits mast cell activation mediated by the high affinity Fc receptor for IgE (Fc⑀RI). In rat basophilic leukemia-2H3 cells expressing transfected mouse gp49B1, mutation of tyrosine to phenylalanine in either of the two immunoreceptor tyrosine-based inhibitory motifs of the gp49B1 cytoplasmic domain partially suppressed gp49B1-mediated inhibition of exocytosis, whereas mutation of both abolished inhibitory capacity. Sodium pervanadate elicited tyrosine phosphorylation of native gp49B1 and association of the tyrosine phosphatases src homology 2 domain-containing phosphatase-1 (SHP-1) and SHP-2 in mouse bone marrow-derived mast cells (mBMMCs). SHP-1 associated transiently with gp49B1 within 1 min after coligation of gp49B1 with cross-linked Fc⑀RI in mBMMCs. SHP-1-deficient mBMMCs exhibited a partial loss of gp49B1-mediated inhibition of Fc⑀RI-induced exocytosis at concentrations of IgE providing optimal exocytosis, revealing a central, but not exclusive, SHP-1 requirement in the counter-regulatory pathway. Coligation of gp49B1 with cross-linked Fc⑀RI on mBMMCs inhibited early release of calcium from intracellular stores and subsequent influx of extracellular calcium, consistent with SHP-1 participation. Because exocytosis is complete within 2 min in mBMMCs, our studies establish a role for SHP-1 in the initial counter-regulatory cellular responses whereby gp49B1 immunoreceptor tyrosine-based inhibition motifs rapidly transmit inhibition of Fc⑀RI-mediated exocytosis.The cross-linking of the high affinity Fc receptor for IgE (Fc⑀RI) 1 on the surface of mast cells activates intracellular signaling pathways that lead to exocytosis of preformed secretory granule components, generation of membrane-derived lipid mediators, and elaboration of immunoregulatory and proinflammatory cytokines. Through the release of their array of bioactive mediators, mast cells have an unusually high proinflammatory potential and contribute to host defense in animal models (1-3) and to the pathogenesis of allergic reactions and bronchial asthma in humans. In addition to Fc⑀RI, mouse mast cells express on their surface gp49B1 (4, 5), an immunoglobulin (Ig) superfamily member with two Ig-like domains (6) and two cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs), which have the amino acid sequence (I/V/S)XYXX(L/V) (7-12). We have shown that when rat IgE bound to Fc⑀RI on mouse interleukin-3-dependent, bone marrow-derived mast cells (mBMMCs) is both cross-linked to itself and coligated with rat anti-gp49B1 mAb bound to gp49B1, by means of F(abЈ) 2 mouse anti-rat IgG (heavy and light chain reactive), the release of secretory granule-and lipid-derived mediators is inhibited (9). As defined by ALIGN amino acid homology analysis, gp49B1 belongs to a family of receptors that has immunoregulatory potential (9, 13). In the mouse, the sole other member of this homology-based family (14) that...

gp49B-Mediated Negative Regulation of Antibody Production by Memory and Marginal Zone B Cells

“…Thus, gp49B may regulate a specific pathway downstream of CD40, namely, a pathway promoting B cell differentiation to plasma cells. gp49B contains two ITIMs in its cytoplasmic tail and has been shown to inhibit cellular activation by recruiting SHP-1 and SHP-2 in mast cells (26)(27)(28)(29). A similar mechanism may regulate the CD40 signaling pathway in MZ and memory B cells.…”

“…We demonstrate in this article that gp49B (also termed Lilrb4) is dominantly expressed on memory and MZ B cells. Although gp49A has a short cytoplasmic tail that lacks any specific signaling motifs, gp49B contains two ITIMs in its cytoplasmic tail that mediate inhibition of cellular activation by recruiting SHP-1 and SHP-2 (26)(27)(28)(29). gp49B has been shown to bind to the integrin a v and b 3 (a v b 3 ), which is expressed on a variety of cells including activated T cells, and this interaction inhibits IgE-dependent degranulation of mast cells (30)(31)(32).…”

Impaired Kit- but Not FcεRI-initiated Mast Cell Activation in the Absence of Phosphoinositide 3-Kinase p85α Gene Products

“…The cells were lysed at 5 ϫ 10 7 cells/ml with 1% Nonidet P-40 extraction buffer (60). For Western blotting, 20 l of lysate (1 ϫ 10 6 cell equivalents) was mixed with 10 l of 3ϫ sample buffer containing 15% ␤-mercaptoethanol and loaded on precast Trisglycine gels (Novex).…”

The gp49B1 Inhibitory Receptor Regulates the IFN-γ Responses of T Cells and NK Cells

“…Although highly homologous to gp49B1 (88% overall amino acid identity), gp49A has a much shorter cytoplasmic domain, lacks the two ITIMs present in gp49B1, and activates mast cells when crosslinked by Ab (18,19,21). gp49B1 inhibits mast cell activation in vitro by recruiting Src homology protein-1 when co-cross-linked with Fc⑀RI (22). Moreover, mast cells in gp49B1-deficient mice exhibit increased sensitivity to Fc⑀RI-dependent mast cell activation that leads to greater tissue inflammation (23).…”