Mechanisms controlling the disassembly of ezrin/radixin/moesin (ERM) proteins, which link the cytoskeleton to the plasma membrane, are incompletely understood. In lymphocytes, chemokine (e.g., SDF-1) stimulation inactivates ERM proteins, causing their release from the plasma membrane and dephosphorylation. SDF-1–mediated inactivation of ERM proteins is blocked by phospholipase C (PLC) inhibitors. Conversely, reduction of phosphatidylinositol 4,5-bisphosphate (PIP2) levels by activation of PLC, expression of active PLC mutants, or acute targeting of phosphoinositide 5-phosphatase to the plasma membrane promotes release and dephosphorylation of moesin and ezrin. Although expression of phosphomimetic moesin (T558D) or ezrin (T567D) mutants enhances membrane association, activation of PLC still relocalizes them to the cytosol. Similarly, in vitro binding of ERM proteins to the cytoplasmic tail of CD44 is also dependent on PIP2. These results demonstrate a new role of PLCs in rapid cytoskeletal remodeling and an additional key role of PIP2 in ERM protein biology, namely hydrolysis-mediated ERM inactivation.

IntroductionAn adequate immune response is the result of a fine balance between a multitude of activating and inhibitory signals, and disruption of this delicate balance can lead to autoimmunity or immunodeficiency. Activation signals can be negatively regulated by cell surface receptors bearing immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic tail. 1 Examples of ITIM-containing receptors expressed on B cells include Fc␥RIIB, CD22, CD72, paired Ig-like receptor (PIR)-B, CD85j, Fc receptorlike (FCRL)4, and CD305. [2][3][4][5] The coligation of the B-cell receptor (BCR) and ITIM-containing receptors results in the attenuation of BCR-mediated signals. 3,5,6 Depending on the developmental stage or activation status, B cells express different sets of inhibitory receptors on their cell surface. 5,7,8 For example, CD305 is highly expressed on naive human B cells, and its expression is low in memory B cells, 5 whereas FCRL4 is mostly expressed on a subset of memory cells and is almost absent on naive B cells. 7 The expression of certain ITIM-containing receptors, such as FCRL4 and CD85j, is increased in specific B-cell subsets that are substantially expanded in certain disease settings, such as in HIV-infected viremic patients with high viral loads 9 and in persons exposed to Plasmodium falciparum. 10 It is acknowledged that the deregulation of the expression of these receptors contributes to the B-cell dysfunctions observed in HIV and malarial chronic infections. 11 CD300a belongs to the CD300 family of activating/inhibitory receptors whose genes are clustered in human chromosome 17. 12 CD300a is a type I transmembrane receptor with an IgV-like extracellular domain and a cytoplasmic tail that has 3 classic ITIM motifs 12,13 and is expressed on cells of both lymphoid and myeloid lineages. 12 Its ligation is capable of inhibiting natural killer cell-mediated cytotoxicity, 13,14 Fc␥RIIa-mediated reactive oxygen species production and Ca 2ϩ flux in neutrophils, 15 Fc⑀RI-mediated activation of mast cells, 16 and eosinophil responses to eotaxin, granulocyte-macrophage colony-stimulating factor, and IL-5. 17 In a murine model of asthma, treatment of mice with a bispecific antibody linking CD300a to CCR3 reversed remodeling and airway inflammation. 18 In another in vivo study, a bispecific antibody fragment linking CD300a to IgE was able to abrogate allergic reactions. 19 Finally, a recent study showed that a bispecific antibody that links kit with CD300a abrogates the allergic reaction induced by stem cell factor in a model of anaphylaxis. 20 All these studies highlight the potential of specifically targeting CD300a for therapeutic purposes.In this study, we demonstrate that CD300a is differentially expressed on human B-cell subsets and can function as a negative regulator of BCR-mediated signaling. Furthermore, we show that the expression of this receptor is deregulated in HIV-infected patients, suggesting a potential role for CD300a in HIV-associated disease progression. The online version of thi...

Human naïve CD4 T cells express low levels of the immunomodulatory receptor CD300a, whereas effector/memory CD4 cells can be either CD300a+ or CD300a−. This suggested that CD300a expression could define a specific subset within the effector/memory CD4 T cell subpopulations. In fact, ex vivo analysis of the IFN-γ producing CD4 T cells showed that they are enriched in the CD300a+ subset. Moreover, stimulated CD4 T cells producing TNF-α and IL-2 besides IFN-γ (polyfunctional) are predominantly CD300a+. In addition to producing markedly higher levels of Th1-associated cytokines, the stimulated CD300a+ CD4 T cells are distinguished by a striking up-regulation of the T-box transcription factor eomesodermin (Eomes), whereas T-bet is up-regulated in both CD300a+ and CD300a− activated CD4 T cells to similar levels. The pleiotropic cytokine TGF-β1 has a determinant role in dictating the development of this Th1 subset, as its presence inhibits the expression of CD300a and down-regulates the expression of Eomes and IFN-γ. We conclude that CD300a+ human Th1 cells tend to be polyfunctional and after stimulation up-regulate Eomes.

B-cell receptor (BCR)-induced activation of phospholipase C-gamma1 (PLCgamma1) and PLCgamma2 is crucial for B-cell function. While several signaling molecules have been implicated in PLCgamma activation, the mechanism coupling PLCgamma to the BCR remains undefined. The role of PLCgamma1 SH2 and SH3 domains at different steps of BCR-induced PLCgamma1 activation was examined by reconstitution in a PLCgamma-negative B-cell line. PLCgamma1 membrane translocation required a functional SH2 N-terminal [SH2(N)] domain, was decreased by mutation of the SH3 domain, but was unaffected by mutation of the SH2(C) domain. Tyrosine phosphorylation did not require the SH2(C) or SH3 domains but depended exclusively on a functional SH2(N) domain, which mediated the association of PLCgamma1 with the adapter protein, BLNK. Forcing PLCgamma1 to the membrane via a myristoylation signal did not bypass the SH2(N) domain requirement for phosphorylation, indicating that the phosphorylation mediated by this domain is not due to membrane anchoring alone. Mutation of the SH2(N) or the SH2(C) domain abrogated BCR-stimulated phosphoinositide hydrolysis and signaling events, while mutation of the SH3 domain partially decreased signaling. PLCgamma1 SH domains, therefore, have interrelated but distinct roles in BCR-induced PLCgamma1 activation.

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