Immune responses must be tightly regulated to avoid hypo-responsiveness on one-hand or excessive inflammation and the development of autoimmunity (hyper-responsiveness) on the other. This balance is at least partially attained through the throttling of activating signals by inhibitory signals. This ideally leads to an adequate immune response against an invader without excessive and extended inflammatory signals that promote the development of autoimmunity. The CD94/NKG2 family of receptors is composed of members with activating or inhibitory potential. These receptors are expressed predominantly on NK cells and a subset of CD8 T cells, and they have been shown to play an important role in regulating responses against infected and tumorigenic cells. Our studies explore all aspects of the biology of these receptors, including ligand and receptor interaction, signaling, receptor synapses, lytic mechanisms, membrane dynamics, and regulation of gene expression. A current emphasis is to understand, at the cell biology and molecular levels, how the the CD94/NKGA inhibitory receptor inactivates signals generated by activation receptors in a dominating manner and by what mechanism this receptor traffics so as to maintain constant presence on the cell surface. Expression of abundant levels of inhibitory receptors, one of which is CD94/NKG2A, must be maintained to suppress unwarranted activation in normal circumstances and to help regulate potentially overzealous responses in combating disease. To maintain cell surface CD94/NKG2A expression levels, NK cells must deal with the fact that CD94/NKG2A is constantly exposed to its ligand, HLA-E, expressed by surrounding cells. In many cases, ligand exposure tends to induce receptor downregulation. We know from previous studies that CD94/NKG2A is long-lived and continuously recycles to the cell surface and that the interaction with ligand does not lead to its downregulation. We investigated CD94/NKG2A endocytosis and found that it occurs by an amiloride-sensitive, Rac1-dependent pinocytic process;however, it does not require clathrin, dynamin, ADP ribosylation factor-6, phosphoinositide-3 kinase or the actin cytoskeleton. Once endocytosed, CD94/NKG2A traffics to early endosomal antigen 1+, Rab5+ early endosomes. It does appear in Rab4+ early/sorting endosome, but, in the time period examined, fails to reach Rab11+ recycling or Rab7+ late endosomes or lysosome-associated membrane protein-1+ lysosomes. These results indicate that CD94/NKG2A utilizes a previously undescribed endocytic mechanism coupled with an abbreviated trafficking pattern, perhaps to insure surface expression. The uptake of solutes and particles by cells, as well as most surface receptors, occurs through pinocytosis. Macro- and micropinosomes share the characteristic of engulfment of the extracellular fluid, but are morphologically distinguished by their size, greater or lesser than 0.2 um, respectively. It is not yet clear that how many types of pinocytic vesicles exist and how many pathways are involved in their endocytosis;however, it is clear that distinguishing these pathways by the size of the endocytic vesicle utilized is somewhat arbitrary. Keeping this in mind, the endocytosis of CD94/NKG2A is clearly a fluid-phase pinocytic process as it is co-endocytosed with fluid-phase markers. The fact that CD94/NKG2A endocytic vesicles are much greater in diameter (0.5-1.5 um) than micropinosomes (<0.2 um) clearly lead us to morphologically characterize them as macropinosomes. The fact that internalized CD94/NKG2A colocalizes with dextran and lucifer yellow, and that its endocytosis is amiloride sensitive and Rac1 dependent supports this conclusion. The cell adhesion molecules ICAM-1 and platelet endothelial cell adhesion molecule-1 are internalized by a macropinocytic mechanism that is independent of clathrin, caveolin and PI3K activity;however, this internalization process requires actin and dynamin activity. The fact that CD94/NKG2A internalization does not require actin, dynamin or Arf6 and is insensitive to PI3K inhibition and PKC stimulation clearly distinguishes it from previously discussed macropinocytic mechanisms. However, despite the fact that CD94/NKG2A-containing endocytic vesicles are too large to fit the definition of a micropinocytosis, they do share, in addition to PI3K independence, the notable feature of actin independency. We arbitrarily chose to term CD94/NKG2A endocytosis as macropinocytic-like realizing that it is clearly biochemically distinguishable from previously described macropinocytic mechanisms. NKG2D/DAP10 is an activation receptor expressed by NK and subsets of T cells, whose ligands include MHC class I chain-related (MIC) protein A and protein B and UL16-binding proteins that are often up-regulated by stress or pathological conditions. DAP10 is required for NKG2D/DAP10 cell surface expression and signaling capacity. Little is known about the mechanisms that regulate DAP10 gene expression. We are currently investigating how the expression of DAP10 is regulated by cytokines, thereby regulating the expression of NKG2D. In addition to initiating signaling cascades leading to mast cell mediator release, aggregation of the high affinity IgE receptor (FceRI) leads to rapid internalization of the cross-linked receptor. However, little is known about the trafficking of the internalized FceRI. Here we demonstrate that in RBL-2H3 cells, aggregated FceRI appears in the early endosomal antigen 1 (EEA1+) domains of the early endosomes within 15 minutes after ligation. Minimal co-localization of FceRI with Rab5 was observed by 30 minutes, followed by its appearance in the Rab7+ late endosomes and lysosomes at later time points. During endosomal sorting, FceRI a and g subunits remain associated. In Syk-deficient RBL-2H3 cells, the rate of transport to lysosomes is markedly increased. During the initial 30 minutes of endocytic traffic of antigen-ligated receptor, the signaling potential of early endosomes, as assessed by protein tyrosine phosphorylation, was enhanced. Taken together, our data demonstrate time-dependent sorting of aggregated FcRI within the endosomal-lysosomal network, and that Syk may play an essential role in regulating the trafficking and retention of FceRI in endosomes. Furthermore, our data suggest that receptor trafficking to early endosomes may be accompanied by ongoing signal propagation.

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Peruzzi, Giovanna; Femnou, Laurette; Gil-Krzewska, Aleksandra et al. (2013) Membrane-type 6 matrix metalloproteinase regulates the activation-induced downmodulation of CD16 in human primary NK cells. J Immunol 191:1883-94
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