The ras-GAP associated protein, p62, is a major tyrosine phosphoprotein in transformed and growth factor treated cells. Although its exact function is not known, it can bind directly to src-family tyrosine kinases and has been implicated as a linker protein bridging activated src family tyrosine kinases with downstream effectors. One novel feature of p62, revealed by its predicted amino acid sequence, is the presence of an RNA-binding region, the KH domain. As p62 becomes tyrosine phosphorylated when src-kinases become activated, we compared the RNA binding ability of p62 in both its phosphorylated and unphosphorylated state. The ability of p62 to bind RNA was severely impaired when p62 was tyrosine phosphorylated. This suggests that the ability of p62 to bind RNA is regulated by tyrosine phosphorylation and implicates the regulation of RNA as a component of tyrosine kinase signaling pathways.

Murine natural killer cells (NK) express lectin-like activation and inhibitory receptors, including the CD94/NKG2 family of receptors that bind Qa-1, and the Ly-49 family that recognizes major histocompatibility complex class I molecules. Here, we demonstrate that cross-linking of NK cells with a new specific anti–Ly-49H mAb induced NK cell cytotoxicity and cytokine production. Ly-49H is expressed on a subset of NK cells and can be coexpressed with Ly-49 inhibitory receptors. However, unlike Ly-49 inhibitory receptors, Ly-49H is not detectable on naive splenic CD3+ T cells, indicating that Ly-49H may be an NK cell–specific activation receptor. In further contrast to the stochastically expressed Ly-49 inhibitory receptors, Ly-49H is preferentially expressed with the Ly-49D activation receptor, and expression of both Ly-49H and Ly-49D is augmented on NK cells that lack receptors for Qa-1 tetramers. On developing splenic NK1.1+ cells, Ly-49D and Ly-49H are expressed later than the inhibitory receptors. These results directly demonstrate that Ly-49H activates primary NK cells, and suggest that expression of Ly-49 activation receptors by NK cells may be specifically regulated on NK cell subsets. The simultaneous expression of multiple activation receptors by individual NK cells contrasts with that of T cell antigen receptors and is relevant to the role of NK cells in innate immunity.

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Murine NK cells express inhibitory receptors belonging to the C-type lectin-like (Ly-49, CD94/NKG2) and Ig superfamily-related (gp49) receptors. The murine gp49B receptor displays structural homology with human killer inhibitory receptors, and was previously identified to be a receptor on mast cells and activated NK cells. The gp49B receptor is highly related to gp49A, a receptor with unknown function. In this study, using a novel mAb produced against soluble gp49B molecules that cross-reacts with gp49A, we examined the cellular distribution and function of these receptors. gp49 is constitutively expressed on cells of the myeloid lineage throughout development, as well as on mature cells. Importantly, gp49 is not expressed on spleen- and liver-derived lymphocytes, including NK cells, but its expression is induced in vitro on NK cells following IL-2 stimulation, or in vivo by infection with murine CMV. Molecular studies revealed that both the immunoreceptor tyrosine-based inhibitory motif-containing gp49B as well as immunoreceptor tyrosine-based inhibitory motif-less gp49A receptors are up-regulated on NK cells following murine CMV infection. When co-cross-linked with NK1.1, gp49B can inhibit NK1.1-mediated cytokine release by NK cells. Taken together, these studies demonstrate that the expression of gp49B on NK cells is regulated, providing the first example of an in vivo activation-induced NK cell inhibitory receptor, in contrast to the constitutively expressed Ly49 family.

Mouse gp49B is an immunoreceptor tyrosine-based inhibitory motif-bearing receptor identified on mast cells and NK cells. In this report, however, we show that this receptor is expressed on macrophages accumulating in the uterine metrial gland in midgestation, along with gp49A that has a very homologous extracellular domain with gp49B but has a short cytoplasmic domain without ITIM. Culture of bone marrow cells in the conditioned medium of the metrial gland resulted in the selective proliferation of macrophages expressing both Fcγ-activating receptors and gp49B inhibitory receptor. Stimulation of macrophages with immobilized IgG, but not with anti-FcγRII/III, induced a considerable amount of TNF-α and IL-10 production, suggesting that the high-affinity receptor for IgG (FcγRI) can transmit activating signals in cytokine production of macrophages. Furthermore, coligation of gp49B with FcγRI resulted in the inhibition of TNF-α production. Thus, our data provide evidence that gp49B is an endogenous negative regulator of macrophage activation and may regulate the function of macrophages during pregnancy.

Inhibitory receptors on hemopoietic cells critically regulate cellular function. Despite their expression on a variety of cell types, these inhibitory receptors signal through a common mechanism involving tyrosine phosphorylation of the immunoreceptor tyrosine-based inhibitory motif (ITIM), which engages Src homology 2 (SH2) domain-containing cytoplasmic tyrosine or inositol phosphatases. In this study, we have investigated the proximal signal-transduction pathway of an ITIM-bearing receptor, gp49B, a member of a newly described family of murine NK and mast cell receptors. We demonstrate that the tyrosine residues within the ITIMs are phosphorylated and serve for the association and activation of the cytoplasmic tyrosine phosphatase SHP-1. Furthermore, we demonstrate a physiologic association between gp49B and SHP-1 by coimmunoprecipitation studies from NK cells. To address the mechanism of binding between gp49B and SHP-1, binding studies involving glutathione S-transferase SHP-1 mutants were performed. Utilizing the tandem SH2 domains of SHP-1, we show that either SH2 domain can interact with phosphorylated gp49B. Full-length SHP-1, with an inactivated amino SH2 domain, also retained gp49B binding. However, binding to gp49B was disrupted by inactivation of the carboxyl SH2 domain of full-length SHP-1, suggesting that in the presence of the phosphatase domain, the carboxyl SH2 domain is required for the recruitment of phosphorylated gp49B. Thus, gp49B signaling involves SHP-1, and this association is dependent on tyrosine phosphorylation of the gp49B ITIMs, and an intact SHP-1 carboxyl SH2 domain.

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